Arh Hig Rada Toksikol 65/2

ARHIV ZA HIGIJENU RADA I TOKSIKOLOGIJU ARCHIVES OF INDUSTRIAL HYGIENE AND TOXICOLOGY

THE OFFICIAL JOURNAL OF Croatian Medical Association – Croatian Society on Occupational Health Croatian Society of Toxicology Slovenian Society of Toxicology Croatian Radiation Protection Association

PUBLISHER Institute for Medical Research and Occupational Health, Zagreb, Republic of Croatia EDITOR IN CHIEF Nevenka Kopjar ASSISTANT EDITORS Irena Brčić Karačonji Ivan Kosalec Jelena Macan REGIONAL EDITOR FOR SLOVENIA Marija Sollner Dolenc MANUSCRIPT EDITOR & EDITORIAL ASSISTANT Dado Čakalo COPY EDITORS Dado Čakalo Makso Herman Željana Pavlaković TECHNICAL EDITING & LAYOUT Nevenka Kopjar Makso Herman STATISTICS EDITOR Jelena Kovačić CROATIAN LANGUAGE REVISION Ivanka Šenda SUBSCRIPTIONS Vesna Lazanin EXECUTIVE EDITORIAL BOARD Ivan Bešlić (Croatia) Irena Brčić Karačonji (Croatia) Selma Cvijetić Avdagić (Croatia) Domagoj Đikić (Croatia) Zdenko Franić (Croatia) Azra Huršidić Radulović (Croatia) Ivan Kosalec (Croatia)

Jelena Kovačić (Croatia) Zrinka Kovarik (Croatia) Jernej Kužner (Slovenia) Ana Lucić Vrdoljak (Croatia) Jelena Macan (Croatia) Marin Mladinić (Croatia) Mirjana Pavlica (Croatia)

Alica Pizent (Croatia) Biserka Radošević-Vidaček (Croatia) Marija Sollner Dolenc (Slovenia) Maja Šegvić Klarić (Croatia) Želimira Vasilić (Croatia)

ADVISORY EDITORIAL BOARD Mohammad Abdollahi (Iran) Gjumrakch Aliev (USA) Jiri Bajgar (Czech Republic) Janusz Blasiak (Poland) Stephen W. Borron (USA) Vlasta Bradamante (Croatia) Metka V. Budihna (Slovenia) Petar Bulat (Serbia) P. Jorge Chedrese (Canada) Miroslav Cikrt (Czech Republic) Miran Čoklo (Croatia) Bhupendra P. Doctor (USA) Metoda Dodič Fikfak (Slovenia) Jagoda Doko Jelinić (Croatia) Vita Dolžan (Slovenia) Damjana Drobne (Slovenia) Hugh L. Evans (USA) Radovan Fuchs (Croatia)

Lars Gerhardsson (Sweden) Jasminka Godnić-Cvar (Austria) Milica Gomzi (Croatia) Andrew Wallace Hayes (USA) Michael C. Henson (USA) Yun-Hwa Peggy Hsieh (USA) Jasminka Ilich-Ernst (USA) Ljiljana Kaliterna Lipovčan (Croatia) Vladimir Kendrovski (Macedonia) Sanja Kežić (The Netherlands) Lisbeth E. Knudsen (Denmark) Krista Kostial (Croatia) Blanka Krauthacker (Croatia) Samo Kreft (Slovenia) Marcello Lotti (Italy) Ester Lovšin Barle (Slovenia) Richard A. Manderville (Canada) Velimir Matković (USA)

Domenico Franco Merlo (Italy) Saveta Miljanić (Croatia) Ana Mišurović (Montenegro) Kenneth A. Mundt (USA) Michael Nasterlack (Germany) Krešimir Pavelić (Croatia) Maja Peraica (Croatia) Martina Piasek (Croatia) Mirjana Radenković (Serbia) Zoran Radić (USA) Miloš B. Rajković (Serbia) Peter Raspor (Slovenia) Zvonko Rumboldt (Croatia) Miloš P. Stojiljković (Serbia) Marko Šarić (Croatia) Krešimir Šega (Croatia) Robert Winker (Austria)

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Covered by Science Citation Index Expanded, PubMed, and Scopus This journal is supported by the Ministry of Science, Education and Sports of the Republic of Croatia

ISSN 0004-1254

ARHIV ZA HIGIJENU RADA I TOKSIKOLOGIJU

ARCHIVES OF INDUSTRIAL HYGIENE AND TOXICOLOGY

Arh Hig Rada Toksikol • Vol. 65 • No. 2 • pp. 139-249 • ZAGREB, CROATIA 2014

CONTENTS Review Low-grade chronic inflammation perpetuated by modern diet as a promoter of obesity and osteoporosis

Jasminka Z. Ilich, Owen J. Kelly, Youjin Kim, and Maria T. Spicer

139

Hana Brborović, Ognjen Brborović, Vlatka Brumen, Gordana Pavleković, and Jadranka Mustajbegović

149

Roshan Kumar Sahu, Bhupendra Singh, Shubhini A. Saraf, Gaurav Kaithwas, and Kamal Kishor

157

Photochemical toxicity of drugs intended for ocular use

Sara Nafisi, Reza Heidari, Mohammad Ghaffarzadeh, Mojtaba Ziaee, Hossein Hamzeiy, Alireza Garjani, and Mohammad Ali Eghbal

169

Cytoprotective effects of silafibrate, a newlysynthesised siliconated derivative of clofibrate, against acetaminophen‑induced toxicity in isolated rat hepatocytes

Elizabeta Galić, Antonija Tadin, Nada Galić, Vilena Kašuba, Marin Mladinić, Ružica Rozgaj, Dolores Biočina-Lukenda, Ivan Galić, and Davor Želježić

179

Micronucleus, alkaline, and human 8-oxoguanine glycosylase 1 modified comet assays evaluation of glass-ionomer cements - in vitro

Nataša B. Sarap, Marija M. Janković, Dragana J. Todorović, Jelena D. Nikolić, and Milojko S. Kovačević

189

Environmental radioactivity in southern Serbia at locations where depleted uranium was used

Barbara Jeršek, Nataša Poklar Ulrih, Mihaela Skrt, Neda Gavarić, Biljana Božin, and Sonja Smole Možina

199

Effects of selected essential oils on the growth and production of ochratoxin A by Penicillium verrucosum

Daniela Jakšić Despot and Maja Šegvić Klarić

209

A year-round investigation of indoor airborne fungi in Croatia

János Varga, Sándor Kocsubé, Gyöngyi Szigeti, Nikolett Baranyi, Csaba Vágvölgyi, Daniela Jakšić Despot, Donát Magyar, Martin Meijer, Robert A. Samson, and Maja Šegvić Klarić

219

Occurrence of black Aspergilli in indoor environments of six countries

Irena Brčić Karačonji and Nataša Brajenović

225

Emil Srebočan

231

Original articles Are nurse presenteeism and patient safety culture associated: a cross-sectional study

Professional paper Evaluation of amphetamine-type stimulant abuse through hair analysis: Results from 12 years of work Letter to the Editor Inconsistency in the article “Metal bioaccumulation in common carp and rudd from the Topolnitsa reservoir, Bulgaria”

Zvonko Rumboldt

233

Nada Oršolić

237

A20

Viewpoint Što je to plagijat u znanosti?

Note Razvoj Zavoda za animalnu fiziologiju Prirodoslovno-matematičkog fakulteta Sveučilišta u Zagrebu - znanost i nastava u prošlosti i sadašnjosti New editions (in Croatian)

Cover page: The cover page shows Penicillium chrysogenum, a known producer of penicillin. The strain is stored in the Microbial Collection of the Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb. Photographed by Maja Šegvić Klarić Disclaimer: This photo is intended to evoke the content of this issue of the journal. It is not intended for instructional or scientific purposes.

139

Ilich JZ, et al. LGCI PROMOTES OBESITY AND OSTEOPOROSIS Arh Hig Rada Toksikol 2014;65:139-148

DOI: 10.2478/10004-1254-65-2014-2541

Review

Low-grade chronic inflammation perpetuated by modern diet as a promoter of obesity and osteoporosis Jasminka Z. Ilich1, Owen J. Kelly2, Youjin Kim3, and Maria T. Spicer1 Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, Florida1, Adult Clinical Nutrition, Abbott Nutrition, Columbus, Ohio2, USA, Bayer CropScience Ltd. Seoul, South Korea3 Received in May 2014 CrossChecked in May 2014 Accepted in June 2014

Some of the universal characteristics of pre-agricultural hominin diets are strikingly different from the modern human diet. Hominin dietary choices were limited to wild plant and wild animal foods, while the modern diet includes more than 70Â % of energy consumed from refined sugars, refined vegetable oils, and highly processed cereals and dairy products. The modern diet, with higher intake of fat has also resulted in a higher ratio of omega-6 (n-6) to omega-3 (n-3) polyunsaturated fatty acids (PUFA), contributing to low-grade chronic inflammation (LGCI) and thus promoting the development of many chronic diseases, including obesity and osteoporosis. In this review, we describe the changes in modern diet, focusing on the kind and amount of consumed fat; explain the shortcomings of the modern diet with regard to inflammatory processes; and delineate the reciprocity between adiposity and inflammatory processes, with inflammation being a common link between obesity and osteoporosis. We present the evidence that overconsumption of n-6 PUFA coupled with under-consumption of n-3 PUFA results in LGCI and, along with the increased presence of reactive oxygen species, leads to a shift in mesenchymal stem cells (precursors for both osteoblasts and adipocytes) lineage commitment toward increased adipogenesis and suppressed osteoblastogenesis. In turn, high n-6 to n-3 PUFA ratios in the modern diet, coupled with increased synthesis of pro-inflammatory cytokines due to adiposity, propagate obesity and osteoporosis by increasing or maintaining LGCI. KEY WORDS: adipocytes; cytokines; eicosanoids; mesenchymal stem cells; omega-3 fatty acids; omega-6 fatty acids; osteoblasts; osteoclasts; polyunsaturated fatty acids

Abbreviations: 5-LOX=5-Lipoxygenase; AA=Arachidonic acid; AHA=American Heart Association; ALA=Alpha-linoleic acid; CLA=Conjugated Linolenic Acid; COX-2=Cyclooxygenase-2; CRP=C-reactive protein; DHA=Docosahexanoic Acid; EPA=Eicosapentanoic Acid; IGBP-3=Insulin like growth factor binding protein-3; IL-1=Interleukin-1; IL-6=Interleukin-6; LA=Linolenic Acid; LBT4=Leukotriene B4; LBT5=Leukotriene B5; LGCI=Low grade chronic inflammation; MSC=Mesenchymal stem cells; MUFA=Monounsaturated Fatty Acids; n-3=Omega-3; n-6=Omega-6; NHANES=National Health and Nutrition Examination Survey; OPG=Osteoprotegerin; PDE=Phosphodiesterase; PGE2=Prostaglandin E2; PGE3=Prostaglandin E3; PUFA=Polyunsaturated Fatty Acids; RANK=Receptor activator of nuclear factor kappa-B; RANKL=Receptor activator of nuclear factor kappa-B ligand (RANKL); SFA=Saturated Fatty Acids; TNF-Îą=Tumor Necrosis Factor-alpha; TXA2=Thromboxane A2

Dietary intake and other lifestyle factors (e.g. physical activity, smoking) are environmental modifiers that have changed dramatically with agriculture and animal husbandry, about 10,000 years ago (1, 2).

However, a number of anthropological, nutritional, and genetic studies indicate that even more drastic changes

happened very recently, some 200 years ago with the onset of industrial revolution, and that these changes could be associated with many chronic diseases or the so-called diseases of civilization (1, 3).

By examining the dietary habits along the evolutionary timeline, one can easily notice the

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difference between the pre-agricultural hominine and modern human diet. Hominin dietary choices were limited to wild plant and animal foods. In contrast, 72Â % of the energy consumed by modern humans includes refined sugars, refined vegetable oils, highly processed cereals and dairy products, as well as high consumption of alcohol (4).

The objectives of this review are to: a) describe the changes in modern diet with regard to several nutrients, particularly addressing the type and amount of consumed fat; b) explain the shortcomings of the modern diet with regard to inflammatory processes; and c) delineate the reciprocity between adiposity and inflammatory processes and the link that connects obesity, osteoporosis, and inflammation. We present the hypothesis that omega-6 (n-6) polyunsaturated fatty acids (PUFA) propagate obesity and osteoporosis by increasing/maintaining low-grade chronic inflammation (LGCI), ultimately shifting the commitment of mesenchymal stem cells (MSC) toward increased adipogenesis.

Discordance between current and past dietary habits along the evolutionary timeline Our genetic makeup has not changed over the last few centuries, but the environment and dietary behaviors have, substantially. The spontaneous mutation rate for nuclear DNA is estimated at 0.5Â % per million years; thus, very little change has occurred in our genes compared to our Paleolithic ancestors of 40,000 years ago, when our genetic profile was

established. Therefore, the profound environmental and dietary changes that began with the introduction of agriculture and animal husbandry some 10,000 years ago, occurred too recently on the evolutionary timeline for the human genome to adapt, contributing to many of the chronic diseases in modern societies (5).

Some nutrients exhibit high discordance regarding their intake in the hominines compared to modern humans that may have contributed to inflammation and subsequent chronic diseases, including obesity and osteoporosis. Of note are the lower consumption of fat, carbohydrates, and sodium (high amounts of the latter might result in negative consequences on bone health via increased calcium excretion) and much higher consumption of calcium and vitamin C (both important in bone health and the former one in weight management). Additionally, with the high intake of plant foods, hominines were consuming much higher amounts of fiber (has a role in weight management) and phytochemicals, known for their anti-oxidative and anti-inflammatory properties. Table 1 compares the intake of hominines and current intake in the USA, as well as the current recommendations of selected nutrients in the USA (adapted from 2, 6, 7).

Changes in fat intake and their subsequent effect on omega-6 (n-6) and omega-3 (n-3) PUFA ratio Among the most salient changes were those in the dietary fat intake, specifically in the type and amount of fatty acids (1, 3, 8). The amount and kind of fat reflected the consumption of primarily lean meat from

Table 1 Estimated intake of selected nutrients and foods by hominines from the late Paleolithic (~40,000 years ago) and modern USA population and current recommendations

Nutrients/ Foods

Late Paleolithic

Current USA Intake

Current USA recommendations

Fat (% of energy)

21

33

20-35

Carbohydrates (% of energy)

46

50

45-65

Protein (% of energy)

33

15

10-35

Alcohol (serving per day)

Minimal

N/A

1-2

P/S ratio

1.4

0.7

N/A

Cholesterol (mg per day)

520

261

<300

100-150

16

38

Sodium (mg per day)

690

3463

<2300

Calcium (mg per day)

1500-2000

1029

1000-1300

440

87

75-95

Fiber (mg per day)

Vitamin C (mg per day) Adapted from references (2, 6, 7)

P/S - polyunsaturated to saturated fatty acid ratio; N/A - data not available

141

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wild animals, low in saturated fatty acids (SFA) and high in both PUFA and monounsaturated fatty acids (MUFA), with low n-6 to n-3 PUFA ratio. Prior to the Neolithic (agricultural) period, or some 40,000 years ago in the Paleolithic, all animal foods consumed by hominines were limited to wild animals (9).

Approximately 50 % of fatty acids in the adipose tissue of wild animals were SFAs, while the dominant type in the muscle and all other organs were PUFA and MUFA. However, for most of the year animals’ subcutaneous and visceral body fat stores were usually depleted, and it was likely that PUFA and MUFA were the predominant fats of the carcass available for hominin consumption. Therefore, high amounts of SFA were probably not available to hominines feeding on these wild animals. Moreover, the consumption of seeds, nuts, various greens, and other wild plants provided the hominines with higher intake of PUFAs and MUFAs and probably with an optimal ratio of n-6 to n-3 (3, 8).

Subsequent changes that occurred with the beginning of animal husbandry and agriculture (in the Neolithic period, some 10,000 years ago) allowed for slaughtering of animals at the peak of body fat accumulation, while the production of grains and other agricultural products with higher SFA content led to an increasing consumption of n-6 PUFAs, creating a less favorable n-6 to n-3 ratio. Technological advances in the early and mid 19th century generated large quantities of grain (especially corn) for animals and humans, which changed the composition of the meat in the cattle, resulting in “marbled meat” characterized by excessive triglyceride accumulation and fat infiltration into the muscular tissue and higher SFA content (10). This and the modernization of food processing and preservation, as well as changes in food choices have altered the ratio between n-6 and n-3 fatty acids in the modern diet and resulted in its low n-3 fatty acid content (8, 9), favoring the development of chronic diseases.

According to numerous anthropological and epidemiological studies, humans evolved on a diet with a n-6 to n-3 PUFA ratio of approximately 1-2:1 (3), whereas this ratio in the current Western-type diet has increased to approximately 10-15:1 (1, 3, 8), as shown in Table 2 (adapted from 1, 3, 8). This suggests that even the current dietary guidelines (except for the recommendations from the American Heart Association for severe coronary patients, who need to lower their triglycerides) fall short of the intake of prehistoric humans. Table 3 (adapted from 1, 3, 8, 11) shows

Table 2 Ratio between n-6 and n-3 polyunsaturated fatty acids in different population groups

n-6/n-3 ratio

Population group Paleolithic/Neolithic

0.8-1

Mediterranean (prior to 1960)

1-2

Current Northern Europe

15

Current USA Adapted from references (1, 3, 8)

15-20

different recommendations for eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) as well as the n-6 to n-3 ratio. The Lyon Diet Heart Study (12) showed that the ratio of 4:1 could be cardioprotective.

The subjects in this study consumed less linoleic acid (n-6) and more of oleic (n-9) MUFA and α-linolenic acid (n-3), which resulted in higher plasma levels of oleic acid, linolenic acid, and EPA and lower levels of linoleic and arachidonic acid (AA), which was ultimately associated with a reduced risk of recurrent heart disease.

Pro- and anti-inflammatory metabolites of n-6 and n-3 fatty acids It is known that elevated n-6 PUFA levels stimulate pro-inflammatory processes and promote the pathogenesis of many diseases such as cardiovascular, neurological, autoimmune, as well as cancer, arthritis, and diabetes (3, 8). Recently however, it has been realized that various pro-inflammatory actions may Table 3 Intake of eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), the n-6 to n-3 ratio reported by NHANES, and recommendations

Intake recommendations NHANES (2009-2010) Dietary Guidelines (2010) AHA (healthy population) AHA (coronary patients) AHA (severe coronary patients)

EPA + DHA (g per day)

n-6/n-3 ratio

0.12

9:1

0.25

8:1

0.5

7:1

1

6:1

2-4

3:1

3

N/A

FDA GRAS level

Adapted from references (1, 3, 8, 11) NHANES - National Health and Nutrition Examination Survey;

AHA - American Heart Association

142 foster obesity and osteoporosis as well (11). In contrast, n-3 fatty acids and their derivatives have been shown to promote anti-inflammatory processes and to have some beneficial health effects on cardiovascular disease (8, 9), cognition in aging, function in the brain grey matter, and the development of eye retina (2, 8).

Eicosanoids, the metabolic products of the sequential elongation and desaturation processes of both n-6 and n-3 PUFA, are the signaling molecules responsible for the immune and inflammatory reactions in the body (13). They originate from the essential fatty acids, linoleic (n-6) and alpha linolenic (n-3). The major metabolites of n-6 PUFA are proinflammatory eicosanoids and include prostaglandin E2 (PGE2), leukotriene B4 (LBT4), and thromboxane A2 (TXA2), generated by lipid-oxidizing enzyme lipoxygenase-5 (5-LOX) and cyclooxygenase-2 (COX-2) (13). The major metabolites of n-3 PUFA are the anti-inflammatory eicosanoids and include prostaglandin E3 (PGE3), leukotriene B5 (LBT5), resolvins, and protectins, also generated by COX-2 and 5-LOX. During metabolism, n-6 and n-3 PUFAs compete for the desaturation enzymes (13, 14), and the higher amount of n-6 PUFA in the modern diet shifts the balance toward the production of proinflammatory eicosanoids (11). Figure 1 illustrates the metabolic pathways of the n-6 and n-3 PUFA series in generating various types of eicosanoids.

Acute and low-grade chronic inflammation and their resolutions The breakdown of fatty acid metabolites and the conversion of eicosanoids generates different pro- and anti-inflammatory products (Figure 2). The proinflammatory lipoxins A4 and B4 are the metabolites of AA, and their action is focused on neutrophil regulation (15), while the E-series resolvins (both E1 and E2) are metabolized from EPA by 5-LOX, and their action is anti-inflammatory (16). Protectins, the D-series resolvins (D1 and D2), and the macrophagespecific maresins are the metabolites of DHA (17), and these are associated with resolving the inflammation (18). Overall, n-3 PUFA are metabolized into antiinflammatory eicosandoids which are important to resolve inflammation otherwise triggered by any insult on the cell, ranging from a bruise to a bacterial infection or even by inflammatory signals from neighboring cells.

Acute inflammation activates the inflammatory cascade of prostaglandins, leukotrienes, and other inflammatory cytokines such as tumor necrosis factor-

Ilich JZ, et al. LGCI PROMOTES OBESITY AND OSTEOPOROSIS Arh Hig Rada Toksikol 2014;65:139-148

alpha (TNF-α), interleukin-6 (IL-6), and interferongamma which recruit neutrophils and macrophages.

When the insult is dealt with, either via apoptosis and phagocytosis of the infected cells and foreign bodies or via cleaning the local inflammation, resolvins, protectins, maresins, and lipoxins all work together to stop further recruitment of immune cells and the propagation of pro-inflammatory cytokines, so that the cell can return to its basal state (19, 20).

In contrast, LGCI is a state of persistent and unresolved inflammation, where pro- and antiinflammatory cytokines are elevated, and inflammation is not resolved (21). In the clinical context, this state results from the continuous presence of specific immune cells leading to 2-4-fold elevations in the circulating levels of pro- and anti-inflammatory cytokines, and more are recruited in a perpetual cycle (21). The exact PUFA metabolite composition of this unresolved or surplus inflammation is unknown (17), but it is likely that there is a bias towards the proinflammatory eicosanoids, particularly with higher n-6 PUFAs intake (Figure 3).

The detrimental role of low-grade chronic inflammation in obesity and osteoporosis Obese individuals have abnormal circulating levels of TNF-α, IL-6, C-reactive protein (C-RP), adiponectin, and leptin, as these molecules are overexpressed in adipose tissue (22-24). PGE2, a metabolite of AA, has been found to mediate locally the biological action of TNF-α and Interleukin-1 (IL-1) in the cases of fever and local inflammation. Leptin and adiponectin are adipokines produced by adipose tissue that mediate chronic inflammation. Leptin has been found to stimulate inflammatory responses (25, 26), while adiponectin acts as an anti-inflammatory adipokine (27). The constant presence of these inflammatory cytokines associated with increased adiposity promotes persistent LGCI in obese individuals (1), while adipose tissue itself increases their production, resulting in a perpetual feedback cycle from one to another (adiposity↔inflammation). For more extensive review see Kelly et al. (11).

In the bone, inflammation also plays a detrimental role and is interconnected with adiposity. It is well established that IL-6 stimulates osteoclastogenesis (formation of bone-resorbing cells) and is considered an osteoresorptive factor (28). Simultaneously, higher amount of fat mass activates osteoclasts via increase in other cytokines like IL-1 and TNF-α, as shown in Figure 4. Osteoclasts are derived from the same

Ilich JZ, et al. LGCI PROMOTES OBESITY AND OSTEOPOROSIS Arh Hig Rada Toksikol 2014;65:139-148

143

Figure 1 Metabolism of essential linoleic (n-6) and alpha linolenic (n-3) fatty acids. An overproduction of arachidonic acid originating from the high intake of linoleic acid will metabolize into pro-inflammatory eicosanoids, which are implicated in many inflammatory and autoimmune disorders. Eicosapentanoic acid (EPA) and docosahexanoic acid (DHA), which are the derivatives of the essential n-3 alpha linolenic fatty acid (created from multiple elongation and desaturation reactions), form anti-inflammatory eicosanoids

hematopoietic stem cells as the immune cells (29). In LGCI accompanied by increased T-cell activation, osteoclastogenesis and bone resorption are accelerated, as the osteoclasts are programmed to respond to inflammatory signals. Therefore, in states such as LGCI, osteoclastogenesis and bone resorption are increased (11), leading to a perpetual feedback cycle (bone resorptionâ&#x2020;&#x201D;inflammation).

Mesenchymal stem cells (MSC) are the common precursors for osteoblasts (bone forming cells) and adipocytes (30). However, inflammation may deregulate the MSC lineage commitment (11, 31).

Even under normal homeostatic conditions, MSC slightly favors adipogenic differentiation. In LGCI, pro-inflammatory mediators in the presence of a high n-6 to n-3 ratio and reactive oxygen species (ROS), stimulate adipogenesis and suppress osteoblastogenesis, ensuring that MSC are committed to becoming adipocytes. In addition, high levels of IL-6, C-RP, and

TNF-Îą are associated with lower muscle mass in older people (32). Since muscle and bone mass decline with age, and fat mass increases, it is possible that agerelated MSC lineage commitment dysfunction occurring in LGCI and coinciding with obesity causes both osteopenia (loss of bone) and sarcopenia (loss of muscle). For the extensive review on the connection between bone, muscle, and fat, see Ilich et al. (31).

Recently, we investigated the effect of n-6 and n-3 PUFAs in different ratios on MSC proliferation and differentiation and found that the n-6 to n-3 ratio of 4:1 resulted in maximal osteoblastogenesis and minimal adipogenesis in a cell culture system (33).

This ratio is higher than in the prehistoric humans (e.g.

1:1), and is feasible for modern humans with some dietary modifications. Figure 5 illustrates the loop effect of modern (A) and prehistoric (B) diet on adipogenesis, osteoblastogenesis, and osteoclastogenesis.

144 Proposed mechanism of action The enzyme COX-2 has a higher affinity for n-6 than n-3 PUFAs and the former will be metabolized at a higher rate (13, 14). However, higher presence of n-3, especially at the 1:1 ratio, would increase the odds of more n-3 being metabolized, which would lower the production of inflammatory eicosanoids. Some COX-2 inhibitors have been observed to decrease osteoblast differentiation and increase adipogenesis (34). This indicates that COX-2 and its lipid products may have a role in promoting MSC differentiation into adipocytes rather than osteoblasts, and that the ratio between n-6-derived COX-2 and n-3-derived COX-2 products may be important in MSC regulation.

Another proposed mechanism concerns the receptor activated nuclear factor kappa-B (RANK) signaling and osteoprotegerin (OPG). RANK binds to its ligand (RANKL), which is secreted by preosteoblasts, and activates osteoclastogenesis and bone resorption. Conversely, OPG, which is also produced by preosteoblasts, inhibits osteoclastogenesis by binding to RANKL, and thus promotes osteoblastogenesis. Upregulated pro-inflammatory cytokines, including TNF-Îą, IL-1, and IL-6 in obese

Ilich JZ, et al. LGCI PROMOTES OBESITY AND OSTEOPOROSIS Arh Hig Rada Toksikol 2014;65:139-148

state seem to mediate osteopenia and osteoporosis by regulating the RANKL/RANK/OPG pathway (31, 35).

CONCLUSION Humans evolved on a diet low in saturated fat, carbohydrates, and sodium and much higher in calcium, vitamin C, fiber, and phytochemicals. Modern humans, however, consume high amounts of n-6 PUFA, about 10-15 higher than hominines. This elevates the ratio between n-6 to n-3 PUFA that stimulates proinflammatory processes and LGCI and promotes the pathogenesis of many chronic diseases, including obesity and osteoporosis. In contrast, n-3 PUFA and their derivatives, consumed by hominines in higher amounts, have been shown to promote anti-inflammatory processes and to have multiple beneficial health effects.

Obese individuals, who consume greater amounts of n-6 PUFA and exhibit higher circulating levels of pro-inflammatory cytokines (TNF-Îą, IL-6, C-RP), create the environment of a persistent LGCI, resulting in a perpetual feedback cycle. Furthermore, some cytokines (e.g. IL-6) stimulate osteoclastogenesis and

Figure 2 Further breakdown of fatty acid metabolites and the conversion of eicosanoids to pro- and anti-inflammatory products and their roles in the resolution of inflammation. PUFA - polyunsaturated fatty acids; AA - arachidonic acid; EPA - eicosapentanoic acid; DHA - docosahexanoic acid

Ilich JZ, et al. LGCI PROMOTES OBESITY AND OSTEOPOROSIS Arh Hig Rada Toksikol 2014;65:139-148

145

Figure 3 Hypothetical representation of acute versus low-grade chronic inflammation. The acute process is linear, since the inflammation is resolved, whereas the chronic process is a constant cycle in which the inflammation is persistent and unresolved. Cytokines are represented by colored shapes. Adapted from (11)

Figure 4 The increase in fat mass leads to overproduction of interleukin-6 (IL-6), interleukin-1 (IL-1), and tumor necrosis factor alpha (TNF-Îą). These cytokines increase bone resorption by stimulating the osteoclasts and inhibiting the osteoblasts

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Figure 5 The loop effect of modern (A) and Paleolithic (B) diet on adipogenesis, osteoblastogenesis, and osteoclastogenesis.

Over-consumption of n-6 PUFA coupled with under-consumption of n-3 PUFA results in increased synthesis of proinflammatory lipid mediators PGE2 and LTB4 by Cox-2 and 5-Lox, leading to peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which increases adipogenesis. This in turn produces more TNF-α, resulting in elevated osteoclastogenesis, and increases Cox-2 expression directly or indirectly via the platelet activating factor (PAF). Increased leptin production by adipose tissue can lower bone formation through its hypothalamic interactions. Furthermore, the elevated PGE2 and LTB4 result in an increase in the insulin-like growth factor-binding protein-3 (IGFBP-3), which inactivates the insulin-like growth factor-1 and promotes the differentiation of MSC to adipocytes. The net result of these events is an increased susceptibility to obesity and osteoporosis. For further review, see (11, 31)

are considered osteoresorptive. Both osteoblasts and adipocytes share MSC as their common precursor in the bone microenvironment. In conditions of LGCI, pro-inflammatory mediators in the presence of a high n-6 to n-3 ratio and reactive oxygen species stimulate adipogenesis and suppress osteoblastogenesis, shifting MSC differentiation toward adipocytes. The presented evidence shows that the increased synthesis of proinflammatory cytokines, mediated by the high n-6 to n-3 ratios, propagate obesity and osteoporosis by increasing or maintaining LGCI. In view of the recognized changes in dietary habits of modern humans compared to our ancestors, it would be prudent to decrease the consumption of n-6 fatty acids toward a more beneficial n-6 to n-3 PUFA ratio of 4:1 (as opposed to the current 15:1) in order to reduce obesity and improve bone health.

Acknowledgement This work was supported by the Hazel K. Stiebeling Professorship Award. The authors appreciate the help of Zein Al-Zomut in drafting the manuscript.

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De Gomez Dumm IN, Brenner RR. Oxidative desaturation of alpha-linoleic, linoleic, and stearic acids by human liver microsomes. Lipids 1975;10:315-7. PMID: 1134219 Nigam S, Fiore S, Luscinskas FW, Serhan CN. Lipoxin A4 and lipoxin B4 stimulate the release but not the oxygenation of arachidonic acid in human neutrophils: Dissociation between lipid remodeling and adhesion. J Cell Physiol 1990;143:512-23. PMID:2162850 Tjonahen E, Oh SF, Siegelman J, Elangovan S, Percarpio KB, Hong S, Arita M, Serhan CN. Resolvin E2: Identification and anti-inflammatory actions: Pivotal role of human 5-lipoxygenase in resolvin E series biosynthesis. Chem Biol 2006;13:1193-202. doi: 10.1016/j.chembiol.2006.09.011 Serhan CN, Chiang N, Van Dyke TE. Resolving inflammation: Dual anti-inflammatory and pro-resolution lipid mediators.

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Sažetak Stalna kronična upala niskoga stupnja zbog suvremenog načina prehrane potiče pretilost i osteoporozu Pojedina univerzalna svojstva prehrane hominina prije pojave poljodjelstva izrazito se razlikuju od suvremene prehrane. Prehrambene navike hominina vjerojatno su bile ograničene na plodove divljeg bilja i lovinu; više od 70 % moderne prehrane odnosi se na konzumaciju rafiniranih šećera, biljnih ulja, žitarica i mliječnih prerađevina. Moderna je prehrana s višim unosom masti također dovela do nepovoljnijeg omjera omega-6 i omega-3 višestruko nezasićenih masnih kiselina (engl. krat. PUFA), koji pridonosi održavanju kronične upale niskoga stupnja, a time i nastanku mnogih kroničnih bolesti, uključujući pretilost i osteoporozu. U ovom se preglednom članku opisuju promjene uslijed modernog načina prehrane, s posebnim osvrtom na vrste i količine konzumirane masti. Također se objašnjavaju nedostatci moderne prehrane s obzirom na upalne procese te međusobna povezanost između pretilosti i upalnih procesa, koji su usto i poveznica između pretilosti i osteoporoze. U članku se iznose saznanja o tome da pretjerana konzumacija omega-6 masnih kiselina uz nedostatnu konzumaciju omega-3 masnih kiselina dovodi do kronične upale niskoga stupnja i povišenih vrijednosti reaktivnih kisikovih čestica (ROS), a time i do pomaka u mezenhimskih matičnih stanica (prekursora osteoblasta i adipocita) prema povećanoj adipogenezi i smanjenoj osteoblastogenezi. Uz povećanu sintezu upalnih citokina zbog pretilosti, moderna prehrana s nepovoljnim omjerom omega-6 i omega-3 kiselina u korist prvih nastavlja poticati pretilost i osteoporozu jer održava i pogoršava kroničnu upalu.

KLJUČNE RIJEČI: adipociti; citokini; ikosanoidi; mezenhimske matične stanice; omega-3 masne kiseline; omega-6 masne kiseline; osteoblasti; osteoklasti; višestruko nezasićene masne kiseline

CORRESPONDING AUTHOR: Jasminka Z. Ilich, PhD, RD, FACN 418 SAN, Nutrition, Food and Exercise Sciences Florida State University, 120 Convocation Way Tallahassee, Fl 32306-1491, USA Email: jilichernst@fsu.edu

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DOI: 10.2478/10004-1254-65-2014-2462

Original article

Are nurse presenteeism and patient safety culture associated: a cross-sectional study Hana Brborović1, Ognjen Brborović2, Vlatka Brumen3, Gordana Pavleković2, and Jadranka Mustajbegović1 Department of Occupational Health and Sports Medicine1, Department of Social Medicine and Organization of Health Care2, University of Zagreb School of Medicine, Andrija Štampar School of Public Health, Department of Managerial Systems & Occupational Environment3, University of Applied Health Studies, Zagreb, Croatia Received in November 2013 CrossChecked in November 2013 Accepted in April 2014

Working as a nurse involves great dedication and sacrifice: working night shifts, working overtime, and coming to work sick. The last is also known as presenteeism. Research has shown that poor nurse performance can affect both caregiver’s and patient’s safety. The aim of this cross-sectional study was to investigate whether nurse presenteeism affected patient safety culture and to look deeper into the characteristics of nurse presenteeism and patient safety culture in Croatia. The study was conducted in one general hospital in Croatia over April and May 2012 and specifically targeted medical nurses as one of the largest groups of healthcare professionals. They were asked to fill two questionnaires: the six-item Stanford Presenteeism Scale (SPS-6) and the Hospital Survey on Patient Safety Culture (HSOPSC). We found no association between presenteeism and patient safety culture. Overall positive perception of safety was our sample’s strength, but other dimensions were positively rated by less than 65 % of participants. The lowest positive response concerned “nonpunitive response to error”, which is consistent with previous studies.

Presenteeist nurses did not differ in their characteristics from nurses without presenteeism (gender, age, years of experience, working hours, contact with patients and patient safety grades). Our future research will have to include a broader healthcare population for us to be able to identify weak spots and suggest improvements toward high-quality and cost-effective health care.

KEY WORDS: Hospital Survey on Patient Safety Culture; occupational health; sickness; Stanford Presenteeism Scale

Working as a nurse involves great dedication and sacrifice: working night shifts, working overtime, and coming to work sick. The last is also known as presenteeism. Presenteeism implies limited job performance due to a health problem (1-4). Research has shown that in occupations such as nursing, poor performance at work can have serious consequences for patient safety (5-7). Patient safety culture (PSC) is a set of individual and group values, attitudes, perceptions, competencies, and behavioural patterns towards health and safety (8). The World Health

Organization (WHO) has described PSC in five points: 1) all healthcare workers accept responsibility for the safety of themselves, their co-workers, patients, and visitors; 2) patient safety is a priority above financial and operational goals; 3) identification, communication, and resolution of safety issues are encouraged and rewarded; 4) learning from accidents without people being blamed; 5) appropriate resources, structure, and accountability are provided to maintain effective safety systems (9). We can say that PSC is actually a prerogative for patient safety. Studies have shown that

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up to 16.6 % of patients in acute care hospitals experience one or more adverse events (10-17) that are harmful or potentially harmful such as incorrect or late diagnosis, prescribing and medication, loss of medical documentation during patient’s transfer, errors in postoperative treatment, errors in the application of medical devices (such as needlestick injuries) (18).

When adverse events do not cause death or permanent damage to health, they lead to extended stays at the hospital and higher cost of treatment (19, 20).

There are 30,000 trained nurses in Croatia, 7,000 of whom hold a diploma degree. Most work in public health care.

Previous research has shown that healthcare workers are at increased risk of working sick and that teachers and nurses have the highest rates of presenteeism related to chronic diseases (21-23).

Letvak et al. (6) showed that nurse presenteeism was associated with an increase in medication errors and patient falls, as well as with poorer self-reported quality of care. A systematic review by Gartner et al.

(7) showed that chronic mental diseases, which are often a cause of presenteeism, were negatively associated with patient safety.

However, little is known about presenteeism among nurses in Croatia, hospital nurses in particular.

The aim of our study was to get a glimpse into presenteeism rates among Croatian hospital nurses and see if their presenteeism affects patient safety.

PARTICIPANTS AND METHODS This cross-sectional study included 148 nurses (of 194 invited) from the general hospital in Požega, Croatia. The hospital was selected based on a convenience sample of registered nurses. As the study was conducted in April and May 2012 while the first investigator attended postgraduate studies at the Zagreb University School of Medicine, we obtained an approval from the School’s Ethics Committee as well as from the hospital’s review board. All participants signed informed consents.

Survey and data collection In this study we used two questionnaires: the sixitem Stanford Presenteeism Scale (SPS-6) and the Hospital Survey on Patient Safety Culture (HSOPSC).

Both were translated into Croatian by one translator for each questionnaire and then back-translated into

English by an independent translator for each questionnaire, who was blinded to the original questionnaire.

The SPS-6 questionnaire consists of six items through which the nurses were asked to evaluate their performance in the face of a health problem (33) using a Likert scale from 1 to 5, where 1 corresponds to strong disagreement and 5 to strong agreement with the statement. Scores for items 1, 3, and 4 are then reversed, so that score 1 equals 5, 2 equals 4, and so on. The sum of all scores can range from 6 to 30.

Higher scores indicate better performance at work (24). For statistical analysis however, we needed a clear cut-off score, to divide respondents into groups whose performance was affected by presenteeism and those whose performance was not affected.

Accordingly, we set cut-off score for presenteeism in the lower quartile, which was 18. Furthermore, in a personal communication, the main author of SPS-6 also suggested 18 as a cut-off score (25).

The second, HSOPSC, questionnaire is a selfreporting tool used worldwide for assessing safety culture in hospitals (8). It has shown moderate-tostrong validity and reliability (26). The results of psychometric analyses for the Croatian version of HSOPSC have been presented and discussed elsewhere (27). Our translation of the US questionnaire consists of 42 items grouped in 12 dimensions and two single-item measures. Eight dimensions measure PSC at unit level, two at hospital level, and two are outcome measures. For three of the 12 dimensions frequency is reported on a five-point scale from “never” to “always”, whereas for the remaining nine dimensions agreement with the statement is reported on a five-point Likert scale from “strongly disagree” to “strongly agree”. Safety grade is a single-item evaluation of patient safety in the nurse’s unit and is scored on a scale from A to F, where higher score indicates higher patient safety level. The second single item measures the frequency of adverse event reports filed by the respondents over the past 12 months, and is scored as follows: no adverse event reports - 0 points; 1 to 2 reports - 1 point; 3 to 5 reports - 2 points; 6 to 10 reports - 3 points, 11 to 20 reports - 4 points, and 21 or more reports - 5 points.

The questionnaires were distributed to the nurses at weekly educational meetings and the responses were anonymous.

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Statistical analysis For all analyses, statistical significance was set at the p value of <0.05. Normality of distribution was tested using the Kolmogorov-Smirnov normality test.

Descriptive analysis included participants’ age, SPS-6 score, years of working experience, the number of working hours, direct contact with the patients, and the number of reported adverse events. Binary logistic regression was used to establish whether presenteeism was associated with patient safety culture. The results were adjusted for age, sex, department, years of working experience, number of working hours a week and direct contact with patients. ANOVA was used to compare SPS-6 scores across departments. Fisher’s exact test was used to compare general characteristics between the participants above and below the cut-off score. Analysis was performed using the SAS version 9.1.3 (SAS Institute Inc., SAS Campus Drive, Cary, NC, USA), and data were processed using the Microsoft Excel Data Entry and Reporting Tool (Microsoft Corporation, Mountain View, CA, USA).

RESULTS AND DISCUSSION Of 194 distributed questionnaires 148 were completed and analysed. Age distribution was not normal [Kolmogorov-Smirnov test, D(139)=0.178, p<0.001] and its median was 45±9.24 years (range: 22-59 years). SPS-6 total scores were normally distributed [D(150)=0.094, p=0.58; KolmogorovSmirnov test] with the mean of 21.3±4.58 and range between 7 and 30. Across hospital departments, mean SPS-6 scores were all above the cut-off score of 18, suggesting that presenteeism was not departmentspecific [Table 1; differences not statistically significant F(6,143)=1.77, p=0.109, ANOVA].

Table 2 shows that the participants divided along the SPS-6 cut-off score did not differ in gender, age,

years of experience, working hours, or contact with patients (p<0.05).

Binary logistic regression in nurses with SPS-6 score 18 and lower showed no association with patient safety culture [chi-square(11)=8.93, p=0.628]. Nurses having the SPS-6 score below the cut-off did not significantly differ in patient safety grades from the nurses scoring 19 and above [chi-square(3)=1.64, p=0.66] (Figure 1).

Even though the overall positive perception of “in-hospital patient transfer” and safety were our sample’s strength, other dimensions had positive responses below 65 %. The lowest positive response rate concerned “nonpunitive response to error”, which is consistent with other studies (Figure 2) (28, 29).

This may be due to the perception that errors shall be punished (so called cultural blame), as lower error rates are seen in units with less positive climate (29, 30). The same goes for the dimension “feedback and communication about error” and “frequency of adverse reporting” (28, 29). The questionnaire also confirmed the perception that hospitals in Croatia are understaffed with nurses. This perception coincides with the public perception, as hospital understaffing with healthcare personnel, nurses and physicians in particular, has been a burning issue in the country for at least 20 years. Our findings on hospital management support for patient safety indirectly corroborate earlier studies with medical nurses, in which poor work organisation had been recognised as stressful and therefore a predictor of low performance at work (31, 32).

Limitations of this study and the questionnaires This study had several limitations. We are aware that the convenient sample and the relatively small number of participating nurses may have biased the results so that they can not be generalised. However, we feel that this convenience sample can represent an average hospital in Croatia. Our results may be biased

Table 1 Mean SPS-6 scores obtained across hospital departments

Departments Surgery

Nonsurgery

Paediatrics

Obstetrics

Respondents 43 48 16 12 Mean SPS-6 21.42 21.04 23 18.62 score (±SD) (4.76) (4.41) (3.31) (4.81) Non-surgical departments included internal medicine and neurology ICU - Intensive Care Unit

Psychiatry

Anaesthesiology

ICU

7

7

15

22.86

18.43

22.53

(5.05)

(2.55)

(4.32)

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Table 2 General characteristics of the participants with SPS-6 score 18 or lower and higher than 18

Men Women Total (N) Median age Years of working experience 1 to 5 years 6 to 10 years 11 to 15 years 16 to 20 years 21 years or more Total (N, %) Number of working hours Less than 20 hours per week 20 to 39 hours per week 40 to 59 hours per week 60 to 79 hours per week 80 to 99 hours per week 100 hours per week or more Total (N, %) Direct interaction or contact with patients Yes No Total (N, %)

by the small number of presenteeist nurses. We chose the four-week period in the spring because it is usually not the time when working population takes a vacation but save it for the holiday season or the summer. We expected our population to be well in their working routine and perhaps experiencing their acute or chronic health problems. We also expected that the four-week frame would suffice to recruit enough participants who had experienced presenteeism and still be able to recall these experiences (24). It is however true that if we asked them to refer to a longer time frame, responses may have been different, but this would be departing from SPS-6.

