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 HONORARY EDITOR Prof Dr Marko Šarić, F.C.A. 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)
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ISSN 0004-1254
ARHIV ZA HIGIJENU RADA I TOKSIKOLOGIJU
ARCHIVES OF INDUSTRIAL HYGIENE AND TOXICOLOGY
Arh Hig Rada Toksikol • Vol. 66 • No. 3 • pp. 181-232 • ZAGREB, CROATIA 2015
CONTENTS Original articles In vitro cytotoxicity evaluation of different pulp capping materials: a comparative study
Claudio Poggio, Matteo Ceci, Alberto Dagna, Riccardo Beltrami, Marco Colombo, and Marco Chiesa
181
Mert Gürkan and Sibel Hayretdağ
189
Acute toxicity of maneb in the tadpoles of common and green toad
Karlo Jurica, Irena Brčić Karačonji, Sandra Šegan, Dušanka Milojković Opsenica, and Dario Kremer
197
Quantitative analysis of arbutin and hydroquinone in strawberry tree (Arbutus unedo L., Ericaceae) leaves by gas chromatography-mass spectrometry
Carla Viegas; Raquel Sabino, Daniel Botelho, Mateus dos Santos, and Anita Quintal Gomes
203
Assessment of exposure to the Penicillium glabrum complex in cork industry using complementing methods
Howard J. Mason, Ian Smith, Siti Marwanis Anua, Nargiz Tagiyeva, Sean Semple, and Graham Devereux
209
Levels of house dust mite allergen in cars
Irena Manfredo
213
Zvonko Rumboldt
219
Sayed Mahdi Marashi and Zeynab Nasri-Nasrabadi
221
Ivan Sabolić and Tvrtko Smital
225
Case report Accidental discovery of asbestos-related occupational pleural disease in unemployed carpenter: a healthcare safety net that needs mending Letters to the Editor Hydroxyethyl starch should not be used to manage severe aluminium phosphide poisoning Response to Professor Rumboldt’s reaction to our letter on hydroxyethyl starch use in managing aluminium phosphide poisoning Note The 2nd Croatian Symposium on Membrane Transporters (2. hrvatski simpozij o transporterima): Membrane Transporters in Toxicological and Pharmacological Research
228
Abstracts from the 2nd Croatian Symposium on Membrane Transporters
A13
New editions (in Croatian)
Cover page: Double staining of sodium D-glucose cotransporter SGLT1 and tubulin in human bronchiolus. SGLT1 is present in the apical membrane domain of Clara cells (red fluorescence), whereas tubulin is localized in the cilia of the ciliated cells (green fluorescence). Courtesy of Ivana Vrhovac, Davorka Breljak, and Ivan Sabolić, Molecular Toxicology Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia. (See abstract Vrhovac I, et al. Inhibitors of glucose transporter SGLT1 in the treatment of diabetes mellitus will not act only in the kidneys; the transporter is also present in other rodent and human organs. Arh Hig Rada Toksikol 2015. Disclaimer: This photo is intended to evoke the content of this issue of the journal. It is not intended for instructional or scientific purposes.
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Poggio C, et al. In vitro cytotoxicity evaluation of different pulp capping materials Arh Hig Rada Toksikol 2015;66:181-188
Original article
DOI: 10.1515/aiht-2015-66-2589
In vitro cytotoxicity evaluation of different pulp capping materials: a comparative study Claudio Poggio1, Matteo Ceci1, Alberto Dagna1, Riccardo Beltrami2, Marco Colombo1, and Marco Chiesa1 Department of Clinical-Surgical, Diagnostic and Pediatric Sciences – Section of Dentistry1, Department of Brain and Behavioral Sciences2, University of Pavia, Pavia, Italy [Received in November 2014; CrossChecked in November 2014; Accepted in August 2015] Direct pulp capping covers the exposed surface of the pulp to maintain its vitality and preserve its functional and biologic activity. The aim of the present study was to compare the biocompatibility effects of seven different pulp-capping materials in vitro: Dycal®, Calcicur®, Calcimol LC®, TheraCal LC®, ProRoot MTA®, MTA-Angelus®, and Biodentine®. Using the Transwell insert methodology by Alamar blue test, we evaluated the cytocompatibility of the above mentioned materials towards murine odontoblasts cells (MDPC-23) at three different times (24, 48, and 72 h). For additional control, the cell viability at 72 hours was also assessed by MTT assay. Morphological analysis of murine odontoblasts was assessed by Confocal Laser Scanning Microscope. The results indicate significantly different biocompatibility among materials with different composition. Biodentine® and mineral trioxide aggregate (MTA)-based products showed lower cytotoxicity, varying from calcium hydroxide-based materials, which exhibited higher cytotoxicity. Although our findings are limited to in vitro conditions, the observation that Biodentine® caused a cytotoxic effect similar to MTA suggests that it may be considered an alternative in pulp-capping treatment, as calcium hydroxide-based materials present higher cytotoxic effects. KEY WORDS: Alamar blue test; biocompatibility; dental material; MTT test; murine odontoblast Direct pulp capping involves the application of a dental material to seal communications between the exposed pulp and the oral cavity (mechanical and carious pulp exposures) in order to protect the dental pulp complex and preserve its vitality (1). Several materials such as calcium hydroxidebased ones and more recently mineral trioxide aggregate (MTA) are commonly used for this purpose (2, 3). Calcium hydroxide is the most popular agent for direct and indirect pulp capping (4, 5). Nevertheless, calcium hydroxide has certain drawbacks, such as poor bonding to dentin, material reabsorption, high solubility, and mechanical instability. In addition, the formation of reparative dentine may not be due to the bioinductive capacity of the material but due to a defence mechanism of the pulp induced by the irritant nature of calcium hydroxide (6, 7). The high pH (12.5) of calcium hydroxide suspensions causes liquefaction necrosis at the surface of the pulp tissue with the formation of a necrotic layer at the material-pulp interface (6). Mineral trioxide aggregate (MTA) cements are therapeutic, endodontic repair calcium silicate materials (7). These materials promote the proliferation/differentiation of human dental pulp cells (8-10) and show calcified tissue-conductive activity (11, 12). Compared to calcium hydroxide materials, Correspondence to: Claudio Poggio, Department of Clinical-Surgical, Diagnostic and Pediatric Sciences – Section of Dentistry, Policlinico “San Matteo”, Piazzale Golgi 3, 27100 Pavia, Italy, E-mail: c.poggio@unipv.it
MTA has an enhanced interaction with dental pulp tissue (13) with less pulp inflammation (14, 15). Several new calcium silicate-based materials have recently been developed (16-18) with the aim of improving some MTA drawbacks such as its difficult handling property (19) and long setting time (16). Biodentine® (Septodont) is one such material and it is claimed to be used for dental restoration in addition to endodontic indications similar to those of MTA. This agent is characterised by the release of calcium hydroxide in solution (20, 21), which when in contact with tissue fluids forms hydroxyapatite (22-24). As pulp capping materials will be in direct contact with pulp tissue for long periods of time, their biocompatibility is of particular importance. The aim of the present in vitro study was therefore to evaluate the possible cytotoxic effects of seven different commercially available pulp-capping materials on murine odontoblasts.
MATERIALS AND METHODS Dental Materials Seven pulp-capping materials were selected for this study: Dycal ® (Dentsply), Calcicur ® (Voco Gmbh), Calcimol LC® (Voco Gmbh), TheraCal LC® (Bisco), ProRoot MTA® (Dentsply), MTA-Angelus® (Angelus), and
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Biodentine® (Septodont). The components of each pulpcapping material and their manufacturers are reported in Table 1. Odontoblast