The authors of the SPS-6 questionnaire acknowledge that work experiences in the past month may be affected by many environmental as well as personal factors and may change from time to time (24). Due to these factors we may not have recruited all respondents who have experienced presenteeism over the last month.

SPS-6 SCORE >18

≤18

13 97 110 45

5 33 38 47

11 16 19 17 47 110 (74.3 %)

4 3 7 5 15 38 (25.7 %)

2 4 100 2 1 1 110 (74.3 %)

0 5 32 1 0 0 38 (25.7 %)

4 106 110 (74.3 %)

0 38 38 (25.7 %)

Our research was anonymous to make participants as comfortable with responding honestly as possible.

However, it is possible that some of our participants were not fully honest and that some nurses who had experienced presenteeism in the previous month did not wish to respond because they did not feel comfortable with telling the truth. Another limitation is that answering to HSOPSC may be tiring, and respondents may lose interest and answer questions offhandedly (33).

For this reason we decided that another questionnaire which examines health problems, along with SPS-6 questionnaire would be too timeconsuming and negatively affect the response rate.

However, our future research should go into that direction. Another reason why we opted not to investigate specific health issues which had led to presenteeism is that research done so far has already provided a good insight on this subject in the field of nursing (4, 21-23, 34-37).

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Figure 1 Distribution of patient safety grades between presenteeists (SPS-6 score ≤18) and non-presenteeists (SPS-6 score >18)

Figure 2 Patient safety culture rating by presenteeists (SPS-6 score ≤18) and non-presenteists (SPS-6 score >18)

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We also acknowledge that HSOPSC measures patient safety culture of the department or the hospital and that it may not relate to presenteeism of individual nurse, whereas SPS-6 is an individual measure. To overcome this limitation we reported our results at the department level.

Tools employed in this research were used “as is”, i.e. in their original format to screen the target population, in an attempt to identify weak spots, but have not succeeded in it.

Our future research will therefore have to include a broader healthcare population for us to be able to identify the weak spots and suggest improvements toward high-quality and cost-effective health care.

Conflict of interest

8.

9.

10.

11.

12.

None to declare.

Acknowledgements We wish to thank all the participants in this study.

This study was funded from the project “Health at the workplace and healthy working environment” (approved by the Ministry of Science, Education and Sports under grant no. 108-1080316-0300).

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Sažetak Presječno ispitivanje povezanosti prezentizma i kulture bolesničke sigurnosti u medicinskih sestara Zanimanje medicinske sestre uključuje veliku predanost i požrtvovnost: medicinske sestre rade noćne smjene, rade prekovremeno i dolaze na posao i kada su bolesne. Naziv za ovu zadnju pojavu je prezentizam.

Neka su istraživanja pokazala da smanjena radna sposobnost medicinskih sestara zbog bolesti može ugroziti sigurnost pružatelja zdravstvene usluge i bolesnika. Cilj ovog presječnog istraživanja bio je ispitati obilježja prezentizma i kulture bolesničke sigurnosti u medicinskih sestara u Hrvatskoj te utječe li njihov prezentizam na kulturu bolesničke sigurnosti. Istraživanje je provedeno u jednoj općoj bolnici u Hrvatskoj tijekom travnja i svibnja 2012., i bilo je posebno ciljano prema medicinskim sestrama s obzirom na to da čine jednu od najvećih skupina zdravstvenih djelatnika. Ispitanici su zamoljeni da ispune dva upitnika: Stanfordsku ljestvicu prezentizma (SPS-6) i Upitnik o kulturi bolesničke sigurnosti u bolnici (HSOPSC). Nije nađena povezanost između prezentizma i kulture bolesničke sigurnosti. Najveći broj pozitivnih odgovora imala je dimenzija “Opća percepcija bolesničke sigurnosti”; ostale dimenzije imale su pozitivne odgovore ispod 65 %. Najmanji broj pozitivnih ocjena imala je dimenzija “Nekažnjavajući pristup neželjenom događaju”, što je u skladu s prethodnim istraživanjima. U pogledu spola, dobi, godina iskustva, radnih sati, kontakata s bolesnicima i njihove sigurnosti, medicinske sestre s prezentizmom nisu se nimalo razlikovale od medicinskih sestara bez prezentizma.

KLJUČNE RIJEČI: bolest; medicina rada; Stanfordska ljestvica prezentizma; Upitnik o kulturi bolesničke sigurnosti u bolnici

CORRESPONDING AUTHOR: Hana Brborović, MD University of Zagreb School of Medicine Andrija Štampar School of Public Health Department of Environmental and Occupational Health Rockefellerova 4, HR-10000 Zagreb, Croatia E-mail: hana.brborovic@snz.hr

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DOI: 10.2478/10004-1254-65-2014-2461

Original article

Photochemical toxicity of drugs intended for ocular use Roshan Kumar Sahu1,2, Bhupendra Singh1, Shubhini A. Saraf1, Gaurav Kaithwas1, and Kamal Kishor3 Department of Pharmaceutical Sciences, School for Biosciences & Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow1, CSIR-Open Source Drug Discovery Unit, Council of Scientific and Industrial Research Anusandhan Bhawan, New Delhi2, Indian Institute of Technology, Kanpur3, India Received in November 2013 CrossChecked in November 2013 Accepted in April 2014

The present investigation was undertaken to evaluate the possible ocular phototoxicity of drugs used in ophthalmic formulations. Sulphacetamide, ketoconazole, voriconazole, diclofenac, and ketorolac were assessed in the concentrations available in the market for their ocular use. The suitable models viz Hen’s Egg Test Chorioallantoic Membrane (HET-CAM) test, Isolated Chicken Eye (ICE) test, and Red Blood Cell (RBC) haemolysis test as recommended by ECVAM, ICCVAM, and OECD guidelines were performed.

Results of HET-CAM and ICE tests suggest that sulphacetamide is moderately toxic in the presence of light/UV-A and very slightly irritant without irradiation. Ketoconazole and voriconazole were found slightly irritant in presence of light/UV-A and non-irritant in dark. Diclofenac and ketorolac demonstrated slight irritancy in the light and were found to be non-irritant in dark. The results suggest that some of the drugs have potential toxic effect in the presence of light. The extent of phototoxicity might get extended when used for longer time. The recommendation is that these drugs should be stored and used in the dark for a specified time and be labelled with specific instructions for patients, especially for those working longer in the sunlight.

KEY WORDS: antibacterial agents; antifungal agents; HET-CAM Test; ICE Test; NSAIDs; RBC Test

In the regulatory context the term ‘eye irritation’ is generally defined as the development of undesirable changes in the eye after the application of a test substance to the anterior surface of the eye, which are reversible within 21 days of treatment. The Organisation for Economic Co-operation and Development (OECD) defined the substances that cause reversible tissue changes to the eye as ocular irritants and severity of the irritancy depends on the change/damage to the eye caused by the substance (1). The changes that are not reversible over the mentioned period are classified as ocular corrosives. The toxic reaction/ocular corrosion develops due to photodegradation of pharmaceutically active compounds or sometimes excipients present in

ocular formulations when exposed to UV radiations (2, 3).

Antibacterial agents either alone or in combination with non-steroidal anti-inflammatory drugs (NSAIDs) are the first line therapy for ophthalmic use. Among these, drugs such as sulphacetamide, ketoconazole, voriconazole, diclofenac, and ketorolac are frequently used. This investigation is an attempt to examine ocular phototoxicity of the above mentioned drugs that show photodegradation but no data has been available to support this. Five drugs primarily belonging to the category of antibacterial agents and NSAIDs, intended for ocular use, were investigated for their possible irritancy/toxicity to the eye, using

158 the methods approved by international regulatory agencies viz the OECD, International Coordination Committee for the Validation of Alternative Methods (ICCVAM), and European Centre for the Validation of Alternative Methods (ECVAM).

MATERIALS AND METHODS Chemicals Sodium salt of sulphacetamide (N-[(4aminophenyl)sulfonyl]acetamide) (CAS number- 14480-9) was obtained from East India Pharmaceutical Works Limited, Kolkata, India as a gift sample.

Ketoconazole (1-[4-(4-{[(2R,4S)-2-(2,4Dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3dioxolan-4-yl]methoxy}phenyl)piperazin-1-yl]ethan1-one) (CAS number 65277-42-1) was procured from Biochemix Pharmaceuticals, Haryana, India.

Voriconazole ((2R,3S)-2-(2,4-difluorophenyl)-3-(5fluoropyrimidin-4-yl)-1-(1H-1,2,4-triazol-1-yl)butan2-ol) (CAS number 137234-62-9) was procured from Auro Labs, Tamil Nadu, India as sterile lyophilised dried powder. Sodium salt of diclofenac (2-(2-(2,6-dichlorophenylamino)phenyl)acetic acid ) (CAS number 15307-86-5) was procured from Zee Labs Limited, Haryana, India and ketorolac tromethamine ((±)-5-benzoyl-2,3-dihydro-1Hpyrrolizine-1-carboxylic acid, 2-amino-2(hydroxymethyl)-1,3-propanediol) (CAS number 74103-07-4) was obtained from Sun Pharma Limited, Mumbai, India. Fluorescein sodium (3’,6’-dihydroxyspiro[2-benzofuran-3,9’-xanthene]1-one) (CAS number 2321-07-5) was obtained from Garuda Chemicals, Mumbai, India. Sodium chloride (Loba Chemie, Mumbai India) (0.9 %) and sodium hydroxide (Loba Chemie, Mumbai, India) (0.1 %) (Loba Chemie, Mumbai India) were used as negative and positive controls in all the tests performed. Sodium hydroxide is recommended to be used as positive control in standard protocols (4-6). Concentrations of drugs for the experiments were decided according to the concentrations available in the market. Three concentrations of each drug were freshly prepared in normal saline before the experiment: sulphacetamide (10, 20, and 30 % w/v), ketoconazole (1, 2, and 3 % w/v), voriconazole (0.5, 1, and 1.5 % w/v), diclofenac (0.05, 0.1, and 0.15 % w/v), and ketorolac (0.2, 0.4, and 0.5 % w/v). The highest concentration of each

Kumar Sahu R, et al. PHOTOTOXICITY OF DRUGS FOR OCULAR USE Arh Hig Rada Toksikol 2014;65:157-167

drug was made with an intention to observe the toxic profile at higher concentrations as these concentrations might be or might not be available in many countries’ markets.

Toxicity testing Hen’s Egg Test, Isolated Chicken Eye Test, and Red Blood Cell Lysis test were applied to profile the possible toxicity of selected chemicals. The methods have been approved by various national and international regulatory agencies like ICCVAM, ECVAM and the Japanese Centre for the Validation of Alternative Methods (JaCVAM). These agencies are primarily working with the objective to develop and validate methods that are an alternative to the existing animal experiments and are not related to ethical issues. The selected methods are based on animals that are available in slaughterhouses (eggs from egg farms and chicken from local slaughterhouses) and consumed as food. Hence no ethical issues are concerned with our investigation.

Hen’s Egg Test (HET-CAM Test) Chicken eggs were procured from local poultry and egg hatching farm at Lucknow, India. Fresh (not older than seven days), fertile, and clean eggs weighing between 50 and 60 g were used. Eggs were candled prior to use and nonviable or defective eggs were discarded. Excessively misshapen eggs or eggs with cracked or thin shells were not used. Eggs were placed in the incubator at 37±0.5 °C and 60±2 % relative humidity. The day on which the eggs were placed in the incubator was considered as day one.

Hand rotation was carried out five times per day until the day eight. Four eggs for each concentration of each case (light and dark) were grouped [total eggs = 4 (each concentration) x15 (concentrations) x2 (light and dark)=120 eggs were successfully grouped]. On day nine, eggs were removed for the assay. The region of air cell was cut and membrane was pared off. Using 0.9 % NaCl, membrane was moistened using a disposable glass pipette. Eggs were placed in the incubator for 30 min. The inner membrane was carefully removed with forceps. Approximately 0.3 mL of tested substances was placed directly onto the clear Chorioallantoic Membrane (CAM) surface. The surface was observed for changes for 5 min (300 s) by taking photographs (Canon EOS 1100D Camera, 12.2 x) at different time points (0, 60, 120, 180, 240, and 300 s). Endpoints (surface changes) that were

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observed were: haemorrhage (bleeding from the vessels); vascular lysis (blood vessel disintegration); and coagulation (intra- and extra-vascular protein denaturation).

The UV-A light (10 watts, Phillips, India) was used to evaluate possible phototoxicity. UV-A bulb was assembled inside the incubator where the temperature was maintained. The scoring of surface changes was carried out according to the scoring table given in the protocol (Table 1). The observations of surface changes were translated into quantitative scoring system using Irritation Score (IS) formula given below (4): Haemorrhage time = observed start (in seconds) of haemorrhage reactions on CAM Lysis time = observed start (in seconds) of vessel lysis on CAM Coagulation time = observed start (in seconds) of coagulation formation on CAM Phototoxicity index was also measured using the formula (7): Phototoxicity index= {[Mean score (light)-Mean score (dark)]/Mean score (dark)}x100 Isolated Chicken Eye Test (ICE Test) Chicken eyes were collected from chickens obtained from local slaughterhouse at Lucknow, India, where they are killed for human consumption (chicken used in the study were approximately seven weeks old weighing approximately 1.5-2.5 kg). Only the eyes of healthy animals considered suitable for entry into the human food chain were used. Eyes were isolated from the eye socket without any damage. A drop of sodium fluorescein 2 % w/v was put onto the corneal surface for 10-20 s, and then immediately rinsed with 20 mL isotonic saline. Cornea was examined for potential

damage. The final number of eyes used was 120 (groups and number of subjects were same as in the HET-CAM Test). Each eye was placed in an absorbent pad and the nictitating membrane was cut as well as other connective tissue. Eyes were placed in a superfusion chamber at a temperature of 32 °C with water pump according to the OECD ICE test protocol-438 (6). The entire cornea was supplied with isotonic saline from a bent stainless steel tube at a rate of 0.10-0.15 mL min-1 via a peristaltic pump. Once all eyes have been examined and approved, they were placed at 32±1.5 °C for 45-60 min to be equilibrated to the test system prior to dosing. The whole system was kept in an incubator to maintain the temperature.

After the equilibration period, a zero reference measurement was recorded for corneal thickness and corneal opacity to serve as a baseline (i.e., time = 0).

The fluorescein retention score determined at dissection was used as the baseline measurement.

Liquid test substance was applied at 0.03 mL with a micropipette so that the entire surface of the cornea was evenly covered. Test material was applied for a total of 10 s and then rinsed with 20 mL isotonic saline at room temperature. After the rinse step, the eye was returned to the superfusion apparatus. Treated corneas were evaluated prior to treatment and starting at 0, 30, 60, 90, 120, 150, 180, 210, 240 min (±5 min) after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, and fluorescein retention.

Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter (PachPen Pachymeter, Accutome, The Netherlands). It is expressed as a percentage and is calculated from corneal thickness measurements according to the following formula (5):

Table 1 Classification scheme for irritation score in HET-CAM Test

Photochemical damage

Score

Irritancy level

No visible membrane discoloration

Up to 0.9

Practically none

Just visible membrane discoloration

1-4.9

Slight

Visible membrane discoloration/haemorrhage, structures are covered partially

8-8.9

Moderate

9 and above

Strong

Visible membrane discolouration/ haemorrhage, structures are entirely covered

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The mean percentage of corneal swelling for all test eyes was calculated for each observation time point. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score is then given for each test substance.

Corneal opacity is calculated by using the area of the cornea that is most densely opacified for scoring.

Pictures of eyes were taken (Canon EOS 1100D Camera, Taiwan, China) and the opacity was observed visually. The mean corneal opacity value for all test eyes was calculated for each observation time point.

The test was performed as dark and light groups (light specifications were kept identical to the HET-CAM Test). The mean fluorescein retention values for all test eyes were observed for the 30-min observation time point. Scoring of opacity and fluorescein retention was carried out using standard scoring tables (Table 2 and Table 3) (5).

Red Blood Cell Haemolysis Test Fresh blood samples taken from hens weighing 2 kg were obtained from the local slaughter house

(Lucknow, India). Blood samples were kept in tubes containing 3.2 % sodium citrate (SD Fine Chemicals, Mumbai, India) solution as anti-coagulating agent.

Repetitive centrifugations at 2000 rpm (approx. 3757 g) for 10 min (Remi CPR-30 Plus, Mumbai, India) were carried out to remove plasma, white blood cells (WBC), and to separate red blood cells (RBC). Citrate buffer (SD Fine Chemicals, Mumbai, India) was used to store the RBCs and prevent coagulation and was used as a vehicle for RBC suspension. RBC was stored at -20 °C until use. The principle of this method is that the addition of a toxic chemical in RBC solutions causes RBC lysis and the haemoglobin released is measured at particular wavelength using colorimeter.

Various concentrations of test samples in phosphate buffer saline (PBS) were incubated with a defined quantity of RBC suspension for 10 min with constant shaking, at room temperature. The incubation period was terminated by high speed centrifugation. The resulting supernatant was then monitored photometrically at 530 or 560 nm (Photocolorimeter 1313, Environmental and Scientific Instruments Co.,

Table 2 Corneal opacity scores

Score

Observation

0

No opacity

0.5

Very faint opacity

1 2 3

Scattered or diffuse areas; details of the iris are clearly visible Easily discernible translucent area; details of the iris are slightly obscured Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible

4

Complete corneal opacity; iris invisible

Table 3 Fluorescein retention scores

Score 0 0.5

Observation No fluorescein retention Very minor single cell staining

1

Single cell staining scattered throughout the treated area of the cornea

2

Focal or confluent dense single cell staining

3

Confluent large areas of the cornea retaining fluorescein.

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Haryana, India) against the blank. The total haemoglobin release by positive control was taken as 100 % and the tests were evaluated relative to that.

The corresponding concentration at the 50 % response was considered as H50 value.

Protein Denaturation Test A 1 % solution of test in PBS was incubated with defined quantity of RBC suspension for 10 min with constant shaking at room temperature. The resulting supernatants were monitored at 575 and 540 nm respectively. The relation between the effective concentration of 50 % haemolysis and protein denaturation (relative to sodium dodecyl sulphate denaturation) is known as Lysis/Denaturation Ratio (L/D) (8). The results obtained were compared for each case as per the evaluation table given in the protocol (8). The test was performed as dark and light group. The irritancy level was scored using standard scoring table (Table 4) (8).

Statistical analysis All data were expressed as mean scores (Mean ± SEM, n=4). For the statistical comparison the results were compared with negative and positive controls using Dunnett’s Test (**P<0.01, *P<0.05 were considered as significant levels). Mean values of light and dark cases were compared using student’s t-test.

Data were analysed using GraphPad Prism Software (California, USA).

RESULTS HET-CAM Test Image recordings were carried out at different time points to observe the changes on the membrane (Figure

1). For sulphacetamide, mean irritation scores in light case were 4.9, 7.7, and 9.8 for 10, 20, and 30 % w/v respectively, indicating the concentration dependent irritancy. For ketoconazole these values were 5.8, 5.4, and 5.8 at 1, 2, and 4 % respectively. For voriconazole the classification was slight, moderate, and slight irritancy while in diclofenac moderate, slight, and slight irritancy were observed. On the contrary, ketorolac demonstrated slight irritancy at all the concentrations tested (Table 5). In all treatments, the mean irritation score was found to be higher in light in comparison to dark case.

ICE Test Results from light case suggest the irritant potential of sulphacetamide, in a concentration-dependent manner. Ketoconazole showed moderate, slight, and moderate level of irritancy at 1, 2, and 4 % w/v concentration respectively. Voriconazole showed very slight level of toxicity at 0.5 and 1.5 % concentrations.

Slight level of irritancy was observed in case of diclofenac as well. Ketorolac was found to be very slightly irritant at 0.2 and 0.5 % w/v concentrations respectively (Table 6).

RBC Test None of the drugs were found to be haemolytic at all concentrations tested in both light and dark cases in the RBC test. Haemolysis was observed at very high concentrations of tested drugs (Table 7).

Combined phototoxicity classification Combined data comparison (Table 8) suggest that drugs show their possible phototoxicity on HET-CAM and ICE tests only but not on RBC test at the used concentrations. This investigation suggests that drugs exhibit phototoxicity potential at the concentrations that are available in the market.

Table 4 Irritancy level classification in RBC test (8)

Eye irritation level

Lysis/Denaturation Ratio (L/D)

Non irritant

>100

Slightly irritant

>10

Moderately irritant

>1

Irritant

>0.1

Very irritant

<0.1

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Table 5 Mean score values and irritancy classification (4) of drugs (HET-CAM Test) Data presented as mean±SEM, (n=4)

Treatment Negative control (NaCl, 0.9 %) Positive control (NaOH, 0.1 %) Sulphacetamide (10 %) Sulphacetamide (20 %) Sulphacetamide (30 %) Ketoconazole (1 %) Ketoconazole (2 %) Ketoconazole (4 %) Voriconazole (0.5 %) Voriconazole (1 %) Voriconazole (1.5 %) Diclofenac (0.05 %) Diclofenac (0.1 %) Diclofenac (0.15 %) Ketorolac (0.2 %) Ketorolac (0.4 %) Ketorolac (0.5 %)

HET-CAM Test Light

Dark

Phototoxicity index

Mean score (IS)

Irritancy classification

Mean score (IS)

Irritancy classification

0

None

0

Slight

None

11.8±0.8

Strong

19.0

Strong

0.60

1.6±0.52 2.4±0.19** 3.5±0.39** 1.1±0.35 0.6±0.35 1.8±0.42* 1.2±0.40 1.1±0.67 2.2±0.24** <0.1 <0.1 <0.1 <0.1 0.4±0.2 0.8±0.5

Slight Slight Slight Slight None Slight Slight Slight Slight None None None None None None

4.9±0.44** (s) 7.7±0.62** (s) 9.8±0.29** (s) 5.8±0.24** (s) 5.4±0.39** (s) 5.8±1.5** (s) 4.4±0.86* (s) 6.4±0.64** (s) 4.0±0.85* 5.4±0.67** 4.6±0.99** 1.9±1.2 1.7±0.97 8±1.6 (s) 2.8±1.6

Slight Moderate Strong Moderate Moderate Moderate Slight Moderate Slight Moderate Slight Slight Slight Slight Slight

2.10 2.24 1.77 4.06 7.44 2.25 2.69 4.70 0.84 76.78 65.35 25.71 23.21 5.59 2.27

Statistical significance compared to negative control using Dunnett’s test. *P<0.05, **P<0.01.

IS = Irritation Score; (s) = significant difference between dark and light mean values (Student’s t test)

DISCUSSION

Figure 1 Images of HET-CAM Test (Light case): (A) Normal (B) Toxic

Phototoxicity is an important concern that must be considered prior to drug prescription/administration.

It might cause severe cellular damage and disrupt the normal function of tissue/organ. Cells that are stressed (loaded with multiple functions at a time) and continuously change their function are more sensitive to phototoxicity (9).

Most of the antibacterial agents and NSAIDs including sulphacetamide, ketoconazole, voriconazole, diclofenac, and ketorolac are reported to get sensitised and degraded in the presence of light which might cause irritation leading to toxicity upon continuous usage (10-16). As we found, very few studies available suggested the possible phototoxicity of the investigated drugs and there were no suitable studies to validate this problem. Most of the studies were carried out by chemical analysis groups. Literature review suggested that the extent of phototoxic side-effects of these drugs depends upon their chemical structure, absorption spectra, binding of the drug to tissue, and the ability

% Swelling

Mean opacity score

Fluorescein retention score

Dark

3.75±0.48 0.5± 0.01 0.87±0.02** 2 ±0.04** 0.25± 0.01 0.25±0.03 0.375±0.01 0.5±0.05 0.375±0.02 0.5±0.04 0.875±0.02** 0.625±0.01 0.5±0.02 0.625±0.08 0.5±0.08 0.875±0.04**

29.88±2.1 13.21±2.7** 9.71±1.9 24.45±3.42** 6.64±0.56 10.27±1.39* 13.68±4.3** 6.22±0.93 14.30±2.74** 7.26±1.09 9.98±1.9 7.86±2.48 6.81±1.31 15.48±1.77** 12.92±4.16** 10.96±2.32*

Mean opacity score 0.25 ±0.02

% Swelling

0.38±0.06

0.25±0.01

0.5±0.02

0.25±0.01

0

0

0.25±0.01

0

0.125±0.02

0.125±0.02

0.125±0.02

0

0.375±0.04

0.625±0.01

0.25±0.01

2±0.4

0.125±0.06

Fluorescein retention score

Light

0.25±0.06

ICE Test

Irritancy classification

Negative control 2.24±0.3 0 0 None (NaCl, 0.9 %) Positive control 20.69±2.1 1.25±0.2** 0.875±0.04 Strong (NaOH, 0.1 %) Sulphacetamide 4.18±1.22 0.375± 0.03** 0 None (10 %) Sulphacetamide 1.70±0.03 0.5 ±0.01 0 None (20 %) Sulphacetamide 2.89±0.9 0 0.375±0.02 None (30 %) Ketoconazole 0 0 0 None (1 %) Ketoconazole 3.79±0.6 0 0.125±0.06 None (2 %) Ketoconazole 1.11±0.4 0.125±0.02 0.125±0.04 None (4 %) Voriconazole 2.62±0.3 0.375±0.05* 0.125±0.02 None (0.5 %) Voriconazole 0.68 0 0 None (1 %) Voriconazole 0 0.125±0.04 0.25±0.02 None (1.5 %) Diclofenac 0.06 0.25±0.02* 0 None (0.05 %) Diclofenac 0.12±0.06 0.125±0.02 0 None (0.1 %) Diclofenac 2.54±0.52 0.25±0.01* 0.25±0.08 None (0.15 %) Ketorolac 0 0.25±0.01* 0.5±0.08 None (0.2 %) Ketorolac 0 0.25±0.01* 0.25±0.01 None (0.4 %) Ketorolac 0.74±0.06 0.5±0.03** 0.375±0.02 None (0.5 %) Statistical significance compared to negative control using Dunnett’s test, *P<0.05, **P<0.01

Treatment

Table 6 Mean score values and irritancy classification of drugs (ICE Test)

Very slight

None

Very slight

None

Very slight

Very slight

Slight

Moderate

Slight

Slight

Moderate

Slight

Moderate

Slight

None

Strong

None

Irritancy classification

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Table 7 H50/DI ratio and irritancy classification of different treatment

Dark

Light

Mean H50 value (mg mL-1)

DI value

H50/ DI ratio

Irritancy classification

Mean H50 value (mg mL-1)

DI value

H50/ DI ratio

Irritancy classification

None

0

0

None

None

0

0

None

Positive control

0.92

59

0.016

Irritant

0.63

96

0.0

Very irritant

Sulphacetamide

Very high

Very high

None

Very high

Very high

1.98

None

Ketoconazole

Very high

5.79

None

Very high

15.31

Voriconazole

Very high

3.48

None

Very high

9.65

Diclofenac

Very high

2.56

None

Very high

5.33

Ketorolac

Very high

5.23

Very high Very high Very high Very high Very high

None

Very high

7.26

Treatment Negative control

H50 – haemoglobin release at 50 % in Red Blood Cell Haemolysis Test, DI – denaturation index

to cross blood-ocular barrier (17). It is worthwhile to mention that the literature is available to suggest the photosensitivity of these agents; however no literature elaborates on the ocular toxicity of these agents. The present study was undertaken to evaluate ocular phototoxicity of these drugs.

Sulphacetamide displayed ‘slight’ irritation potential in HET-CAM test (dark) in a concentration

Very high Very high Very high Very high

HET-CAM

ICE

RBC

Sulphacetamide (10 %)

Slight

None

None

Sulphacetamide (20 %)

Moderate

Slight

None

Sulphacetamide (30 %)

Strong

Moderate

None

Ketoconazole (1 %)

Moderate

Slight

None

Ketoconazole (2 %)

Moderate

Moderate

None

Ketoconazole (4 %)

Moderate

Slight

None

Voriconazole (0.5 %)

Slight

Slight

None

Moderate

Moderate

None

Voriconazole (1.5 %)

Slight

Slight

None

Diclofenac (0.05 %)

Very slight

Very slight

None

Diclofenac (0.1 %)

Very slight

Very slight

None

Diclofenac (0.15 %)

Very slight

Very slight

None

Ketorolac (0.2 %)

None

None

None

Ketorolac (0.4 %)

Very slight

Very slight

None

Ketorolac (0.5 %)

Very slight

Very slight

None

Voriconazole (1 %)

None None None

independent manner. However, in light case it showed concentration dependent toxicity and reached ‘strong irritant’ category at higher concentrations. These results suggest that in the presence of light, sulphacetamide is more toxic than in the dark, depending on the concentration. In ICE test similar results were observed except that the extent of toxicity was lower than in HET-CAM test. In the presence of

Table 8 Combined phototoxicity classifications

Treatment

None

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Kumar Sahu R, et al. PHOTOTOXICITY OF DRUGS FOR OCULAR USE Arh Hig Rada Toksikol 2014;65:157-167

light, sulphacetamide exists in triplet state and releases electrons. It produces SO, C6H4SO2, and NHR radicals (10). Photodegradation is reported to occur through the supply of hydrated electrons to the tissue. These electrons interfere with bimolecular biosynthesis and cause cellular damage and modifications like depolymerisation of hyaluronic acid (11). These intermediate radicals have the ability to cause phototoxic and photoallergic effects, as observed in our study (12, 18).

Ketoconazole was found to be ‘moderately irritant’ at all concentrations in HET-CAM test whereas in ICE it displayed ‘slight’ to ‘moderate’ effect at different concentrations. Main photo-degradation products of ketoconazole are (cis-1-acetyl-4-{4-((2-(2chlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3dioxolan-4-yl)methoxy)phenyl}piperazine and (cis1-acetyl-4-{4-((2-(4-chlorophenyl)-2-(1H-imidazol1-ylmethyl)-1,3-dioxolan-4-yl)methoxy)phenyl} piperazine (13). Possible mechanisms to explain ketoconazole phototoxicity include generation of singlet oxygen, superoxide anion radical, and other free radicals which cause cellular damage (14).

Ketoconazole is said to lose its pharmacological activity after photodegradation (19).

Voriconazole also exhibited results similar to ketoconazole in HET-CAM and ICE tests. Voriconazole is reported to produce 1-(2, 4-difluorophenyl)-2(1H-1, 2,4-triazol-1-yl)-1-ethanone as the main photodegradation product and its major metabolites are di-hydroxyl and N-oxide metabolites (20). Reports are available on the skin phototoxicity of voriconazole which suggest that the most likely mechanisms of its phototoxicity are related to either voriconazole (superoxide anion radical, singlet oxygen) or its N-oxide metabolite, and retinoidal metabolic interference (15, 21, 22).

Diclofenac and ketorolac have shown slight irritancy at all concentrations in HET-CAM test whereas in ICE test both demonstrated slight to none irritancy level in light conditions. These responses were concentration independent in both tests. Under photocatalytic conditions diclofenac releases degradation products through various chemical mechanisms: photocyclisation (2-(8-chloro-9Hcarbazol-1-yl) acetic acid, 1-chloro-8-methyl-9Hcarbazole; decarboxylation (2,6-dichloro-N-otolylbenzenamine); and dehalogenation. The products after photocyclisation are reported to cause cell lysis.

N-centred carbazolyl radical is also formed in photodegradation (16). These radical intermediates

are responsible for the phototoxic effects of diclofenac sodium by hydrogen abstraction from the biomolecules, which initiates a further toxic effect. Thus, the photosensitising and phototoxic properties of diclofenac are associated with degradation products produced by light exposure (23). Ketorolac demonstrated none to very slight irritancy in a concentration independent manner. Results suggest that no significant level of phototoxicity is achieved by ketorolac. In the presence of light, triplet excited state of ketorolac is observed, which is followed by oxidation (24). This single mechanism could be accounted for the observed very slight level of toxicity by ketorolac in our investigation.

Conclusion Our findings suggest that the investigated drugs have phototoxicity potential at the concentrations that are available in the market. This investigation also indicates that the antibacterial agents and NSAIDs having phototoxic potential can be harmful, if used for long time. The results demonstrate that the analysed drugs intended for ocular use are phototoxic and therefore should be stored and used in dark. We used models which are ex-vivo in nature. The findings have to be validated further using more pre-clinical and clinical research.

REFERENCES 1. Organisation for Economic Cooperation and Development (OECD). Guidelines for the Testing of Chemicals No. 405: Acute Eye Irritation/Corrosion. Paris: OECD; 2002.

2. Furrer P, Mayer JM, Gurny R. Ocular tolerance of preservatives and alternatives. Eur J Pharm Biopharm 2002;53:263-80. doi: 10.1016/S0939-6411(01)00246-6 3. Benavides T, Martínez V, Mitjans M, Infante MR, Moran C, Clapés P, Clothier R, Vinardell MP. Assessment of the potential irritation and photoirritation of novel amino acidbased surfactants by in vitro methods as alternative to the animal tests. Toxicology 2004;201:87-93. doi: 10.1016/j. tox.2004.04.003 4. ICCVAM Test Method Evaluation Report: Appendix G-ICCVAM Recommended HET-CAM Test Method Protocol, 2006 [displayed 2 May 2014]. Available at http://ntp.niehs. nih.gov/iccvam/docs/ocutox_docs/oteval/appg-508.pdf 5. ICCVAM-Recommended Protocol for Using the Isolated Chicken Eye (ICE) Test Method Protocol, 2012 [displayed 2 May 2014]. Availalble at http://ntp.niehs.nih.gov/iccvam/ docs/protocols/iceprotocol16jul12v2-508.pdf 6. Organisation for Economic Cooperation and Development (OECD). Guidelines for the Testing of Chemicals No 438: Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants. Paris: OECD; 2009.

166 7. Velpandian T, Bankoti R, Humayun S, Ravi AK, Kumari SS, Biswas NR. Comparative evaluation of possible ocular photochemical toxicity of fluoroquinolones meant for ocular use in experimental models. Indian J Exp Biol 2006;44:38791. PMID: 16708892 8. INVITTOX Protocol No 37, European Centre for the Validation of Alternative Methods RBC Test System, Ispra: ECVAM DB-ALM; 2010.

9. Editors. Artifacts of light. Nat Meth. 2013;10:1135. doi: 10.1038/nmeth.2760 10. Ahmad T, Ahmed I. Multicomponent spectrophotometric assay of sulphacetamide and photodegradation products. Pak J Pharm Sci 1990;3:29-39. PMID:16414659 11. Miyazaki T, Yomota C, Okada S. Depolymerization of hyaluronate by the phototoxic drugs phenothiazines and sulphacetamide. Colloid Polymer Sci 2000;278:84-9. doi: 10.1007/s003960050014 12. Jordan L. Holtzman. Role of reactive oxygen and metabolite binding in drug toxicity. Life Sci 1982;30:1-9. doi: 10.1016/0024-3205(82)90629-4 13. Mhaske RA, Sahasrabudhe S. Identification of Major Degradation Products of Ketoconazole. Sci Pharm 2011;79:817-36. doi: 10.3797/scipharm.1107-18 14. Mohamed KN. Severe photodermatitis during ketoconazole therapy. Clin Exp Derm 1988;13:54. PMID: 3208444 15. Murayama N, Imai N, Nakane T, Shimizu M, Yamazaki H. Roles of CYP3A4 and CYP2C19 in methyl hydroxylated and N-oxidized metabolite formation from voriconazole, a new antifungal agent, in human liver microsomes. Biochem Pharmacol 2007;73:2020-6. doi: 10.1111/j.1365-2125.2009.03534.x 16. Vogna D, Marotta R, Napolitano A, Andreozzi R, d’Ischia M. Advanced oxidation of the pharmaceutical drug diclofenac with UV/H2O2 and ozone. Water Res 2004;38:41422. doi: 10.1016/j.watres.2003.09.028

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17. Roberts JE. Ocular phototoxicity. J Photochem Photobiol B 2001;64:136-43. doi: 10.1016/S1011-1344(01)00196-8 18. Motten AG, Chignell CF. Spectroscopic studies of cutaneous photosensitizing agents–III. Spin trapping of photolysis products from sulphanilamide analogs. Photochem Photobiol 1983;37:17-26. PMID: 6300940 19. Staub I, Flores L, Gosmann G, Pohlmann A, Fröehlich PE, Schapoval EES, Bergold AM. Photostability studies of ketoconazole: isolation and structural elucidation of the main photodegradation products. Latin Am J Pharm 2010;29:1100-6.

20. Adams AIH, Grace G, Paulo HS, Ana MB. LC stability studies of voriconazole and structural elucidation of its major degradation product. Chromatographia 2009;69(Supplement):115-22. doi: 10.1365/s10337-009-1082-3 21. Epaulard O, Saint-Raymond C, Villier C, Charles J, Roch N, Beani JC, Leccia MT. Multiple aggressive squamous cell carcinomas associated with prolonged voriconazole therapy in four immunocompromised patients. Clin Microbiol Infect 2010;16:1362-4. doi: 10.1111/j.1469-0691.2009.03124.x 22. Scholz I, Oberwittler H, Riedel KD, Burhenne J, Weiss J, Haefeli WE, Mikus G. Pharmacokinetics, metabolism and bioavailability of the triazole antifungal agent voriconazole in relation to CYP2C19 genotype. Br J Clin Pharmacol 2009;68:906-15. doi: 10.1111/j.1365-2125.2009.03534.x 23. Encinas S, Bosca F, Miranda MA. Phototoxicity associated with diclofenac: a photophysical, photochemical, and photobiological study on the drug and its photoproducts.

Chem Res Toxicol 1988;11:946-52. doi: 10.1021/tx9800708 24. Leo G, Hi-Shi C, Johanson D. Light degradation of ketorolac tromethamine. Int J Pharmaceutics 1988;41:105-13. doi:10.1016/0378-5173(88)90142-1.

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167

Sažetak Fotokemijska toksičnost lijekova namijenjenih okularnoj uporabi Cilj ovog ispitivanja bio je istražiti moguću okularnu fototoksičnost lijekova koji se koriste u oftalmološkim formulacijama. Sulfacetamid, ketokonazol, vorikonazol, diklofenak i ketorolak ispitivani su u koncentracijama koje su dostupne na tržištu u njihovom obliku koji je namijenjen za okularnu uporabu.

Primijenjeni su testovi iritacije na kokošjem jajetu (Hen’s Egg Test Chorioallantoic Membrane - HETCAM), izoliranom kokošjem oku (Isolated Chicken Eye - ICE) i test hemolize crvenih krvnih stanica (Red Blood Cell - RBC) prema preporukama ECVAM-a, ICCVAM-a i OECD-a. Rezultati HET-CAM i ICE testova upućuju na umjerenu toksičnost sulfacetamida u prisutnosti svjetla/UV-A te vrlo blagu iritaciju u mraku. Ketokonazol i vorikonazol pokazali su blagu iritaciju u prisutnosti svjetla/UV-A te nikakvu iritaciju u mraku. Diklofenak i ketorolak pokazali su blagu iritaciju na svjetlu i nikakvu iritaciju u mraku. Rezultati upućuju na moguću toksičnost nekih od navedenih lijekova u prisutnosti svjetla. Razmjer fototoksičnosti može biti povećan pri produljenoj uporabi lijeka. Preporučuje se čuvati navedene lijekove na zatamnjenome mjestu te ih označiti odgovarajućim uputama za korištenje, osobito za osobe koje su dulje vrijeme izložene sunčevoj svjetlosti.

KLJUČNE RIJEČI: antibakterijski lijekovi; antimikotici; HET-CAM test; ICE test; nesteroidni protuupalni lijekovi; RBC test

CORRESPONDING AUTHOR: Roshan Kumar Sahu CSIR-Open Source Drug Discovery Unit, Council of Scientific and Industrial Research Anusandhan Bhawan, 2, Rafi Marg, New Delhi, India Email: roshansahu@osdd.net

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Nafisi S, et al. EFFECTS OF SILAFIBRATE AGAINST APAP IN RAT HEPATOCYTES Arh Hig Rada Toksikol 2014;65:169-178

DOI: 10.2478/10004-1254-65-2014-2434

Original article

Cytoprotective effects of silafibrate, a newly-synthesised siliconated derivative of clofibrate, against acetaminophen‑induced toxicity in isolated rat hepatocytes Sara Nafisi1,2,3,4, Reza Heidari5, Mohammad Ghaffarzadeh6, Mojtaba Ziaee1,2, Hossein Hamzeiy1,3, Alireza Garjani2,3, and Mohammad Ali Eghbal2,3 Biotechnology Research Center1, Drug Applied Research Center2, Pharmacology and Toxicology Department, School of Pharmacy3, Students’ Research Committee4, Tabriz University of Medical Sciences, Tabriz, Pharmaceutical Sciences Research Center (PSRC), Shiraz University of Medical Sciences, Shiraz, Iran5, Chemistry and Chemical Engineering Research Center of Iran, Tehran6, Iran Received in August 2013 CrossChecked in August 2013 Accepted in January 2014

Acetaminophen (N-acetyl para amino phenol, APAP) is a widely used antipyretic and analgesic drug responsible for various drug-induced liver injuries. This study evaluated APAP-induced toxicity in isolated rat hepatocytes alongside the protective effects of silafibrate and N-acetyl cysteine (NAC). Hepatocytes were isolated from male Sprague-Dawley rats by collagenase enzyme perfusion via the portal vein. This technique is based on liver perfusion with collagenase after removing calcium ions (Ca2+) with a chelator.

Cells were treated with different concentrations of APAP, silafibrate, and NAC. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation, and mitochondrial depolarisation were measured as toxicity markers. ROS formation and lipid peroxidation occurred after APAP administration to rat hepatocytes.

APAP caused mitochondrial depolarisation in isolated cells. Administration of silafibrate (200 µmol L-1) and/or NAC (200 µmol L-1) reduced the ROS formation, lipid peroxidation, and mitochondrial depolarisation caused by APAP. Cytotoxicity induced by APAP in rat hepatocytes was mediated by oxidative stress. In addition, APAP seemed to target cellular mitochondria during hepatocyte damage. The protective properties of silafibrate and/or NAC against APAP‑induced hepatic injury may have involved the induction of antioxidant enzymes, protection against oxidative stress and inflammatory responses, and alteration in cellular glutathione content.

KEY WORDS: drug-induced liver injury (DILI); fibrates; mitochondria; oxidative stress; reactive oxygen species (ROS)

Acetaminophen (N-acetyl para amino phenol, APAP) is a widely used analgesic and antipyretic drug, which if applied excessively can cause severe hepatic damage or even death in experimental animals and individuals (1). At recommended doses, APAP is mainly metabolised by sulphation and glucuronidation (2). A small proportion of APAP is metabolised through cytochrome CYP2E1 and, to a lesser extent, CYP1A2 and CYP3A4, which produce the reactive metabolite N‑acetyl‑p‑benzoquinoneimine (NAPQI).

This reactive intermediate is efficiently detoxified by conjugation with glutathione (3). However, in overdoses, a large amount of APAP is metabolised through the P450 family of cytochromes leading to GSH depletion by NAPQI conjugation followed by the covalent binding of NAPQI to proteins (4).

Cellular mitochondria seem to be the target for acetaminophen-induced hepatotoxicity (5).

Although the precise mechanism of APAP hepatotoxicity is not fully understood, some studies

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(5, 6) have suggested that NAPQI exerts a cytotoxic effect through its covalent binding to cytosolic or microsomal proteins and membrane components, inducing oxidative stress in cells, inhibition of mitochondrial respiration, and depletion of cellular ATP. Research on acetaminophen-induced hepatotoxicity and finding new approaches to prevent it is a dynamic field of research due to high incidence of hepatotoxicity induced by this drug accidentally or in suicidal attempts (6,7).

Silafibrate (ethyl 2-(4-(trimethylsilyl) phenoxy)2-methylpropanoate) (Figure 1) is a siliconated derivative of the drug clofibrate (8). Fibrates belong to the peroxisome proliferator-activated receptor α (PPAR-α) drug group, which has been reported to have anti-inflammatory effects (9). Furthermore, PPAR ligands’ antioxidant and oxidative stress modulating properties have been shown in previous studies (10,11). It has been reported that the trimethylsilyl group in silafibrate (Figure 1) increases the anti‑inflammatory properties of clofibrate and enhances its pharmacological effect on PPAR receptors (8). It has also been shown that PPAR receptors have a crucial role in preventing APAPinduced hepatic damage (12). The role of silafibrate, as a new and more potent analogue of clofibrate (8) against APAP-induced hepatotoxicity in isolated rat hepatocytes was evaluated in this study. The protective role of N-acetylcysteine (NAC) as a gold standard treatment for acetaminophen-induced hepatotoxicity (13), was also studied and compared with silafibrate in this investigation.

Cell death, oxidative stress induction (ROS formation), lipid peroxidation, and mitochondrial injury were assessed as toxicity markers induced by acetaminophen (5, 6).

Figure 1 Chemical structure of silafibrate (left) and acetaminophen (right)

MATERIAL AND METHODS Chemicals Acetaminophen was purchased from Medisca Pharmaceutique Incorporation (Montreal, Canada).

N-acetyl cysteine (NAC) and (4-(2-hydroxyethyl) 1-piperazine-ethanessulfonic acid (HEPES) were obtained from Acros (New Jersey, USA). Albumine bovine type was purchased from Roche Diagnostics Corporation (Indianapolis USA). Rhodamine 123 and Collagenase from Clostridium histolyticum were obtained from Sigma Aldrich (St. Louis, MO, USA).

Trichloroacetic acid (TCA), ethyleneglycolbis (ρ‑aminoethylether)-N,N,N′,N′‑tetra acetic acid (EGTA), and Trypan Blue were obtained from Merck (Darmstadt, Germany). A siliconised analogue of clofibrate, silafibrate, was synthesised in the Chemistry and Chemical Engineering Research Center, Tehran, Iran. Thiobarbituric acid (TBA) was obtained from SERVA (Heidenberg, New York). All salts used for preparing buffer solutions were of analytical grade and obtained from Merck (Darmstadt, Germany).

Hepatocyte preparation Male Sprague-Dawley rats (three animals for each test, N=15) weighing 250–300 g were housed in plastic cages at ambient temperature (25±3 °C). Animals had free access to food and water. Collagenase perfusion method was used to isolate rat hepatocytes (14). This technique is based on liver perfusion with collagenase after the removal of calcium ion (Ca2+) with a chelator (EGTA 0.5 mol L-1). The liver was perfused with different buffer solutions through the portal vein.

Collagenase-containing buffer solution destructed liver interstitial tissue and caused hepatocytes to be easily isolated. Isolated hepatocytes (10 mL, 106 cells mL-1) were incubated in the Krebs-Henseleit buffer (pH 7.4) under an atmosphere of 95 % O2 and 5 % CO 2, in 50 mL round bottom flasks which were continuously rotating in a water bath at 37 °C. For more precise details on isolated rat hepatocytes preparation, readers are referred to Moldéus et al (15).

As CYP2E1, the acetaminophen-metabolising enzyme expression is low in rat liver (16), we used the enzyme induction method by administering β‑naphtoflavone (40 mg kg-1, i.p., for three consecutive days) (17) to accelerate acetaminophen‑induced toxicity in rat hepatocytes. After this, hepatocytes were isolated and used. All animals received humane care and were used

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according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” (18), which was approved by the local ethic committee in Tabriz University of Medical Sciences, Tabriz, Iran.

Different concentrations of acetaminophen were added to the cellular media to find the dose response of the drug in rat hepatocytes and the LC50 concentration (the concentration which leads to 50 % cell death after 120 min of incubation) for the next experiments.

Cell viability Trypan blue dye exclusion staining was used to assess the percentage of dead cells (19). Hepatocyte viability was determined at different time intervals to evaluate the effect of acetaminophen on cell viability.

LC 50 dose of the drug was determined and the protective effects of silafibrate and NAC against cell death induced by acetaminophen were tested.

Hepatocytes were at least 85 % viable before their use, as recommended in previous studies (20-27).

Reactive oxygen species (ROS) formation To determine the extent of ROS generated during acetaminophen metabolism, 2,7‑dichlorofluorescein diacetate (DCFH-DA; 1.6 µmol L-1) was added to the hepatocyte incubate. DCFH-DA was first hydrolysed to non-fluorescent DCFH in hepatocytes. DCFH then reacted with ROS to form the highly fluorescent DCFH. 1 mL (approximately 106 cells) of hepatocyte suspension was taken and the fluorescence intensity was measured using a Jasco® FP‑750 spectrofluorometer (Jasco Corporation, Tokyo, Japan) with excitation and emission wavelengths of 500 nm and 520 nm, respectively (28).

Lipid peroxidation measurement Hepatocyte lipid peroxidation was determined by measuring the amount of thiobarbituric acid reactive substances (TBARS) formed during the decomposition of lipid hydroperoxides. After treating 1 mL aliquots of hepatocyte suspension (10 6 cells mL -1) with trichloroacetic acid (70 % w/v) and boiling the supernatant with thiobarbituric acid (0.8 % w/v) for 20 min, the absorbance of the observed colour was determined using an Ultrospec ® 2000 UV spectrophotometer at 532 nm (Pharmacia Biotech Cambridge, England) (29).

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Mitochondrial membrane potential Mitochondrial membrane potential was assessed as an indicator of toxicity induced by methimazole or N-methylthiourea. The fluorescent dye, rhodamine 123, was used as a probe to evaluate the mitochondrial membrane potential in rat hepatocytes. Samples (1 mL) were taken from the cell suspension at scheduled time points, and centrifuged at 1000 g for 1 min. The cell pellet was then resuspended in 2 mL of fresh incubation medium containing 1.5 µmol L-1 rhodamine 123 and gently shaken in a thermostatic water bath at 37 °C for 10 min. Hepatocytes were separated by centrifugation (402 g for one min) and the amount of rhodamine 123 appearing in the incubation medium was measured fluorimeterically at 490 nm excitation and 520 nm emission wavelengths using a Jasco® FP-750 spectrofluorometer (30).

Statistical analysis Results are given as mean±SE for at least three independent experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc test. P<0.05 was considered as significant difference.

RESULTS In isolated rat hepatocytes, acetaminophen hardly caused cytotoxicity and very high concentrations of the drug were needed to induce toxicity (Figure 2).

Hence, to accelerate the acetaminophen-induced toxicity, we used the enzyme-induced hepatocyte model. The LC50 of acetaminophen was found to be 750 µmol L-1 (Figure 2). Different compounds (APAP in combination with proposed protective agents such as silafibrate and/or NAC) were added to the incubation medium, aimed to determine their ability to modulate the toxic response of acetaminophen.

Silafibrate and/or NAC caused no significant toxicity in hepatocytes as compared to the control cells when administered alone at given concentrations.

Administration of NAC (200 µmol L -1 ) or silafibrate (200 µmol L-1) effectively reduced cell death (Figure 3), a significant amount of formed ROS (Figure 4), and lipid peroxidation (Figure 5) caused by acetaminophen. Acetaminophen also caused mitochondrial depolarisation in rat hepatocytes (Figure 6) and NAC or silafibrate proved to have a preventive role in this regard (Figure 6).

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Figure 2 Dose-response of acetaminophen-induced cytotoxicity in isolated rat hepatocytes.

BNF: β-naphtoflavone * Indicates the significantly higher cell death than that of control group (p<0.05).

Hepatocytes cell death was not significant in the control group at different time intervals as assessed by trypan blue exclusion test.

Figure 3 The protective effect of N-acetyl cysteine (NAC) and silafibrate against acetaminophen‑induced cytotoxicity in rat hepatocytes. * Indicates significantly higher cytotoxicity as compared to the control group (p<0.05). a

Indicates significantly lower cytotoxicity as compared to the acetaminophen‑treated group (p<0.05)

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Figure 4 Acetaminophen‑induced ROS formation in isolated rat hepatocytes and the effect of NAC and silafibrate administration. * Significantly higher ROS level than control group (p<0.05). # Significantly lower ROS level than acetaminophen‑treated hepatocytes (p<0.05)

DISCUSSION Drug-induced liver disease is the most common cause of acute liver failure, and acetaminophen accounts for the bulk of these (6). Incubation of enzyme-induced rat hepatocytes with acetaminophen caused cell death concentration-dependently. The LC50 for acetaminophen was 750 µmol L-1. Acetaminophen caused ROS formation, lipid peroxidation, and mitochondrial depolarisation in isolated rat hepatocytes. Administration of NAC and/or silafibrate diminished the toxic effects of acetaminophen in rat hepatocytes.

In previous studies, it has been shown that administration of PPAR ligands such as clofibrate diminished acetaminophen-induced hepatotoxicity in in vivo models (31). In addition, it has been found that PPARs null mice (PPARs -/-) were more susceptible to hepatotoxicity induced by acetaminophen (32).

Furthermore, the protective effects of PPAR ligands in in vitro experiments have been shown (33).

However, the mechanism(s) underlying the hepatoprotection afforded by peroxisome proliferators have yet to be clarified, but the induction of antioxidant enzymes (34), alteration in cellular glutathione content (35), and protection against oxidative stress and inflammatory responses (36-38)

are the proposed protective mechanisms. It has been found that PPAR ligands have a role in modulating oxidative stress and its deleterious consequences in different tissues such as liver (39), nervous (40), and vascular systems (41). These mechanisms could be involved in the protective properties of silafibrate against APAP-induced cytotoxicity in freshly-isolated rat hepatocytes. Currently there is no data available on the direct effect of silafibrate on ROS in biological systems. However, the chemical structure of this drug and its direct effects on reactive species might also be attributed to its protective properties in isolated rat hepatocytes. This characteristic might arise from high electron-donating nature of trimethylsilyl (TMS) group in the drug.

NAC is a standard clinical treatment against APAPinduced hepatotoxicity (42). It provides protection mainly by counteracting oxidative stress (43), and scavenging APAP reactive metabolite (44). Moreover, it replenishes hepatic glutathione reservoirs as a crucial defence barrier against xenobiotics (44).

We found that acetaminophen caused ROS formation in isolated rat hepatocytes (Figure 4). Since PPARα ligands showed anti-oxidative stress properties in previous studies (36, 37, 45), one of the mechanisms by which the drug silafibrate may protect rat hepatocytes against acetaminophen might be its effect

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Figure 5 Lipid peroxidation caused by acetaminophen (APAP) in isolated rat hepatocytes and the protective role of N-acetyl cisteine (NAC) and silafibrate. * Significantly higher TBARS level than control group (p<0.05). §

Significantly lower TBARS level than acetaminophen‑treated group (p<0.05)

on alleviating oxidative stress (Figure 4). The effect of silafibrate on lipid peroxidation induced by acetaminophen could be attributed to its effect on reducing the ROS level, which is a major cause of lipid peroxidation in cells (46).

It has been shown that oxidative stress is one of the major causes of mitochondrial damage (47). A part of silafibrate protection against acetaminophen‑induced cytotoxicity may be due to its effect in preventing mitochondrial injury caused by this drug. In the current investigation we found that silafibrate as a new and more potent analogue of clofibrate (8) showed protective effects against APAP-induced cytotoxicity.

These results might provide new therapeutic strategies against APAP-induced hepatotoxicity.

CONCLUSION Silafibrate as a newly synthesised analogue of clofibrate protected rat hepatocytes against acetaminophen-induced toxicity. The protective effects of silafibrate could be attributed to its role in counteracting oxidative stress and/or its consequences such as lipid peroxidation and mitochondrial damage.

The induction of antioxidant enzymes (34), alteration in cellular glutathione content (35), and protection

against oxidative stress and inflammatory responses (36-38) might be involved in the cytoprotective properties of silafibrate. On the other hand, the duration of study in this in vitro model of isolated rat hepatocytes might not be sufficient to observe some silafibrate protective properties, such as induction of antioxidant enzymes. Future in vivo investigations will provide more insights on silafibrate hepatoprotective properties.

Acknowledgements This study was carried out at Biotechnology and Drug Applied Research Centres (DARC) of Tabriz University of Medical Sciences. The authors thank Biotechnology Research centre and Drug Applied Research Center for providing financial supports and facilities to carry out this investigation. This paper was a part of the Pharm.D. thesis for Sara Nafisi and was supported technically by the students’ research committee of Tabriz University of Medical Sciences, Tabriz, Iran. The authors thank students’ research committee for their support.

Conflict of interest The authors declare no conflict of interest.

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Figure 6 Acetaminophen‑induced mitochondrial depolarisation. NAC and silafibrate administration effectively prevented mitochondrial injury caused by acetaminophen. * Indicates significantly lower membrane potential as compared to the control group (p<0.05). § Indicates a significantly higher membrane potential as compared to the acetaminophen‑treated group (p<0.05) REFERENCES 1. Amar PJ, Schiff ER. Acetaminophen safety and hepatotoxicity - where do we go from here? Expert Opin Drug Saf 2007;6:341-55. doi: 10.1517/14740338.6.4.341 2. Dai G, He L, Chou N, Wan YJ. Acetaminophen metabolism does not contribute to gender difference in its hepatotoxicity in mouse. Toxicol Sci 2006;92:33-41. doi: 10.1093/toxsci/ kfj192 3. James LP, Mayeux PR, Hinson JA. Acetaminophen-induced hepatotoxicity. Drug Metab Dispos 2003;31:1499-506. doi: 10.1124/dmd.31.12.1499 4. Hinson JA, Nelson SD, Mitchell JR. Studies on the microsomal formation of arylating metabolites of acetaminophen and phenacetin. Mol Pharmacol 1977;13:62533. PMID: 18660 5. Bruno M. Inhibition of protein phosphatase activity and changes in protein phosphorylation following acetaminophen exposure in cultured mouse hepatocytes. Toxicol Appl Pharmacol 1998;153:119-32. PMID: 9875306 6. Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Hynan LS, Reisch JS, Schiødt FV, Ostapowicz G, Shakil AO, Lee WM. Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study. Hepatology 2005;42:1364-72. doi: 10.1002/hep.20948 7. Schilling A, Corey R, Leonard M, Eghtesad B. Acetaminophen: Old drug, new warnings. Cleve Clin J Med 2010;77:19-27. doi: 10.3949/ccjm.77a.09084 8. Ziaee M, Samini M, Bolourtchian M, Ghaffarzadeh M, Ahmadi M, Eghbal MA, Khorrami A, Andalib S, MalekiDizaji N, Garjani A. Synthesis of a novel siliconized analog of clofibrate (Silafibrate) and comparison of their antiinflammatory activities. Iran J Pharm Res 2011;11:91-5.

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Sažetak Citoprotektivni učinci silafibrata, novosintetiziranog silikoniranog derivata klofibrata protiv acetaminofenom izazvane toksičnosti u izoliranim hepatocitima štakora Acetaminofen (N-acetil-para-aminofenol, APAP) često je korišteni antipiretik i analgetik koji može izazvati oštećenja jetara. Na modelu izoliranih hepatocita štakora istražili smo toksične učinke APAP-a i protektivne učinke silafibrata i N-acetilcisteina (NAC). Hepatociti su izolirani iz mužjaka štakora soja Sprague-Dawley perfuzijom jetara i uvođenjem enzima kolagenaze putem portalne vene. Ta se tehnika zasniva na perfuziji jetara kolagenazom nakon uklanjanja kalcijevih iona (Ca2+) kelatorom. Stanice su tretirane različitim koncentracijama APAP-a, silafibrata i NAC-a. Kao markeri toksičnosti mjereni su smrt stanica, stvaranje reaktivnih kisikovih vrsta (ROS), lipidna peroksidacija i depolarizacija mitohondrija. Primjena APAP-a u štakora izazvala je stvaranje ROS-ova i lipidnu peroksidaciju. APAP je uzrokovao depolarizaciju mitohondrija u izoliranim stanicama. Primjena silafibrata (200 µmol L-1) i/ili NAC-a (200 µmol L-1) smanjila je stvaranje ROS-a, lipidnu peroksidaciju i depolarizaciju mitohondrija uzrokovanu APAP-om. Utvrdili smo da je citotoksičnost APAP-a posredovana oksidativnim stresom. Nadalje, čini se da su mitohondriji ciljni stanični organeli za oštećenja hepatocita izazvanih APAP-om. Moguće je da su protektivna svojstva silafibrata i/ili NAC-a protiv APAP‑om induciranog oštećenja jetara uključivala i indukciju antioksidacijskih enzima, zaštitu od oksidativnog stresa i upalnih odgovora te promjenu razine staničnoga glutationa.

KLJUČNE RIJEČI: fibrati; mitohondriji; oksidativni stres; oštećenje jetara izazvano lijekom; reaktivne kisikove vrste (ROS)

CORRESPONDING AUTHOR: Mohammad Ali Eghbal Tabriz University of Medical Sciences Pharmacology and Toxicology Department School of Pharmacy Tabriz, Iran E-mail: m.a.eghbal@hotmail.com

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DOI: 10.2478/10004-1254-65-2014-2392

Original article

Micronucleus, alkaline, and human 8-oxoguanine glycosylase 1 modified comet assays evaluation of glass-ionomer cements - in vitro Elizabeta Galić1, Antonija Tadin2, Nada Galić3, Vilena Kašuba4, Marin Mladinić4, Ružica Rozgaj4, Dolores Biočina-Lukenda2, Ivan Galić2, and Davor Želježić4 Health Center of Split-Dalmatia County1, Study of Dental Medicine, School of Medicine, University of Split2, Split, Department of Endodontics and Restorative Dental Medicine, School of Dental Medicine, University of Zagreb3, Mutagenesis Unit, Institute for Medical Research and Occupational Health4, Zagreb, Croatia Received in April 2013 CrossChecked in May 2013 Accepted in April 2014

The purpose of this study was to evaluate the genotoxic potential of components leached from two conventional self-curing glass-ionomer cements (Fuji IX and Ketac Molar), and light-curing, resin modified glass-ionomer cements (Vitrebond, Fuji II LC). Evaluation was performed on human lymphocytes using alkaline and hOGG1 modified comet, and micronucleus assays. Each material, polymerised and unpolymerised, was eluted in extracellular saline (1 cm2 mL-1) for 1 h, 1 day, and 5 days. Cultures were treated with eluates using final dilutions of 10-2, 10-3, and 10-4. Alkaline comet assay did not detect changes in DNA migration of treated cells regardless of the ionomer tested, polymerisation state, and elution duration. Glass ionomers failed to significantly influence micronucleus frequency. No oxidative DNA damage in treated lymphocytes was observed using hOGG1 modified comet assay. Obtained results indicate high biocompatibility of all tested materials used in the study under experimental conditions.

KEY WORDS: biocompatibility; cytotoxicity; DNA damage; genotoxicity; lymphocytes

The most important requirement for a material to be used in medical applications is its biocompatibility.

Dental materials can lead to cell damage of the pulp, gingiva, and oral mucosa. Various tests have been applied to assess the risk of such damage (1-4).

Due to their properties, glass ionomer cements (GICs) are widely used as restorative materials in dentistry. Since their commercial introduction, GICs have been available in essentially two types: ‘conventional’ glass ionomers (CGICs) and resinmodified glass ionomer cements (RMGICs) (5).

CGICs are composed of fine calcium fluoroaluminosilicate glass powder and aqueous solutions of polyacrylic acid and its copolymers.

RMGICs are usually formulated from fluoro-

aluminosilicate glasses, photo-initiators, polyacrylic acid, water, and a water-soluble methacrylate monomer, such as hydroxyethyl methacrylate (HEMA) (6, 7).

Despite the improved mechanical properties of RMGICs their biocompatibility is still controversial (4, 5). Most of the studies suggested that CGICs revealed minimal toxicity in vitro or in vivo, whereas RMGICs showed significant cytotoxicity and genotoxicity (8, 10-12). There are sufficient data demonstrating that the liberated substances may diffuse pulp ward in high quantities (triethyleneglycoldimethacrylate - TEGDMA, HEMA), particularly through thin dentin layers or after acid-etching of dentin. It appears that high quantities of these

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components may result in pulpal inflammation (13).

De Souza Costa et al. (10) reported that Vitrebond reduced viability of odontoblasts by 74.6 %, Fuji II LC by 45.5 %, Fuji IX by 29.5 %, and Ketac Molar by 32.5 %. However, Koulaouzidou et al. (14) found that Fuji II LC showed only minimal cytotoxicity and mild antiproliferative effect on three cell lines (L929, BHK21/C13, and RCP-C2A). Do Nascimento et al.

(15) reported that Vitrebond could cause marked inflammatory pulpal reactions and a large necrotic zone. Six et al. (16) used Fuji IX on rats’ upper molars and found that after 8 days it caused disruption of the odontoblastic layer, blood vessels’ dilatation, and inflammatory reaction of the pulp with presence of bacteria in dentinal tubules. However, after 30 days, pulpal tissue was again healthy and looked normal (16).

Thus, the aim of this in vitro study was to comparatively evaluate possible genotoxicity of monomers released from two types of commercially available groups of glass ionomers, CGICs and RMGICs, with respect to their polymerisation state and time elapsed from the setting of the materials. To detect possible differences in their mechanism of actions two different cytogenetic techniques were applied on human lymphocyte cultures: comet and micronucleus assay. In addition, human 8-oxoguanine glycosylase 1 (hOGG1) enzyme modified comet assay was performed to evaluate possible oxidative DNA damage.

MATERIALS AND METHODS Blood sampling Each material was tested on lymphocytes of three young, healthy, non-smoking voluntary donors. The donors were one male and two females with mean age 29.33±1.69. According to a questionnaire filled by donors, in 12 months prior to blood sampling, they had not been exposed to any physical or chemical agents that might have interfered with the results of genotoxicity testing. Blood was drawn by antecubital venipuncture into heparinised vacutainers (Becton Dickenson, Plymouth, UK). All donors were acquainted with the study and they willingly signed permissions to use their blood samples for scientific purposes. All described procedures were in accordance with the high ethical standards, complied with the principles laid down in the Declaration of Helsinki.

Preparation of glass ionomer materials Four different glass ionomers were tested: Fuji IX (GC Corp, Tokyo, Japan) and Ketac Molar (3M/ESPE, St. Paul, MN, USA) as conventional GICs, and Fuji II LC (GC Corp, Tokyo, Japan) and Vitrebond (3M/ ESPE, St. Paul, MN, USA) as resin-modified GICs. The composition of selected materials as per their manufacturers is presented in Table 1. Ionomers were polymerised in aseptic conditions as recommended by the manufacturers, in 4 mm in diameter and 2 mm thick rounded plates. CGICs were polymerised chemically in the air, and RMGICs with the Elipar

Table 1 Glass-ionomers used in the present study (according to manufacturers’ data sheets)

Glass ionomer group

Resin-modified glass ionomer cements

Conventional glass ionomer cements

Ionomer tested

Components

Fuji II LC (GC, Tokyo, Japan)

Powder: fluoroaluminosilicate glass Liquid: polyacrylic acid and copolymer of acrylic and maleic acid, 2-hydroxyethyl-methacrylate (HEMA), initiator, water.

Vitrebond (3M ESPE, St. Paul, MN, USA)

Powder: SiO2, AlF3, ZnO, Na3AlF6, SP-940S glass powder, disodiumhydrogenphosphate, diphenyliodoniumchloride Liquid: conacid-isocyanatoethyl-methacrylateacrylic acid , HEMA, bornandion, di-tert-butyl-p-cresol

Fuji IX (GC, Tokyo, Japan) Ketac Molar (3M ESPE, St. Paul, MN, USA)

Powder: fluoroaluminosilicate glass, polyacrylic acid powder Liquid: polyacrylic acid, polybasic carboxylic acid Powder: fluoroaluminosilicate glass, ZnO, polycarboxylic acid, pigments Liquid: water, copolymer acrylic calcium, maleic acid, tartaric acid, benzoic acid

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TriLight halogen curing unit (650 mW cm-2) from a distance of 2 mm for 40 s. Polymerised ionomers were eluted for 1 h, 1 day, and 5 days in extracellular saline (ECS), consisting of 40 mmol L-1 NaCl, 3 mmol L-1 KCl, 1 mmol L-1 CaCl2, 1 mmol L-1 MgCl2, 10 mmol L-1 HEPES, adjusted to pH 7.3–7.4 with NaOH. Material surface vs. ECS volume ratio was 1 cm2 mL-1 (17).

The elutions of unpolymerised glass ionomers in ECS were set up in the same manner.

Initiation of cell cultures and micronucleus assay To initiate the cell cultures, 0.5 mL of blood was introduced into a cell culture flask (Nunc GmbH, Wiesbaden, Germany) containing 5 mL of RPMI medium (Gibco, Carlsbad, USA) supplemented with 15 % foetal bovine serum (Sigma, St. Louis, USA), 10 μg mL-1 of phytohemagglutinin (Murex, Dartford, UK), 2.5 g L-1 of phenol red pH indicator (Sigma, St. Louis, USA), 100 IU of penicillin (Sigma, St. Louis, USA), and 100 IU of streptomycin (Sigma, St. Louis, USA). Duplicate cell cultures per donor were treated with the following four dilutions of each glass ionomer eluate: 10 -1, 10 -2, 10 -3, and 10 -4. Dilutions were prepared in RPMI medium, and pH values were adjusted at 7.2. The same treatment scheme was used for each of the three elution times (1 h, 1 day, and 5 days). GIC eluates were initiated 5 days, 1 day, and 1 h prior to blood sampling. The whole blood cultures were treated for 72 h. Simultaneously, negative control cultures were treated with the same volume of physiological solution of ECS, whereas the positive controls were treated with bleomycin (Sigma, St. Louis, USA) at the final concentration of 30 μg mL-1 for the 5 last hours of cultivation. Cytochalasin B (Sigma, St. Louis, Germany) was added at a final concentration of 6 μg ml-1 to the cultures after 44 h of incubation. Cultures were incubated at 37 °C for 72 h.

After incubation, the same culture was simultaneously subjected to analysis by all techniques.

For micronucleus assay, cells were centrifuged, pretreated with physiological solution, and fixed with 3:1 (v/v) methanol/acetic acid solution. They were then air-dried and stained with 5 % Giemsa (Sigma, St. Louis, USA) (18). For each elution time and dilution tested, 1000 binucleated lymphocytes per duplicate slide per donor were analysed scoring the number of micronuclei. We applied the scoring criteria described by Fenech et al. (19).

Cytotoxicity testing Using the trypan blue exclusion technique, the relative cell viability was tested 72 h after the cultures were initiated. For all tested ionomers, cytotoxicity of dilution 10-1 of 1-day eluates appeared to be higher than 17 %. Therefore, only the cultures treated with dilutions of 10-2, 10-3, and 10-4 were analysed using micronucleus, alkaline, and hOGG1 modified comet assays for all elution periods. After the treatment of cell cultures with ionomer eluates pH indicator contained within the medium did not show any changes in pH value. After the end of cultivation period, pH of each cell culture was additionally checked by a SevenEasy pH meter (Metler-Toledo GmbH, Schwertzenbach, Switzerland) and it was continuously 7.27±0.04, which is, according to the manufacturer, the regular pH value for RPMI cell culture medium.

Alkaline and hOGG1 modified comet assay Following the treatment, cultures were centrifuged, supernatant removed, and both alkaline and hOGG1 comet assays were performed on whole blood samples in accordance with the protocols by Singh et al. (20) and Smith et al. (21), respectively. All the chemicals were obtained from Sigma Chemical Company (Sigma-Aldrich, Munich, Germany).

Normal melting point agarose (NMP) 0.6 % was layered on slides precoated with 1 % NMP. Blood samples (8 µL) were mixed with 0.5 % low melting point agarose, placed on the slides, and immersed in standard ice-cold lysis solution (pH 10) and stored overnight at 4 °C. For the alkaline comet assay the standard procedure was followed (20). For the hOGG1 modified comet assay, following the lysis slides were rinsed in three changes of neutralisation buffer (40 mmol L-1 HEPES, 0.1 mol L-1 KCl, 0.5 mmol L-1 EDTA, pH 7.4) for 5 min at room temperature. One parallel slide was treated with hOGG1 enzyme in neutralisation buffer (V=60 µL; final dilution 1:1000).

The other parallel slide was treated with 60 µL of neutralisation buffer only. Treatment was performed in a humidified chamber for 10 min at 37 °C and followed by 15 min of denaturation (1.5 mol L-1 NaCl, 1 mmol L -1 Na 2EDTA, pH 12.1) and 20 min of electrophoresis at 0.7 V cm-1, 300 mA. The slides were neutralised in three changes of buffer (0.4 mol L-1 Tris-HCl, pH 7.5), 5 min each, stained with ethidium bromide (20 g mL-1) and examined using a 250x magnification fluorescence microscope (Olympus,

182 Japan). A total of 100 comets per concentration tested were scored on each duplicate slide. The edges of the gel, occasional dead cells, and superimposed comets were avoided. Tail length and tail intensity were measured using Comet Assay IV analysis system (Perceptive Instruments Ltd., Suffolk, UK). Oxidative DNA damage was calculated as per the following description. For each donor, mean tail intensity was calculated separately for the slides treated with hOGG1 enzyme and for the slides treated with buffer.

Further difference between those two values was obtained for each donor, indicating oxidative DNA damage. Results of hOGG1 comet assay were presented as mean tail lengths only, since in this type of assay modification percentage of DNA in tail is the most informative parameter (21).

Statistical analysis Differences in the number of micronuclei treated and control lymphocytes were evaluated using chisquare and Fisher’s LSD test. Regarding the comet

Galić E, et al. IN VITRO EVALUATION OF GLASS-IONOMER CEMENTS Arh Hig Rada Toksikol 2014;65:179-188

assay endpoints, to normalise the distribution and to equalise the variances, a logarithmic transformation of data was applied. Differences between the groups were tested using ANOVA followed by Duncan’s test.

To evaluate oxidative DNA damage, differences between the means for the slides treated with enzyme and the corresponding slides treated with buffer only were tested using ANOVA followed by Duncan’s post-hoc analysis. The level of significance was set at 0.05.

RESULTS Micronucleus assay did not show statistically increased micronucleus frequency for any of the eluates tested. A slightly increased number of micronuclei was detected in the cultures treated with 10-2 dilution of 1-h eluates of unpolymerised Vitrebond and Ketac Molar (Figure 1).

Figure 1 Number of micronuclei in lymphocyte cultures treated with different dilutions (10-2, 10-3, and 10-4) of initial glass ionomer eluates (1 cm2 mL-1 of extracellular saline) after 1 h, 24 h, and 5 days of elution. One thousand binucleated lymphocytes were analysed per treatment on each of three blood samples p - polymerised, n - unpolymerised

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Eluates of polymerised forms of these ionomers did not influence micronucleus frequency. Further, 10-2 and 10-3 dilutions of 1-day eluates of unpolymerised Ketac Molar, and the polymerised form of GC Fuji II LC slightly, but insignificantly elevated the number of micronuclei (Figure 1).

Similarly, in the culture treated with dose eluates, a slightly increased micronucleus frequency compared to 1-h elution was found. Dilution 10-2 of 5-day eluates of polymerised Vitrebond and GC Fuji IX GP, and unpolymerised Ketac Molar induced further insignificant elevation of MN (Figure 1).

Neither did the comet assay show statistically increased tail length or Olive tail moment for any of the eluates of the tested glass ionomers, polymerised or unpolymerised. Again, slightly elevated DNA migration was detected in lymphocytes treated with 10 -2 dilution of 1-h of both unpolymerised and polymerised Vitrebond, unpolymerised Ketac Molar, and polymerised Fuji II LC (Figures 2, 3). Later

eluates did not show any differences regardless of their dilution or polymerisation state.

Application of hOGG1 modified comet assay did not indicate an increase in the 8-oxoguanine formation due to possible oxidative stress induced by resin monomers released form the tested glass ionomers (Table 2). A slight increase in tail intensity was observed for all tested materials 24Â h after the initial setting, and only for the lowest eluate dilution (10-2).

However, the increase was simultaneously observed for the slides treated with hOGG1 enzyme and the corresponding buffer treated slides. We did not observe any statistical difference in the percentage of DNA in tails due to hOGG1 treatment.

DISCUSSION The study presents the results of genotoxicity evaluation of eluates of four glass ionomers: Fuji II LC and Vitrebond as resin-modified glass ionomer

Figure 2 Tail length values for lymphocyte cultures treated with different dilutions (10-2, 10-3, and 10-4) of initial glass ionomer eluates (1 cm2 mL-1 of extracellular saline) after 1 h, 24 h, and 5 days of elution. One hundred comets were analysed per treatment on each of three blood samples. p - polymerised, n - unpolymerised

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Figure 3 Mean tail moment values for lymphocyte cultures treated with different dilutions (10-2, 10-3, and 10-4) of initial glass ionomer eluates (1 cm2 mL-1 of extracellular saline) after 1 h, 24 h, and 5 days of elution. One hundred comets were analysed per treatment on each of three blood samples. p - polymerised, n - unpolymerised

cements, and Fuji IX and Ketac Molar as conventional glass ionomer cements. We aimed to detect possible differences in the mechanisms of their genotoxicity that may arise due to differences in their composition and initiation of polymerisation (chemical or photoactivation). To get more information on their biocompatibility, glass ionomer cements were tested both in their unpolymerised and polymerised form. In this assessment two cytogenetic techniques were used: alkaline comet and micronucleus assay. Comet assay detects primary DNA damage that could result from direct interaction of monomers, initiators or any other leached components with genomic DNA. That type of damage does not require cell division to be expressed and evaluated (22). Modification of the comet assay procedure by exposing the isolated DNA to hOGG1 enzyme allows to specifically detect 8-oxoguanine base that arises as a result of oxidative DNA damage. Micronucleus assay detects DNA lesions that are not repaired properly and after replication result in the formation of acentric chromosome fragments. It also detects damage of the cell spindle disabling chromosomes to be attached

properly and chromatids to be divided between the newly forming cells. Thus, contrary to the comet assay, micronucleus assay enables detection of both types of genotoxic agents, clastogens and aneugens (18).

It has been proven that due to incomplete polymerisation, residual monomers (HEMA, BisGMA) are leached from dental materials (23). In our study we did not detect any genome damage in the above mentioned period of time and eluate dilutions using comet assay. However, micronucleus assay indicated a slight but insignificant increase in the micronucleus frequency in lymphocytes treated with the lowest dilution (10-2) of 1-day eluate of polymerised GC Fuji II LC, and 5-day eluates of polymerised Vitrebond and GC Fuji IX GP. Unpolymerised Vitrebond and Ketac Molar insignificantly elevated micronuclei incidence as early as after 1-h elution indicating faster leaching of active components from unpolymerised materials. Unpolymerised Ketac Molar slightly influenced micronuclei induction throughout the entire period of 5-day elution indicating persistence of its leached active components, presumably ZnO that was suspected to be clastogenic and that was not

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Table 2 Results of hOGG1 modified comet assay presenting mean tail intensity values (% DNA in comet’s tail). The results obtained for the slides treated with buffer and the corresponding slides treated with enzyme are shown for each treatment. One hundred comets were analysed per treatment on each of three blood samples

Glass ionomer

Form

Eluate dilution 10 10-3 10-4 10-2 10-3 10-4 10-2 10-3 10-4 10-2 10-3 10-4 10-2 10-3 10-4 10-2 10-3 10-4 10-2 10-3 10-4 10-2 10-3 10-4 -2

Unpolymerised Vitrebond Polymerised

Unpolymerised Ketac Molar Polymerised

Unpolymerised GC Fuji IX GP Polymerised

Unpolymerised GC Fuji II LC Polymerised Negative control (22 μL extracellular saline) Positive control (0.1 mmol L-1 H2O2 10 min)

Oxidative DNA damage after elution of glass ionomers (mean±SD) 1 h

1 day

5 days

0.70±0.32 0.16±0.08 0.14±0.06 0.18±0.08 0.12±0.04 0.24±0.07 0.16±0.03 0.02±0.00 0.03±0.01 0.27±0.09 0.04±0.00 0.14±0.05 0.01±0.00 0.14±0.02 0.07±0.05 0.14±0.04 0.15±0.04 0.01±0.01 0.13±0.05 0.09±0.03 0.08±0.03 0.11±0.03 0.02±0.01 0.04±0.03 0.02±0.02

1.98±0.94 1.86±0.98 1.02±0.86 1.18±0.80 1.33±0.82 0.92±0.47 1.53±0.68 0.95±0.23 1.31±0.54 1.40±0.86 0.90±0.32 0.13±0.06 1.52±0.27 1.07±0.09 0.86±0.14 0.97±0.07 1.13±0.74 0.98±0.55 0.93±0.28 1.20±0.23 0.87±0.17 1.04±0.19 1.17±0.41 0.92±0.47 0.81±0.18

0.12±0.06 0.08±0.02 0.06±0.00 0.05±0.03 0.06±0.01 0.04±0.01 0.34±0.11 0.13±0.05 0.11±0.01 0.16±0.03 0.22±0.08 0.18±0.06 0.18±0.03 0.24±0.09 0.06±0.02 0.11±0.03 0.14±0.02 0.12±0.02 0.01±0.02 0.14±0.03 0.12±0.03 0.14±0.03 0.10±0.02 0.11±0.02 0.03±0.01

16.4±1.821

28.07±1.201

15.7±1.051

statistically significant oxidative DNA damage, p<0.05 between the corresponding slides treated with hOGG1 enzyme and buffer only. Oxidative DNA damage was calculated as difference in tail intensity values between hOGG1 and buffer treated slides.

1

contained in other glass-ionomers (24). However, the observed effects could not at all be considered biologically relevant. Considering the effect of Vitrebond and Fuji II LC on micronucleus frequency, it would be mainly mediated by HEMA that is known to be genotoxic (25). Contrary to Kleinsasser et al.

(25) that reported increased DNA migration ability of human lymphocytes exposed to HEMA, we did not detect any effect of ionomers containing this monomer on comet assay endpoints. Thus, the observed fluctuations in the number of micronuclei, although biologically insignificant, might have been mediated by slight anuegenic effect of monomers. Vitrebond’s genotoxicity could be additionally mediated by dipheniliodiumchloride (DPICI) that is used as the

initiator. Leyhausen et al. (26) reported that DPICI that was leached from polymerised Vitrebond exhibited genotoxic activity. These results may suggest that even from the unpolymerised form of ionomers, after mixing powder and liquid components HEMA could not be leached in the amounts needed to induce significant DNA damage under the experimental conditions used in our evaluation. The choice of solvent vehicle used in elution might also be critical. Kleinsasser et al. (25) used organic solvent DMSO, and we used extracellular saline as the main polar solvent saline solution. Ribeiro et al. (27) published the results on genotoxic evaluations of Vitrebond and Ketac Molar on Chinese hamster ovary (CHO) and mouse lymphoma (L5178Y) cell lines

186 using comet assay. Unlike in our study, they tested the components separately without mixing them. The results showed that powders from assay did not show genotoxic effects. The authors observed increased DNA migration only for the highest concentration of Ketac Molar powder phase (100 µg mL -1 and 1000 µg mL-1), and the highest concentration of Vitrebond liquid phase (1 %). No measurable genotoxicity was found in the liquid from Ketac Molar in all dilutions tested (0.01–1 %). In the study of Bakopoulou et al. (28), 72 h-eluates of Ketac Cem significantly increased the frequency of sister chromatid exchanges and chromosomal aberrations in human lymphocyte cultures.

Stea et al. (29) evaluated genotoxicity of Vitrebond eluates in human lymphocytes cultures using sister chromatid exchange (SCE) assay. The authors reported increased SCE frequency for 10-2 eluate dilution of unpolymerised Vitrebond and 10-1 of the polymerised one. Bakopoulou et al. (28) showed Vitrebond’s eluate to be very cytotoxic, as the first series of dilutions of both materials caused a complete mitotic arrest in lymphocyte cultures but the authors used ten-fold lower dilutions of eluate in culture treatment than we did in the present study. Also, the eluate derived from Vitrebond specimens (5 mm in diameter and 2 mm thick) caused extensive genotoxicity after 24 h, increasing the frequencies of sister chromatid exchange and the number of chromosomal aberrations (28). Of all the observed insignificant effects on micronucleus frequency, the one caused by Fuji IX was the lowest.

The main deficiency of ex vivo tests is their questionable clinical relevance. Although the effects detected in in vitro studies could not be directly extrapolated into in vivo conditions, they are informative and could indicate a potential degree of health risk that may arise from using certain chemical compounds in dental restoration. In vitro studies provide limited responses due to the lack of biological and physiological components that cannot be replicated in the laboratory. The largest limitations of this study were low concentrations used to reproduce the situation in the mouth and the type of cell on which we carried out the research. It would have been a better solution to have used oral cells that are the first line of defence in the oral cavity in direct contact with the tested material. We could suggest that under the conditions presented in this study, in non-toxic concentrations range, the evaluated glass ionomer cements (Vitrebond, Ketac Molar, Fuji IX GP, and

Galić E, et al. IN VITRO EVALUATION OF GLASS-IONOMER CEMENTS Arh Hig Rada Toksikol 2014;65:179-188

Fuji II LC) did not exhibit any biologically relevant genotoxic activity on peripheral blood lymphocytes in vitro measured by comet and micronucleus assays, nor were they able to induce oxidative DNA damage.

With respect to such results, a slight and insignificant increase in the micronucleus number might raise suspicion of their restricted aneugenicity. Altogether, under the experimental conditions used in the study, the tested glass ionomers showed a satisfying level of biocompatibility.

Acknowledgements This investigation was supported by the Croatian Ministry of Science, Education, and Sports as part of the Themes No: 022-0222148-2137 and No: 0650650444-0418.

Conflict of interest The authors declare no conflict of interest.

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Sažetak Procjena in vitro toksičnosti stakleno-ionomernih cemenata primjenom mikronukleus testa, alkalnog komet testa i komet testa modificiranog hOGG1 enzimom Svrha istraživanja bila je procijeniti genotoksični potencijal komponenata koje izlučuju dva konvencionalna samopolimerizirajuća stakleno-ionomerna cementa (Fuji IX i Ketac Molar) te svjetlosno polimerizirajući i smolom modificirani stakleno-ionomerni cementi (Vitrebond, Fuji II LC). Istraživanje je provedeno na ljudskim limfocitima primjenom alkalnog komet testa, komet testa modificiranog hOGG1 enzimom te mikronukleus testa. Svaki materijal, polimerizirani i nepolimerizirani, eluiran je u fiziološkoj otopini (1 cm2 mL-1) tijekom jednog sata, jednog dana i tijekom 5 dana. Kulture limfocita tretirane su eluatima u razrjeđenjima 10-2, 10-3 i 10-4. Alkalnim komet testom nisu zabilježene promjene u migraciji DNA iz tretiranih stanica bez obzira na ispitani ionomer, vrstu polimerizacije i trajanje elucije. Izloženost staklenim ionomerima nije značajno utjecala na učestalost mikronukleusa. Primjenom hOGG1 modificiranog komet testa nije zamijećeno oksidativno oštećenje DNA u tretiranim limfocitima. Dobiveni rezultati upućuju na visoki stupanj biokompatibilnosti svih testiranih materijala koji su se koristili u eksperimentalnim uvjetima.

KLJUČNE RIJEČI: biokompatibilnost; citotoksičnost; genotoksičnost; limfociti; oštećenje DNA

CORRESPONDING AUTHOR: Antonija Tadin Department of Endodontics and Restorative Dental Medicine Study of Dental Medicine, School of Medicine, University of Split Soltanska 2, 21 000 Split, Croatia E-mail: atadin@mefst.hr

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Sarap NB, et al. RADIOACTIVITY IN SOUTHERN SERBIA Arh Hig Rada Toksikol 2014;65:189-197

DOI: 10.2478/10004-1254-65-2014-2427

Original article

Environmental radioactivity in southern Serbia at locations where depleted uranium was used Nataša B. Sarap, Marija M. Janković, Dragana J. Todorović, Jelena D. Nikolić, and Milojko S. Kovačević University of Belgrade, Vinča Institute of Nuclear Sciences, Radiation and Environmental Protection Department, Belgrade, Serbia Received in July 2013 CrossChecked in February 2014 Accepted in April 2014

In the 1999 bombing of the Federal Republic of Yugoslavia, NATO forces used ammunition containing depleted uranium. The cleaning of depleted uranium that followed was performed in southern Serbia by the Vinča Institute of Nuclear Sciences between 2002 and 2007 at the locations of Pljačkovica, Borovac, Bratoselce, and Reljan. This paper presents detailed results of radioactivity monitoring four years after cleaning (2011), which included the determination of gamma emitters in soil, water, and plant samples, as well as gross alpha and beta activities in water samples. The gamma spectrometry results showed the presence of natural radionuclides 226Ra, 232Th, 40K, 235U, 238U, and the produced radionuclide 137Cs (from the Chernobyl accident). In order to evaluate the radiological hazard from soil, the radium equivalent activity, the gamma dose rate, the external hazard index, and the annual effective dose were calculated. Considering that a significant number of people inhabit the studied locations, the periodical monitoring of radionuclide content is vital.

KEY WORDS: environmental samples; gamma spectrometry; gross alpha activity; gross beta activity

Over the last 40 years, Europe has experienced several major radioactive fallouts, from atmospheric nuclear tests in North Africa during the 1960s, the Chernobyl accident in 1986, the Acerinox accident in 1998 to the wars in former Yugoslavia in the 1990s (1). Depleted uranium (DU) came into military use in the early 1990s and was first used in 1991 in the Gulf War (2, 3). The DU used during the wars in former Yugoslavia represents the most recent source of radioactivity released into the environment, following NATO air strikes (1, 4-6). NATO and Serbian Military (VS) data on DU use are in large discordance, but overlap for at least four locations (5, 6) in southern Serbia: Pljačkovica, Borovac, Bratoselce, and Reljan (Figure 1).

Uranium is a naturally occurring ubiquitous heavy metal found in various chemical forms in all

soils, rocks, seas, and oceans. It is also present in drinking water and food. Natural uranium consists of a mixture of three different isotopes: 238U (99.27 %), 235U (0.72 %), and 234U (0.0054 %) (7).

It is both chemically toxic and radioactive, and being water-soluble, it is easily taken up by plants, thus entering food chains and soil/water systems (8). Metallic uranium used for military purposes is practically insoluble in water and body fluids.

However, while exposed to air and water, uranium is slowly oxidised to a +4 or +6 state (9).

The uranyl ion easily forms complexes, mainly carbonate [UO2(CO3)2]-2, which are well dissolved in water and body fluids. As the result of these chemical processes (weathering), uranium dispersed into the environment in a metallic form can slowly migrate to drinking water and plants and enter the

190

Figure 1 Information on locations in southern Serbia which are targeted depleted uranium. GPS coordinates of locations: Pljačkovica (420 34′ 47″ N; 210 53′ 50″ E); Borovac (420 23′ 45″ N; 210 45′ 09″ E); Bratoselce (420 20′ 40″ N; 210 45′ 23″ E); Reljan (420 18′ 53″ N; 210 45′ 58″ E)

human food chain. However, the weathering of DU penetrators strongly depends on the type of soil, its humidity and temperature (9). Depending on the physicochemical characteristics of the medium and chemical form in which uranium is bound, there are various scenarios for environmental contamination.

Quantitative determination of the DU fraction in environmental samples is difficult, primarily because of the relatively large content of natural uranium present in the environment. Based on the obtained concentrations of 235U and 238U and their ratio, a conclusion whether this ratio presents the natural activity ratio of uranium isotopes or possible presence of DU can be reached.

Considering the fact that people live and work on the locations examined here, it is necessary to periodically monitor the content of radionuclides in samples from these locations, especially because of the use of DU. Therefore, one of the aims of this study was to offer a detailed approach to perform such monitoring.

MATERIALS AND METHODS Environmental monitoring Regular radioactivity monitoring in Serbia includes spectrometric measurements of gamma emitters in soil, water and plant samples, as well as determination of the gross alpha and beta activities

Sarap NB, et al. RADIOACTIVITY IN SOUTHERN SERBIA Arh Hig Rada Toksikol 2014;65:189-197

in water samples. The cleaning of DU left over after NATO air strikes, along with detailed dosimetric screening and decontamination, was performed by the Radiation and Environmental Protection Department of the Vinča Institute of Nuclear Sciences from 2002 to 2007 at Pljačkovica, Borovac, Bratoselce, and Reljan, located in southern Serbia, in coordination with the Agency for Radiation Protection and Nuclear Safety of Serbia (10) (Figure 1). Because of the loose soil at these locations, projectiles containing DU were found already at a depth of one meter. The discovered projectiles, contaminated soil, and all radioactive materials were disposed of as radioactive waste.

After the cleaning, the terrain was aligned and another dosimetric screening was performed. In 2011, the Department collected and analysed samples from these locations in order to determine the activity concentrations of radionuclides and verify that the fields were successfully cleaned of DU. Sampling and sample preparation Soil samples were collected at 5 measuring points for each location (east, west, north, south, and centre) at a depth of 10-15 cm using a probe. The sampling depth was chosen based on the fact that these measurements follow the migration of radionuclides by depth (11). About 2 kg of samples were placed into polyethylene bags and transported to the laboratory. After removing the stones and vegetation, all soil samples were dried to 105 °C, sieved, placed in plastic 500 mL Marinelli beakers and left to reach radioactive equilibrium for four weeks (12).

The plant samples were taken differently and sampling included the presence of plant species (hay and grass). About 1 kg of samples was placed into polyethylene bags and transported to the laboratory.

Plant samples were dried at room temperature over a few days, ashed at 450 °C during 24 h, placed in plastic boxes of 100 mL and left to reach radioactive equilibrium for four weeks (12).

Water samples were taken from public fountains and wells from where the inhabitants draw water.

These samples (20 L each) were collected directly from the springs in plastic bottles and tightly closed.

For gamma spectrometric measurements, a volume of 17 L of water samples was acidified to pH 2, evaporated to 200 mL under an infrared lamp, and then poured into 200 mL cylindrical polyethylene vials. The samples were then stored to reach radioactive equilibrium (12). For measurements of

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gross alpha and beta activity, a volume of 3 L of water samples was evaporated to dry residue under an infrared lamp. The remainder was heated to dryness at 450 °C (13, 14). The residues were transferred quantitatively to a stainless-steel planchet and measurements were performed immediately after preparation.

Plant samples were concentrated by mineralisation and water samples by evaporation in order to maximize the detection efficiency, which is higher in geometries that are not collected in a large volume because the self-absorption effect is smaller.

Procedure for gamma spectrometry Gamma spectrometric measurements were performed using HPGe Canberra detectors (Canberra Industries, Meriden, Connecticut, USA) with counting efficiency of 18 and 20 %. Due to the low activity in the samples and to reduce the uncertainty of measurement, the samples were measured 60,000 s.

The calibration of detectors for water and plant sample measurement was performed using a cylindrical polyethylene bottle of 200 mL and a plastic box of 100 mL in volume, respectively. This secondary reference material was obtained from the primary reference liquid radioactive material (9031− OL−116/08, type ERX, Czech Metrological Institute, Prague, Czech Republic) spiked with a series of radionuclides (241Am, 109Cd, 139Ce, 57Co, 60Co, 88Y, 113 Sn, 85Sr, 137Cs, and 210Pb) with total activity of 114.9 kBq on 3 March 2008. The calibration of detectors for measurement of soil samples was performed using a silicone resin matrix in the geometry of a plastic Marinelli beaker of 500 mL in volume, (9031−OL−208/08, type ERX, Czech Metrological Institute, Prague, Czech Republic) spiked with a series of radionuclides (241Am, 109Cd, 139 Ce, 57Co, 60Co, 203Hg, 88Y, 113Sn, 85Sr, and 137Cs) with total activity of 40.624 kBq on 15 April 2008.

The spectra were analysed using the program GENIE 2000 (Canberra Industries, Meriden, Connecticut, USA). The activity of 226Ra and 232Th was determined by their decay products: 214Bi (609 keV, 1120 keV, and 1764 keV), 214Pb (295 keV and 352 keV) and 228Ac (338 keV and 911 keV), respectively. 235U was determined via 186 keV corrected for 226Ra. 238U was determined via 234Th (63 keV) or by 234Pa (t1/2=1.17 min, 1000 keV). The activities of 40K and 137Cs were determined from its 1460 keV and 661 keV, respectively. The activities of 210 Pb and 7Be were determined from its 46 keV and

477 keV, respectively. The background spectrum was recorded regularly after or before the sample counting, with an empty 200 mL cylindrical polyethylene bottle, 100 mL plastic box, and 500 mL plastic Marinelli beaker.

The accuracy and reproducibility of gamma spectrometry systems were verified on weekly basis by a quality control (QC) procedure. QC procedure consisted of the following steps: total background count rate was monitored to verify that the detector and shield have not been contaminated by radioactive materials; the total activity of the calibration source [60Co and 133Ba issued by the Czech Metrological Institute and traceable to BIPM (Bureau International des Poids et Measures)] was used to check the efficiency calibration and the general operating parameters of the gamma spectrometry system (source positioning, contamination, library values, and energy calibration); the detector-shield background, full energy peak efficiency, peak shape, and peak drift were measured to confirm whether they were within the warning and acceptance limits; the acceptance limits were set according to the manufacturer’s specifications (peak shape and drift, full energy peak efficiency) and previous experiences (detector shield background); the specific activity of the radionuclides in the samples (A) was calculated using the equation:

(1)

where N is count rate of the sample, t ‒ counting time (s), Pγ ‒ probability of gamma decay (%), ef ‒ detector efficiency (%), V and m are the volume (L) and mass (kg) of the sample.

Minimum detectable activity (MDA) was calculated by the equation (2):

(2)

where LLD is the detection limit, and B is background.

The combined measurement uncertainty of results was calculated at the 95 % level of confidence (k=2).

Minimum detectable activity was inversely proportional to the measurement time of samples (equation 2), which is very important for spectrometry of gamma emitters in environmental samples. Also, in order to reduce the measurement uncertainty of the count rate below the photo peak of a certain energy that corresponds to radionuclides with low activities (such as 235 U), it is necessary to increase the

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measurement time. Uncertainty measurement of count below the photo peak affects the overall uncertainty of the results, which can be calculated by the equation (3):

where is the activity of the sample (Bq L‒1), V is the volume of the sample (L) that corresponds to the mass of solid residue, and I is given by the formula:

where N is the count rate for the sample (s‒1), B is background (s‒1) and ef is the efficiency of the detectors for alpha and beta measurements.

Minimum detectable activity was calculated by the equation (6):

where ∆/N is relative uncertainty of count which is corrected on background, ∆t/t relative uncertainty of time measurement, ∆Pγ/Pγ relative uncertainty of the gamma decay probability (table value), ∆ef/ef relative uncertainty of detector efficiency, ∆V/V (∆m/m) relative uncertainty of volume determination (or mass determination), σn uncertainty due to the fitting and due to the unfairness in the calibration.

Procedure for gross alpha and gross beta activity Gross alpha and beta activity in water samples were determined by α/β low-level proportional counter Thermo Eberline FHT 770 T (ESM Eberline Instruments GmbH, Erlangen, Germany). The counting time was 7,200 s.

Calibration was performed by using a standard source of 90Sr (EM 145, Prague, Czech Republic) with an activity of 189.4 Bq on 1 August 2011 for beta activity and a standard source of 241Am (EM 445, Prague, Czech Republic) with an activity of 224 Bq on the 1 August 2011 for alpha activity. The counting gas was a mixture of 90 % argon and 10 % methane.

The counting efficiencies for the system were 26 % for alpha and 35 % for beta. The background of each detector was determined by counting an empty planchet for 3,600 s.

Quality of sampling and measurement and the calculated uncertainty are very important for predicting the dose for a population. The accuracy and reproducibility of a gas proportional counter were verified on a periodic basis (every week). Calibration was done every week with calibration standards and efficiency was checked. Total background count rate without a source was monitored to verify that the detector and shield have not been contaminated by radioactive materials. Alpha and beta efficiencies of gas proportional counter were checked with 241Am and 90 Sr sources, respectively.

Gross alpha and beta activity was calculated using the following formula:

(4)

MDA =

LLD

V

(5)

(6)

where LLD is the detection limit (s‒1) and V is the volume of the sample (L). Measurement uncertainty determined as expanded measurement uncertainty was 30−40 % for alpha and 15 % for beta.

Radium equivalent activity The distribution of 226Ra, 232Th, and 40K in soil is not uniform. Uniformity with respect to exposure to radiation has been defined in terms of radium equivalent activity (Raeq) in Bq kg−1 to compare the specific activity of materials containing different amounts of 226Ra, 232Th, and 40K (8). It is calculated using the following equation (15, 16): (7) where ARa, ATh, and AK are the specific activities of 226 Ra, 232Th, and 40K in Bq kg−1, respectively.

Gamma dose rate calculation The external gamma dose rate in the air 1 m above ground level was calculated from the measured specific activities of 226Ra, 232Th, and 40K in soil assuming that the other radionuclides, such as 137Cs, 90 Sr, and the 235U series, can be neglected as they contribute very little to the total dose from environmental background (8, 17). The calculations were performed according to the following equation (18): (8) •

where D is the dose rate in nGy h−1.

Calculation of external hazard index The external hazard index, Hex, was defined as (15):

Sarap NB, et al. RADIOACTIVITY IN SOUTHERN SERBIA Arh Hig Rada Toksikol 2014;65:189-197

(9)

The value of this index must be less than unity in order to keep the radiation hazard insignificant. The maximum value of Hex equal to unity corresponds to the upper limit of radium equivalent activity (370 Bq kg−1) (8).

Calculation of annual effective dose To estimate the annual effective dose, the following must be taken into account: the conversion coefficient from absorbed dose in air to effective dose and the indoor occupancy factor (8). Using the dose rate data obtained from the concentration values of natural radionuclides in soil, adopting the conversion factor of 0.7 Sv Gy−1 (18) from the absorbed dose rate in air to the effective dose received by adults, and considering that people in Serbia on average spend 20 % of their time outdoors, the annual effective doses were calculated by (8): (10)

where DE is the annual effective dose in nSv.

RESULTS AND DISCUSSION The results of measurements of gamma emitters (mean activity concentrations) in soil samples collected at different locations in southern Serbia are presented in Table 1 along with a comparison with relevant literature. The activity concentrations of the radionuclides ranged between 16-102 Bq kg−1 for 226 Ra, 21-90 Bq kg−1 for 232Th, 184-979 Bq kg−1 for 40 K, 2.9-19 Bq kg−1 for 137Cs, 1.1-7.4 Bq kg−1 for 235U, and 21-95 Bq kg−1 for 238U. The man-made radioisotope 137Cs, which has a generally negative impact on the environment, was identified in all of the samples and these values were in good agreement with those obtained in other regional studies (8, 19, 20). From the late 1980s, most 137 Cs originates from the Chernobyl nuclear accident.

The highest concentrations of 226Ra, 40K, 137Cs, 235U, and 238U were measured for Bratoselce, and the lowest for Borovac. Based on the measured concentrations of 226Ra, 232Th, and 40K, radium equivalent activity, gamma dose rate, external hazard index and annual effective dose are calculated and presented in Table

193 2. The radium equivalent activity values for the soil samples varied from 60 to 260 Bq kg−1. These values were below the recommended level (370 Bq kg-1) (21).

The mean gamma dose rate in air was calculated to be 78 nGy h−1 and was insignificantly higher than the global average (57 nGy h−1) (18). The calculated values of outdoor annual effective dose ranged from 0.03 to 0.15 mSv, which is close to the global average value (0.07 mSv) (18). The external hazard index obtained in this study ranged from 0.16 to 0.70. Since these values were lower than unity (18), we can conclude that the radiation hazard in the studied area was low.

The results of gamma emitter measurements in water samples from southern Serbia are presented in Table 3. 226Ra was detected in all of the water samples, except for Bratoselce. 40K was detected in water samples from Pljačkovica and Reljan. The obtained values were low and mainly typical for water samples, while the activity concentrations for other radionuclides were below the detection limit.

The results on gross alpha and beta activity in the same water samples are presented in Table 4. Gross alpha activity ranged between <MDA−0.024 Bq L−1, while gross beta activity ranged from 0.07 to 0.36 Bq L−1. The obtained results showed that the natural activity concentrations of alpha and beta emitting radionuclides in water samples from southern Serbia were within recommended levels (0.5 and 1.0 Bq L−1, respectively) (22). The gross alpha activity (Table 4) was lower than the 226Ra concentration in the tested samples (Table 3), as the determination of 226 Ra via daughters in cylindrical polyethylene bottle often leads to erroneous results. Gaseous Rn escapes from the water to a gaseous phase and remains partially present in the bottle, while its solid progenies 214 Bi and 214Pb can non-uniformly deposit on the walls of a bottle.

The results of gamma emitters in plant samples are presented in Table 5. In addition to the radionuclides already mentioned, the plant sample also revealed a presence of 210Pb and 7Be. The obtained results for 210 Pb are characteristic for plant samples and higher than values for other natural radionuclides. This was expected because a plant can absorb 210Pb from soil and air. The activity concentrations of the radionuclides ranged between 1.8-4.5 Bq kg -1 for 226Ra, 2.13.2 Bq kg-1 for 232Th, 284-434 Bq kg-1 for 40K, <MDA1.3 Bq kg-1 for 137Cs, 63-123 Bq kg-1 for 7Be, and 7-50 Bq kg -1 for 210 Pb. The obtained activity concentrations for 235U and 238U in plant samples from

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Table 1 Activity concentrations of radionuclides in soil samples in southern Serbia and comparison with literature (Bq kg−1)

Location Pljačkovica Borovac Bratoselce Reljan Average Republic of Srpska (8) Croatia (19) Slovenia (19) Vojvodina (Serbia) (20) World (18)

Ra

226

27±5 16±3 102±28 35±6 45

Th

232

K

40

137

Cs

32±4 498±44 14±1 21±3 184±19 3±1 58±6 979±133 19±2 90±11 941±64 5±1 50 651 10 Comparison with literature

235

U

238

U

235

U/238U

2.2±0.3 1.1±0.2 7.4±0.8 2.9±0.4 3.4

33±7 21±5 95±18 49±12 57

0.067 0.052 0.059 0.059 /

47

41

536

26

3.4

64

/

74 73.8

62 /

650 318

39 25.1

5.4 /

110 69.5

/ /

40

53

554

12

/

51

/

35

30

400

/

/

35

/

Table 2 Radium equivalent activity, gamma dose rate, annual effective dose, and external hazard index at four locations in southern Serbia

Location Pljačkovica Borovac Bratoselce Reljan Average

Radium equivalent activity (Bq kg−1) 111 60 260 236 167

Gamma dose rate (nGy h−1) 53 27 123 110 78

Annual effective dose (mSv) 0.07 0.03 0.15 0.13 0.10

External hazard index 0.30 0.16 0.70 0.64 0.45

Table 3 Activity concentrations of radionuclides in water samples from southern Serbia (Bq L−1)

Location Bazovnik Pljačkovica draw well house Borovac fountain courtyard 1 Bratoselce courtyard 2 fountain Reljan courtyard

Ra <0.02 0.055±0.007 <0.02 0.033±0.005 <0.02 <0.02 0.018±0.003 0.040±0.007 226

Th <0.02 <0.02 <0.02 <0.02 <0.03 <0.02 <0.02 <0.02 232

K <0.13 0.17±0.03 <0.13 <0.13 <0.13 <0.13 <0.13 0.15±0.03 40

Cs <0.005 <0.005 <0.005 <0.005 <0.004 <0.005 <0.004 <0.005 137

U <0.007 <0.007 <0.007 <0.007 <0.009 <0.007 <0.009 <0.007 235

Table 4 Gross alpha and gross beta activity concentrations of water samples from southern Serbia (Bq L−1)

Location Pljačkovica Borovac Bratoselce Reljan

Bazovnik draw well house fountain courtyard 1 courtyard 2 fountain courtyard

Gross alpha activity

Gross beta activity

0.009±0.001 0.020±0.003 <0.006 0.009±0.001 <0.006 <0.006 <0.006 0.024±0.004

0.16±0.02 0.29±0.04 0.07±0.01 0.09±0.01 0.30±0.05 0.29±0.04 0.11±0.02 0.36±0.05

U <0.15 <0.15 <0.15 <0.15 <0.12 <0.15 <0.15 <0.15 238

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Table 5 Activity concentrations of gamma emitters in plant samples from southern Serbia (Bq per kg of dry matter)

Radionuclide

Location Pljačkovica

Ra 1.8±0.2 Th 3.0±0.6 40 K 434±43 137 Cs 1.0±0.1 235 U <0.3 238 U <4 7 Be 123±12 210 Pb 50±6 Three plant samples were taken from each location 226 232

Borovac

Bratoselce

Reljan

2.3±0.5 3.2±0.6 308±32 1.3±0.3 0.3±0.1 4.4±1.5 116±15 32±4

4.5±0.7 2.1±0.5 284±31 <0.4 <0.2 <2.2 108±10 33±4

2.0±0.4 3.2±0.8 431±45 0.8±0.2 0.3±0.1 3.5±1.4 63±7 7±2

Pljačkovica and Bratoselce were below the detection limit.

CONCLUSION The results of gamma spectrometric measurements of soil samples showed that the radioactivity levels in samples were similar to values characteristic for other locations in world where depleted uranium had not been used (8, 19, 20, 23‒29).

According to the calculated values of external hazard index, there is no risk for the population living in the investigated area. Measurements of radioactivity in environmental samples at these locations in southern Serbia should, however, continue.

5.

6.

7.

8.

9.

10.

Acknowledgement This work was partially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, under Project no. III 43009.

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197

Sažetak Radioaktivnost u životnoj sredini južne Srbije na lokacijama na kojima je korišten osiromašeni uran U napadu na Saveznu Republiku Jugoslaviju 1999. godine NATO-ove su snage koristile municiju s osiromašenim uranom. Od 2002. do 2007. Laboratorij za zaštitu od zračenja i zaštitu životne sredine Instituta Vinča proveo je akciju čišćenja osiromašenog urana na lokacijama Pljačkovica, Borovac, Bratoselce i Reljan. U ovom se radu iznose detaljni rezultati praćenja radioaktivnosti u južnoj Srbiji četiri godine nakon spomenute akcije čišćenja (2011), koji uključuje određivanje gama zračenja u uzorcima zemljišta, vode i biljki te određivanje ukupne alfa i beta aktivnosti u uzorcima vode. Rezultati gama spektrometrije pokazali su prisutnost prirodnih radionuklida: 226Ra, 232Th, 40K, 235U, 238U i proizvedenog radionuklida 137Cs. Za procjenu radijacijskog rizika iz zemljišta izračunati su ekvivalent radija, brzina apsorbirane doze gama zračenja, indeks radijacijskog rizika uslijed vanjskog izlaganja i efektivna doza zračenja. Imajući u vidu da na ispitivanim područjima živi znatan broj ljudi, iznimno je važno da se ondje periodično prati sadržaj radionuklida.

KLJUČNE RIJEČI: uzorci iz životne sredine; gama spektrometrija; ukupna alfa aktivnost; ukupna beta aktivnost

CORRESPONDING AUTHOR: Nataša B. Sarap University of Belgrade, Institute Vinča, Radiation and Environmental Protection Department, Mike Petrovića Alasa 12−14, 11001 Belgrade, Serbia E−mail: natasas@vinca.rs

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Jeršek B et al. INHIBITION OF FUNGAL GROWTH BY ESSENTIAL OILS Arh Hig Rada Toksikol 2014;65:199-208

DOI: 10.2478/10004-1254-65-2014-2486

Original article

Effects of selected essential oils on the growth and production of ochratoxin A by Penicillium verrucosum Barbara Jeršek1, Nataša Poklar Ulrih1,2, Mihaela Skrt1, Neda Gavarić3, Biljana Božin3, and Sonja Smole Možina1 Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana1, Centre of Excellence for Integrated Approaches in Chemistry and Biochemistry of Proteins (CipKeBip)2, Ljubljana, Slovenia, Department of Pharmacy, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia3 Received in January 2014 CrossChecked in January 2014 Accepted in May 2014

Essential oils from oregano (Origanum vulgare L.), mint (Mentha piperita L.), fennel (Foeniculum vulgare Mill.), and pine (Abies alba Mill.) needles and cones, and their active substances thymol, carvacrol, menthol, and anisaldehyde were tested for antifungal activity against Penicillium verrucosum. The lowest minimal inhibitory concentrations (MICs) were achieved for essential oil of oregano, followed by carvacrol, thymol, and menthol. These antifungal components were further investigated, as the main aim of our study was to elucidate the effect of natural antifungals on ochratoxin A production. During 21 days of exposure, the growth of P. verrucosum, and subsequently the production of ochratoxin A, was fully inhibited by thymol at ½ MIC (0.0625 mg mL-1), but menthol at ¼ and ½ MIC (0.1875 and 3750 mg mL-1) showed no growth inhibition. After 21 days of incubation, the greatest inhibitory effect on ochratoxin production (inhibition was 96.9 %) was also achieved with thymol at ¼ MIC (0.0313 mg mL-1). Essential oil of oregano (¼ MIC, 0.2930 µL mL-1) and carvacrol (½ MIC, 0.1953 µL mL-1) stimulate production of ochratoxin A at 13.9 % to 28.8 %, respectively. The observed antifungal effects depended on the agent, the concentration used, and the time of interaction between the agent and P. verrucosum. Our results indicate the possibility of using oregano essential oil as a substitute for artificial preservatives in certain foods, but further research is needed.

KEY WORDS: anisaldehyde; carvacrol; food spoilage; menthol; minimal inhibitory concentration; natural antifungal agents; oregano; thymol

Moulds of the Penicillium, Aspergillus, Fusarium and Alternaria genera are known food spoilage microorganisms and mycotoxin producers that can cause significant economic losses (1-3). Ochratoxin A (OTA) is a very important mycotoxin, as it has teratogenic, embryotoxic, genotoxic, neurotoxic, immunosuppressive, and nephrotoxic effects (4). The International Agency for Research on Cancer (IARC) classified OTA as possibly carcinogenic to humans (group 2B) (5, 6). It can originate from producers such as Aspergillus ochraceus and Aspergilli from the

section Nigri (A. carbonarius), Penicillium verrucosum, and Penicillium nordicum (5, 7).

Potential contamination with Aspergillus and Penicillium moulds and their growth depend on temperature, moisture content, and the time over which a food is exposed to adverse conditions. These processes can also be affected by the presence of spores, mechanical and insect damage, mineral nutrient availability, pH and carbon dioxide levels, and chemical and physical treatments applied to food (8). The presence of moulds in food does not

200 necessarily mean that a food contains mycotoxins, and vice versa - mycotoxins can also be found in food without any presence of moulds. Given that OTA is a very stable mycotoxin that is not destroyed during standard food-production processes, it can be found both in raw foods and processed food products. OTA is most frequently found in cereals and cereal products, dried vine fruit, wine, coffee, pulses, beer, grape juice, cacao products, nuts, and spices (5).

Traditionally, control of mould growth and OTA production in foods has been performed by applying different chemical additives. Over the last decade, studies have investigated different natural antimicrobials that might replace these traditionally used chemical additives (9-11). Chemical preservatives have become less effective and can cause the development of resistant fungal strains (12-14). There are also increasing consumer demands to reduce the usage of synthetic fungicides and pesticides, and these requirements have led researchers to investigate naturally occurring compounds as alternatives to the use of chemical agents.

Different plant extracts and essential oils have already been shown to have antimicrobial/antifungal effects (14-18). These are commonly related to plant origin, extraction type, and chemical structure of their active substances. Prior to their application, potential natural antifungals need to be evaluated for their activity in order to determine their effectiveness not only for fungal growth inhibition but also for inhibition of mycotoxin production.

The aim of the present study was to investigate the antifungal effects of selected essential oils and their active substances through the evaluation of P. verrucosum growth and OTA production.

MATERIALS AND METHODS Essential oils and their active substances Essential oils of oregano (Origanum vulgare L.), mint (Mentha × piperita L.), fennel (Foeniculum vulgare Mill.), and pine (Abies alba Mill.) needles and cones (Amadea natura d.o.o., Zagreb, Croatia) were tested for antifungal activity against P. verrucosum.

Furthermore, thymol, carvacrol, menthol, and anisaldehyde (Sigma-Aldrich, St. Louis, MO, USA) were used for testing as pure active substances of these essential oils. Dimethyl sulfoxide (DMSO) (Merck,

Jeršek B et al. INHIBITION OF FUNGAL GROWTH BY ESSENTIAL OILS Arh Hig Rada Toksikol 2014;65:199-208

Darmstadt, Germany) was used as the solvent. DMSO was diluted as described in the section on the determination of minimal inhibitory concentrations (MICs).

Plant material Leaves of cultivated plants of mint (Mentha x piperita L., Lamiaceae) were collected in July of 2010 in the Vojvodina province, Republic of Serbia.

Oregano herb (Origanum vulgare L., Lamiaceae) and fennel fruits (Foeniculum vulgare L., Apiaceae) were obtained from the Institute for Studies on Medicinal Plants “Dr Josif Pancic” in Belgrade in 2010. The collected plant material was air-dried and samples were stored in double paper bags in a dry place without direct sunlight until hydrodistillation.

Isolation of essential oils Essential oils were obtained by hydrodistillation according to European Pharmacopoeia IV (2002) (19), with n-hexane as a collecting solvent. The solvent was removed under vacuum and essential oils were dehydrated with anhydrous Na2SO4.

GC/MS analysis Qualitative and quantitative analysis of oregano essential oil was carried out using a Hewlett-Packard 5973–6890 GC-MS system, operating in electron ionization (EI) mode at 70 eV, equipped with a splitsplitless injector (200 °C) and a flame ionization detector (250 °C). Helium was used as carrier gas (1 mL min-1), and the capillary columns used were HP 5MS (30 m×0.25 mm; film thickness 0.25 μm). The temperature programs were 60–280 °C at a rate of 3 °C min-1 and 60–260 °C at a rate of 3 °C min-1, respectively; the split ratio was 1:10. Coelution and MS analysis identified the separated compounds by comparing their relative retention times with those of authentic samples (Carl Roth GmbH, Karlsruhe, Germany). For the components for which authentic substances were not available the identification was based on matching their retention indices and mass spectra with NIST/NBS, Wiley libraries spectra, and literature data (20).

Maintenance of mould strain and preparation of spore suspension The experimental work was carried out with Penicillium verrucosum CBS 302.48 from the collection of the Centraalbureau voor Schimmelcultures

Jeršek B et al. INHIBITION OF FUNGAL GROWTH BY ESSENTIAL OILS Arh Hig Rada Toksikol 2014;65:199-208

Fungal Biodiversity Centre of the Royal Netherlands Academy of Arts and Sciences (CBS-KNAW, Utrecht, The Netherlands). The strain was maintained on malt extract agar (Merck, Darmstadt, Germany) plates and stored in a refrigerator (1-4 °C). For each experiment, P. verrucosum was sub-cultured on malt extract agar plates (Merck, Darmstadt, Germany) and incubated at 25 °C for 10 to 14 days, to allow significant sporulation. The conidia were removed from the malt extract agar plates and suspended in 1 mL saline solution (NaCl, 8.5 g L-1, in water) containing Tween 80 (Merck, Hochenbrunn, Germany) (0.1 %; v/v). The spore suspensions were adjusted to the final concentration of 105-106 spore mL-1 using a BürkerTürk counting chamber (Brand, Germany).

Determination of minimal inhibitory concentrations (MICs) The purpose of determining the MICs was to identify the concentrations of essential oils and their active substances that inhibit the growth of P. verrucosum and the production of OTA. The microdilution assay was used with RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) to determine the MICs of the essential oils and their active substances (18, 22, 23). These agents were dissolved in DMSO to prepare stock concentrations of 32 mg mL-1 for thymol and menthol and 100 μL mL-1 for carvacrol, anisaldehyde, and the essential oils.

These stock solutions were further diluted with RPMI-1640 medium (1:4 v/v) to 8 mg mL-1 for thymol and menthol and 25 μL mL-1 for the other agents. The solutions of the essential oils and their active substances (50 μL) were then serially diluted with RPMI-1640 medium in 96-well microtitre plates and 50 μL of P. verrucosum spore suspension was added to each well. The positive control encompassed three wells containing 50 μL of RPMI-1640 medium with 50 μL of spore suspension alone, whereas the negative control was three wells containing 50 μL RPMI-1640 medium and the antifungal agents alone (50 μL). Three wells were also used solvent control to determine if DMSO affects the growth of P. verrucosum. The preparation procedure was the same as for the dilutions of the antifungal agents, except that initially only 100 μL of DMSO solution was added.

Once prepared, these plates were shaken on a microplate shaker (Eppendorf, Hamburg, Germany) for 1 min and incubated at 25 °C for 48 h. As an indicator of growth, 10 μL p-iodonitrotetrazolium violet (Sigma Aldrich, Steinheim, Germany) (2 mg mL-1, in water)

201 was added to the microplate wells, which were additionally incubated at 25 °C for 48 h.

The MICs were determined as the lowest concentration of an antifungal agent that inhibited P. verrucosum growth, whereby the solution in the well remained clear, with no change in colour. The selected unit for MIC was μL mL-1 for essential oil of oregano, essential oil mint, essential oil pine needles, essential oil pinecones, essential oil of fennel, carvacrol and anisaldehyde as these essential oils and active substances were in liquid state and mg mL-1 for thymol and menthol since they were obtained in a solid state.

Effects of essential oils and their active substances on P. verrucosum and production of ochratoxin A To determine the effects of the selected essential oils and their active substances on the growth of P. verrucosum and formation of OTA, Czapek Yeast Extract agar (CYA) (23) was used. Each agent was tested at ½ MIC and ¼ MIC, previously determined in the microdilution assays.

After autoclaving, the CYA medium was cooled to 45 °C and solutions of the essential oil and their active substances were prepared for testing, which was performed on 10 Petri plates. Negative control plate contained only CYA medium, while the solvent control plates contained CYA medium with the two highest DMSO concentrations used to dissolve the tested substances (0.2 % of DMSO for control samples at thymol and menthol; 0.4 % for other active substances). All of the CYA plates were three-point inoculated with the P. verrucosum spore suspension using a split needle and incubated at 25 °C. The diameters of the colonies were measured after 7, 10, 14, 17, and 21 days of incubation. In parallel, sampling for thin-layer chromatography (TLC) and highperformance chromatography (HPLC) was performed from the corresponding CYA plates. The formation of the OTA was determined qualitatively by TLC and quantitatively by HPLC. The antifungal activities were evaluated according to the mean fungal growth rates and are expressed as mean±SD (mm per day).

The screening for OTA production was performed using TLC. After 14, 17, and 21 days of incubation of the CYA plates, three agar plugs were removed from the central, sub-central, and boundary areas of the colonies and transferred to TLC plates using the agarplug method described by Samson and Hoekstra (23).

For the HPLC screening method (24), three-point inoculated CYA agar plates were analysed after 7, 14, 17, and 21 days of P. verrucosum growth at 25 °C. At

202 each time, three agar plugs were removed from the central, sub-central, and boundary areas of the colony and introduced into a 1.5 mL tube. Then, 0.5 mL of methanol (Merck, Darmstadt, Germany) was added to each tube, which was shaken at 270 rpm for 60 min.

From each of the test tubes, the supernatant was evaporated under a gentle stream of nitrogen and the residue was reconstituted in 0.2 mL mobile phase for HPLC determination. Solutions of OTA standards and extracts were analysed using an HPLC system that consisted of a 1260 Infinity model G1312B binary pump, a model G1367E autosampler, and a model G1321B fluorescence detector (Agilent Technologies, Inc., Wilmington, DE, USA). Data signals were acquired and processed on a PC running Agilent Chemstation (Agilent Technologies, Inc., Wilmington, DE, USA). The HPLC analyses were carried out using a ThermoHypersil-Keystone (ThermoHypersil, UK) Lichrosorb RP18 column (4.6x250 mm; 10 µm particle size). The separation conditions for the OTA standard solutions and extracts were as follows: column temperature 25 °C; injection volume 20 µL; flow rate 0.8 mL min-1; time 60 min. The excitation and emission wavelengths were set at 330 nm and 460 nm, respectively. The mobile phase consisted of acetonitrile: water: acetic acid (57:41:2 v/v/v).

Acetonitrile was HPLC grade from Sigma-Aldrich (St. Louis, MO, USA), and acetic acid was analytical grade from Merck (Darmstadt, Germany).

Chromatographic peaks were identified by comparing retentions and fluorescence spectra of the samples with those of the standard compounds. Quantitation was carried out by external standardization. Calibration standards in the range 0.2-1.0 µg mL-1 were prepared by serial dilution from a 10 µg mL-1 mixed standard.

Calibration standards were prepared in HPLC diluent.

Five concentration points were assayed in triplicate.

The OTA levels in the extracts were calculated from the calibration curve y=388.8x-34.499, where y is the area response and x the concentration of OTA in µg mL-1. The correlation factor was 0.9982. The limit of quantification (LOQ) and the limit of detection (LOD) were calculated on the basis of the standard deviation of the response and the slope obtained from the linearity plot of the standard mixture of OTA. LOQ and LOD were calculated as 3.3α/S and 10α/S, respectively, where α is the standard deviation of the y-intercept and S is the slope of regression line. The LOQ and the LOD were 0.220 µg mL-1 and 0.073 µg mL -1, respectively. The OTA recovery was determined as an average value 99.96 %. Since all of

Jeršek B et al. INHIBITION OF FUNGAL GROWTH BY ESSENTIAL OILS Arh Hig Rada Toksikol 2014;65:199-208

the tested agents were dissolved in DMSO, to determine their exact effect, results obtained for the solvent controls had to be subtracted from the values obtained by our measurements for P. verrucosum growth and OTA production. Than the inhibition (I) of growth and inhibition of OTA production were calculated as I (%)=[(K–O)/K]x100, where K was a value taken from the control sample I (CYA medium), and O from the sample where the essential oil or its active substances were added. In cases where the production of OTA was higher than that of the control samples, the stimulation of OTA production was calculated as S (%)=[(O–K)/O]x100 (9, 14). Inhibition of growth was calculated from results obtained for P. verrucosum growth as diameter of colony (mm) and inhibition (or stimulation) of OTA production was calculated from results obtained for P. verrucosum production of OTA (µg mL-1).

Statistical analysis The experimental data for growth inhibition (I) were evaluated statistically using the SAS⁄STAT program (SAS Software, 1999; SAS Institute Inc., Cary, NC, USA). The basic statistical parameters were calculated by the MEANS procedure. The data were tested for normal distributions and analysed using the general linear model (GLM) procedure. The statistical model included the main effect of time of incubation (14, 17, 21 days) and antifungal agent and its concentration (essential oil of oregano, thymol, carvacrol, menthol at ½ and ¼ MIC). The means were obtained using Duncan’s procedure and compared at the 5 % probability level.

RESULTS AND DISCUSSION Essential oil of oregano thymol, carvacrol and menthol were selected for full testing of antifungal effects against P. verrucosum growth and OTA production. The criteria for selection were I) selection of one essential oil that has the lowest MIC among tested essential oils and II) selection of 3 active components that are present in tested essential oils and have the lowest MICs. According to these criteria, the essential oil of oregano and thymol and carvacrol as its main active components were selected. In addition, menthol as the third most active pure component was selected as well.

Jeršek B et al. INHIBITION OF FUNGAL GROWTH BY ESSENTIAL OILS Arh Hig Rada Toksikol 2014;65:199-208

MICs of essential oils and their active substances Based on the obtained MICs, oregano essential oil and carvacrol had the highest antifungal effects on P. verrucosum (Table 1).

Table 1 Minimal inhibitory concentrations (MICs) for essential oils and their active substances determined against P. verrucosum

Essential oil/active substance Essential oil of oregano Essential oil mint Essential oil pine needles Essential oil pinecones Essential oil of fennel Thymol Carvacrol Menthol Anisaldehyde

MIC 1.1719 μL mL-1 6.2500 μL mL-1 9.3750 μL mL-1 6.2500 μL mL-1 2.343 μL mL-1 0.1250 mg mL-1 0.3906 μL mL-1 0.7500 mg mL-1 0.7813 μL mL-1

P. verrucosum growth and OTA production in the presence of oregano essential oil and active substances The effects of oregano essential oil (see Table 2 for chemical composition), thymol, carvacrol, and menthol on P. verrucosum growth are shown in Figure 1. Menthol at concentrations of ½ MIC and ¼ MIC had no effects on the growth of P. verrucosum. Growth was slower in the presence of oregano essential oil (½ MIC, ¼ MIC) and carvacrol (¼ MIC), as well as

203 clearly slower in the presence of thymol (¼ MIC) and carvacrol (½ MIC). Thymol at ½ MIC led to full growth inhibition.

Table 3 shows the results regarding OTA production, which was followed over the same 21-day incubation period in CYA. OTA production was inhibited, in all tested periods, by oregano oil only at ½ MIC. After 14 and 17 days of incubation, the essential oil of oregano at ¼ MIC resulted in a 66.6-92.1 % inhibition of OTA production.

Surprisingly, after 21 days, oregano essential oil led to a 28.8 % stimulation of OTA production.

When thymol at ½ MIC was added to the CYA growth medium, P. verrucosum was fully inhibited and there was no OTA production. The lower concentration of thymol (¼ MIC) resulted in an 83.3 % inhibition of P. verrucosum growth and an >95 % inhibition of OTA production after 21 days.

Carvacrol at ½ MIC showed inhibition of P. verrucosum growth over the 21 days of incubation and a 13.9 % OTA production stimulation. At its lower concentration (¼ MIC), carvacrol still had an inhibitory effect on P. verrucosum growth and on OTA production.

At both concentrations, menthol had no effects on P. verrucosum growth, although there were high levels of OTA production inhibition.

In general, the P. verrucosum growth inhibition (Table 4) was the greatest in the first days of the incubation and then reduced regardless of the

Figure 1 Mean P. verrucosum growth rates (mm per day) over a 21-day incubation with different antifungal agents at ½ MIC and ¼ MIC. The bars indicate standard deviations for experiments carried out in duplicate

204

Jeršek B et al. INHIBITION OF FUNGAL GROWTH BY ESSENTIAL OILS Arh Hig Rada Toksikol 2014;65:199-208

Table 2 Chemical composition of oregano essential oil

Pick No.

Compounds

Monoterpene Hydrocarbones 3 β-Myrcene 4 γ-Terpineneb Oxygenated Monoterpenes 5 trans-Sabinene hydrate 6 Linaloolb 7 Borneolb 8 α-Terpineolb Aromatic Oxygenated Monoterpenes 9 Thymol methyl eter 10 Carvacrol methyl eter 11 Thymolb 12 p-Cymene-7-ol 13 Carvacrolb Sesquiterpene Hydrocarbons 14 α-Caryophyllene 15 trans Caryophylleneb 16 Aromadendrene 17 α-Himachalene 18 α-Amorphene 19 β-Himachalene 20 β-Bisabolene 21 δ-Cadinene Oxygenated Sesquiterpenes 22 Caryophyllene oxide 23 α-Cadinol 24 Farnesolb 25 cis-Lanceol 27 Cedran-diol Diterpenes 28 Abietatriene Aromatic Hydrocarbons 1 Anisole Aliphatic compounds 2 1-Octen-3-ol 26 Octadecane Identified compounds (%)

R.I.a 0991 1060 1098 1097 1169 1189 1245 1290 1291 1299 1409 1419 1441 1451 1485 1505 1506 1523 1583 1654 1725 1761 1898 2057 0.918 0979 1800

Percentage (%) 5.1 0.9 4.2 11.4 4.8 6.1 0.5 59.9 0.6 0.9 4.5 0.5 53.4 8.7 0.2 1.6 0.4 0.2 0.2 1.6 4.5 8.3 4.5 3.3 0.1 0.3 0.1 0.5 0.5 1.1 1.1 0.2 0.2 95.2

Retention indices (R.I.) relative to C9-C24 n-alkanes on the HP 5MS column, bidentification based on retention times of authentic compounds on HP 5MS column a

antifungal agent tested. OTA production prior to day 14 of incubation (for thymol at ¼ MIC and carvacrol at ½ MIC also after 14 and 17 days could not be determined because the colonies were too small).

Inhibition of OTA production did not accompany growth inhibition, which showed very different data (from >95 % inhibition to >25 % stimulation), which indicates the complexity of the P. verrucosum response mechanisms to these antifungals.

Our data show that, among the tested essential oils and active substances, the lowest MICs were obtained for oregano oil, thymol, carvacrol, and menthol.

Akgül and Kivanç (15) have already shown that the essential oil of oregano, thymol and carvacrol have strong antifungal activities, as they almost completely inhibited the growth of different Aspergillus, Mucor and Penicillium strains at 0.025 % (w/v) and 0.05 % (w/v). Sokolić-Mihalak et al. (18) investigated the effects of the essential oil of Thymus serpyllum L. and thymol on ochratoxigenic strains of Aspergillus, demonstrating that the MICs for their A. niger and A. ochraceus strains ranged from 0.625-250 µL mL-1 for the essential oil and 0.0078-0.125 mg mL-1 for thymol.

Vasquez Beatriz et al. (17) reported that thymol inhibited Penicillium citrinum growth over 10 days

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Table 3 Mean P. verrucosum production of OTA during the 21-day incubations with the different antifungal agents at ½ MIC and ¼ MIC

Average production of OTA (µg mL-1) at different incubation period 14 days 17 days 21 days

Antifungal agent (concentration) Control I

0.177±0.025

0.266 ± 0.021

0.081±0.001

Control II

0.135±0.002

0.308 ± 0.011

0.144±0.013

Essential oil of oregano (½ MIC)

0.187±0.012

0.314 ± 0.013

0.196±0.017

Essential oil of oregano (¼ MIC)

0.194±0.028

0.320 ± 0.042

0.254±0.042

Thymol (½ MIC)

n.a.

n.a.

n.a.

Thymol (¼ MIC)

n.a.

n.a.

0.128±0.017

Carvacrol (½ MIC)

n.a.

n.a.

0.240±0.029

Carvacrol (¼ MIC)

0.189±0.034

0.308±0.023

0.185±0.011

Menthol (½ MIC)

not done

0.278±0.011

0.177±0.008

Menthol (¼ MIC)

0.151±0.039

not done

0.156±0.013

OTA, ochratoxin A; Control I, production of OTA by P. verrucosum on MEA; Control II, production of OTA by P. verrucosum on MEA with addition of DMSO in the concentration used for dilution of antifungal agent; n.a., not applicable as there was no growth or the colonies were too small;

The values are means of two repetitions ± standard deviations

Table 4 Mean P. verrucosum growth inhibition and inhibition of OTA production during the 21-day incubations with the different antifungal agents at ½ MIC and ¼ MIC

Incubation period (days) 17

14 Antifungal agent (concentration)

21

Growth Inhibition (%)

Inhibition of OTA production (%)

Growth Inhibition (%)

Inhibition of OTA production (%)

Growth Inhibition (%)

Inhibition of OTA production (%)

Essential oil of oregano (½ MIC)

31.7±1.9A, z

70.6

24.3±2.4B,w

97.9

21.0±1.5C,z

72.6

<0.0001

Essential oil of oregano (¼ MIC)

31.7±5.8A, z

66.6

22.9±5.3B,w

92.1

17.9±2.6B,w

28.8 stim.

0.0082

100.0±0.0A, x 71.7±1.9A, y

n.a. n.a.

100.0±0.0A,x 58.1±2.0B,z

n.a. n.a.

100.0±0.0A, x 41.0±2.5C, y

n.a.

96.9

<0.0001 <0.0001

98.3±1.9A, x

n.a.

66.2±1.9B,y

n.a.

42.1±1.4C, y

13.9 stim.

<0.0001

15.4±1.9A, w

69.2

5.4±6.5B, t

100.0

8.4.±1.6B,t

50.0

0.0225

1.6±1.9A, t

not done

1.3±2.6A,ut

100.0

0.5±1.0A, u

59.3

0.7118

0.0±0.0A, t

87.7

0.0±0.0A,u

not done

0.0±0.0A, u

81.5

/

Thymol (½ MIC) Thymol (¼ MIC) Carvacrol (½ MIC) Carvacrol (¼ MIC) Menthol (½ MIC) Menthol (¼ MIC) ptime of incubation

<0.0001

<0.0001

p

<0.0001

OTA, ochratoxin A stim. - stimulation of OTA production; n.a., not applicable as there was no growth or the colonies were too small; The values are means of four repetitions ± standard error.

Level of significance: highly statistically significant: p≤0.001. Means with a different superscript within a row (A, B, C) differ significantly (p≤ 0.05).Means with a different superscript within column (x, y, z, w, t, u) differ significantly (p≤0.05)

206 of incubation at 200 mg mL -1, but not at lower concentrations (100 and 50 mg mL-1). Murthy et al.

(14) reported approx. a 60 % inhibition of growth of the OTA-producing strains of A. ochraceus, A. niger and Penicillium sp. by the essential oil of Indian borage after 5 to 7 days of incubation. This plant contains the same active substances (thymol and carvacrol) as oregano (16, 25). The inhibitory effects of the essential oil of oregano on P. verrucosum growth were shown in the present study, and these effects might also be mediated by the content of the above mentioned isolated compounds.

Carvacrol was determined as the dominant compound in investigated oregano essential oil, followed by thymol, linalool, bisabolen, and caryoplyllene. This combination of different monoand sesquiterpene compounds clearly contributed to its antifungal and OTA production inhibitory activity.

However, isolated compounds, especially thymol exhibited higher antifungal activity and notable inhibition of OTA production which may indicate that synergism is not involved in these activities. Also, despite similar effects, comparisons of studies are often difficult, or even impossible, because of the origins of the essential oils, or because of the different methodologies used for the evaluation of these antifungal effects. Also, the compositions of the essential oils are highly dependent on the plant varieties, and within the same variety, they can also depend on the geographical origin, geographical location, climatic conditions, and method of extraction (12).

CONCLUSIONS Our data indicate that natural antimicrobial agents, like essential oils and their isolated constituents can be used as antifungals. However, prior to application, they need to be evaluated for their activity in terms of inhibition of both growth and toxin production. The essential oil of oregano (½ MIC) and thymol (¼ and ½ MIC) and carvacrol (¼ MIC) can be safely used as substitutes for chemical additives in foods for the partial or complete inhibition of the growth of P. verrucosum and for the inhibition of OTA production over 21 days of incubation. However, these were in vitro results and behaviour of the essential oils and active agents in foods could be a lot different depending on many intrinsic and extrinsic factors.

Jeršek B et al. INHIBITION OF FUNGAL GROWTH BY ESSENTIAL OILS Arh Hig Rada Toksikol 2014;65:199-208

Acknowledgements The authors would like to thank the Slovenian Research Agency and Ministry of Agriculture and Environment of the Republic of Slovenia for financing the project CRP V4-1079. The authors also thank Dr Lea Demšar from Biotechnical Faculty for statistical analysis, Nina Hostnik for experimental work and Dr Ksenija Markov from Faculty of Food Technology and Biotechnology, University of Zagreb, for providing some of the tested essential oils.

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Available at http://eur-lex.europa.eu/LexUriServ/ LexUriServ.do?uri=OJ:L:2010:050:0008:0012:EN:PDF 4. Ochratoxin A. In: Evaluation of Certain Food Additives and Contaminants. Sixty-eight report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Geneva: WHO, 2007. p. 169-80.

5. European Food Safety Authority (EFSA). Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. EFSA J 2006;365:1-56.

6. Ochratoxin A. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Some Naturally Occurring Substances: Food Items, and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. Vol. 56. Geneva: WHO, IARC; 1993. p. 489-521.

7. Pitt JI, Marta H, Taniwaki M, Cole B. Mycotoxin production in major crops as influenced by growing, harvesting, storage and processing, with emphasis on the achievement of Food Safety Objectives. Food Control 2013;32:205-15. doi: 10.1016/j.foodcont.2012.11.023 8. Amézqueta S, González-Peñas E, Murillo-Arbizu M, López de Cerain A. Ochratoxin A decontamination: a review. Food Control 2009;20:326-33. doi: 10.1016/j.foodcont.2008.05.017 9. Angelini P, Nellini R, Pagiotti A, Vianello B. Antimicrobial activities of various essential oils against foodborne pathogenic or spoilage moulds. Ann Microbiol 2006;56:659. doi: 10.1007/BF03174972

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10. Dobre AA, Gagiu V, Niculita P. Preliminary studies on the antimicrobial activity of essential oils against food borne bacteria and toxigenic fungi. Food Technol 2011;35:16-26.

11. Jayasena DD, Jo C. Essential oils as potential antimicrobial agents in meat and meat products: A review. Trends Food Sci Technol 2013;34:96-108. doi: 10.1016/j.tifs.2013.09.002 12. da Cruz Cabral L, Fernández Pinto V, Patriarca A. Application of plant-derived compounds to control fungal spoilage and mycotoxin production in foods. Int J Food Microbiol 2013;166:1-14. doi: 10.1016/j.ijfoodmicro.2013.05.026 13. Kumar A, Shukla R, Singh P, Dubey NK. Chemical composition, antifungal and antiaflatoxigenic activities of Ocimum sanctum L. essential oil and its safety assessment as plant based antimicrobial. Food Chem Toxicol 2010;48:539-43. doi: 10.1016/j.fct.2009.11.028 14. Murthy PS, Ramalakshmi K, Srinivas P. Fungitoxic activity of Indian borage (Plectranthus amboinicus) volatiles. Food Chem 2009;114:1014-8. doi: 10.1016/j. foodchem.2008.10.064 15. Akgül A, Kivanç M. Inhibitory effects of selected Turkish spices and oregano components on some foodborne fungi.

Int J Food Microbiol 1998;6:263-8. PMID: 2978951 16. D’Antuono LF, Galletti GC, Bocchini P. Variability of essential oil content and composition of Origanum vulgare L. populations from a north Mediterranean area (Liguria Region, northern Italy). Annal Botan 2000;86:471-8. doi: 10.1006/anbo.2000.1205 17. Vazquez Beatriz I, Fente C, Franco CM, Vazquez MJ, Cepeda A. Inhibitory effects of eugenol and thymol on Penicillium citrinum strains in culture media and cheese. Int J Food Microbiol 2001;67:157-63. doi: 10.1016/S01681605(01)00429-9

207 18. Sokolić-Mihalak D, Frece J, Slavica A, Delaš F, Pavlović H, Markov K. The effects of wild thyme (Thymus serpyllum L.) essential oil components against ochratoxin-producing Aspergilli. Arh Hig Rada Toksikol 2012;63:457-62. doi: 10.2478/10004-1254-63-2012-2309 19. European Pharmacopeia. Vol. 2.8.12. 4th ed. Strasbourg Cedex: Council of Europe; 2002. p. 183-4.

20. Adams RP. Identification of Essential Oil Components by Gas Chromato- graphy/Mass Spectroscopy. 4th ed. Carol Stream (IL, USA): Allured Publishing Co.; 2007.

21. Clinical and Laboratory Standards Institute. M38-A2.

Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; Approved standard. 2nd ed. 2008 [displayed 27 March 2014]. Available at http://shopping. netsuite.com/c.1253739/site/Sample_pdf/M38A2_sample. pdf 22. Mavri A, Abramovič H, Polak T, Bertoncelj J, Jamnik P, Smole Možina S, Jeršek B. Chemical properties and antioxidant and antimicrobial activities of Slovenian propolis. Chem Biodivers 2012;9:1545-8. doi: 10.1002/ cbdv.201100337 23. Samson RA, editor. Introduction to Food- and Airborne Fungi. Utrecht: Centraalbureau voor schimmelcultures; 2000.

24. Bragulat MR, Abarca ML, Cabañes FJ. An easy screening method for fungi producing ochratoxin A in pure culture. Int J Food Microbiol 2001;71:139-44. PMID: 11789931 25. Stefanakis MK, Touloupakis E, Anastasopoulos E, Ghanotakis D, Katerinopoulos HE, Makridis P. Antibacterial activity of essential oils from plants of the genus Origanum.

Food Control 2013;34:539-46. doi: 10.1016/j. foodcont.2013.05.024

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Jeršek B et al. INHIBITION OF FUNGAL GROWTH BY ESSENTIAL OILS Arh Hig Rada Toksikol 2014;65:199-208

Izvleček Vpliv izbranih eteričnih olj na rast in tvorbo ohratoxina A pri plesnih vrste Penicillium verrucosum Eterična olja origana (Origanum vulgare L.), mete (Mentha piperita L.), janeža (Foeniculum vulgare Mill.), in borovih (Abies alba Mill.) iglic in storžev, ter njihove sestavine timol, karvakrol, mentol in anisaldehid smo testirali kot snovi z protiglivno aktivnostjo proti plesnim vrste Penicillium verrucosum. Najnižje minimalne inhibitorne koncentracije (MICs) smo določili eteričnemu olju origana, ter karvakrolu, timolu, in mentolu. Rast plesni vrste P. verrucosum je bila popolnoma inhibirana po 21 dneh inkubacije v prisotnosti ½ MIC timola (0,0625 mg mL-1), medtem ko so eterično olje origana (½ MIC, ¼ MIC), timol (¼ MIC) in karvakrol (½ MIC, ¼ MIC) inhibirali rast plesni od 8,3 % do 70.8 %. Dodatek mentola v koncentracijah ¼ MIC in ½ MIC (0.1875 mg mL-1 in 0.3750 mg mL-1) pa ni imel inhibitornega vpliva na rast plesni vrste P. verrucosum. Popolno inhibicijo tvorbe ohratoksina A smo določili le v primeru, ko je bila tudi rast teh plesni popolnoma inhibirana (pri ½ MIC timola). Največjo inhibicijo tvorbe ohratoksina A (96,9 %) po 21 dnevih inkubacije smo določili pri ¼ MIC timola (0.0313 mg mL-1). Pospešeno tvorbo ohratoksina A (od 13,9 % do 28,8 %) pa smo določili pri ¼ MIC eteričnega olja origana (0.2930 mg mL-1) in ½ MIC karvakrola (0.1953 mg mL-1). Protiglivni vplivi so bili odvisni od testirane snovi, njene koncentracije ter časa interakcij med protiglivno snovjo in plesnimi vrste P. verrucosum. Rezultati nakazujejo možnosti uporabe eteričnega olja origana v živilih namesto kemijskih konzervansov. Ker pa so živila kompleksna okolja v katerih je potrebno upoštevati več dejavnikov, so potrebne nadaljnje raziskave.

KLJUČNE BESEDE: eterična olja; karvakrol; mentol; MIC; naravna protiglivna snov; origano; timol

CORRESPONDING AUTHOR: Barbara Jeršek Department of Food Science and Technology Biotechnical Faculty, University of Ljubljana Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia E-mail: barbka.jersek@bf.uni-lj.si

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Jakšić Despot D and Šegvić Klarić M. INDOOR AIRBORNE FUNGI IN CROATIA Arh Hig Rada Toksikol 2014;65:209-218

DOI: 10.2478/10004-1254-65-2014-2483

Original article

A year-round investigation of indoor airborne fungi in Croatia Daniela Jakšić Despot and Maja Šegvić Klarić Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia Received in December 2013 CrossChecked in December 2013 Accepted in April 2014

This study assessed the composition of aeromycota at a grain mill and four dwellings (two apartments and two basements) as well as in outdoor air during one year in Zagreb, Croatia. The incidence of Aspergilli from sections Flavi, Nigri, and Versicolores was also assessed. Airborne fungi were collected using an air-sampler and DG-18 agar plates. The average concentrations of airborne fungi in the grain mill ranged from 14,310 to 40,000 cfu m-3, which was above the hazardous level (104 cfu m-3), whereas the values statistically estimated using Feller’s correction were up to six times higher. Concentrations in the apartment (163-1244 cfu m-3) were lower than in outdoor air (286-2090 cfu m-3) and lower than in the basement (697-1203 cfu m-3), except in the warmer period of the year when they were similar. The most abundant species throughout the year were Cladosporium spp. (90-100 %), Penicillium spp. (40-100 %), and Alternaria spp. (10-100 %), which are common for temperate climates. Aspergilli from the Flavi (50100 %) and Nigri (15-40 %) sections as well as A. ochraceus (15-60 %) and Eurotium spp. (85-100 %) were the most abundant at the grain mill and were rarely found in outdoor air. In the basement, Aspergilli (Versicolores) were more abundant than in the apartment. The excess of aeromycoparticles in the grain mill throughout the year may have represented a serious health risk to mill workers. This is the first Croatian one-year study of indoor airborne fungi in a grain mill and dwellings; however monitoring should continue over a longer period.

KEY WORDS: Flavi; grain mill; Nigri; residential areas; Versicolores

Many studies have recently investigated the levels of airborne fungi in indoor environments and their role in the initiation and/or progression of chronic respiratory diseases like asthma and chronic rhinosinusitis (1-6). In addition, the European Environment Agency (EEA) has published a report on air quality in Europe in 2012 (7), where the most severe health effects were attributed to air pollution by particulate matter (PM). The highest mortality caused by PM clearly stems from particle fractions 2.5 µm in diameter, which represents 40-80 % of PM mass concentration in ambient air in Europe. Fungal spores are regularly found in outdoor and indoor air

as part of PM. Their size ranges usually from 1 to 10 µm with variations even among the same species.

Besides fungal spores, fragments of mycellium may also be released in the air with sizes even smaller than fungal spores (8-10). Inhalation of these particles represents a threat to human health due to possible deposition in the lower respiratory system and interactions with lung tissue. No uniformness in the suggested guidelines for acceptable levels of fungi in indoor environments has yet been achieved and there is no dose-response relationship between the concentration of airborne fungi and adverse health effects. Moreover, the immunocompetence of an

210

Jakšić Despot D and Šegvić Klarić M. INDOOR AIRBORNE FUNGI IN CROATIA Arh Hig Rada Toksikol 2014;65:209-218

exposed individual plays a crucial role in both the type and intensity of possible effects.

Airborne fungi are of special concern in industrial environments such as grain mills or agricultural warehouses, where their concentration may be up to a hundred thousand times higher than in outdoor air.

It has been suggested that occupational concentrations of airborne fungi above 10 4 cfu m -3 should be considered a health hazard in non-sensitized subjects, although this does not mean that such a working environment will necessarily lead to any respiratory symptoms (9-13). Considering the limited number of studies addressed at the fungal burden in occupational environments and urban homes in our country, the aim of the present study was to investigate the composition and concentration of aeromycota in industrial and residential environments in Zagreb, Croatia, for which purpose measurements were performed in a grain mill, an apartment and a basement.

Special attention was paid to the occurrence of Aspergilli from sections Flavi, Nigri, and Versicolores for several reasons. In immunocompromised patients, most invasive respiratory infections are caused by Aspergillus fumigatus, A. flavus, A. terreus, and A. niger sensu lato (14, 15). Aspergillus flavus and A. parasiticus from the Flavi section are important producers of the highly toxic and carcinogenic aflatoxin B1 and these species grow on almost all crops (16). Aspergillus versicolor is the most frequently reported Versicolores fungus from damp indoor environments and its presence might be related to Sick Building Syndrome (17-20). Moreover, it represents a special threat for human health due to its very small spores and release of mycellium fragments (10) as well as ability to produce sterigmatocystin (21, 22).

Finally, Aspergillus section Nigri, or black aspergilli, which can easily be isolated from occupational and residential environments, have been reported to produce ochratoxins and fumonisins (23).

This is the first quantitative and qualitative assessment of indoor airborne fungi in a grain mill and dwellings over a one-year period in Croatia.

MATERIALS AND METHODS Sampling and determination of airborne fungi Airborne fungi were collected over a one-year period (2012) in two-month intervals at a grain mill

(GM) situated near Zagreb, Croatia, and in residential locations which included two apartments (AP) and two basements (BS). The differences between the chosen apartments and basements were considered irrelevant for this study. Samples of outdoor air (ODA) were also collected. The floor area of the APs was approximately 70 m2 each, both inhabited and without visible mould growth. The BS’s were situated in the same dwellings as the APs. These were unoccupied and served as a repository for the inhabitants. Visible mould growth as well as typical mouldy odour was observed in both basements. The GM was situated in an industrial area 40 km outside Zagreb and served for processing cereals, mainly corn and wheat, as well for short-term flour storage. It was organised as a building consisting of a big reception and depository at the ground floor, milling site located at the first floor, and sieving site located at the second floor. The air was quite saturated with grain particles, but no mouldy odour was observed. Samplings were done in the middle of the working week in the morning during the most intensive and constant milling operations at 20 locations, including site of grain/flour exchange, site of flour storage, site of sieving, and site of milling.

Twenty samples were taken from the APs at locations: kitchen, dining room, living room, bedroom, and bathroom. Samples were also taken from 10 locations in the BS, two at each site. The total number of samples was 420. Air temperature (°C) and relative humidity (%) were measured by thermo-hygrometer (Boneco, Widnau, Switzerland) at every location during each sampling. Fungi were sampled 1 m above ground using a Mas 100 Eco air sampler (Merck, Darmstadt, Germany) with 400 holes (hole to agar impactor) and dichlorane 18 % glycerol agar (DG-18) plates (25).

The impaction velocity of the sampler was approximately 10.8 m s-1 and airflow rate 100 L min-1. Because of the high contamination level, a volume of 10 L (sampling time 6 s) was chosen for sampling in the GM, while 50 L min-1 (sampling time 30 s) was applied at the other sampling locations. After field sampling, the plates were incubated for 5 days at 25±2 °C, after which the developed fungal colonies were counted.

The concentrations of airborne fungi were expressed as colony forming units (cfu) per volume of air sampled (expressed in cubic meter), i.e. cfu m-3.

Feller’s correction (probable statistical total) was applied to all of the samples (24). The airborne fungi were identified on the basis of their macro- and microscopic characteristics after subculturing on Czapek, Malt Extract and Potato Dextrose agar (Fluka,

Jakšić Despot D and Šegvić Klarić M. INDOOR AIRBORNE FUNGI IN CROATIA Arh Hig Rada Toksikol 2014;65:209-218

Sigma-Aldrich, Steinheim, Germany), according to the manuals (25, 26). In order to separate the aspergilli of interest, they were isolated on Czapek Yeast Agar [CYA, (26)] and Malt Extract Agar (MEA; Becton Dickinson, MD, USA) and incubated at 25 °C in the dark for seven days (Aspergillus section Flavi and section Nigri) and ten to fourteen days for Aspergillus section Versicolores. The identification of Aspergillus species was conducted according to Pitt and Hocking (25) and de Hoog et al. (26).

Statistics Concentrations of airborne fungi at each location are represented as mean±SD of cfu m-3. For each plate, Feller’s correction (probable statistical total) was applied - as the number of viable particles impinged on a plate increases, the probability of the next particle going into an “empty hole” decreases. The probable number of viable particles calculated from Feller’s formula (Pr=N 1/N + 1/N-1 + 1/N-2 + 1/N-r+1), given by the manufacturer (Merck KgaA, Darmstadt, Germany; Pr=probable statistical total; r=Number of cfu counted; N=total number of holes in the sampling head).

The Kolmogorov-Smirnov test was used to verify whether the variables were normally distributed.

Concentrations of airborne fungi were logarithmically transformed to normalise distribution. When normality was achieved, ANOVA and Tukey multiple comparison tests were used. Otherwise, Kruskal-Wallis test was applied, followed by Dunn’s multiple comparison test.

Spearman’s correlation coefficients between average fungal concentrations at each location and ambient parameters (temperature and relative humidity) were also calculated.

RESULTS AND DISCUSSION Data related to average concentrations of airborne fungi (cfu m-3) and average temperature and relative humidity specific for each sampling site and each period of year are presented in Table 1. Levels of aeromycota in the GM (up to 40,000 cfu m-3) were significantly higher during the whole year than at the other sampling sites (p<0.0001). Statistically corrected concentrations at the GM were up to 6 times higher than the measured values. Compared to observations from studies on similar occupational environments such as another grain mill [max. concentration

211

1.7x104 cfu m-3 (27)], rice mill [mean 13.71x10348.42x103 cfu m-3; (28)], outdoor agricultural area [mean 72-1796 cfu m-3; (29)], agricultural non-point source during wheat harvesting season [mean 105106 cfu m-3; (30)], or sawmills (mean 1700-7300 cfu m-3; (13)] our analysis showed an overload with airborne fungi in the GM throughout the entire year. Taking into account statistically estimated values (up to 2.6x105 cfu m-3), levels of airborne fungi in the GM were 20 times higher than the occupational concentration of airborne fungi considered hazard to human health (>10,000 cfu m-3). Variations of airborne fungi levels in the GM showed no correlation to both temperature and relative humidity (Table 2). This was not surprising because the measured levels were the result of fungal contamination of grains that developed during storage, before delivery to the GM. Seasonal variations of airborne fungi in the AP and BS did not exhibit a similar pattern. In the BS, airborne fungi exceeded 1000 cfu m-3 from May to November while the levels in January, March, September, and November were significantly higher as compared to the AP (p<0.001). Variations of airborne fungi levels in the BS also did not show a correlation to temperature and relative humidity (Table 2). Taken together, these observations suggest a more pronounced fungal contamination in the BS probably due to poor maintenance and ventilation.

Seasonal variations of airborne fungi levels in the AP and ODA showed a similar pattern and positively correlated to temperature changes (R=0.6661, R=0.5409) but not to relative humidity. However, the levels of aeromycota in the AP obtained in January, March, July, and September were significantly lower than in ODA (p<0.001).

The observed concentrations of fungi in ODA (240-2090 cfu m -3) were twice as high as the concentrations observed in similar investigation conducted in 2002/03 in Austria [100-1000 cfu m-3; (31)]. In that same investigation, apartments without visible mould growth had a maximum of fungal spore concentration of 300 cfu m-3. The highest observed concentration in the AP from our investigation was 13 times higher (4000 cfu m-3) and more than 16 times higher (5060 cfu m-3) in the BS. Our previous yearround investigation, which was also performed in 2002/03, at outdoor air locations in Zagreb (32) showed that airborne fungi peaked in August and September (up to 400 cfu m-3), which was five times lower than the levels measured in that same period in this study. Since Zagreb and the Austrian region

161600±127186* 249925±57579* 160725±128276* 262800±0* 48980±92190* 6940±3241* 164±105.8 184±128.7 1462±1460 1078±506.3 477±192 224±109.1 804±1199*b 916±1218*a 1275±1108 1123±557.3 1172±1035*a 1539±2205*a; ***b 288±71.2**b 1586±3163**a 1506±696***b 2426±323.3**a;***a 914±288.9**a 240±62.54

20-460 40-520 320-4000 140-1660 200-960 40-420 40-3600 100-3800 240-3320 480-2140 340-3800 180-5060 200-440 180-5880 800-2600 1800-2500 640-1520 120-340

200-460 180-10600 840-3140 2040-3000 660-1680 120-340

40-4780 100-5140 240-4280 500-2480 340-5140 180- 8000

20-480 40-540 320-5540 480-2140 200-1020 40-420

4200-262800 5300-262800 4300-262800 262800-262800 8200-262800 1700-15100

11.9 13.4 22 27.3 34.6 2.8

9.9 11.2 18.3 21 20.7 13.9

18.3 18.8 23.9 26.9 28.5 18.6

6.2 8.0 24 24.4 25 10.2

Temperature (°C)

*p<0.0001 GM compared to all other sampling sites; *a p<0.0001 BS vs. AP; *b p=0.0008 BS vs. AP; **a p<0.0001 ODA vs. AP; **b p=0.0008 ODA vs. AP; ***ap<0.0001 ODA vs. BS; ***b p=0.0465 ODA vs. BS; ***c p<0.0001 BS vs. ODA

January March May July September November

4000-40000 5000-40000 4100-40000 40000-40000 7400-40000 1700-12600

Concentration of airborne fungi (cfu m-3) (mean±SD) Min-max (mean±SD)* Min-max* Absolute values Calculated values

GM 25630±16440* 38250±7826* 26745±16719* 40000±0* 14310±11222* 6270±2661* AP January 163±102.7 March 182±123.3 May 1244±1014 July 997±448.5 September 462±176.9 November 223±107.1 BS January 697±931.4*b March 797±917.6*a May 1123±853.6 July 1034±464.4 September 1041±751*a November 1203±1444*a; ***b ODA January 286±66.2**b March 884±1711**a May 1350±550.6***b July 2090±235.6**a;***a September 860±250.5**a November 240±62.54

Sampling period

35 33 32 35 22 46

29.5 35.5 47.5 54.5 39 40

41 36 42 37 30.5 49

34 29 36 42 31 66

Relative humidity (%)

Table 1 Quantitative composition of airborne fungi throughout one year at locations as follows: grain mill (GM), apartments (AP), basements (BS), outdoor air (ODA)

212 Jakšić Despot D and Šegvić Klarić M. INDOOR AIRBORNE FUNGI IN CROATIA Arh Hig Rada Toksikol 2014;65:209-218

213

Jakšić Despot D and Šegvić Klarić M. INDOOR AIRBORNE FUNGI IN CROATIA Arh Hig Rada Toksikol 2014;65:209-218

Table 2 Correlation between concentration of airborne fungi (cfu m-3), temperature, and relative humidity

Sampling site

Grain mill

Apartments

Basements

Outdoor air

Parameter

T (°)

rh (%)

T (°)

rh (%)

T (°)

rh (%)

T (°)

rh (%)

p-value (two-tailed)

0.8998

0.2336

0.0476

0.8479

0.2309

0.1730

0.0957

0.6215

R square

0.0045

0.3295

0.6661*

0.01035

0.3325

0.4068

0.5409

0.06659

T-temperature rh-relative humidity

*p<0.05 significant positive correlation

investigated in the previously mentioned study are in the same climate zone, we can speculate that the higher levels of airborne fungi in ODA and in the AP and BS in this study could be related to current climate changes. However, the sample size and number of locations prevent us from reaching a firm conclusion.

The qualitative composition of airborne fungi at each site and in each sampling period is presented in Table 3. The qualitative composition of airborne fungi in the GM differed from the other sampling locations.

The most dominant species in the GM belonged to Aspergillus spp. and Eurotium spp. (85-100 %).The most prevalent fungal genera in the residential areas were similar to those in ODA: Cladosporium spp (45100 % in AP and 55-95 % in BS), Penicillium spp.

(85-100 % in AP and 70-100 % in BS), and Aspergillus spp. (20-80 % in AP and 50-95 % in BS).

High frequencies of Aspergillus spp., Eurotium spp., Cladosporium spp., Penicillium spp. and Alternaria spp. at places where people live and work raises concern because all of them have proven to be causative agents for fungal allergies and other respiratory disorders (27, 33). Aspergilli of interest (sections Flavi, Nigri, and Versicolores) were frequent only at certain locations. We did not specify any species from these sections because the applied methods were sufficient only for sectional classifications. Due to morphological similarities among species from each section, specific DNA sequence data is required (16, 34-38).

Species belonging to section Flavi were very frequent in the GM (50-100 %) and present throughout the year. The sampled plates after incubation sometimes revealed overgrowth by these species, indicating a very high concentration of their viable conidia in the air. Eurotim spp. was also dominantly present in the GM (90-100 % samples) throughout the year and their mycellia usually covered the entire plate. Black aspergilli were isolated from GM samples and the peak was observed in May (55 %). As for the other mould genera, Cladosporium spp. was frequent

in GM samples (60-95 %) followed by Wallemia spp.

(40-90 %), Ulocladium spp. (5-80 %), and Alternaria spp. (5-10 %). Our failure to isolate the aforementioned species from samples taken in certain periods may have been due to overgrowth by section Flavi and/or Eurotium species.

We can also speculate on the observed frequencies of section Versicolores isolates. These species are known for their very small conidia (around 3 μm) and even if sampling is successful, their growth is too slow which causes them to become overwhelmed by other species. Despite this, we occasionally succeeded in isolating these species from GM samples (5-40 %).

Versicolores species reached their peak in November, which was probably due to the fact that other airborne fungi were at their lowest concentrations. Considering the very high concentration of airborne fungi in the GM, exposed workers are under risk. Moreover, due to the enormous amount of fungi belonging to section Flavi, there is the possibility of toxigenic strains and consequently aflatoxin B1 (AFB1). Recently it has been proven that black aspergilli are able to produce fumonisins in high quantities (23). Consequently these mycotoxins might be inhaled through grain and flour dust or via conidia of black aspergilli. Well-known producers of fumonisins belong to the genus Fusarium and they are present in high amounts at places where maize is processed and stored (26). Because Fusarium spp. conidia are not likely to be airborne, we managed to isolate these species only in September (10 %).

However, the presence of Fusarium spp. is expected throughout the year and so is the presence of fumonisins in maize.

Significant differences have been observed in the composition of aeromycota in residences compared to ODA. Sometimes, plates taken in the BS were overwhelmed in growth by Aspergillus section Versicolores. This indicated drastic fungal contamination with these species at particular sites, since a frequency of A. versicolor above 20 % is considered an indicator of indoor fungal contamination

10

40

80

40

0

100

0

0

0

0

60

0

10

90

0

0

100

10

0

0

Alternaria spp.

Aspergillus ochraceus

Section Flavi

Section Nigri

Section Versicolores

Aspergillus spp.

Aspergillus terreus

Aureobasidium spp.

Botrytis spp.

Chrysonilia sitophila

Cladosporium spp.

Culvularia spp.

Epicoccum spp.

Eurotium spp.

Fusarium spp.

Mucor spp.

Penicillium spp.

Rhizopus stolonifer

Ulocladium spp.

Wallemia spp.

30

20

0

90

0

0

60

0

0

45

0

0

0

0

80

45

6.2

0

0

15

0

2

30

50

0

85

0

0

40

5

0

55

0

5

0

0

95

60

5.2

0

20

25

0

3

January (%)

10

0

0

90

0

0

50

0

0

100

0

0

5

0

90

0

0

0

0

20

0

4

60

5

0

95

0

0

100

40

0

55

0

0

0

5

95

5

15

50

15

10

0

1

1 - grain mill, 2 - apartment, 3 - basement, 4 - outdoor air

0

1

Absidia spp.

Airborne fungi

5

0

0

100

0

0

20

5

0

65

0

0

0

0

45

40

10

0

0

0

0

2

20

45

0

100

0

0

65

0

0

95

5

0

0

0

65

65

0

5

40

40

0

3

March (%)

Table 3 Qualitative composition of airborne fungi at selected locations

0

30

0

100

0

0

0

30

0

100

0

0

0

0

0

0

0

0

0

10

0

4

90

65

0

100

0

0

100

0

0

95

0

0

0

0

95

25

55

70

35

0

0

1

30

30

0

95

0

0

45

0

20

100

0

0

0

0

20

10

5

0

5

5

0

2

15

60

0

100

0

0

85

0

0

95

0

0

0

0

80

35

0

5

10

15

0

3

May (%)

0

60

0

70

0

0

20

0

10

100

0

0

10

0

0

0

0

0

0

60

0

4

40

35

0

100

0

0

100

0

0

0

0

0

0

0

100

0

30

100

60

0

0

1

10

95

0

90

0

0

50

15

0

95

0

0

0

0

45

20

0

10

10

65

0

2

15

50

5

80

5

0

85

0

0

80

0

0

0

0

95

50

5

0

30

20

0

3

July (%)

10

90

0

40

0

0

60

0

0

100

0

0

0

0

20

0

10

10

0

100

0

4

50

80

0

80

0

0

100

0

0

70

0

0

0

0

100

25

20

90

35

0

0

1

10

25

10

85

0

0

15

0

0

90

0

0

0

0

60

50

25

5

15

65

0

2

0

25

0

70

0

0

80

10

0

75

0

0

0

0

100

50

5

10

35

40

5

3 0

4

0

30

0

70

0

0

0

30

0

100

0

0

0

0

40

10

0

0

10

10

September (%)

85

25

0

95

10

10

100

0

0

65

0

0

0

5

85

40

15

55

35

5

0

1

15

5

0

100

0

0

25

0

0

65

0

0

0

0

75

65

5

0

0

0

0

2

55

35

0

90

0

0

70

0

0

80

0

0

0

0

50

45

15

15

25

10

0

3

0

0

30

0

0

90

0

0

0

0

30

10

0

30

0

0

0

4

50

0

0

100

November (%)

214 Jakšić Despot D and Šegvić Klarić M. INDOOR AIRBORNE FUNGI IN CROATIA Arh Hig Rada Toksikol 2014;65:209-218

Jakšić Despot D and Šegvić Klarić M. INDOOR AIRBORNE FUNGI IN CROATIA Arh Hig Rada Toksikol 2014;65:209-218

(21). One of the mould sources in the air of indoor environments is damp building material. Aspergillus species are often found on substrates such as walls of residentces and working interiors, where their survival depends only on water activity (aw). Aspergilli are primary colonizers and can be customized to aw values below 0.8, which allows them to succeed in damp indoor environments more likely than other moulds (22, 39). Aspergillus versicolor is the most frequently reported fungal species in section Versicolores from damp indoor environments and its presence might be related to Sick Building Syndrome (17-20). Recent publications based on the molecular identification of Aspergillus species (Versicolores) indicated that Aspergillus versicolor sensu stricto were not common in buildings. However, it has also been reported that Aspergilli from this section isolated from indoor air are highly diverse in temperate regions (36), so we cannot exclude the presence of A. versicolor sensu stricto in our isolates. Considering the variety of recently identified Aspergillus species from this section (36), it is likely that more than one was able to produce sterigmatocystin. Therefore, their presence in high concentrations in indoor air could represent a serious threat to human health. Samples from the AP did not indicate overgrowth by these species, although their presence was persistent. Relative humidity measured at the AP in periods when indoor spaces are heated (January, November) ranged from 41 to 49 % and a high rate of condensation on windows was observed. In that same period, the ambient temperature was 18.3-18.6 °C and these conditions were considered supportive for sporulation and mycotoxin production

by A. versicolor (40).

CONCLUSIONS Considering the observed and analysed data, there is a threat of chronic exposure to secondary metabolites (sterigmatocystin, fumonisin, and aflatoxin). These metabolites can accumulate in spores and mycellium fragments in unknown concentrations. When people are exposed to high concentrations of mycotoxins, especially from contaminated food, one can reasonably easily establish a connection with a certain disease.

However, when they are present in very low concentrations, it is very difficult to study and evaluate their effects. Moreover, there are many unknown facts about these effects when inhalation is the primary means of exposure. However, there is a remarkable

215

amount of studies proving toxic, mutagenic, and carcinogenic properties of mould secondary metabolites on respiratory surrogate tissues, cell lines, and animal respiratory system, as well as case-reports addressing adverse health effects in humans (4, 41-46).

Therefore, monitoring of airborne fungi in occupational as well as living environments is of great importance in the prevention of unfavourable effects on human health.

Acknowledgements The study was done within the scientific project No.006-0061117-1242, financially supported by the Ministry of Science, Education and Sports of Republic of Croatia.

Conflict of interest The authors declare that they have no conflict of interest.

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Nielsen KF. Mycotoxin production by indoor molds. Fungal Genet Biol 2003;39:103-17. PMID: 12781669 Varga J, Kocsubé S, Suri K, Szigeti Gy, Szekeres A, Varga M, Tóth B, Bartók T. Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin. Int J Food Microbiol 2010;143:143-9. doi: 10.1016/j.ijfoodmicro.2010.08.008 Feller W. An Introduction to the Probability Theory and Its Application. New York: John Wiley and Sons Inc; 1950.

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27. Lugauskas A, Krikštaponis A, Šveistyte L. Airborne fungi in industrial environments- potential agents of respiratory diseases. Ann Agric Environ Med 2004;11:19-25. PMID: 15236494 28. Desai MR, Ghosh SK. Occupational exposure to airborne fungi among rice mill workers with special reference to aflatoxin producing A. flavus strains. Ann Agric Environ Med 2003;10:159-62. PMID: 14677906 29. Adhikari A, Sen MM, Gupta-Bhattacharya S, Chanda S. Airborne viable, non-viable, and allergenic fungi in a rural agricultural area of India: a 2-year study at five outdoor sampling stations. Sci Total Environ 2004;326:123-41. doi: 10.1016/j.scitotenv.2003.12.007 30. Hameed AAA, Khodr MI. Suspended particulates and bioaerosols emitted from an agricultural non-point source.

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35. Godet M, Munaut F. Molecular strategy for identification in Aspergillus section Flavi. FEMS Microbiol Lett 2010;304:15768. doi: 10.1111/j.1574-6968.2009.01890.x 36. Jurjević Ž, Peterson SW, Horn BW. Aspergillus section Versicolores: nine new species and multilocus DNA sequence based phylogeny. IMA Fungus 2012;3:59-79. doi: 10.5598/ imafungus.2012.03.01.07 37. Jurjević Ž, Peterson SW, Stea G, Solfrizzo M, Varga J, Hubka V, Perrone G. Two novel species of Aspergillus section Nigri from indoor air. IMA Fungus 2012;3:159-73. doi: 10.5598/ imafungus.2012.03.02.08.

38. Nielsen KF, Mogensen JM, Johansen M, Larsen TO, Frisvad JC. Review of secondary metabolites and mycotoxins from the Aspergillus niger group. Anal Bioanal Chem 2009;395:1225-42. doi: 10.1007/s00216-009-3081-5 39. Schulz T, Senkpiel K, Ohgke H. Comparision of the toxicity of reference mycotoxins and spore extracts of common indoor moulds. Int J Hyg Environ Health 2004;207:267-77. doi: 10.1078/1438-4639-00282 40. Vujanović V, Smoragiewicz W, Krzysztyniak K. Airborne fungal ecological niche determination as one of the possibilities for indirect mycotoxin risk assessment in indoor air. Environ Toxicol 2001;16:1-8. PMID: 11345539 41. May JJ, Stallones L, Darrow D, Pratt DS. Organic dust toxicity (pulmonary mycotoxicosis) associated with silo unloading.

Thorax 1986;41:919-23. PMID: 3590053 42. Straus DC. Molds, mycotoxins, and sick building syndrome.

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43. Piecková E, Wilkins K. Airway toxicity of house dust and its fungal composition. Ann Agric Environ Med 2004;11:67-73.

PMID: 15236501 44. Piecková E. Adverse health effects of indoor moulds. Arh Hig Rada Toksikol 2012;63:545-59. doi: 10.2478/10004-1254-632012-2221.

45. Jakab GJ, Hmieleski RR, Zarba A, Hemenway DR, Groopman JD. Respiratory aflatoxicosis: suppression of

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pulmonary and systemic host defenses in rats and mice. Toxicol Appl Pharmacol 1994;125:198-205. PMID: 8171428 46. Kelman BJ, Robbins CA, Swenson LJ, Hardin BD. Risk from inhaled mycotoxins in indoor office and residential environments. Int J Toxicol 2004;23:3-10. doi: 10.1080/10915810490265423

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Sažetak Jednogodišnje istraživanje razine plijesni u zraku unutarnjih prostora Cilj rada bio je ispitati varijacije učestalosti (%) i koncentracija (cfu m-3) plijesni u zraku mlina žitarica i četiriju stambenih prostora (dva stana i dva podruma) u odnosu na vanjski zrak tijekom jedne godine u Zagrebu (Hrvatska). Učestalost Aspergillus vrsta iz sekcija Flavi, Nigri i Versicolores također je ispitana.

Plijesni iz zraka su uzorkovane pomoću uređaja Air-sampler MAS 100 Eco u kojem su postavljene DG18-agarske ploče. Prosječna koncentracija aerogenih plijesni u mlinu bila je između 14.310 i 40.000 cfu m-3, što je veće od koncentracije (104 cfu m-3) koja se smatra opasnom za zdravlje. Procijenjene vrijednosti koncentracija plijesni u zraku mlina, dobivene Felerovom korekcijom, čak su šest puta veće od izmjerenih koncentracija. U stanovima (163-1244 cfu m-3) koncentracije aerogenih plijesni bile su manje nego u vanjskom zraku (286-2090 cfu m-3) i podrumima (697-1203 cfu m-3), izuzev u toplijim mjesecima kada su izmjerene vrijednosti bile slične. Tijekom godine dominirale su vrste iz rodova Cladosporium spp.

(90-100 %), Penicillium spp. (40-100 %), i Alternaria spp. (10-100 %), koje su uobičajene u područjima s umjerenom klimom. Aspergile iz sekcija Flavi (50-100 %) i Nigri (15-40 %), A. ochraceus (15-60 %) i Eurotium spp. (85-100 %) dominirali su u zraku mlina, a u uzorcima vanjskog zraka rijetko su detektirane.

Vrste Aspergillus iz sekcije Versicolores s većom učestalošću nađene u podrumima nego u stanovima.

Veliko opterećenje mlina aerogenim plijesnima tijekom cijele godine može biti opasno za zdravlje radnika.

Ujedno, ovo je prvo jednogodišnje ispitivanje sezonske varijabilnosti u sastavu plijesni u zraku mlina žitarica i stambenih prostora u Hrvatskoj. Međutim, monitoring treba nastaviti tijekom dužeg razdoblja.

KLJUČNE RIJEČI: Flavi; mlin; Nigri; stambeni objekti; Versicolores

CORRESPONDING AUTHOR: Maja Šegvić Klarić Department of Microbiology, Faculty of Pharmacy and Biochemistry University of Zagreb, Croatia E-maiI: msegvic@pharma.hr

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Varga J, et al. DISTRIBUTION OF BLACK ASPERGILLI IN INDOOR AIR Arh Hig Rada Toksikol 2014;65:219-223

DOI: 10.2478/10004-1254-65-2014-2450

Original article

Occurrence of black Aspergilli in indoor environments of six countries János Varga1, Sándor Kocsubé1, Gyöngyi Szigeti1, Nikolett Baranyi1, Csaba Vágvölgyi1, Daniela Jakšić Despot2, Donát Magyar3, Martin Meijer4, Robert A. Samson4, and Maja Šegvić Klarić2 Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary1, Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia2, National Institute of Environmental Health, Budapest, Hungary3, CBS Fungal Biodiversity Centre, Utrecht, The Netherlands4 Received in October 2013 CrossChecked in October 2013 Accepted in January 2014

Black Aspergilli (Aspergillus section Nigri) are widely distributed in various habitats. They act as food spoilage organisms, human pathogens, and mycotoxin producers and are frequently encountered in indoor environments. Black Aspergilli, specifically A. niger, A. welwitschiae, and A. carbonarius, produce different ochratoxins and fumonisins. Ochratoxins are known to induce renal disorders following inhalation, which necessitates the determination of potential mycotoxin-producing species in our environment. This paper aimed to compare the diversity and species distribution of black Aspergilli in the indoor environments of six different countries using morphological and molecular methods. A total of 178 black Aspergillus isolates were identified from six countries. In contrast with results from previous studies, A. niger was not the only black Aspergillus detected in indoor air. Species distribution differed among countries, although the distribution in European countries (Croatia, Hungary, the Netherlands, and Turkey) with a temperate climate was considerably similar. The highest species diversity was observed in indoor samples from Thailand, while the lowest was found in Algeria. Potentially ochratoxin- and fumonisin-producing fungi were detected in the indoor air of all six countries. Further studies need to clarify the effect of these fungi and their mycotoxins on human and animal health.

KEY WORDS: Aspergillus section Nigri; indoor fungi; calmodulin gene; sequence-based identification; species distribution

Moulds are widely distributed in indoor and outdoor environments. They are essential components of our ecosystem providing decomposition of many organic substances necessary for plant, animal, and human life (1). However, excessive exposure to moulds has been a health issue for humans for many years, as they are frequently found in homes, office buildings, schools, vehicles, and other locations where organic matter and water are left unattended. They are common in household dust and can cause allergic symptoms or invasive infections in humans with weak immune systems. They can also be harmful considering their toxin-producing abilities (2).

Black Aspergilli (Aspergillus section Nigri) are important in food mycology, medical mycology, and biotechnology, frequently occurring in indoor environments (1, 3). Many species cause food spoilage, but are also used in the fermentation industry to produce various enzymes and organic acids (4).

Some species also act as plant pathogens, including A. carbonarius and A. niger in grapes, and A. welwitschiae in onions and the plant Welwitschia mirabilis (5, 6). Moreover, several species can produce mycotoxins including ochratoxins and fumonisins that can be harmful for both humans and animals (4, 6-9).

Although data regarding fumonisin absorption

220 following inhalation and/or dermal exposure are not available (10), ochratoxin A exposure via inhalation has been described in the literature to cause kidney disease in animals and humans (11), which is why precise identification at the species level is of immense importance. Recent data indicate that Aspergillus section Nigri comprises 25 species (4, 12-14), which can be divided into five main clades. The A. niger clade includes 10 biseriate species and was divided into three subclades based on ß-tubulin and calmodulin sequence data: the A. tubingensis, A. niger, and A. brasiliensis subclades (13). Recently A. awamori was described (15) and later renamed A. welwitschiae, while A. acidus was renamed A. luchuensis (16). Some of these species are able to produce carcinogenic mycotoxins including ochratoxins and fumonisins (4, 17). Black Aspergilli isolated from indoor air are usually referred to as A. niger (18). Black Aspergilli were found in indoor air in several countries including Great Britain, the Czech Republic, Egypt, Saudi Arabia, the US, Canada, and Slovakia (18). In general, they occur more frequently in tropical or subtropical areas (18, 19).

Although they are common in indoor environments, the species composition of black Aspergilli is unknown. That is why the aim of this work was to compare the diversity and species distribution of black Aspergilli in indoor environments of various countries using morphological and molecular methods.

MATERIALS AND METHODS The samples used were obtained from six different countries as part of interlaboratory cooperation and comparison. Samples of airborne fungi were collected in Croatia, Hungary, the Netherlands, Turkey, Algeria, and Thailand between 2010 and 2013 using standard methods. The Petri plate sedimentation method or airsamplers using dichloran 18% glycerol agar (DG18) plates were used for sampling airborne fungi (1, 20). The plates were incubated at 25±2 °C and the developed fungal colonies were counted 5 days later.

Airborne fungi were identified on the basis of their macro- and microscopic characteristics after subculturing on Czapek Yeast Agar (CYA) and Malt Extract Agar (MEA) (1). Black Aspergillus isolates (n=178) were purified and cultivated on CYA and MEA plates at 25 °C in the dark for seven days.

Morphological identifications were carried out according to literature (1, 4, 21).

Varga J, et al. DISTRIBUTION OF BLACK ASPERGILLI IN INDOOR AIR Arh Hig Rada Toksikol 2014;65:219-223

For sequence-based identification, the cultures used for the molecular studies were grown on malt peptone (MP) broth for 2 days, and DNA was extracted from the mycelia using the Masterpure™ yeast DNA purification kit (Epicentre Biotechnologies, Madison, WI, USA) according to the manufacturer’s instructions.

A part of the calmodulin gene was amplified and sequenced as described previously (4). The calmodulin sequences were compared using nucleotide-nucleotide BLAST (blastn) with default settings (22) with the Genbank database and our own sequence database.

Species identification was determined from the lowest expected value of BLAST output.

RESULTS AND DISCUSSION In this preliminary study, the species distribution of black Aspergilli was examined in various indoor environments of six countries using sequence-based methods. Since the sampling methods varied, a detailed analysis of the quantitative distribution of species was not possible. Consequently, we instead concentrated on the species distribution of the isolates.

Altogether, 178 black Aspergillus isolates were obtained and examined using morphological methods.

In accordance with the literature, these isolates could not be reliably assigned to species based on their macro- or micromorphological features, with the exception of A. carbonarius, which produces larger conidial heads and conidia than the other species (4).

However, the isolates were successfully assigned to species using partial sequence data of the calmodulin gene, which is in agreement with previous studies (4, 13).

The observed species distributions were highly variable in the examined countries (Figure 1). The examined isolates from Thailand showed the greatest diversity; 22 isolates belonged to 7 different species among which 2 isolates represented a new species related to Aspergillus saccharolyticus (23) and will be characterized in future studies, especially regarding their genetic and chemical profiles. The smallest diversity was observed in the Algerian dataset, where only 2 species could be observed. Interestingly, A. carbonarius, commonly found in various types of environments and significant for its ability to produce high quantities of ochratoxin A (4, 24), was observed only in this country. The diversity of black Aspergilli was similar in European countries. Four species were identified in Hungary and the Netherlands while 5

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Hungary (n=73)

Croatia (n=41)

3%

2% 12%

18% 33%

2%

A. niger A. welwitschiae

42%

A. tubingensis A. luchuensis 42%

A. niger A. welwitschiae A. tubingensis A. luchuensis A. piperis

46%

Netherlands (n=14)

Turkey (n=12) 8%

21%

8%

29%

A. niger A. welwitschiae

25%

A. tubingensis A. luchuensis 29%

51%

A. welwitschiae A. tubingensis A. luchuensis A. uvarum A. aculeatinus

8%

21%

Thailand (n=22)

Algeria (n=16) 9%

14%

5%

14% 22% 5%

A. niger A. tubingensis A. neoniger A. fijiensis A. trinidadensis A. japonicus new sp.

38%

A. welwitschiae A. carbonarius 62%

31%

Figure 1 Distribution of black Aspergillus species in indoor environments in different countries based on their calmodulin sequence data. n â&#x20AC;&#x201C; represents the number of strains in section Nigri

species were identified in Croatia. Interestingly, the species distributions were very similar: A. niger, A. tubingensis, A. luchuensis, and A. welwitschiae were identified in all three countries. In Turkey, A. tubingensis, A. luchuensis, and A. welwitschiae were also present, whereas A. niger was not detected.

However, in this country the sample size was relatively small, since only 12 black Aspergilli could be isolated.

In summary, in contrast to previous studies, A. niger was not the only black Aspergillus present in indoor air. Species diversity was different in the countries examined, although the species distributions in European countries with temperate climate were highly similar. A. niger, A. tubingensis, A. luchuensis, and A. welwitschiae were identified in three of the

European countries, as well as in Turkey, with the exception of A. niger, indicating the significant role of climatic conditions on the distribution and occurrence of indoor fungi. At least one of the potentially ochratoxin- and fumonisin-producing fungi A. niger, A. welwitschiae, and A. carbonarius was detected in all six countries. Further studies are needed to clarify the real impact of these fungi and their mycotoxins on human and animal health.

Acknowledgements The research of S.K., Sz.Gy., N.B. and Cs.V. was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TĂ MOP 4.2.4.A/2-11-1-2012-0001

222 “National Excellence Program”. The relating research groups were also supported by the project TÁMOP 4.2.2.A-11/1/KONV-2012-0035 and by the Hungarian Scientific Research Fund (OTKA; grant reference number No. K84122 and K84077), providing infrastructure and research equipment.

REFERENCES 1. Samson RA, Houbraken J, Thrane U, Frisvad JC, Andersen B. Food and indoor fungi. Utrecht: CBS-KNAW Fungal Biodiversity Centre; 2010.

2. Jarvis BB, Miller JD. Mycotoxins as harmful indoor air contaminants. Appl Microbiol Biotechnol 2005; 66:367-72.

3. Schwab CJ, Straus DC. The roles of Penicillium and Aspergillus in sick building syndrome. Adv Appl Microbiol 2004; 55:215-38.

4. Samson RA, Noonim P, Meijer M, Houbraken J, Frisvad JC, Varga J. Diagnostic tools to identify black Aspergilli. Stud Mycol 2007; 59:129-45.

5. Varga J, Kocsubé S, Szigeti G, Man V, Tóth B, Vágvölgyi C, Bartók T. Black Aspergilli and fumonisin contamination of onions purchased in Hungary. Acta Aliment 2012; 41:41423.

6. Nielsen KF, Mogensen JM, Johansen M, Larsen TO, Frisvad JC. Review of secondary metabolites and mycotoxins from the Aspergillus niger group. Anal Bioanal Chem 2009; 395:1225–46.

7. Frisvad JC, Larsen TO, Thrane U, Meijer M, Varga J, Samson RA, Nielssen KF. Fumonisin and ochratoxin production in industrial Aspergillus niger strains. PLoS ONE 2011; 6:e23496.

8. Šegvić Klarić M. Adverse effects of combined mycotoxins.

Arh Hig Rada Toksikol 2012; 63:519-30.

9. Piecková E. Adverse health effects of indoor moulds. Arh Hig Rada Toksikol 2012; 63:545-9.

10. European Commission. Opinion of the Scientific Committee on Food on fusarium toxins part 3: Fumonisin B1 (FB1). European Commission Health and Consumer Protection Directorate-General, Brussels, Belgium; 2000.

Varga J, et al. DISTRIBUTION OF BLACK ASPERGILLI IN INDOOR AIR Arh Hig Rada Toksikol 2014;65:219-223

11. Di Paolo N, Guarnieri A, Garosi G, Sacchi G, Mangiarotti AM, Di Paolo M. Inhaled mycotoxins lead to acute renal failure. Nephrol Dial Transplant 1994; 9(Suppl 4):116-20.

12. Samson RA, Houbraken JAMP, Kuijpers AFA, Frank JM, Frisvad JC. New ochratoxin or sclerotium producing species in Aspergillus section Nigri. Stud Mycol 2004; 50:45-61.

13. Varga J, Frisvad JC, Kocsubé S, Brankovics B, Tóth B, Szigeti G, Samson RA. New and revisited species in Aspergillus section Nigri. Stud Mycol 2011; 69:1-17.

14. Jurjević Z, Peterson SW, Stea G, Solfrizzo M, Varga J, Hubka V, Perrone G. Two novel species of Aspergillus section Nigri from indoor air. IMA Fungus 2012; 3:159-73.

15. Perrone G, Stea G, Epifani F, Varga J, Frisvad JC, Samson RA. Aspergillus niger contains the cryptic phylogenetic species A. awamori. Fungal Biol 2001; 115:1138-50.

16. Hong SB, Lee M, Kim DH, Varga J, Frisvad JC, Perrone G, Gomi K, Yamada O, Machida M, Houbraken J, Samson RA. Aspergillus luchuensis, an industrially important black Aspergillus in East Asia. PLoS ONE 2013; 8:e63769.

17. Varga J, Kocsubé S, Suri K, Szigeti Gy, Szekeres A, Varga M, Tóth B, Bartók T. Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin. Int J Food Microbiol 2010; 143:143-9.

18. Piecková E. Aspergillus sp. in dwellings and health implications of indoor fungi. [Displayed 29 September 2013].

Available at http://www.aspergillus.org.uk/indexhome. htm?secure/articles/./pieckova.html~main 19. Scott JA. Studies on indoor fungi. Ph D Thesis. Toronto: University of Toronto; 2001.

20. Amend SA, Seifert KA, Samson RA, Bruns TD. Indoor fungal composition is geographically patterned and more diverse in temperate zones than in the tropics. Proc Natl Acad Sci USA 2010; 107:13748-53.

21. Raper KB, Fennell DI. The genus Aspergillus. Baltimore: Williams and Wilkins; 1965.

22. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–10.

23. Sørensen A, Lübeck PS, Lübeck M, Nielsen KF, Ahring BK, Teller PJ, Frisvad JC. Aspergillus saccharolyticus sp. nov., a black Aspergillus species isolated in Denmark. Int J Syst Evol Microbiol 2011; 61:3077-83.

24. Varga J, Kozakiewicz Z. Ochratoxin A in grapes and grapederived products. Trends Food Sci Technol 2006; 17:72-81.

Varga J, et al. DISTRIBUTION OF BLACK ASPERGILLI IN INDOOR AIR Arh Hig Rada Toksikol 2014;65:219-223

223

Sažetak Rasprostranjenost crnih plijesni roda Aspergillus u unutarnjem prostoru u šest država Crne plijesni roda Aspergillus široko su rasprostranjene u različitim životnim prostorima. Razgrađivači su hrane, ljudski patogeni i proizvođači mikotoksina. Također ih se često može pronaći u unutarnjim prostorima, a pojedine vrste poput A. niger, A. welwitschiae i A. carbonarius proizvode ohratoksine i fumonizine.

Udisanje ohratoksina uzrokuje poremećaje bubrežne funkcije, pa je iznimno važno odrediti plijesni u našem okolišu koje bi ih mogle proizvoditi. U ovom se radu uspoređuje raznolikost i rasprostranjenost vrsta crnih plijesni roda Aspergillus u unutarnjem prostoru šest različitih zemalja morfološkim i molekularnim metodama. Analizirano je ukupno 178 uzoraka. Za razliku od prethodnih studija, u zraku unutarnjih prostora nije pronađena samo A. niger. Rasprostranjenost vrsta u europskim zemljama (Hrvatska, Mađarska, Nizozemska i Turska), gdje je klima umjerena, bila je umnogome slična. Najveća raznolikost uočena je u uzorcima iz Tajlanda, a najmanja u Alžiru. Da bi se razjasnili štetni učinci navedenih plijesni i mikrotoksina na zdravlje ljudi i životinja, potrebna su daljnja istraživanja.

KLJUČNE RIJEČI: gen za kalmodulin; identifikacija zasnovana na slijedu baza; plijesni unutarnjih prostora; rasprostranjenost vrsta

CORRESPONDING AUTHOR: János Varga Department of Microbiology Faculty of Science and Informatics, University of Szeged Közép fasor 52, 6726 Szeged, Hungary E-mail: jvarga@bio.u-szeged.hu

Brčić Karačonji I and Brajenović N. DETECTION OF AMPHETAMINES THROUGH HAIR ANALYSIS Arh Hig Rada Toksikol 2014;65:225-230

225

DOI: 10.2478/10004-1254-65-2014-2514

Professional paper

Evaluation of amphetamine-type stimulant abuse through hair analysis: Results from 12 years of work Irena Brčić Karačonji and Nataša Brajenović Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia Received in March 2014 CrossChecked in March 2014 Accepted in June 2014

Hair analysis is a reliable tool for detecting long-term exposure to illegal drugs, including amphetaminetype stimulants, over periods from a few weeks to a few months, depending on the length of the hair used for analysis. Between 2000 and 2012, over 600 hair samples were analysed at the Institute for Medical Research and Occupational Health, Croatia (IMROH) for the presence of amphetamine-type stimulants.

IMROH has used the same procedure for testing hair samples for amphetamine-type stimulants for over twelve years. It was found to be reliable for confirming repeated abuse of amphetamine-type stimulants.

Gas chromatography/mass spectrometry (GC/MS) was used to determine amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA-Ecstasy), and 3,4-methylenedioxyethylamphetamine (MDEA) in hair. Hair samples were either taken at the Institute, delivered by mail or a third person brought them to the laboratory. In most cases, the hair samples were tested anonymously. A total of 23 % of the tested samples were positive for one or more amphetamine-type stimulant. MDMA was the most frequently detected substance, whereas the most frequent combination was amphetamine with MDMA. Our results could indicate a trend in amphetamine-type stimulant abuse among young people in the Republic of Croatia.

KEY WORDS: GC/MS; MDMA; methamphetamine; quantitative determination

Amphetamine-type stimulants usually refer to synthetic substances like amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA-Ecstasy), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxyethylamphetamine (MDEA) that act as central nervous system stimulants. According to data provided by the European Monitoring Centre for Drugs and Drug Addiction (1), approximately one quarter of Europe’s adult population have used an illicit drug at some point in their lives and approximately 3.8 % of all Europeans aged 15-64 have consumed amphetamines at least once in their lifetime (2). A study conducted by the Ivo Pilar Institute of Social Science on illicit drug use in the general Croatian population (4,756 respondents) showed that 16 % of

adults aged 15-64 reported taking at least one type of drug of abuse in their lifetime (3). Amphetamine-type stimulants are the second most frequently used illicit drugs after cannabis: the lifetime prevalence of taking cannabis in Croatia is 15.6 %, followed by amphetamines (2.6 %) and ecstasy (2.5 %). The main concern is the higher lifetime prevalence (25.7 %) of taking an illegal drug among the 15-34 age group. In the same age group, the lifetime prevalence rate of taking amphetamine-type stimulants was two times higher than among all adults (aged 15-64). Around 23 % of respondents who had taken an amphetaminetype stimulant at least once reported their first experience with these illegal drugs at an age between 16 and 17. The second main concern is that, after years of declines in the quantity of amphetamine-type

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Brčić Karačonji I and Brajenović N. DETECTION OF AMPHETAMINES THROUGH HAIR ANALYSIS Arh Hig Rada Toksikol 2014;65:225-230

stimulants seized, a rise was recorded in 2011 (4). In a comprehensive study performed in a Zagreb wastewater treatment plant in 2009, a consumption of 1–3 kg per year amphetamine-type stimulants was estimated based on their analysis in wastewater. The authors concluded that the amphetamine-type drug consumption rate in the city of Zagreb (3.6–9.7 mg per day in 1000 inhabitants) is comparable to that in Italy and Switzerland, but significantly lower than in UK and Spain (5).

Due to the abovementioned reasons, the early identification of drug abuse in the general population is critical in planning preventative action and reducing the damage caused by illicit drug use. Unlike urine, in which the generally accepted detection time for amphetamine-type stimulants is 1 to 3 days, hair provides information on long-term exposure to amphetamine-type stimulants over a period from a few weeks to a few months, depending on the length of the hair collected (6).

Since 2000, the Institute for Medical Research and Occupational Health in Zagreb, Croatia (IMROH) has provided quantitative hair analyses for amphetaminetype stimulants for a wide variety of customers including private customers, addiction prevention centres, hospitals, courts, and employers. The aim of this paper is to provide an overview of amphetaminetype stimulant abuse in Croatia, as well as to evaluate the different characteristics of the hair samples analysed at the Institute with regard to how and from whom they were received.

MATERIALS AND METHODS Hair samples Hair samples (n=666) from various sources were received or taken in the laboratory. A strand of hair of approximately 5 mm in diameter was cut from close to the scalp at the vertex posterior area, folded in aluminium foil or paper, and the proximal and distal ends were marked. We analysed hair segments 2-4 cm long from the proximal end, which represent approximately 2-4 months of hair growth as stated in the literature (7). The study was conducted according to the ethical standards of the Helsinki Declaration and was approved by the IMROH Ethics Committee.

The investigation presented in this paper was performed within a wider research project entitled

”Identification of drug abuse by comparative analysis of biological specimens” and funded by the Ministry of Science and Technology of the Republic of Croatia.

Hair analysis Hair samples were analysed according to a previously validated procedure (8). The gas chromatography/mass spectrometry (GC/MS) method was developed for determining amphetamine, methamphetamine, MDMA, MDA, and MDEA in hair. Over the years, a few procedural modifications were done (reagent and standard suppliers were changed and deuterated internal standards were purchased). All of the changes were fully validated before routine laboratory work. In brief, the hair was washed in dichloromethane, dried, cut into very small pieces, and 50 mg of hair was used for analysis. Hair samples were submitted to alkaline digestion with 1 mol L-1 sodium hydroxide at 70 °C for 20 min prior to extraction with ethyl acetate (2x1 mL). The extract was evaporated to dryness in the presence of a 100 μL mixture of methanol:hydrochloric acid (99:1, v/v) to prevent a loss of volatile amphetamine and derivatizated with 50 μL of heptafluorobutyric anhydride (HFBA) at 60 °C for 30 min. The derivatizated samples were evaporated to dryness, and reconstituted in 100 μL of ethyl acetate for injection of 1 μL onto a gas chromatograph Varian 3400 CX with Saturn ion trap mass spectrometer equipped with HP-5MS capillary column (5 % diphenyl-95 % dimethylpolysiloxane, 30 m, 0.25 mm ID, 0.25 μm film thickness; J&W Agilent Technologies; Santa Clara, USA). Three ions for the analytes and two ions for the internal standards were monitored in the selected ion monitoring (SIM) mode. The following ions were used for each drug: a m p h e t a m i n e - H F B A , m / z 2 4 0 , 11 8 , 9 1 ; methamphetamine-HFBA, m/z 254, 210, 118; MDAHFBA, m/z 135, 162, 240; MDMA-HFBA, m/z 162, 254, 210; MDEA-HFBA, m/z 268, 135, 162; amphetamine-d5-HFBA, m/z 244, 92; and MDMA-d5HFBA, m/z 258, 165. The underlined ions were used for quantitation.The standards were prepared in blank hair extracts. Each case sample batch included standards and positive and negative control. The precision expressed as relative standard deviation (RSD) was <9 % and the accuracy was >85 % for all of the 5 analytes. The limit of detection ranged from 0.01 ng mg-1 to 0.05 ng mg-1. The confirmation cut-off for amphetamine-type stimulants was 0.2 ng mg-1.

External quality assessment was performed through participation in the Proficiency Testing programme

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organised by the Society of Hair Testing (9). For the purpose of this study, all data above the limit of detection were included.

RESULTS AND DISCUSSION During the period from 28 April 2000 to 10 December 2012, 666 hair samples were analysed. A large majority of our customers were parents who had suspected their children of taking drugs – primarily amphetamine-type stimulants. In most instances, a third person (e.g., parent) brought the hair sample for analysis (40 %). In other cases, hair samples were taken at IMROH by trained personnel (37 %) or delivered by mail (23 %). In 73 % of cases, hair samples were tested anonymously (e.g., the hair samples were received under only the first or a fictitious name, or a number). Hair analysis requests from private customers accounted for approximately 92 % of the total number of samples. Less than 8 % of cases included hair samples tested for official purposes [addiction prevention centres and hospital requests (3.8 %), workplace drug testing (2.7 %), and court proceedings (1.4 %)]. The probable reason for such a low percentage of official requests in Croatia compared to e.g. United Kingdom (10) is that hair testing for illegal drugs has not yet been included in any Croatian law or regulation.

The main drawbacks in case of sampling hair from a third person are the lack of control regarding protocols and the ability to collect hair ”next to the

scalp”. LeBeau et al. (11) suggested that, beside the variability in the growth rate, inconsistent collection may have an important impact on the interpretation of results.

Among customers who could be identified by gender (they came personally to the laboratory) males were more likely to be positive (23 % of positive males vs. 17 % of positive females), which is in accordance with studies by Strote at al. (12), Martins et al. (13), and Molinaro et al. (14), who reported a higher prevalence of illegal drug use among males. Also, males were 2.5 times more frequent clients compared to females.

Table 1 summarises the positive results of hair testing (n=155), in which one or more type of drug were found. A total of 23 % of the tested samples were positive for one or more amphetamine-type stimulant.

Among the hair samples received, 25 % of samples from users who provided their identity tested positive compared to 20 % of positive hair samples received anonymously. Considering single drug use, MDMA was the most frequent detected substance (in 10 % of the samples), followed by amphetamine (5 %). The most frequent combination was amphetamine with MDMA (5 % of all hair samples). Methamphetamine was detected in only 1 % of hair samples. In less than 2 % of hair samples, we detected methamphetamine + MDMA, amphetamine + methamphetamine, amphetamine + methamphetamine + MDMA or amphetamine + MDMA + MDA + MDEA. According to surveys conducted on a large sample size in Norway (15), Australia (16), United States (13),

Table 1 Distribution of positive hair samples (n=155) based on the presence of certain amphetamine-type stimulants and their combinations in the 2000-2012 period

Amphetamine-type stimulant(s) in hair samples

a

Number of positive samples

MDMA(+MDAa)

64

Amphetamine+MDMA(+MDAa)

36

Amphetamine

34

Methamphetamine

9

Methamphetamine+MDMA(+MDAa)

5

Amphetamine+Methamphetamine

3

Amphetamine+Methamphetamine+MDMA

3

Amphetamine+MDMA+MDA+MDEA

1

not detected in all samples; MDMA - 3,4-methylenedioxymethamphetamine;

MDA - 3,4-methylenedioxyamphetamine; MDEA - 3,4-methylenedioxyethylamphetamine

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and Taiwan (17), MDMA appeared to be the most commonly used illegal drug in adolescents and young adults. The prevalence of MDMA in our study was not surprising since most of our clients were adolescents and young adults considered to be frequent consumers of MDMA. In addition, MDMA is easily available and relatively cheap. Growing concerns have been raised about ecstasy use during adolescence because young ecstasy users tend to be more naive and more vulnerable to the harmful effects. The small number of samples positive for methamphetamine could be explained by its low accessibility and relatively high price (4). The levels detected for amphetamine-type stimulants are presented in Table 2.

The measured concentrations were in accordance with already published concentration ranges (10, 18, 19). Among the 109 cases positive for MDMA, its metabolite MDA was detected in 23 cases. The MDA/ MDMA ratio in hair ranged from 0.01 to 0.25, which was in agreement with earlier observations (20). In most of the MDMA positive samples (73.4 %), MDMA was found in mass fractions up to 5.0 ng mg-1, which is in accordance with previously reported results (21).

Amphetamine was found in 77 cases, 36 of which exhibited a co-occurrence with MDMA, which is generally the most frequent combination. The probable reasons for the wide range of mass fractions for amphetamine and MDMA could be the different purity, frequency, and amount consumed. Also, the different hair treatments in 10 % of the analysed hair samples could be the reason for a slight decrease of drug content in those hair samples, which has been documented by several authors (18, 22, 23).

Interpretation of hair analysis results is a very challenging process due to several possible sources

of error. In addition to different hair growth rate and errors in sampling, a possible problem in hair analysis could be false positive hair testing result caused by passive exposure to drug from the environment.

Therefore, a decontamination step using appropriate solvents is necessary prior to the analysis of hair samples (24). In addition, another problem is the lack of correlation between frequency of drug use and its concentration in hair due to the different incorporation rate of drug in the hair, different hair melanin content, use of hair treatments (bleaching and dyeing), etc.

Pharmacogenetic variations also play an important role in the interpretation of hair analysis results (25).

CONCLUSIONS It is evident that hair is a reliable biological marker for cumulative exposure to illicit drugs. IMROH has used the same hair sample testing procedure for amphetamine-type stimulants for over twelve years.

This method was found to be reliable for confirming repeated amphetamine-type stimulant abuse. Our study can serve to provide a preliminary idea about the trend of amphetamine-type stimulant abuse among adolescents and young adults in Croatia.

Acknowledgements Author’s attendance of the TIAFT2013 Congress (where this study was presented) was supported by a donation from Biovit, Croatia. The authors also wish to thank Ms Vesna Triva for her most skilful technical assistance.

Table 2 Mass fraction of certain analytes determined in hair samples

Number of positive samples

Mass fraction (ng mg-1) median (range)

MDMA

109

2.30 (0.45-90.8)

Amphetamine

77

1.51 (0.15-118.7)

MDA

23

0.81 (0.18-4.64)

Methamphetamine

20

0.67 (0.10-2.01)

MDEA

1

2.13 (-)

Analyte

MDMA - 3,4-methylenedioxymethamphetamine;MDA - 3,4-methylenedioxyamphetamine;

MDEA - 3,4-methylenedioxyethylamphetamine

Brčić Karačonji I and Brajenović N. DETECTION OF AMPHETAMINES THROUGH HAIR ANALYSIS Arh Hig Rada Toksikol 2014;65:225-230

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Sažetak Evaluacija zlouporabe stimulansa amfetaminskog tipa putem analize kose: rezultati dvanaestogodišnjeg rada Analiza kose pouzdan je način za detektiranje dugotrajnog konzumiranja ilegalnih droga, uključujući stimulanse amfetaminskog tipa u razdoblju od nekoliko tjedana do nekoliko mjeseci, ovisno o duljini kose koja se koristi za analizu. Od 2000. do 2012. u Institutu za medicinska istraživanja i medicinu rada analizirano je više od 600 uzoraka kose na prisutnost stimulansa amfetaminskog tipa. Vezani sustav plinski kromatograf/spektrometar masa koristio se za određivanje masenog udjela amfetamina, metamfetamina, 3,4-metilenedioksiamfetamina (MDA), 3,4-metilenedioksimetamfetamina (MDMA-Ekstazi) i 3,4-metilenedioksietilamfetamina (MDEA) u kosi. Uzorci kose uzimani su u Institutu, dostavljeni su poštom ili ih je treća osoba donijela u laboratorij. U većini slučajeva uzorkovanje i analiza kose obavljeni su anonimno. Dvadeset tri posto analiziranih uzoraka kose bilo je pozitivno na jedan ili više stimulansa amfetaminskog tipa. Najčešće je detektiran MDMA, a najčešća kombinacija stimulansa bila je amfetamin s MDMA. U Institutu se postupak za testiranje uzoraka kose na prisutnost stimulansa amfetaminskog tipa provodi više od 12 godina i smatra se pouzdanim načinom potvrde ponavljanog konzumiranja tih stimulansa.

Naši rezultati mogu upućivati na trend zloporabe stimulansa amfetaminskog tipa među mlađom hrvatskom populacijom.

KLJUČNE RIJEČI: GC/MS; kvantitativno određivanje; MDMA; metamfetamin

CORRESPONDING AUTHOR: Irena Brčić Karačonji, PhD Institute for Medical Research and Occupational Health, Ksaverska c. 2, POB 291, HR-10001 Zagreb, Croatia E-mail: ibrcic@imi.hr

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Srebočan E. INCONSISTENCY IN RECENTLY PUBLISHED ARTICLE Arh Hig Rada Toksikol 2014;65:231

Letter to the Editor

Inconsistency in the article “Metal bioaccumulation in common carp and rudd from the Topolnitsa reservoir, Bulgaria” Emil Srebočan Veterinary Faculty, University of Zagreb, Zagreb, Croatia

Reading the article by Yancheva et al. (1) published in the last issue of Archives of Industrial Hygiene and Toxicology, I found an inconsistency. The authors determined the concentration of various metals in the water and two species of fish in a copper mine and metallurgic industry area. Table 2 (1) showed the concentrations in the water and it could be seen that the concentration of lead in all of the study periods was below the detection limit. Table 3 (1) showed the concentrations of metals in fish, making it clear that the concentration of lead in organs was particularly high.

In my opinion, such high metal concentrations in organs are not possible under the described conditions; however, the authors did not comment on this discrepancy at all. An amount of up to 5,500 ppb of lead in the liver of carp could not

CORRESPONDING AUTHOR: Prof Emil Srebočan, PhD, DVM Department of Pharmacology and Toxicology Veterinary Faculty, University of Zagreb Croatia e-mail: emil@vef.hr

accumulate unless the water contained some amount of lead; at least a minimum concentration, which would be 10 ppb (detection limit of the instrument).

Therefore, I think there was an error in the measurement of lead in water, which is why I challenge the authors to discuss and explain this discrepancy in their measurements.

REFERENCES 1. Yancheva V, Stoyanova S, Velcheva I, Petrova S, Georgieva E. Metal bioaccumulation in common carp and rudd from the Topolnitsa reservoir, Bulgaria. Arh Hig Rada Toksikol 2014;65:57-66. doi: 10.2478/10004-1254-65-2014-2451

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Rumboldt Z. WHAT IS PLAGIARISM IN SCIENCE? Arh Hig Rada Toksikol 2014:65:233-236

Viewpoint

Što je to plagijat u znanosti?

Zvonko Rumboldt Medicinski fakultet Sveučilišta u Splitu, Split, Hrvatska

Na pisanje ovih redaka potaknuo me zanimljiv prilog o plagiranju iz pera Nenada Raosa (1). Osim toga, član sam Uredničkog odbora Arhiva, premda ne osobito aktivan, što je bio dodatan poticaj za ovaj osvrt. Naime, iz nadahnutog teksta kolege Raosa moglo bi se možda zaključiti da je plagiranje (koristoljubivo otimanje bitnih dijelova tuđeg djela radi lažnog prikazivanja kao da je vlastito) tek manji prijestup koji bitno ne remeti cjelinu učinkovitosti i vjerodostojnosti znanstvenoistraživačkog rada. O tome sam već pisao u više navrata (2-5), no uvijek nekako usput, pa bi se i prema tim napisima mogao steći dojam da se radi o manje važnom problemu koji se ne tiče toliko akademske zajednice koliko eventualnog zakonskoga gonjenja. Držim da je posrijedi upravo suprotno: riječ je o jednom od najtežih zlodjela u znanosti. Naime, cijeli znanstveni sustav počiva na iskrenosti, povjerenju i na stvaralačkoj sumnji u ispravnost vlastitih i tuđih nalaza. Prijevara je prema tome znanosti strana, a sumnja u vjerodostojnost iznesenih podataka izaziva nesigurnost i odgađa primjenu istinskih otkrića, a provjeravanje objavljenih rezultata pretvara plodonosno verificiranje u sumnjičavo isljedništvo, kojemu znanstvenici baš i nisu vješti. Osim toga, zbog naravi znanstvenog istraživanja, krivotvorine se prije ili kasnije otkriju, pa se varanje radi dugoročnijeg probitka tu mahom ne isplati.

Ljestvica najgorih povreda znanstvene čestitosti koja bi njihove vinovnike morala gotovo trenutno isključiti iz akademske zajednice, mogla bi izgledati ovako: • izmišljanje nepostojećih ili “prilagodba” dobivenih podataka (brojeva, grafikona ili slika; engl. data fabrication) radi obmanjivanja znanstvene javnosti i prijetvornog iznošenja vlastitih “rezultata”.

• iznošenje ili prikazivanje tuđih tekstova ili podataka kao vlastitih (plagiranje, tj. krađa ili otimačina) radi stjecanja lažnog znanstvenog ugleda ili akademskog napredovanja (engl. plagiarism).

• lažno autorstvo, tj. dopisivanje među autore osoba koje nisu suštinski pridonijele ostvarenju određenog djela (engl. guest authoring) radi ostvarivanja intelektualnog, društvenog, a nerijetko i materijalnog koristoljublja. Zrcalna slika ovoga je “skriveno autorstvo” (engl. ghost authoring), gdje se osoba ili tvrtka koja stvarno napiše rad ne navodi, nego se autorstvo pripisuje drugima (npr. skupini zvučnih istraživača koji su proveli promotivno istraživanje za farmaceutsku industriju i prema njenim uputama).

Nedjela iz prve skupine su, uvjetno rečeno, razmjerno najteža, a ona iz treće najblaža. Ipak, jasno razlučivanje, a posebno stupnjevanje navedenih radnji bitno je otežano iz više razloga: Nelagodno je živjeti i djelovati u sredini koja sve više cijeni individualizam i osobni probitak, bez protuteže u jednakoj razini osobne odgovornosti.

Etičke vrednote se relativiziraju i mnogo toga prolazi što bi u zdravom društvu bilo ne samo moralno nego i praktički sankcionirano.

Krivotvorenje, laž i prijevara postaju u nas sve prihvatljiviji oblici ponašanja, čemu svjedoči i niz otkrivenih plagijata istaknutih političara i znanstvenika.

Spomenimo tek nečasne primjere Ante Đapića i Asima Kurjaka (6-8). Akademsko nepoštenje u našoj sredini ukorijenilo se već tijekom studija (9), pa i ranije (10).

Utvrđeno je da je čak 11 % rukopisa ponuđenih jednom našem časopisu plagijat (10), a urednici tog istog časopisa iznijeli su podatak o nekih 29 % članaka sumnjivog autorstva (obično je to dopisivanje lažnih autora) (12)! Ni druge sredine nisu pošteđene kriminalnih radnji, no ondje su takve rabote izuzeci,

234 a u nas, stjecajem nesretnih okolnosti, postaju gotovo pravilo i temelj uspješnosti.

Nečasni “autori” nerijetko rabe sva tri navedena prijestupa. Plagiranje se danas lakše otkriva nizom računalnih programa (npr. eTBLAST, Grammarly, Turnitin ili CrossCheck, kojim se služi i ovaj Arhiv), ali i varalice pronalaze sve prepredenije načine zamagljivanja tragova, tako da je riječ o stalnoj “igri policajaca i lopova”. Prokazati izmišljene podatke još je teže: obično su lukavo sročeni u prividnu vjerodostojnost pa ih otkrivaju tek dobro upoznati “žviždači”, koji na žalost u nas ne mogu očekivati gotovo nikakvu potporu akademske pa ni šire društvene zajednice, a neugodne su im posljedice zbog “izdaje” neizbježne. Slično vrijedi i za “gostujuće autore”, jer se taj postupak krivotvorenja u našoj sredini navodno dobrohotno, a zapravo licemjerno, gura pod tepih i ocjenjuje kao “kolegijalnost”.

Stupnjevanje takvih rabota doista je teško jer prosudbe ne treba uvelike temeljiti na kvantitativnim podacima, nego na kvalitativnom odvagivanju utemeljenom na moralnim počelima, na dobrom ukusu i na osjećaju mjere. Primjerice, kako odrediti u koliko se zasebnih članaka smiju podijeliti rezultati određenog istraživanja a da se ne pretvore u salami science?

Koliko se puta smiju ponoviti određena istraživanja da bi ih se još uvijek moglo zvati verificirajućima, a ne redundantnima? Koji se dio tuđeg teksta smije navesti bez citiranja izvora, a koliki uz navođenje autora? Koja je to granica okolnosti, kakvoće ili količine preko koje se korištenje tuđeg djela treba smatrati plagijatom? Je li moguće sve te nedoumice kodificirati tako da budemo bar pravilni ako već ne možemo biti pravedni? Teško. Razlikuje se doduše bezobrazno plagiranje (engl. blatant plagiarism) od navodno blažeg, složenijeg prepisivanja u obliku kolaža iz više izvora (engl. patchworking). Postoji li među tim prijevarama bitna razlika? Ipak, ulažu se napori za stupnjevanje takvih povreda znanstvene čestitosti. Primjerice, neki razlikuju tri razine plagiranja. Manjim (minor) prekršajem smatra se ako je prepisano do 10 % teksta, 10-25 % umjerenim (moderate), a više od 25 % teškim (major) plagijatom s odgovarajućom razinom sankcija (13-17). Jesmo li spremni provesti u djelo takva ili slična mjerila?

Ipak, imajmo u vidu da je riječ o svjetskom problemu koji je u našem podneblju tek nešto jače izražen. Literatura s tog područja doista je bogata.

Primjerice, baza podataka PubMed navodi 1.193 rada s ključnom riječi plagiarism, a pregršt zanimljivih podataka može se naći na internetu, npr. u Wikipediji.

Rumboldt Z. WHAT IS PLAGIARISM IN SCIENCE? Arh Hig Rada Toksikol 2014:65:233-236

Što bismo mogli poduzeti? Ne baš puno i ne baš lako, ali ipak...

Prije svega bi trebalo osigurati društvenu klimu moralne odgovornosti, koja je sama po sebi razumljiva.

Zato valja stvoriti kritične mase građana, političara i znanstvenika kojima takvi stavovi nisu nametnuti nego su dio njihova svjetonazora i temeljnih vrednota.

Takav odgoj počinje u obitelji, a nastavlja se tijekom školovanja, od dječjeg vrtića do poslijediplomskih studija. Vrlo težak zadatak, zar ne?

Drugo, moramo svjedočiti vlastitim primjerom (engl. role model). To se danas zanemaruje u širokom rasponu od osnovne škole do akademskih zajednica.

Zaboravlja se da školovanje nije samo obrazovni nego i odgojni proces (u čemu nam bolonjski proces baš ne pomaže). Naše bi zauzimanje trebalo ići od svakodnevnih sitnica pristojnog ponašanja (npr. pljuvanje, bahato razbacivanje opušaka, omota ili otpadaka hrane, vođenje pasa na obavljanje nužde u javne parkove, arogantno parkiranje automobila...) do općih moralnih stavova čestitosti, dosljednosti i istinoljubivosti. Doista, učestalost se nečasnih radnji na fakultetima smanjuje ako nastavnici u tom smislu pokazuju jasne i dosljedne stavove (9, 10). Nažalost, naši se sveučilišni profesori prečesto nađu među osvjedočenim plagijatorima i dokazanim trgovcima ispitima...

Treće, valja jasno definirati povrede znanstvene čestitosti te koje će i kakve korake u tom smislu poduzeti uredništva časopisa, a kakve je mjere predvidjela akademska zajednica (8, 13-17). Iako se takvi stavovi ponegdje mogu naći i u pisanom obliku, u pravilu skriveni, vrlo općeniti i deklarativni (5), konkretni su postupci prije iznimka nego pravilo. Četvrto, moramo postati kritičniji prema vlastitoj akademskoj sredini ako joj zaista želimo dobro. To se u prvom redu odnosi na recenziju i uređivanje znanstvenih i stručnih časopisa, ali i drugih publikacija, npr. zbornika ili kvalifikacijskih radova. Ocjenjivački posao recenzenta je zahtjevan (u prvom redu mora utvrditi izvornost i mogućnost provjere podnesenog istraživanja), često neugodan, a društveno gotovo obezvrijeđen, pa ne začuđuje da je u nas većina recenzija površna i neodgovorna, razapeta između dviju krajnosti: udvorničke snishodljivosti i bahata ismijavanja (2-5). Riječ je o prerijetkoj, usto poderanoj mreži, kroz koju plagijati lako prolaze. Ni drugdje nije sjajno, što – uz niz ostalih – ocrtava nedavni primjer. Jedan je novinar sročio formalno uredno napisan rad, ali s

Rumboldt Z. WHAT IS PLAGIARISM IN SCIENCE? Arh Hig Rada Toksikol 2014:65:233-236

apsurdnom metodologijom, potpuno izmišljenim podacima i pod nizom lažnih imena autora, koji je uputio na ocjenu radi objavljivanja uredništvima 304 znanstvena online časopisa, u rasponu od agronomije, biologije i kemije do farmacije i medicine (20). Rukopis je odbilo samo 98 časopisa, njih čak 157 prihvatilo, a preostalih 49 još nije odgovorilo do trenutka objavljivanja te “diverzije” (20). Dakle, najmanje 51,6 % uredništava nesmotreno se i neodgovorno ulovilo u tu stupicu!

Zbunjujuće, zar ne? Koliko dakle smijemo vjerovati netom objavljenom znanstvenom radu?

Peto, u nas se prikrivanje znanstvene prijevare još uvijek smatra kolegijalnošću, a otkrivanje nečasnih radnji izdajom. Nedavno je tako jedan član povjerenstva za izbor u znanstveno-nastavno zvanje uočio da je jedini kandidat (to je isto jedna od naših specifičnosti!) praktički prepisao kvalifikacijski rad drugog kolege, koji je obranjen nekoliko godina ranije. Recenzent je utvrdio da je najmanje 44 % spornog teksta doslovno prepisano, a ostatak djelomično, uz sumnjivu vjerodostojnost iznesenih brojčanih podataka. Nakon niza samo nama sličnih peripetija i promjena nekoliko povjerenstava konačno je zaključeno da je inkriminirani rad “vrlo sličan do identičan”, ali da “pitanje etičnosti treba u svakom slučaju razriješiti” ... netko drugi. Tko? Taj je stav potvrdilo i etičko povjerenstvo našeg ministarstva znanosti. Ni meritum stvari ni sumnju u krivotvorenje iznesenih podataka nije dakako nitko provjeravao. Kandidat je elegantno promaknut u više znanstveno-nastavno zvanje, nakon čega je, osokoljen takvim zaključivanjem utužio recenzenta za klevetu i pravomoćno mu je za “duševne boli” dosuđena odšteta od više desetaka tisuća kuna.

Pogodite: kako će se u sličnim okolnostima ponašati idući recenzent?

Smatram da bismo se ipak, u svakom pojedinom slučaju, trebali držati moralnih počela istinoljubivosti i dosljednosti. Evo primjera mog nedavnog odgovora jednom našem uredništvu glede očitog plagijata (točne sam podatke izostavio jer nisu bitni i uz malo se truda ipak mogu pronaći, a osobno neću biti izvrgnut bijesu krivotvoritelja): “Prema našem dogovoru ... pažljivo sam pregledao kopiju članka A i kopiju članka B i našao da su odista gotovo identični. Tekst drugog članka je praktički u cijelosti preuzet iz prvoga, dijelom tehnikom “fotokopiranja”, a dijelom tehnikom “kolaža” (tj. pojedine su rečenice malo izmiješane uz promjenu reda riječi, par glagola ili pridjeva). Različiti su jedino

235 brojevi ispitanika ...; slika 1 i 2 u drugom članku preuzete su iz sl. 1 u prvom uz promjenu predznaka (preinaka negativiteta u pozitivitet), a nova je i tbl. 1, za koju nije prikazana značajnost opaženih razlika, premda se to u tekstu tvrdi. Sažetak ... je isto tako prepisan, mada u presloženom obliku, a referencije su identične, osim što je u drugom članku izbačena ref. 15 iz prvoga, koja se citira kao nova ref. 6.

Nema dakle sumnje da je riječ o plagijatu (jesu li možda i podaci izmišljeni valja tek provjeravati!). Po mom mišljenju Uredništvo ... treba: Objaviti izjavu o zabrinutosti (ili rabiti neki pogodniji izraz; engl. editorial expression of concern) zbog kasno utvrđenih činjenica.

Uputiti pismo odgovora kolegama (vjerojatno i nepoznatim piscima upozorenja iz NIH, SAD, te prvom autoru oba članka – A i B), a od autora drugog članka (B) tražiti obrazloženje i povlačenje rada.

Publicirati da se članak povlači/briše (engl. editorial note of paper retraction).

Obavijestiti dekana medicinskog fakulteta C, ravnatelje odgovarajućih bolnica te etička povjerenstva, ne samo radi uvida i provjere tog članka, već i cjelokupnog opusa autora B ...”.

Plagiranje je u Republici Hrvatskoj doduše i zakonski sankcionirano Zakonom o autorskom pravu i srodnim pravima (18) te Zakonom o znanstvenoj djelatnosti i visokom obrazovanju (19), ali od toga je mala korist jer se propisi provode neravnomjerno, a primjenjuju selektivno. Da bi se stvorila prijeko potrebna razina društvene svijesti, naša akademska zajednica mora početi ne samo na riječima nego i djelom poštivati i provoditi temeljne zasade autorstva što ih je svojedobno jasno podastro Edward J. Huth, dugogodišnji urednik poznatog časopisa Annals of Internal Medicine (21), koje ovdje iznosim u nešto skraćenom obliku, a bar formalno ih prihvaća većina časopisa: 1. Preuzimanje javne odgovornosti za djelo.

2. Sudjelovanje u planiranju, analizi i pisanju.

3. Svaku od tih aktivnosti valja pripisati bar jednom autoru.

4. Samo prikupljanje podataka nije dovoljno: intelektualna i tehnička pomoć nisu dostatne za autorstvo, no traže zahvalu (nakon odobrenja) na kraju teksta.

Kad svi mi prihvatimo te stavove u provedbenom smislu, navlastito glede autorske odgovornosti, lakše ćemo se otresti plagiranja i drugih oblika obeščašćivanja znanosti te podignuti ugled i etičku razinu akademske zajednice. Prema nedavnim podacima (22), između

236 399 znanstvenih časopisa, prosječnog čimbenika utjecaja (engl. impact factor, IF) od 6,51, samo ih 35,1 % jasno navodi kriterije kršenja znanstvene čestitosti, od kojih je baš plagiranje najčešće (56,1 % svih zloporaba), a tek 30,8 % spominje i povlačenje takva rada iz bibliografskih baza podataka radi obrane znanstvenog integriteta.

LITERATURA 1. Raos N. Što je plagijat u znanosti? Arh Hig Rada Toksikol 2014;65:129-31.

2. Rumboldt Z. Zajednički nazivnik loših recenzija i pseudoznanosti. Medicina 1986;22:19-24 [isti tekst je s dopuštenjem otisnut u Liječničkom vjesniku (1986;108:4059) i u Radiologia iugoslavica (1987;21:195-9)].

3. Rumboldt Z. Ethical dues in biomedical publications. Acta Med Croat 2000;53:203-6. PMID: 11379487 4. Rumboldt Z. Publiciranje i akademsko napredovanje u nas.

Treba li što mijenjati? Liječničke novine 2007;7:60-1.

5. Rumboldt Z. Neke natuknice o etičkim dilemama recenziranja.

Acta Med Croat 2008;62:443-6.

6. Marušić M, Marušić A. Threats to integrity of the Croatian Medical Journal. Croat Med J 2007;48:779-85. doi: 10.3325/ cmj.2007.6.779 7. Katavić V. Znanstveno-istraživačka čestitost u medicini.

Liječničke novine 2007;7:33-7.

8. Godlee F. Plagiarism and punishment. BMJ 2007;335 doi: http://dx.doi.org/10.1136/bmj.39392.602523.47 9. Kukolja Taradi S, Taradi M, Knežević T, Đogaš Z. Stodents come to medical schools prepared to cheat: a multi-campus investigation. J Med Ethics 2010;36:666-70. doi: 10.1136/ jme.2010.035410 10. Kukolja Taradi S, Taradi M, Đogaš Z. Croatian medical students see academic dishonesty as an acceptable behaviour: a cross-sectional multicampus study. J Med Ethics 2012;38:376-9. doi: 10.1136/medethics-2011-100015

CORRESPONDING AUTHOR: professor emeritus Zvonko Rumboldt Medicinski fakultet Sveučilišta u Splitu e-adresa: zr@mefst.hr

Rumboldt Z. WHAT IS PLAGIARISM IN SCIENCE? Arh Hig Rada Toksikol 2014:65:233-236

11. Baždarić K, Bilić-Zulle L, Bruminin G, Petrovečki M. Prevalence of plagiarism in recent submissions to the Croatian Medical Journal. Sci Eng Ethics 2012;18:223-39. doi: 10.1007/s11948-011-9347-2 12. Marušić A, Marušić M. Can small journals provide leadership? Lancet 2012;379:1361-3. doi: 10.1016/S01406736(11)61508-0 13. Kjeldsen SE, Narkiewicz K, Cifkova R, Mancia G. ESH statement on detection and punishment of abstract fraud and poster plagiarism. J Hypertension 2006;24:203-4. PMID: 16331121 14. Rosselot Jaramillo E, Bravo Lechat M, Kottow Lang M, Valenzuela Yuraidini C, O’Ryan Gallardo M, Thambo Becker S, Horwitz Campos N, Acevedo Pérez I, Rueda Castro L, Sotomayor MA; Comisión de Etica, Facultad de Medicina, Universidad de Chile. Santiago de Chile. [Plagiarism.

Document from the Ethics Commission of the Medical School, University of Chile, in Spanish]. Rev Med Chil 2008;136:653-8. doi: /S0034-98872008000500016 15. Redman BK, Merz JF. Scientific misconduct: do the punishments fit the crime? Science 2008;321:775. doi: 10.1126/science.1158052 16. Sanctions for authors of plagiarized Surgery for Obesity and Related Diseases article. Surg Obes Relat Dis 2011;7:125-9. doi: 10.1016/j.soard.2010.12.002 17. Ross S, Magee L, Walker M, Wood S. Protecting intellectual property associated with Canadian academic clinical trials - approaches and impact. Trials 2012;13:234. doi: 10.1186/1745-6215-13-243 18. Zakon o znanstvenoj djelatnosti i visokom obrazovanju.

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22. Bosch X. Improving biomedical journals’ ethical policies: the case of research misconduct. J Med Ethics 2014 doi: 10.1136/medethics-2013-101822.

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Note

Razvoj Zavoda za animalnu fiziologiju Prirodoslovnomatematičkog fakulteta Sveučilišta u Zagrebu - znanost i nastava u prošlosti i sadašnjosti Nada Oršolić Zavod za animalnu fiziologiju, Prirodoslovno-matematički fakultet, Zagreb, Hrvatska

Zavod za animalnu fiziologiju Prirodoslovnomatematičkog fakulteta Sveučilišta u Zagrebu obilježio je 2013. godine 50 godina svog postojanja i rada. Namjera je ovog prikaza dati kratki pregled povijesti i sadašnjosti Zavoda, s naglaskom na njegovu znanstvenu, nastavnu i stručnu djelatnost. Za pripremu ovog prikaza djelomično su korišteni materijali sabrani prigodom izrade monografije o Zavodu (1).

Povijest Zavoda za animalnu fiziologiju Zavod za animalnu fiziologiju (osnovan 1963.) jedan je od novijih zavoda Prirodoslovno-matematičkog fakulteta (PMF) Sveučilišta u Zagrebu. Do osnutka PMF-a (1946.) nastava iz prirodoslovlja održavala se u sklopu Mudroslovnog fakulteta, jednog od triju fakulteta utemeljenih odlukom Hrvatskoga sabora o osnutku Sveučilišta 1874. godine.

Međutim, nastava iz biologije, ponajprije iz botanike, postupno se počela održavati tek od 1876. godine. Osobite zasluge u počecima održavanja sveučilišne nastave iz fiziologije imao je profesor Spiridion Brusina. On je u akademskoj godini 1877./1878. držao predavanja iz kolegija Opća zoologija te Morfologija i opća fiziologija, koja su sadržavala gradivo iz fiziologije životinja. Sadržaje iz područja fiziologije u studij Biologije uveo je i Daniel Riessner, doktor medicine i doktor prirodoslovlja, učitelj službene medicine i anatomije čovjeka, također naslovni sveučilišni profesor Pravoslovnog i državoslovnog fakulteta Sveučilišta u Zagrebu. Sve do akademske godine 1939./1840. predavao je nekoliko kolegija: Fiziološka anatomija živčevlja i osjetila čovječjih, Fiziološka anatomija droblja čovjekova, Fiziološka anatomija asimilatornih i rasplodnih ustroja čovječjih, Fiziološka anatomija osjetnih ustroja čovječjih, Anatomija čovjeka (na fiziološkoj osnovi) i Fiziološka anatomija čovjeka. Godine 1927. na Filozofskom fakultetu zaposlio se Krunoslav Babić, koji u svojstvu profesora iz zoologije i predstojnika Zoološko-zootomskog zavoda u akademskoj godini

1928./1929. započinje predavanja iz Opće zoologije i fiziologije.

Osnutkom Prirodoslovno-matematičkog fakulteta (1946.) na Sveučilištu u Zagrebu nastavlja se tradicija održavanja nastave iz fiziologije životinja i čovjeka. U početku su nastavu držali honorarni sveučilišni profesori s drugih fakulteta. Primjerice, Petar Đurić, redoviti profesor Veterinarskog fakulteta, u akademskoj godini 1948./1949. drži predavanja i vježbe iz Fiziologije životinja. Od akademske godine 1950./1951. u vježbama mu pomaže asistent Srećko Vatovec, koji kao docent u akademskoj godini 1955./1956. drži predavanja iz Fiziologije životinja studentima treće godine studija Biologije. U akademskoj godini 1957./1958. nastavu iz kolegija Fiziologija životinja i Praktični rad iz fiziologije životinja vodio je Ivan Ehrlich, redoviti profesor Veterinarskog fakulteta. Za prve asistente iz fiziologije životinja izabrane su Nada Bjelić i Nada Kondić. Od akademske godine 1958./1959. nastavu iz Fiziologije životinja s praktikumom za studente treće godine studija Biologije preuzima dr. Nikša Alegretti, tada docent Medicinskog fakulteta Sveučilišta u Zagrebu. Do 1963. predavao je kolegije: Temelji fiziologije životinja i čovjeka, Izborna poglavlja iz opće fiziologije životinja i Fiziologiju izmjene tvari. Od akademske godine 1961./1962. u studij Biologije uvodi se predmet Anatomija i fiziologija čovjeka, koji je predavao dr. Vitomir Kos, tada asistent u Zavodu za rendgenologiju i radium terapiju Kliničkoga bolničkoga centra Rebro u Zagrebu.

Uskoro je postalo razvidno da zbog povećanih znanstveno-nastavnih potreba Prirodoslovno-matematički fakultet mora osigurati vlastiti kadar iz područja animalne fiziologije. Stoga se 1963. na Biološkom odjelu (danas odsjeku) osniva novi zavod: Zavod za fiziologiju životinja.

Kao njegov prvi predstojnik izabran je liječnik, profesor Borislav Nakić, fiziolog i imunolog, koji je tu dužnost obnašao do svoje iznenadne smrti 1970. godine. Na početku 1964. Zavod mijenja ime u Zavod za animalnu fiziologiju.

Zbog nedostatka prostora, nastava je isprva bila organizirana

238 na Medicinskom fakultetu. Ubrzo se pristupilo uređenju unajmljenog prostora Muzeja za umjetnost i obrt na Rooseveltovu trgu 6, u kojemu su organizirana redovita predavanja i praktikumi za studente biologije. Profesor Nakić okuplja mlade suradnike – Nadu Kondić-Mitin, Andriju Kaštelana i Oskara Springera – te započinje razvijati fiziologiju kao zasebnu struku u okrilju prirodoslovlja. Time se stvaraju uvjeti za sustavni znanstveni i nastavni rad iz fiziologije, imunologije, transplantacijske imunologije, radiobiologije, imunogenetike, ekofiziologije i ekotoksikologije. Početci Zavoda bili su ispunjeni brojnim poteškoćama jer je trebalo uspostaviti sasvim novi sustav, organizirati praktične vježbe iz fiziologije životinja te organizirati uzgajalište pokusnih životinja (visokosrodni sojevi miševa i štakora), koje su prijeko potrebne za znanstveni i nastavni rad iz imunologije, transplantacijske biologije i fiziologije.

Već 1965. godine na PMF-u u Zagrebu započinje redovita nastava iz predmeta Imunologija za studente biologije, koji je predavao prof. dr. sc. Drago Ikić. Takav dodiplomski kolegij tek je kasnije uveden na studije medicine i veterine. Napredak znanstvenih istraživanja u Zavodu pratilo je i uvođenje novih predmeta u visokoškolsku nastavu. Tako se 1967. u poslijediplomski studij Eksperimentalne biologije uvodi predmet Transplantacijska biologija za studente biologije i medicine. U to vrijeme u Zavodu se zapošljavaju novi suradnici: Dušan Volf, Miroslav Hauptfeld i Jozsef Mikuška. Godine 1968. dr. sc.

Borislav Nakić izabran je za redovitog profesora i dekana Prirodoslovno-matematičkog fakulteta Sveučilišta u Zagrebu. Iste godine organizirao je Sveučilišni odjel za transplantacijsku imunologiju, u kojem je kao predstojnik okupio hrvatske imunologe na makroprojektu „Imunobiologija“. Zbog značajnog znanstvenog doprinosa svjetskoj znanosti profesor Nakić postaje članom uglednih znanstvenih društava u inozemstvu, gdje je promicao tzv.

Zagrebačku imunološku školu. Uspješan put osnivača Zavoda za animalnu fiziologiju prekinula je iznenadna smrt 1970. godine.

Od 1970. do 1986. predstojničku dužnost obnašao je dr. sc. Andrija Kaštelan. U tom su se razdoblju u Zavodu zaposlili asistenti Ivan Bašić, Dušan Volf, Ljerka Brkljačić, Vesna Kerhin Brkljačić, Zvezdana Vuk Pavlović, Vera Dolejškova i Dunja Košuta. U sklopu Zavoda nastavljen je intenzivan nastavni i znanstveni rad u području fiziologije, imunologije, transplatacijske imunologije, radiobiologije, neurofiziologije i endokrinologije.

Rad u području transplantacijske biologije, imunogenetike i kliničke transplantacije organa rezultirao je osnivanjem Centara za tipizaciju tkiva, najprije u sklopu Zavoda, a kasnije Centra za tipizaciju tkiva u KBC-u Rebro.

Godine 1986. u taj novoosnovani centar iz Zavoda odlaze prof. dr. sc. Andrija Kaštelan, dr. sc. Ljerka Brkljačić i dr. sc. Vesna Kerhin Brkljačić. Predstojnikom Zavoda postaje prof. dr. sc. Ivan Bašić, koji tu dužnost obnaša u nekoliko navrata sve do 2007. godine. U razdoblju 1988./1989. predstojništvo preuzimaju prof. dr. sc. Dušan Volf i prof. dr. sc. Dunja Košuta. Znanstveni i nastavni rad Zavoda u tom razdoblju uključuje istraživanja fiziologije imunohematopoetskog sustava, funkcioniranja endokrinog sustava s naglaskom na hormone timusa, biologije tumora i

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metastaza, ekotoksikologije te veterinarske imunologije i genetike. U tome razdoblju u Zavodu su radili asistenti Ljubomir Škrinjar, Miroslav Rožić, Nikica Zaninović, Matija Franković, Roland Jurečić, Danko Dobec, Nada Oršolić i Zoran Tadić.

Na početku 1990-ih započinju radovi na uređenju novog prostora u kojemu se Zavod i danas nalazi. Preseljenje iz prostora Muzeja za umjetnost i obrt provedeno je tijekom 1991. godine. Nekoliko godina poslije u Zavodu se zapošljavaju novi asistenti i stručni suradnici: Anica Horvat, Haidi Arbanasić, Domagoj Vučić, Vesna Lacković, Ana Gomerčić, Domagoj Đikić, Ivna Tomašković i Duje Lisičić, a 2006. dr. sc. Dubravka Hranilović, koja je do tada radila u Laboratoriju za neurokemiju i molekularnu neurobiologiju Zavoda za molekularnu biologiju Instituta Ruđer Bošković.

Umirovljenjem prof. dr. sc. Ivana Bašića za predstojnicu Zavoda izabrana je prof. dr. sc. Nada Oršolić, koja vodi Zavod od 2007. do akademske godine 2011./2012. U tom razdoblju u Zavodu su zaposlena još dva znanstvena

Slika 1. Suradnici Zavoda za animalnu fiziologiju 1967. godine (stoje slijeva): Miroslav Hauptfeld, Borislav Nakić, Nada Kondić-Mitin, Ivanka Rumora, Josipa Posenjak, Josipa Zake, Vladimir Bedeković; (sjede slijeva): Vinka Zaninović, Ilona Šmit, Matilda Derikrava, Oskar Springer. Preuzeto iz (1)

novaka: Pavle Josipović i Sofija Blažević te predavačica dr. sc. Julija Erhardt.

U akademskoj godini 2012./2013. ulogu obnašateljice dužnosti predstojnice Zavoda, a zatim i predstojnice preuzima prof. dr. sc. Vesna Benković.

Povijesni prikaz znanstvene djelatnosti U doba osnivanja Zavoda za animalnu fiziologiju Zagreb je bio jako središte imunoloških istraživanja, čiji su nositelji bili dr. sc. Nikša Allegretti (Medicinski fakultet), dr. sc. Veljko Stanković, dr. sc. Vlatko Silobrčić (Institut Ruđer Bošković) i dr. sc. Borislav Nakić (Prirodoslovnomatematički fakultet).

Profesor Nakić bio je član Vijeća Međunarodne udruge imunoloških društava, organizirao je Društvo imunologa Jugoslavije i Hrvatsko društvo imunologa (1968). U tom je razdoblju uspostavljena suradnja Zavoda za animalnu fiziologiju s mnogim vrsnim svjetskim znanstvenicima, primjerice s Dirkom W. van Bekkumom, Leonom O. Jacobsonom, Takashijem Makinodanom, Janine E. Voisin, Nielsom K. Jerneom.

Oršolić N. POVIJEST ZAVODA ZA ANIMALNU FIZIOLOGIJU Arh Hig Rada Toksikol 2014;65:237-249

Šezdesetih godina prošlog stoljeća intenzivno su proučavane osnove transplantacijske imunologije i reakcije odbacivanja presatka od nesrodnog primatelja. Zahvaljujući profesoru Nakiću i suradnicima hrvatska su znanstvena istraživanja u tom području bila na razini svjetskih dostignuća. Nova su saznanja rezultirala pokretanjem inicijative za osnivanjem Laboratorija za tipizaciju tkiva u sklopu Zavoda za animalnu fiziologiju. U tom se razdoblju provode istraživanja transplantacije hematopoetskih tkiva, ali i drugih tkiva i organa, primjerice transplantacije gušterače u svrhu liječenja šećerne bolesti, transplantacije zubnih zametaka i mnoga druga. Profesor Nakić također je istraživao mogućnosti parabiotskog spajanja dvaju štakora kako bi hormon inzulin mogao prelaziti iz jedinke s gušteračom u jedinku bez gušterače. To je, međutim, rezultiralo smrću jednog od štakora uslijed imunološke reakcije. U rješavanju patogeneze parabiotske bolesti i njenih imunoloških mehanizama uključio se i tadašnji student Vlatko Silobrčić. Godine 1968. prof. dr. sc. Borislav Nakić imenovan je redovitim profesorom na Prirodoslovnomatematičkom fakultetu Sveučilišta u Zagrebu. Godinu dana poslije dobio je republičko priznanje – nagradu za znanstvena dostignuća „Ruđer Bošković“. Njegov znanstveni doprinos svjetskoj znanosti omogućio mu je da postane članom Londonskoga kraljevskoga društva za medicinu (engl. Royal Society of Medicine), Društva za transplantaciju (engl. Transplantation Society) i Europskoga društva radiobiologa. Znanstveni opus profesora Nakića obuhvaća 31 znanstveni rad; svi ti radovi prepoznati su kakvoćom ideja i preciznošću, a treba napomenuti da ih znanstvenici diljem svijeta citiraju još i danas (2-10).

Nakon smrti prof. dr. sc. Borislava Nakića istraživanja u području presađivanja tkiva nastavio je prof. dr. sc.

Andrija Kaštelan. Posebice se bavio utvrđivanjem tkivne podudarnosti (kompatibilnosti) davatelja i primatelja u transplantaciji bubrega. Nakon prelaska u Laboratorij za tipizaciju KBC-a Zagreb, prof. dr. sc. Kaštelan započeo je

Slika 2. Djelatnici Zavoda za animalnu fiziologiju 2013. (stoje slijeva): Sofia Blažević, Julija Erhardt, Ružica Duh, Gordana Žakman, Sanja Škalec, Haidi Arbanasić, Hrvoje Jedvaj, Anica Horvat Knežević, Marija Potočić, Ana Bolfek, Valentina Greguraš, Jure Vulić, Ksenija Fučkar Reichel; (sjede slijeva): Zoran Tadić, Ana Galov, Dubravka Hranilović, Vesna Benković, Nada Oršolić i Domagoj Đikić. Preuzeto iz (1)

239 tipizaciju DR lokusa te svaranje uvjeta za još bolje praćenje kompatibilnosti u HLA sustavu između davatelja i primatelja organa (6-9). Njegov znanstvenoistraživački rad iznimno je značajan za razvoj transplantacijske medicine u Hrvatskoj, osobito transplantacije bubrega kao metode liječenja. Svojim je djelovanjem pridonio pripremanju prvog zakona koji je regulirao pitanje transplantacije sa živog i umrlog davatelja. Hrvatska je tada imala vodeću ulogu u donošenju zakonskih akata na polju transplantacije, a svoje su zakone u skladu s hrvatskim modelom donosile i ostale republike. Pitanje davalaštva s umrle osobe temeljilo se na informiranom pristanku. Tih se godina započelo s korištenjem organa od osoba kojima je utvrđena moždana smrt, što je pridonijelo poboljšanju rezultata transplantacije.

Osim što je bio utemeljitelj i voditelj Zavoda (Centra) za tipizaciju tkiva i Klinike za urologiju u KBC-u Rebro, profesor Kaštelan bio je i voditelj Republičkoga referalnog centra za transplantaciju i tipizaciju tkiva te Hrvatskoga registra i banke dobrovoljnih darovatelja koštane srži.

Nadalje, sudjelovao je u izvođenju nastave iz Fiziologije i Imunologije na Medicinskom fakultetu Sveučilišta u Zagrebu te bio voditeljem brojnih doktorskih i magistarskih radova temeljenih na problemima vezanima uz područje transplantacijske imunologije.

Imunološka istraživanja u Zavodu za animalnu fiziologiju s vremenom se proširuju na temeljna istraživanja mehanizama imunohematopoeze i imunomodulacije.

Kasnih sedamdesetih, tijekom osamdesetih i devedesetih godina prošlog stoljeća u laboratoriju prof. dr. sc. Oskara Springera provode se istraživanja endokrinih i staničnih molekularnih signala, usmjerena na otkrivanje djelovanja hormona timusa na hematopetski i imunosni sustav i na imunološki status nakon neonatalne timektomije. Profesor Springer i suradnici u Zavodu započeli su i s potpuno novim područjem istraživanja iz ekotoksikologije. Cilj tih istraživanja bio je utvrditi djelovanje toksikanata iz okoliša, poput rezidualnih pesticida u hrani i vodi, na promjene imunohematopoetskog sustava životinja i ljudi u skladu s tadašnjim znanstvenim trendovima usmjerenima na očuvanje okoliša, ekologiju, zagađenje i utjecaj ksenobiotika na ljudsko zdravlje (8-11). Osim znanstvenog i nastavnog rada, profesor Springer niz je godina bio glavni i odgovorni urednik časopisa Priroda, mjesečnika za popularizaciju prirodnih znanosti i ekologije. Napisao je i veliki broj školskih udžbenika.

Znanstvena djelatnost prof. dr. sc. Ivana Bašića započinje ranih sedamdesetih godina prošloga stoljeća sudjelovanjem u projektima koji su istraživali hematoimunopoiezu i imunomodulaciju biološkog odgovora domaćina tumora i njenu korisnost u kontroli tumorskoga rasta. Rezultati tih istraživanja objavljeni su u prestižnim časopisima (12-15). Početna istraživanja profesora Bašića temelje se na uporabi citostatika u sprječavanju akutnog sindroma GVH (engl. graft versus host), reakcije nakon transplantacije stanica koštane moždine, a potom je proučavao utjecaj tumora na hematopoiezu miša. Zatim su uslijedila istraživanja u području biologije tumora i metastaza te uporabe imunomodulacijskih, kemoterapijskih i radiosenzibilizacijskih sredstava u hematopoiezi, te kontroli tumorskog rasta i metastaziranja tumora (16-19). Bavio se i istraživanjima na području antimutageneze i uporabe

240 radioprotektora (20, 21). U području veterinarske imunologije sa skupinom istraživača s Veterinarskog fakulteta u Zagrebu istraživao je imunološke aspekte virusne bolesti infekciozne anemije konja i njene homologizacije sa AIDS-om (22, 23) te imunosti tek oprašene prasadi (24, 25). Potom središte svoje znanstvene djelatnosti usmjeruje na istraživanje genetike svinja i utjecaja RYR1 gena na malignu hipertermiju u svinja (26, 27) te istraživanje genoma hrvatskoga hladnokrvnjaka, hrvatske autohtone pasmine konja, metodom mikrosatelita (28) kao i na određivanje očinstva u goveda uporabom mikrosatelitskih lokusa (29). Njegova znanstvena djelatnost vezana za tumore i metastaze vidljiva je i u poglavljima udžbenika i knjiga: u knjizi Klinička onkologija (30) nositelj je poglavlja Biologija i metastaziranje tumora, a u knjizi Veterinarska onkologija (31) nositelj je poglavlja Tumor i metastaza; imunobiološka razmatranja i bioterapija. Uredio je i knjigu Pokusni modeli u biomedicini (32). U drugoj polovici devedesetih godina prošlog stoljeća pa do umirovljenja u suradnji s prof. dr. sc. Nadom Oršolić provodi istraživački rad na kemoprevenciji zloćudnih tumora i metastaza u pokusnih životinja uporabom pčelinjih proizvoda. U tom području sa svojom istraživačkom skupinom postiže znakovite znanstvene rezultate iz kojih je proistekao veliki broj znanstvenih i stručnih radova.

Prof. dr. sc. Dušan Volf bavio se istraživanjima fiziologije imuno-hematopoetskog sustava, proučavanjem kimerizma u radijacijskih kimera i genetički imunodeficijentnih sojeva miševa. Proučavanjem regeneracije hematopoietskih stanica u radijacijskih kimera, utjecaja zračenja na hematopoetski sustav i uporabe radioprotektora, profesor Volf proširuje istraživanja Zavoda na područje radiobiologije. Svoj je interes posebice usmjerio na ulogu timusa u diferencijaciji matičnih stanica koštane srži (33-35) te na ontogenezu limfocita B, što je detaljno istraženo u okviru magistarskoga rada Nade Oršolić Karakteristike progenitora B-limfocita induktora kimerizma u miševa CBA/N. Tim istraživanjima utvrđeno je da Peyerove ploče i periferni limfni čvorovi u miša imaju zasebne ontogenetske puteve te da se diferencijacijsko račvanje organspecifičnih usmjerenih prekursorskih populacija odvija još na razini koštane srži. Nadalje, treba istaknuti i njegov znanstveni doprinos u istraživanju dugotrajnih kultura koštane srži za različite stanične linije, praćenje proliferacijske kinetike hematopoetskih stanica frakcioniranih na perkolu te načinima dobivanja obogaćene populacije matičnih stanica i mogućnosti regeneracije hematopoietskih stanica u radijacijskih kimera (36-40).

Prof. dr. sc. Dunja Košuta svoju je znanstvenu djelatnost započela na istraživanju kimerizma u CBA/N životinja (36, 37), a nastavila praćenjem staničnih biljega stimuliranih limfocita u ljudi i životinja pomoću monoklonalnih protutijela. Bavila se istraživanjem imunomodulacijskog učinka lokalnog ozračenja primarnog tumora i pripravka s aktivnošću IL-2 na broj spontanih metastaza u plućima štakora soja Y59, proučavala primjenu peptidoglikanskog polimera i monomera na staničnost limfatičkih organa te njihov odgovor na nespecifične mitogenike fitohemaglutinin i konkanavalin (41, 42).

Znanstvena djelatnost članova Zavoda – danas Današnja se znanstvena djelatnost u sklopu Zavoda može svrstati u nekoliko različitih područja istraživanja:

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- kronične bolesti prouzročene oksidativnim stresom, posebice različiti oblici protutumorskog istraživanja: imunologija i imunomodulacija, kemoprevencija, genotoksičnost, zaštitni učinak od zračenja i kemoterapije, hipertermalna intraperitonealna kemoterapija, tumorski modeli – in vivo i in vitro stanične kulture, križna otpornost tumorskih stanica na lijekove (engl. multi drug resistance, MDR); dijabetes kao bolest oksidativnog stesa, flavonoidi kao fitoestrogeni u prevenciji i terapiji osteoporoze, upalne bolesti kože (voditelj istraživanja: prof. dr. sc. Nada Oršolić; suradnici: prof. dr. sc. Vesna Benković, prof. dr. sc.

Domagoj Đikić, dr. sc. Anica Horvat Knežević) - fiziološki mehanizmi toksičnosti ksenobiotika (voditelji istraživanja: prof. dr. sc. Domagoj Đikić i prof. dr. sc. Vesna Benković; suradnici: prof. dr. sc. Nada Oršolić, dr. sc. Anica Horvat Knežević) - kognicija i percepcija u zmija (voditelj istraživanja: doc. dr. sc. Zoran Tadić; suradnik dr. sc. Duje Lisičić) - neurobiološka osnova poremećaja u ponašanju, prije svega uloga serotoninskog sustava (voditelj istraživanja: prof. dr. sc. Dubravka Hranilović; suradnik dr. sc. Sofija Blažević) - konzervacijska genetika ugroženih vrsta u Hrvatskoj (voditelj istraživanja: doc. dr. sc. Ana Galov i dr. sc. Ivna Kocijan do 2012., suradnik dr. sc. Haidi Arbanasić) Sva spomenuta područja istraživanja znanstvene aktivnosti temelje se na sljedećim znanstvenim projektima: “Kemoprevencija tumora polifenolnim sastavnicama” (engl. Chemoprevention of tumor growth by polyphenolic compounds); projekt je financiralo Ministarstvo znanosti obrazovanja i športa Republike Hrvatske u razdoblju 2007.

– 2013.; nositelj projekta prof. dr. sc. Nada Oršolić. „Imunoadjuvatno i zaštitno djelovanje propolisa u životinja“ (engl. Immunadjuvant and protective effects of propolis in animals); projekt je financiralo Ministarstvo znanosti obrazovanja i športa Republike Hrvatske u razdoblju 2007. – 2013.; nositelj projekta prof. dr. sc. Ivan Bašić, a nakon njegove smrti 2009. nositelj je prof. dr. sc.

Vesna Benković. „Prirodni antioksidansi i kemoterapija“ (engl. Natural antioxidans and chemotherapy); projekt financira Federalno ministarstvo obrazovanja i nauke Republike Bosne i Hercegovine; nositelji projekta prof. dr. sc. Nada Oršolić i doc. dr. Milenko Bevanda. „Kognicija i percepcija u zmija“ (engl. Cognition and Perception in Snakes); projekt je financiralo Ministarstvo znanosti obrazovanja i športa Republike Hrvatske i u razdoblju 2007. – 2013. i Department of Organismic and Evolutionary Biology, Harvard University (SAD); nositelji projekta doc. dr. sc. Zoran Tadić i dr. Anthony Herrel. „Neurobiološka podloga autizma: uloga serotoninskog sustava“ (engl. Neurobiological basis of autism: the role of the serotonin system); projekt je financiralo Ministarstvo znanosti obrazovanja i športa Republike Hrvatske u razdoblju 2007. – 2013.; nositelj projekta prof. dr. sc.

Dubravka Hranilović. „Uloga lipidnih splavi i glikokonjugata u razvoju i regeneraciji živčanih stanica“ (br. 219-0061194-2158); voditelj projekta prof. dr. sc. Marija Heffer, od 2005. do danas; suradnik na projektu prof. dr. sc. Domagoj Đikić.

Oršolić N. POVIJEST ZAVODA ZA ANIMALNU FIZIOLOGIJU Arh Hig Rada Toksikol 2014;65:237-249

„Epigenetika serotoninske signalizacije: istraživanja metilacije gena za serotoninski prijenosnik, monoamin oksidazu B i serotoninski receptor 2A“; bilateralni hrvatskonjemački projekt, voditelji J. Štefulj i P. Zill, od 2013. – 2014.; suradnik na projektu prof. dr. sc. Dubravka Hranilović.

Neuroimaging, neurogenomics and pharmacogenomics of the frontal lobe connectivity: normal development and abnormalities in developmental cognitive disorders; voditelji I. Kostović i P. Rakić, UKF projekt, od 2007. do 2010.; suradnik na projektu prof. dr. sc. Dubravka Hranilović. „In vivo istraživanje niskoaromatskog visokosteranskog derivata naftalana (NAVS)“; istraživanje je provedeno za tvrtku Medicopharmacia (Ljubljana), u razdoblju 2010.

– 2011.; nositelj istraživanja prof. dr. sc. Nada Oršolić.

“Zaštita podvrsta riđovke Vipera berus berus i Vipera berus bosniensis“; projekt Državnog zavoda za zaštitu prirode, od 2011. do 2013.; suradnik na projektu dr. sc. Duje Lisičić. „Istraživanje i zaštita gušterica Algiroides nigropunctatus, Podarcis sicula i Zootoca vivipara“; projekt herpetološke udruge Hyla i Državnog zavoda za zaštitu prirode, od 2010. do 2011.; suradnik na projektu dr. sc. Duje Lisičić. „Monitoring ptičje gripe u Hrvatskoj“; projekt Hrvatskoga veterinarskog instituta, od 2008. do 2012.; suradnik na projektu dr. sc. Duje Lisičić. „Monitoring kosca Crex crex u Hrvatskoj“; projekt Hrvatske akademije znanosti i umjetnosti, od 2006. do danas; suradnik na projektu dr. sc. Duje Lisičić. „Zaštita dugonogog šišmiša (Myotis capaccini) i zaštita krškog staništa u Hrvatskoj“; projekt Hrvatskoga biospeološkog društva, Državnog zavoda za zaštitu prirode, Fonda za zaštitu okoliša i energetsku učinkovitost; od 2007. do 2008.; suradnik na projektu dr. sc. Duje Lisičić. „Ekologija šišmiša u špilji Veternici“: projekt Hrvatskoga biospeološkog društva, Državnog zavoda za zaštitu prirode, Fonda za zaštitu okoliša i energetsku učinkovitost, od 2003. do 2005.; suradnik na projektu dr. sc. Duje Lisičić. „Biologija dinarskog voluhara (Dinaromys bogdanovi) u Hrvatskoj“; projekt prijavljen preko Udruge za biološka istraživanja – BIOM u suradnji sa Zoološkim vrtom u Zagrebu i Fondom za zaštitu okoliša i energetsku učinkovitost, , od 2011. do danas; nositelj projekta dr. sc.

Duje Lisičić.

Veliki dio istraživanja Zavoda temelji se na proučavanju polifenolnih aktivnih sastavnica u biljnoj prehrani i tradicionalnoj medicini koje se rabe u liječenju bolesti uzrokovanih oksidativnim stresom i upalom. Posebno su istražena njihova brojna biološka svojstva, uključujući antioksidativna, imunomodulatorna, protutumorska, protumetastatska protuleukemijska, radioprotektivna, genotoksična, antialergijska te učinak na dijabetes i osteoporozu. U nizu objavljenih radova (43-56) ispitali smo učinke vodene i alkoholne otopine propolisa (hrvatskoga i brazilskoga podrijetla) te njihovih polifenolnih sastavnica (kafeinska kiselina, fenetil kafeat, kvercetin, naringin, naringenin, krizin, kurkumin, krizin), iscrpka zelenog čaja – epigalokatehin galata, taninske i galne kiseline,

241 proantocijanida, ikarina. Voditelj tih istraživanja je prof. dr. sc. Nada Oršolić, koja je s istraživanjem pčelinjih proizvoda započela još tijekom izrade svojega doktorskoga rada te ga nastavila vođenjem brojnih doktorskih, magistarskih i diplomskih radova. Danas sa svojim suradnicima u sklopu Zavoda (prof. dr. sc. V. Benković, dr. sc. A. Horvat Knežević, prof. dr. sc. D. Đikić, dr. sc. D. Lisičić), ali i sa suradnicima izvan Zavoda, vodi projekte temeljene na toj problematici: „Kemoprevencija rasta tumora polifenolnim sastavnicama“, ”Prirodni antioksidansi i kemoterapija“.

Temeljni su ciljevi tih projekata istražiti: a) učinak pčelinjih proizvoda te polifenolnih sastavnica na tumorski rast i metastatsku sposobnost in vivo i protuleukemijsku aktivnost in vitro; b) mehanizme učinkovitosti pčelinjih proizvoda, posebice njihovih polifenolnih/flavonoidnih sastavnica u imunomodulaciji, genotoksičnosti te smrti tumorskih stanica; c) učinak istraživanih pripravaka samih i/ili združenih s kemoterapeuticima; d) ekspresiju P-glikoproteina, glutationa i enzima pridruženih glutationu u staničnim linijama osjetljivima i otpornima na citostatik; e) moguću ulogu P-glikoproteina u međureakciji polifenola i citostatika.

Ta su istraživanja pokazala mogućnosti primjene propolisa i njegovih flavonoidnih sastavnica u svrhu postizanja maksimalne citotoksičnosti na tumorske stanice, smanjenja doze citostatika i cijene koštanja tumorske terapije, smanjenja popratnih toksičnih učinaka citostatika na normalne stanice te poboljšanja imunosnog i hematološkog statusa organizma (43, 50, 57-59). Dobiven je uvid o mogućim mehanizmima istraženih sastavnica, posebice o aktivnosti makrofaga, proizvodnji interleukina (IL-1), NO, citotoksičnosti na tumorske stanice, odnosu i regulaciju T i B stanica te njihov učinak na apoptozu, nekrozu i redoks stanje staničnih linija MCa, HeLa V79 i A549 te na protuleukemijsku aktivnost na staničnim linijama MOLT, JURKAT, HL-60, RAJI, U937. U radovima je dobro definirana i istaknuta preventivna primjena vodene otopine propolisa i polifenolnih sastavnica na rast tumora i metastaza, dan je kritički osvrt na doze te raspravljena mogućnost njihove terapijske primjene s citostaticima.

Pokazan je izravni učinak propolisa i njegovih polifenolnih sastavnica na rast tumora u uvjetima in vivo i in vitro te neizravni mehanizam preko aktivacije makrofaga i proizvodnje različitih čimbenika i citokina. Neki od njih mogu djelovati izravno u doticaju s tumorskim stanicama narušavajući cjelovitost stanica ili stvarajući negativni okoliš za iste, primjerice za TNF, H2O2 i NO ili neizravno lučenje IL-1 i IFN-γ kroz aktivaciju drugih stanica te povećanje učinkovitosti i oboružanosti istih u ubijanju tumorskih stanica (60-71). Dio istraživanja odnosi se na protutumorski i imunomodulacijski učinak meda (71, 72), peluda i matične mliječi (54, 55, 73).

Nadalje, s obzirom na to da mnogi tipovi tumorskih stanica u glodavaca i ljudi iskazuju križnu otpornost na lijekove, što je jedan od najvećih ograničavajućih čimbenika uspješnog liječenja mnogih tumorskih bolesti, provedena su istraživanja mogućega združenog učinka flavonoida, kemoterapeutika i hipertermije. Zloćudne su stanice

242 osjetljivije na hipertermiju od normalnih stanica zbog smanjenog odvođenja topline, slabije opskrbe hranjivim tvarima, hipoksije i acidoze. Hipertermija povećava topljivost kemoterapeutika te njihovu reaktivnost s molekulom DNA, što posljedično dovodi do brojnijih jednolančanih i dvolančanih lomova, stvaranja poprečnih veza unutar i između lanaca DNA te pojačane apoptoze tumorskih stanica. Nadalje, hipertermija uzrokuje strukturne i funkcionalne promjene stanica. Oštećenje stanične membrane i porast pasivnog transporta pridonose povećanju količine kemoterapeutika u stanici. Hipertermija zaustavlja sintezu proteina i enzima te razara lizosomske membrane.

Zbog oštećenja diobenog vretena i enzima, posebice DNA i RNA polimeraze, zaustavljena je sinteza i popravak DNA, što dodatno pospješuje djelovanje kemoterapeutika. Dakle, kemosenzitivni učinak flavonoida i hipertermije pridonose citotoksičnom učinku kemoterapeutika, o čemu govori nekoliko objavljenih radova (74-77).

Dio radova odnosi se na procjenu zaštitnog učinka propolisa i njegovih polifenolnih/flavonoidnih sastavnica (kvercetin, krizin, naringin, naringenin, kafeinska kiselina) od ionizirajućeg zračenja. Izlaganje životinja i ljudi ionizirajućem zračenju u dijagnostici i terapiji uzrokuje doza-ovisne promjene na molekularnoj, substaničnoj i staničnoj razini, na jedinki i populaciji. Oštećenja prouzročena zračenjem, posebice limfoidnog i hematopoetskog sustava, uzrokuju pojačanu osjetljivost jedinke na infekcije, što može dovesti do smrti. Rezultati naših istraživanja upućuju na to da pripravci propolisa imaju brojne pozitivne učinke ako se primijene prije zračenja (preventivno): smanjuju razinu oštećenja DNA leukocita, smanjuju broj jedno- i dvolančanih lomova u molekuli DNA, broj kromosomskih aberacija, ubrzavaju popravak DNA te posljedično značajno povećavaju preživljavanje ž i v o t i n j a . To m u č i n k u z a s i g u r n o p r i d o n o s i imunostimulativno, protubakterijsko i protumikrobno svojsvo propolisa i sastavnih mu flavonoida te njihova sposobnost u procesu zacjeljivanja rana i regeneraciji hematopoietskog sustava. Osim pozitivnih učinaka, zapaženi su i neki negativni učinci flavonoida, na primjer povećanje broja mikronukleusa, što je posljedica prooksidativnog učinka nekih flavonoida u određenim uvjetima (45, 49, 78-83).

Također, dio radova odnosi se na istraživanju učinka pčelinjeg otrova na rast karcinoma dojke u uvjetima in vivo i/ili in vitro te na istraživanju protumetastatskog učinka.

Jasno je pokazano da prisutnost pčelinjeg otrova u tkivu može odgoditi rast tumora i povećati preživljavanje životinja te da se protutumorski učinak temelji na bliskom dodicaju sastavnica pčelinjeg otrova s tumorskim stanicama, njihovoj lizi, apoptozi i nekrozi te modulaciji imunološke reakcije. Dan je kritički osvrt o dozi, načinu unosa, imunoreaktivnosti nositelja tumora, osobitostima pojedinih sastavnica pčelinjeg otrova na modulaciju imunološke reakcije (84). Nadalje, u preglednom radu (85) opisani su molekularni mehanizmi i glavni signalni putevi učinkovitosti pčelinjeg otrova, posebice njegove glavne sastavnice melitina, te fosfolipaze A i apamina na razne stanične linije tumora ljudi i životinja te na tumorskim modelima miša i štakora, kao i mogućnost primjene litičkog peptida melitina

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u terapiji tumora životinja i ljudi preko raznih nosača za lijekove (nanočestice, liposomi, imunokonjugati, virusni vektori).

U novije vrijeme provedena su istraživanja novih protuleukemijskih lijekova kao što su troksacitabin i adafostin na kroničnu mijeloidnu leukemiju (CML) (86, 87). Troksacitabin (β-L-dioksolan citidin, BCH-4556) novi je nukleozidni analog koji u pokusima pokazuje snažnu antitumorsku aktivnost na leukemije i solidne tumore.

Sličan je citarabinu (Ara-C), koji se već dulje vrijeme primjenjuje u liječenju akutne mijeloične leukemije (AML).

U sklopu naših istraživanja ispitana je učinkovitost BCH4556 na stanice CML u uvjetima in vitro i in vivo. Istražen je učinak BCH-4556 samog i združenog s Ara-C, adafostinom (NSC 680410) ili imanitibom (STI-571) na stanične linije AML i CML senzitivne i rezistentne na STI571. Dobiveni rezultati upućuju na to da BCH-4556 ima značajnu protuleukemijsku aktivnost ne samo na stanice AML nego i na stanice CML koje su rezistentne na STI-571.

Onkogen Bcr/Abl za trajno aktiviranu tirozin kinazu uzrokuje Philadelphia-kromosom pozitivnu (Ph+) leukemiju.

STI-571 je kompetitivni inhibitor za ATP-vezno mjesto BCR/ABL, koji pokazuje visoku aktivnost na taj tip leukemije. Naša su istraživanja (87) bila usmjerena na utvrđivanje učinaka adafoostina na proliferaciju, apoptozu i aktivnost enzima tirozin kinaze u Bcr/Abl pozitivnim staničnim linijama (Ph+; p210 Bcr/Abl KBM-5, p210 Bcr/Abl KBM-7, p210 Bcr/Abl K562), ili Ph- (AML-2, AML-3) staničnim linijama.

Također je procijenjena selektivnost adafostina na primarne CML ili AML stanice blasta. Učinkovitost adafostina u STI-571-senzitivnim i -rezistentnim stanicama u sličnim koncentracijskim vrijednostima upozorava na nedostatak križne rezistencije između dva lijeka. Istovremena ili obrada u nizu stanica CML ili AML s adafostinom i drugim kemoterapeuticima ima aditivni učinak na rast stanica.

Metodom klonalnog rasta potvrđena je selektivnost adafostina za CML i AML progenitore u odnosu na kontrolu.

Adafostin pobuđuje apoptozu u Ph+ i Ph- staničnim linijama ovisno o dozi i vremenu putem proizvodnje superoksidnih aniona. Isti je učinak neovisan o aktivnosti Bcr/Abl kinaza.

Obrada stanica s adafostinom pokazuje postupno smanjenje fosforilizacije tirozina u Bcr/Abl i drugim polipeptidima ovisno o vremenu i dozi. Nadalje, rezultati istraživanja upućuju na to da Crk-L ili Cbl proteini nisu potrebni za signalni put Bcr/Abl kinaza i citotoksičnost adafostina.

Dobiveni rezultati govore u prilog da je adafostin obećavajući lijek za oboljele od kronične i akutne mijeloidne leukemije, posebice za oboljele od CML rezistentne na STI -571.

Poznato je da je prehrana važan čimbenik u sprječavanju nastanka bolesti poput tumora, dijabetesa i pretilosti. Dio naših istraživanja imao je za cilj utvrditi učinak propolisa i njegovih polifenolnih/flavonoidnih sastavnica na sprječavanje posljedica dijabetesa na molekularnoj i staničnoj razini metabolički aktivnih organa i tkiva (jetra, bubrezi i krv). To je posebice važno jer tradicionalni biljni pripravci sadržavaju tvari koje imaju sposobnost zaštititi funkciju gušterače, povećati razinu inzulina, inhibirati absorbciju glukoze u crijevima ili utjecati na apsorbciju metabolita u inzulin ovisnim procesima. Osim općenitih povoljnih učinaka, prirodni pripravci imaju znatno manji toksični učinak na organizam u odnosu na sintetske lijekove, lako su dostupni, a nije zanemarivo ni to što nisu skupi.

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Naši rezultati upućuju na to da se zaštitni učinak propolisa i njegovih polifenolnih/flavonoidnih sastavnica (kvercetina, krizina, naringina, naringenina, kafeinske kiseline), kao i učinak zelenog čaja i kurkumina na dijabetes prouzročen aloksanom na molekularnoj i na razini cjelokupnog organizma tkiva, organa i stanica zasniva prije svega na njihovoj protuoksidativnoj, protuupalnoj i regenerativnoj sposobnosti (44, 88-91).

Jedan dio naših istraživanja temelji se na procjeni učinkovitosti meda i flavonoida na mikrofloru probavnog sustava te na proces detoksifikacije prirodnih kancerogena koje svakodnevno unosimo hranom. Med je funkcionalna hrana jedinstvenog sastava, antimikrobnih svojstava i bifidogenog učinka sa sposobnošću inhibicije toksičnog učinka mikotoksina te potiče rast i aktivnost mikroflore probavnog sustava, proliferaciju bifido bakterija i laktobacila. Te bakterije imaju probiotski učinak, rastu u crijevima i proizvode metabolite koji inhibiraju rast patogena i daju otpornost domaćinu, a posebice povećavaju nespecifičnu imunofagocitnu aktivnost cirkulirajućih granulocitnih stanica i monocita/makrofaga. One također luče proteolitičke enzime kojima razgrađuju bakterijske i druge toksine te mogu imati zaštitni učinak protiv kancerogena. Jedan od mehanizama uklanjanja mutagenih mikotoksina svakako je i njihovo vezanje s probiotičkim bakterijama u crijevima. Polifenoli prisutni u hrani mogu utjecati na mikrobni sastav crijevne flore te ublažiti toksičnost reaktivnih radikala, a keliranjem Fe2+ mogu spriječiti prooksidativno djelovanje željeza u kolonu, inhibirati prooksidativno djelovanje lipooksigenaza i ciklooksigenaza te pojačati detoksifikacijski sustav stanica.

Nadalje, naši rezultati upućuju i na to da fenolne sastavnice mogu sudjelovati u regulaciji metabolizma toksina ili u regulaciji protuoksidativnih enzima probavnog sustava (44, 92).

Dio istraživanja koja smo proveli temeljio se na procjeni učinkovitosti flavonoida kao fitoestrogena u sprječavanju posljedica osteoporoze. Na pokusnom modelu štakora istražili smo učinkovitost flavonoida kvercetina, naringenina, krizina, ikarina i proantocijanidina u prevenciji i liječenju osteoporoze izazvane 15-dnevnom intragastričkom primjenom retinoične kiseline. Cilj je bio ustanoviti postoji li razlika u antiosteoporotičnoj aktivnosti između korištenih flavonoida i bisfosfonata alendronata, za koji je već utvrđen pozitivni učinak te je kao antiresorptivni lijek registriran u Republici Hrvatskoj. U istraživanju su praćene promjene težine životinja, provedena su fizikalna mjerenja kosti, mjerena je mineralna gustoća kosti, izmjerene su razine ukupnog kalcija (Ca) i fosfora (P), biokemijskih biljega koštanog preokreta (osteokalcin i C-terminalni križno vezani telopeptid), hematoloških i biokemijskih odrednica, praćena su funkcionalna oštećenja bubrega, jetara i krvnih stanica komet- i mikronukleus-testom te je izmjerena razina lipidne peroksidacije i glutationa. Svi navedeni parametri potvrđeni su i histopatološkim oštećenjima kosti. Rezultati tih istraživanja prikazani su u dva doktorska rada te u radovima u postupku objave (93).

Proveli smo i istraživanja genotoksičnih i citotoksičnih učinaka inhalirajućih anestetika (halotan, sevofluran, izofluran) samih ili združenih s kemoterapeutikom cisplatinom. Uspješnost zahvata u medicinskoj i

243 veterinarskoj praksi zahtijeva što učinkovitije anestetike sa što manjom toksičnošću. Dosadašnja istraživanja upućuju na citotoksične i genotoksične učinke inhalacijskih anaestetika, posebice na stanicama jetara, bubrega i mozga.

Premda se glavnina inhaliranih anestetika uklanja iz organizma respiracijom, jedan se dio metabolizira u jetrima putem obitelji citokrom oksidaza P-450 i izlučuje bubrezima, pa je pitanje njihove toksičnosti oduvijek izazivalo veliko zanimanje istraživača. Mehanizmi toksičnosti inhalacijskih anestetika mogu biti izravni i neizravni, prouzročeni prisutnošću toksičnih metabolita i njihovim kovaletnim vezivanjem na stanične makromolekule (proteine i lipide) i/ili pojavnošću imunosnog odgovora na «modificirane» izmijenjene jetrene proteine prouzročene reaktivnošću metabolita i posljedičnim stvaranjem imunokompleksa i autoprotutijela na vlastite tkivne komponente. Poznato je da toksičnost anestetika djeluje na stanice kao signal koji izaziva mnoge odgovore, uključujući prilagodbu, oštećenje, proliferaciju, apoptozu i nekrozu, što su pokazali i naši rezultati na tumorskim stanicama Ehrlichova ascitesnog tumora (94-97). Najnovija istraživanja prof. dr. sc. Nade Oršolić i suradnika temelje se na utvrđivanju mogućih oštećenja prouzročenih oksidativnim stresom pod utjecajem ionizirajućeg zračenja i anestetika. Istraživanja koja se provode imaju za cilj utvrditi i usporediti učinke međudjelovanja ionizirajućeg zračenja i anestetika: halotana, isoflurana i sevoflurana, primijenjenih pojedinačno u jednoj dozi na modelu Swiss albino miša. Prati se preživljenje, ukupni broj leukocita, razina oštećenja DNA u leukocitima, jetrenim, moždanim i bubrežnim stanicama, njihov antioksidativni status i oblik stanične smrti. Istovremeno se procjenjuju funkcionalna bubrežna i jetrena oštećenja te mogućnosti popravka oštećene DNA spomenutih stanica, regeneracije tkiva i organa te hematopoetskog i imunosnog sustava kroz različito vrijeme (0, 2, 6, i 24 h). Dobiveni rezultati trebali bi dati uvid u međudjelovanje ionizirajućeg zračenja i anestetika radi moguće praktične primjene pri odabiru najmanje štetnog anestetika za planirane zahvate s ionizirajućim zračenjem u veterini i medicini, a u cilju prevencije i očuvanja zdravlja ljudi i životinja.

U okviru projekta „In vivo ispitivanje niskoaromatskog visokosteranskog derivata naftalana (NAVS)“ istraživali smo učinkovitost niskoaromatskog visokosteranskog derivata naftalana (NAVS) na životinjskom modelu štakora kojima je primjenom triju različitih iritanata (heksil salicilat, di-n-propil disulfid, l-bromoheksan) inducirana psorijaza. Ispitana je protuupalna učinkovitost NAVS i njegova mogućnost primjene u liječenju psorijaze sljedećim metodama: određivanjem broja upalnih stanica u trbušnoj šupljini, diferencijalnom analizom stanica trbušne šupljine, funkcionalnom analizom makrofaga, procjenom biokemijskih i hematoloških odrednica kao pokazatelja upale i oštećenja posredovanih upalnim stanicama, termografijom, histopatološkom analizom kože te procjenom aktivnosti proupalnih citokina posrednika patogeneze psorijaze. Nadalje, potencijalni genotoksični i mutageni učinak pripravka procijenjen je primjenom komet-testa i mikronukleus-testa.

Rezultati istraživanja protuupalnog učinka propolisa i flavonoida (kvercetin, krizin, kurkumin i epigalokatehin-

244 3-galat) na modelu miša gdje su psorijazne promjene kože prouzročene heksilom ili propil disulfidom prikazani su u radovima (98-100). Potvrđena je protuupalna i protuoksidativna učinkovitost flavonoida te mogućnost primjene termografije u upalnim bolestima kože.

Zadnjih godina u Zavodu za animalnu fiziologiju započelo se s istraživanjima koja su vezana uz druge grane fiziologije. Članovi Zavoda uvode istraživanja komparativne animalne fiziologije, neurofiziologije i ponašanja životinja, metaboličke fiziologije i toksikologije.

Na spomenuta istraživanja nadovezuju se istraživanja mehanizama fiziološke toksikologije ksenobiotika pod vodstvom prof. dr. sc. Domagoja Đikića. Osim imunotoksikologije, tematika je proširena na više fizioloških mehanizama i integralnu toksikološku fiziologiju.

Proučavaju se fiziološke promjene nastale uslijed izloženosti ksenobioticima u hrani, posebice triazinima, imadazolima, piretroidima, karbamatima i njihovim mješavinama.

Istražuju se sinergistički, antagonistički i potencirajući učinci tih ksenobiotika na biokemijske procese i mehanizme izazivanja oksidativnog stresa, posebice njihova toksikodinamika, neurotoksičnost, hepatotoksčnost, nefrotoksičnost i miotoksičnost. Rezultati istraživanja toksikologije triazina (prometrin) pridonijeli su ne samo razjašnjavanju koliko su ti pesticidi opasni za ljudsku populaciju nego i nedavnoj zabrani uporabe te skupine spojeva u Hrvatskoj. Analizom biokemijskih odrednica (LDH, GGT, ALP, kreatinin, ALT, AST) u krvi miševa utvrđeno je da je nakon oralne primjene prometrina svakih 48 sati u subkroničnim dozama 185, 375 i 555 mg kg-1 tijekom 28 dana najznačajniji učinak povećanje razine GGT i smanjenje razine kreatinina. Nadalje, uočeno je da prometrin ima miotoksični i nefrotoksični potencijal te remeti važne sustave enzima koji sudjeluju u metaboličkim putevima i bioenergetici organizma. U izloženih miševa primjenom komet-testa utvrđeno je povećanje razine oštećenja DNA, koji je bio razmjeran vremenu izloženosti i primljenoj dozi prometrina (101-104).

U pokusima sinergističkih učinaka ksenobiotika najznačajniji je doprinos otkriće potencirajućeg toksičnog učinka niskih doza aneugena karbendazima u kombinacijama s niskim dozama imadazolskih i piretroidnih spojeva (105108). Profesor Đikić uvodi i istraživanja u području fiziologije metabolizma, koja su usmjerena na lipidnu fiziologiju, prehranu i prirast na različitim životinjskim modelima.

Istraživanja su započela suradnjom na istraživanju uloge lipidnih splavi i glikokonjugata u razvoju i regeneraciji živčanih stanica razvijanjem modela inducirane hibernacije u miša. Uz to, prof. dr. sc. Domagoj Đikić provodi i istraživanja dinamike metabolizma kolesterola u mozgu i jetrima ovisno o antioksidativnom statusu tkiva u stanjima hiperkolesteremije na modelu miša i hipometabolizma u žabe vrste Pelophylax ridibundus. U području komparativne fiziologije ističu se i njegovi radovi iz komparativne hematologije nižih kralježnjaka (109, 110). U području fiziologije metabolizma posebno su mu područje zanimanja istraživanja usmjerena na komparativnu termalnu i hibernacijsku (96, 97, 111-113) i lipidnu fiziologiju (114-117) te fiziologiju metabolizma i prirasta biomase (118123) na nekoliko životinjskih modela.

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Istraživanjima neurofiziologije i ponašanja životinja posebice se bave prof. dr. sc. Dubravka Hranilović i doc. dr. sc. Zoran Tadić.

Od 2007. do 2013. u sklopu MZOŠ-ova projekta „Neurobiološka podloga autizma: uloga serotoninskog sustava“, voditeljica kojega je prof. dr. sc. Dubravka Hranilović, istraživana je poremećena homeostaza serotonina kao jedan od aspekata neurobiološke podloge autizma. Istraživanja na ljudskoj populaciji bila su usmjerena na otkrivanje mehanizma nastanka hiperserotoninemije (povišene razine serotonina u krvi) i njene povezanosti s poremećenom funkcijom serotonina kao modulatora razvoja mozga i neurotransmitora, koja se očituje u pojavi autističnih simptoma. Na hiperserotoninemičnoj podskupini autističnih ispitanika uočena je povezanost sa stupnjem poremećaja u govoru, što je upozorilo na mogućnost korištenja hiperserotoninemije kao endofenotipa u istraživanju genetičke podloge autizma (124). Usporedbom aktivnosti proteina koji reguliraju perifernu serotoninsku homeostazu između hiperserotoninemičnih autističnih ispitanika, normoserotoninemičnih autističnih ispitanika i zdravih kontrolnih ispitanika uočena je pojačana aktivnost enzima monoamin-oksidaze i smanjena aktivnost receptora 5HT2A u autističnih ispitanika. Zaključeno je da bi u podlozi povišene razine serotonina u trombocitima mogla stajati promjena u metabolizmu, a ne u aktivnom unosu serotonina u trombocite (125). Profesorica Hranilović i suradnici svoja su istraživanja potom usmjerili i na eksperimentalni model štakora s farmakološki izazvanim promjenama u metabolizmu serotonina, na kojem su istražili izazivaju li povišene koncentracije serotonina tijekom razvoja mozga trajne promjene u razini serotonina, metabolizmu serotonina i aspektima ponašanja reguliranih serotoninom. U štakora obrađenih prekursorom serotonina uočena je prolazna hiperserotoninemija i trajno snižena koncentracija serotonina u mozgu, koja je vjerojatno posljedica manjeg broja serotonergičnih okončina u moždanoj kori. U štakora obrađenih inhibitorom razgradnje serotonina uočena je trajna hiperserotoninemija i drastično smanjena razina serotonina u mozgu, koja je vjerojatno posljedica jakih promjena u sintezi, pohrani i razgradnji serotonina (126). Štakori su također bili podvrgnuti bateriji testova kako bi se utvrdilo u kojoj mjeri promijenjena razina i/ili metabolizam serotonina utječu na ponašanje posredovano serotoninom. Utvrdili su da je intenzitet promjena u ponašanju pratio intenzitet promjena srotoninske homeostaze – životinje tretirane prekursorom serotonina pokazivale su blago smanjenu anksioznost, dok su životinje tretirane inhibitorom razgradnje serotonina pokazale iznimno smanjenu anksioznost i povećanu kognitivnu fleksibilnost, što je u skladu s drastično smanjenom razinom serotonina u njihovu mozgu (127). Najzad, u obrađenim je životinjama istražena ekspresija gena za elemente koji reguliraju serotoninsku neurotransmisiju. Uočeno je da perinatalna primjena prekursora serotonina, kao i inhibitora razgradnje serotonina, uzrokuje (dugo)trajne promjene u ekspresiji tih gena (ponajprije gena za enzim monoamin oksidazu), koje vjerojatno stoje u podlozi opaženih neurokemijskih i bihevioralnih promjena (128). Dosadašnji rezultati upućuju na moguću važnost opisanog modela u

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istraživanjima utjecaja tvari koje povećavaju razinu serotonina na razvoj ljudskog fetusa.

U sklopu istraživanja u Zavodu proučavan je i neuroprotektivni učinak prehrane. Uloga kvercetina u zaštiti stanica i prevenciji neurodegenerativnih poremećaja opisana je u radovima (129, 130).

Istraživanja doc. dr. sc. Zorana Tadića u najvećoj su mjeri usmjerena na ekofiziologiju i ponašanje odnosno na njihove interakcije i evoluciju. Kao odličan model za to pokazale su se primorske gušterice (Podarcis melisellensis), čiji mužjaci na otoku Lastovu imaju polimorfizam trbušne obojenosti: u populaciji postoje mužjaci s narančastim, žutim i bijelim trbuhom. Istraživanja su pokazala da mužjaci narančastog trbuha imaju najjači zagriz i najvišu razinu koritkosterona, ali se razine testosterona ne razlikuju među s k u p i n a m a . Ta k o đ e r n e p o s t o j i r a z l i k a u imnunokompetentnosti između triju skupina mužjaka. Stoga ne postoji ni korelacija između imuniteta i razine testosterona. Međutim, rezultati upozoravaju na mehanistički odnos između testosterona i svojstva reguliranog seksualnom selekcijom (131). Uočeno je da zaraza ektoparazitima među trima skupinama mužjaka nije izražena tijekom sezone aktivnosti, ali se pojačava u jesen, potkraj aktivne sezone.

U jesen se broj krvnih endoparazita (hemogragarina) jako povećava u mužjaka bijelog trbuha, dok je povećanje neznatno u narančastih mužjaka. Zanimljivo je da se imunološki odgovor (testiran miješanom leukocitnom reakcijom – odgovorom na fitohemaglutinin) pojačava u jesen, dok se u narančastih mužjaka on tada znatnije gasi.

Također je utvrđena negativna korelacija između imunološkog odgovora na fitohemaglutinin i zaraze ektoparazitima – krpeljima i grinjama. U ovom se radu prvi put korištenjem kombinacije imunoloških i parazitoloških testova na terenu dokazalo postojanje različitih evolucijski stabilnih strategija i visokoadaptivnih reprodukcijskih svojstava (engl. life-history strategies) u lacertidnih guštera različite boje trbuha (132). Docent Tadić bavi se i proučavanjem ponašanja i fiziologije zmija. U istraživanju arborealnih zmija ljutica svojte Trimeresurus, koje žive u južnoj i jugoistočnoj Aziji i svojim su ponašanjem tzv. sitand-wait predatori, prvim takve vrste u svijetu, proučen je međuodnos funkcionalne morfologije, fiziologije, biomehanike i ponašanja u tih zmija. Pokušalo se odgonetnuti što se s tijelom zmije događa tijekom obrambenog napada. Zanimljivo je da brzina napada i njegova udaljenost ne ovise o tjelesnoj masi, što znači da male zmije napadaju jednako brzo i, proporcionalno, jednako daleko kao i odrasle. Suprotno svim poznatim modelima korištenima u funkcionalnoj morfologiji, ubrzanje tih zmija raste s povećanjem njihove mase. To je posljedica negativnog alometrijskog rasta mase glave u kombinaciji s pozitivnim izometrijskim rastom epaksijalnih mišića prednjeg dijela tijela koji ga ubrzavaju tijekom napada. Također se pokazalo da izraženi spolni dimorfizam tih zmija (mužjaci su značajno kraći i lakši od ženki) utječe i na brzinu napada: ženke jednake duljine tijela kao i mužjaci značajno su masivnije i brže napadaju od mužjaka (133). Urednik časopisa odabrao je ovaj rad kao tzv. priču s naslovnice (engl. cover story) u prvom broju časopisa u 2011. godini jer je to prvi rad u svijetu koji objedinjuje pionirska integrativna istraživanja biomehanike i fiziologije

aborealnih zmija. Rad je i prva priča s naslovnice Biološkog odsjeka PMF-a koja je na njemu u potpunosti osmišljena i napravljena.

U istraživanjima ponašanja i učenja provedenima u našem zavodu uporabljeni su i drugi modeli, što je prikazano u radovima (134, 135).

U Zavodu se provode i istraživanja u području molekularne ekologije. Doc. dr. sc. Ana Galov i dr. sc. Haidi Arbanasić bave se istraživanjem genetičke raznolikosti sisavaca u Hrvatskoj, koristeći se pri tom neutralnim i adaptivnim genetičkim biljezima. Ta su istraživanja u početku bila usmjerena na populacijsku genetiku i proučavanje genetičke varijabilnosti hrvatskih autohtonih pasmina konja pomoću mikrosatelitskih lokusa (28, 136), nakon čega su proširena i na populacije divljih životinja, posebice ugroženih i onih kojima prijeti izumiranje.

Istražena je genetička raznolikost i populacijska strukturiranost u populacijama dobrog dupina iz Jadranskoga mora (Tursiops truncatus) (137), medvjeda (Ursus arctos) (138) i vukova (Canis lupus) koji nastanjuju područje Republike Hrvatske (139), pri čemu se koristilo neutralnim genetičkim biljezima kao što su mikrosatelitski lokusi i kontrolna regija mitohondrijske DNA. Ta istraživačka grupa trenutačno sudjeluje u međunarodnom projektu koji pomoću genetičkih biljega utvrđuje populacijsku strukturu čaglja u Europi (140) i pojavu hibridizacije između čaglja i psa. U novije vrijeme istraživanja su proširena i na područje imunogenetike odnosno glavnog sustava tkivne podudarnosti (engl. major histocompatibility complex, MHC). MHC geni sve se učestalije koriste u suvremenim istraživanjima adaptivne genetičke varijabilnosti u populacijama divljih životinja. Neki od MHC gena pripadaju među najvarijabilnije u genomu sisavaca: kodiraju glikoproteinske receptore na stanicama imunosnog sustava koje prikazuju antigenske strukture limfocitima T, što pokreće specifični imunosni odgovor i stoga ima ključnu ulogu u funkcioniranju imunosnog sustava. Smatra se da je varijabilnost MHC genske regije posljedica adaptivnih procesa unutar neke populacije i pokazatelj je sposobnosti populacije da prepozna raznovrsne antigenske strukture i pokrene odgovarajuću imunosnu reakciju. Provedena su istraživanja MHC-a u domaćih životinja, hrvatskih pasmina konja (141) i magaraca na Balkanu (142) te divljih populacija kao što su vuk (143), dobri dupin i čagalj.

Polazeći od činjenice da pojedini MHC aleli uvjetuju otpornost ili podložnost u odnosu na određene bolesti, uključujući i zarazne bolesti, nedavno su započela istraživanja čiji je cilj utvrditi postojanje moguće povezanosti između određene bolesti i MHC statusa, posebice u konja, a potom i u populacijama divljih životinja.

Nastava kroz povijest i danas Organizirana provedba nastave iz animalne fiziologije i srodnih kolegija na Biološkom odsjeku Prirodoslovnomatematičkog fakulteta Sveučilišta u Zagrebu odvija se od osnutka Zavoda za animalnu fiziologiju 1963. godine. Iste je godine dr. sc. Borislav Nakić izabran za izvanrednog profesora iz Animalne fiziologije za studente eksperimentalnih i profesorskih smjerova na Prirodoslovnomatematičkom fakultetu. Profesor Nakić upamćen je kao

246 vrstan pedagog i profesor koji je mnogo vremena posvetio radu sa studentima i podučavanju mladih znanstvenika, omogućivši im usavršavanje na nekim od najprestižnijih svjetskih znanstvenih institucija. Nastavu nakon smrti profesora Nakića preuzima prof. dr. sc. A. Kaštelan, potom prof. dr. sc. O. Springer, prof. dr. sc. D. Volf i prof. dr. sc. I. Bašić te naposljetku njihovi suradnici i asistenti. U uspješnoj provedbi praktikumske nastave svakako treba istaknuti i značajan doprinos brojnih tehničkih suradnika: Matilda Derikrava, Vlado Bedeković, Bepina Zake, Gordana Žakman, Marija Potočić, Nino Vukmanić i Hrvoje Jedvaj.

U sklopu Zavoda od njegova osnutka provodi se sustavni nastavni rad iz fiziologije, imunologije, transplatacijske imunologije, radiobiologije, neurofiziologije i endokrinologije, neuroimunologije, imunogenetike, ekofiziologije i ekotoksikologije.

Među kolegijima koji su činili okosnicu nastavnog djelovanja Zavoda u okviru diplomskih studija Biologije ističu se Animalna fiziologija I i Animalna fiziologija II, Imunologija, Neurofiziologija i endokrinologija, Neurofoziologija i bioenergetika, Radiobiologija.

Napretkom pojedinih znanstvenih disciplina pokazala se potreba za uvođenjem novih kolegija: Ekotoksikologija, Osnove patofiziologije, Pesticidi u ekosustavu, Komparativna fiziologija, Imunologija i imunogenetika, Ekološka imunologija i dr.

Nastavnici Zavoda za animalnu fiziologiju sudjeluju i u poslijediplomskom studiju Prirodoslovno-matematičkog fakulteta Sveučilišta u Zagrebu (smjerovi: biomedicina, ekologija i toksikologija) s kolegijima: Transplantacijska imunologija, Timus, hematopoeza i imunološki sustav, Ekofiziologija, Ekotoksikologija, Imunohematotoksikologija, Pesticidi i imunološki sustav, Imunobiologija tumora i metastaza, Imunost u domaćih životinja i dr.

Reforma visokoškolske nastave i prilagodba zahtjevima Bolonjskog procesa rezultirali su daljnjim osuvremenjivanjem nastave, pa se na prediplomskim i diplomskim studijskim programima Biološkog odsjeka uvode kolegiji: Laboratorijske životinje u biološkim istraživanjima, Ponašanje životinja, Komparativna imunologija, Osnove patofiziologije, Fiziologija oksidativnog stresa u ljudi i životinja, Fiziološki mehanizmi u toksikologiji, Metode u imunologiji, Biologija tumora, Imunologija tumora i metastaza, Molekularna patologija, Opća onkologija, Neuroimunologija, Toksikologija okoliša, Osnove molekularne ekologije, Fiziologija endokrinog sustava, Fiziologija metabolizma i bioenergetika, i dr.

Nastava iz većine spomenutih kolegija izvodi se na diplomskom studiju Eksperimentalna biologija, modul Fiziologija i imunobiologija.

Osim studenata matičnog fakulteta, u zadnje vrijeme zanimanje za neke od novijih kolegija, posebice za Ponašanje životinja, Fiziologiju oksidativnog stresa u ljudi i životinja i za Fiziološke mehanizmi u toksikologiji, pokazuju i studenti drugih fakulteta Sveučilišta u Zagrebu, osobito studenti psihologije i sociologije Filozofskog fakulteta te studenti Farmaceutsko-biokemijskog fakulteta.

U sklopu Doktorskoga studija biologije suradnici Zavoda osmislili su i predaju nove kolegije: Bioterapija i kemoprevencija tumora, Integrativna fiziologija, Animalni modeli glodavaca u eksperimentalnoj onkologiji,

Oršolić N. POVIJEST ZAVODA ZA ANIMALNU FIZIOLOGIJU Arh Hig Rada Toksikol 2014;65:237-249

Molekularna podloga poremećaja ponašanja, Mutageni i antimutageni.

Znanstveno-nastavno osoblje Zavoda od samog osnutka do danas uspješno vodi dio nastave na drugim fakultetima, primjerice na Prehrambeno-biotehnološkom fakultetu, Filozofskom fakultetu, Veterinarskom fakultetu, Medicinskom fakultetu, Odsjeku za psihologiju Hrvatskih studija Sveučilišta u Zagrebu, Zdravstvenom veleučilištu Zagreb, Prirodoslovno- matematičkom fakultetu i Studiju mora i pomorstva Sveučilišta u Splitu, i dr.

Bogati znanstveno-istraživački i nastavni rad u sklopu Zavoda rezultirao je izradom više od 500 diplomskih radova, pedesetak magistarskih te osamdesetak doktorskih radova pod voditeljstvom i suvoditeljstvom našeg nastavnog osoblja.

Uzgajalište pokusnih životinja Zbog specifičnosti znanstvenoistraživačkog i nastavnog rada, Zavod za animalnu fiziologiju od svog se osnutka bavi i sustavnim uzgojem pokusnih životinja (visokosrodni sojevi miševa i štakora). Danas se u uzgajalištu pokusnih životinja Zavoda uzgaja nekoliko sojeva miševa koji se rabe u znanstvene i nastavne svrhe: CBA, CBA/N, CBA/T6T6, BALB/c, C57/black, Swiss albino i soj štakora Y59. Soj štakora Y59 stvoren je u tom uzgajalištu i registriran u Rat Genome Database Milwaukee, SAD (RGID ID67938).

Zavod posjeduje i najveću akademsku zbirku živih zmija u Europi (http://snakes.biol.pmf.unizg.hr), koju održava doc. dr. sc. Zoran Tadić.

Zbog potreba znanstvenog i nastavnog rada i traženja što pogodnijeg modela u biomedicinskim istraživanjima, osim uzgajališta pokusnih životinja (visokosrodni sojevi miševa i štakora), u novije su vrijeme prostori uzgojnih nastambi dopunjeni uzgajalištem kukaca, gmazova i vodozemaca, osobito za potrebe izvođenja nastave novih kolegija Ponašanje životinja, Komparativna imunologija, Laboratorijske životinje u biološkim istraživanjima Patofiziologija, Fiziologija oksidativnog stresa u ljudi i životinja, Fiziološki mehanizmi u toksikologiji, kako bi se što više povezao znanstveni i nastavni rad te što bolje osposobilo studente za rad u području biomedicine, toksikologije, farmakologije, tumorske imunologije i eksperimentalne biologije, neurofiziologije i ponašanja životinja.

Istraživanja na laboratorijskim životinjama značajno su pridonijela napretku temeljnih bioloških znanosti te velikom broju spoznaja iz biologije i biomedicine. Važno je istaknuti da uporaba životinja, uz primjenu “tri R” načela (engl. replacement = zamjena; reduction = smanjenje; refinement = oplemenjivanje), također ostaje nezamjenjivi dio edukacije velikog broja studenata na svim studijskim programima Biološkog odsjeka PMF-a.

Razvidno je da Biologija, posebice kolegiji i praktikumska nastava te istraživanja u sklopu Zavoda za animalnu fiziologiju, uvelike ovise o pokusima na životinjama i razvoju brojnih animalnih modela. Upravo raznovrsnost primjene različitih modela u istraživanju privlači brojne studente za izradu diplomskih radova, a zanimanje za područje nastavnog i znanstvenog rada Zavoda posebice je vidljivo u manifestaciji Noć biologije, koja okuplja sve znatiželjne ljude, zaljubljenike u biologiju i srednjoškolce zainteresirane za studij

247

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biologije, pri čemu se već godinama bilježi osobito zanimanje za područje fiziologije, imunologije, neurofiziologije i ponašanja životinja.

Nastamba za uzgoj pokusnih životinja (miš i štakor) obnovljena je i registrirana u 2013. pri Ministarstvu poljoprivrede u skladu sa Zakonom o zaštiti životinja (Narodne novine 135/06, 37/13) i Pravilnikom o zaštiti životinja koje se koriste u pokusima ili u druge znanstvene svrhe (Narodne novine, broj 55/13). Voditeljica nastambe je dr. sc. Anica Horvat Knežević.

Uspješnosti održavanja uzgajališta laboratorijskih životinja od osnutka Zavoda do danas pridonijele su i vrijedne timariteljice i laborantice: Vinka Zaninović, Kata Duh, Kata Kokorić, Josipa Posenjak, Nada Bočkal, Emina Bašić, Ružica Duh, Ana Bolfek i Valentina Greguraš.

dobar su pokazatelj vrijednosti originalnih znanstvenih ideja i aktivnosti, koja svakako zavrjeđuje priznanje hrvatske javnosti.

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Stručna aktivnost Osim znanstvenih i nastavnih aktivnosti, u Zavodu za animalnu fiziologiju oduvijek se velika pozornost davala stručnom radu. Suradnici Zavoda aktivno su uključeni u obrazovni program Hrvatskoga biološkog društva, pokret Znanost mladima, čija je svrha bila upoznati učenike osnovnih i srednjih škola u Hrvatskoj s osnovama znanstvenog rada i poticati zanimanje za prirodoslovna istraživanja.

Uz to, u različitim prigodama djelatnici Zavoda održali su stručna predavanja iz problematike naših istraživanja i sudjelovali u provedbi brojnih stručnih studija. Doc. dr. sc.

Zoran Tadić i prof. dr. sc. Nada Oršolić sudjelovali su i u velikom broju emisija o popularizaciji znanosti na Hrvatskoj radioteleviziji, posebice u emisijama Divni novi svijet (HR) i Dobro jutro, Hrvatska (HTV), Govorimo o zdravlju, Med kao lijek, Znanstvena petica, itd.

Tijekom 2012. godine članovi Zavoda za animalnu fiziologiju osmislili su “Program osposobljavanja osoba koje rade s pokusnim životinjama i životinjama za proizvodnju bioloških pripravaka” (www.labanim.hr). U provedbu tečaja uključen je cijeli Zavod, a njegov je glavni koordinator dr. sc. Julija Erhardt. Program osposobljavanja polaznicima daje teoretsko znanje potrebno za rad s pokusnim životinjama te praktično iskustvo u rukovanju i obavljanju osnovnih procedura na životinjama. Programi zadovoljavaju zahtjeve Ministarstva poljoprivrede, koje je odobrilo programe tečajeva te su upisani u evidenciju pri Upravi za veterinarstvo. Program također slijedi preporuke i kriterije Europske asocijacije za laboratorijske životinje (Federation of European Laboratory Science Association, FELASA).

Zaključne napomene Iz ovog je pregleda razvidno da je prvih 50 godina postojanja Zavoda za animalnu fiziologiju rezultiralo njegovim značajnim doprinosom i prepoznatljivošću na području znanstvenih istraživanja te visokoškolskog i stručnog rada ne samo u domaćim nego i u međunarodnim okvirima. Svakako treba istaknuti da su objavljeni znanstveni radovi te osobito veliki broj diplomskih, magistarskih i doktorskih radova pokazatelj uspješne znanstvene, nastavne i stručne aktivnosti članova Zavoda od osnutka do danas. Njihov dosadašnji odjek i citiranost

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NEW EDITIONS Arh Hig Rada Toksikol 2014;65:A20

A20

NEW EDITIONS Environmental Health Criteria 243: Aircraft disinsection insecticides. Geneva: World Health Organization 2013. (International Programme on Chemical Safety – IPCS) 144 str. (engleski jezik) ISBN 978 92 4 157243 9; Cijena 40 CHF (za zemlje u razvoju 28 CHF) Aircraft disinsection insecticides još je jedna publikacija Svjetske zdravstvene organizacije (SZO) iz serije o okolišnim kriterijima i ljudskom zdravlju.

Taj dokument pruža uvid u opći model koji se može koristiti za procjenu izloženosti insekticidima u dezinsekciji zrakoplova. Kao primjeri ljudske izloženosti koriste se dva modela: a) rezidualna dezinsekcija – nanošenje insekticida na površine putničke kabine i teretnog prostora; b) prskanje.

Naravno, postoje i drugi načini zaštite prostora i površina od insekata, no ta dva modela mogu se koristiti u svim procjenama zdravstvenih rizika, uz male izmjene osnovnih vrijednosti određenih parametara. U tim se modelima kao sredstva za The Environmental Behaviour of Radium: Revised Edition (Technical Reports Series No. 476).

Vienna: International Atomic Energy Agency 2014.

– 267 str. (engleski jezik). ISBN 978-92-0-143310-7 Već se godinama međunarodna znanstveno-stručna zajednica bavi istraživanjem utjecaja prirodno radioaktivnog materijala (naturally occurring radioactive materials – NORM) koji sadrži mnoge prirodne izotope U, Th, Ra, Rn, Pb i Po. Izotopi Ra posebno su značajni iz nekoliko razloga: prisutni su u sva tri prirodna niza radioaktivnog raspada; mobilnost Ra u okolišu visoka je u mnogim prirodnim uvjetima; sklonost Ra da se zbog svoje kemijske srodnosti s Ca nakon unosa u tijelo nakuplja u kostima.

Međunarodna agencija za atomsku energiju (International Atomic Energy Agency – IAEA) objavila je reviziju publikacije iz 1990. godine (TRS 310). Budući da su tijekom proteklog razdoblja objavljene mnoge publikacije o temi Ra u okolišu, Agencija je pokrenula reviziju podataka, kojom su obuhvaćene publikacije objavljene do 2013. godine.

TRS 476 sadržava nove informacije o ključnim procesima i modelima prijenosa radija u okolišu (kopnenom i morskom) te koncepte važne za

primjenu insekticida koriste hidraulička prskalica ili spremnik s aerosolom. Kao primjeri procjene rizika za ljudsko zdravlje korišteni su izračuni temeljeni na insekticidima permetrinu, D-fenotrinu i estofenproksu.

Jedan je od ciljeva ove publikacije harmonizirati procjenu zdravstvenih rizika od insekticida koji se koriste u dezinsekciji zrakoplova te stvoriti usporedive podatke koji će se koristiti prilikom registracije i označavanja tih proizvoda. Procjena rizika za ljudsko zdravlje uključuje radnike koji koriste i pripremaju insekticide te putnike u tretiranim zrakoplovima.

Svrha je ovog dokumenta pomoći vladinim organizacijama u zemljama članicama SZO-a prilikom odlučivanja o pitanjima dezinsekcije zrakoplova.

Međutim, valja napomenuti kako regulativa u vezi s odobrenjem proizvoda i metoda koji će se primjenjivati u dezinsekciji zrakoplova ostaju u domeni nacionalnih zakonodavstava.

Marin Mladinić radiološke procjene. Naime, u TRS 310 glavni naglasak bio je na probleme uzrokovane iskapanjem i obradom uranovih ruda, a nova publikacija uključuje utjecaj Ra iz svih NORM industrija. TRS 476 pruža podatke o akcidentalnim i kontroliranim ispustima Ra u okoliš te o prijenosu radionuklida kroz hranidbeni lanac svih živih bića. Uz ostalo, istaknuti su i problemi s korištenjem nekih izotopa radija kao tracera u istraživanjima različitih procesa u okolišu.

Budući da je za pouzdane radiološke procjene rizika te za planiranje remedijacije nužno imati najnovije podatke, u TRS 476 su između ostaloga uključene informacije iz IAEA-inih relevantnih publikacija objavljenih nakon 1991. godine. To uključuje izvještaje o procesima prijenosa u okolišu za čitav niz radionuklida, uključujući i Ra (TRS 419 (2003); TRS 422 (2004) TECDOC-1616 (2009); TRS472 (2010).

Također treba istaknuti da je ova publikacija usmjerena isključivo na Ra i ne sadržava podatke o njegovim potomcima, iako su potomci radija također važni za ukupnu procjenu utjecaja na okoliš (npr. ekshalacija 222Rn i 220Rn iz tla).

Tomislav Bituh

PRINT Denona, Zagreb

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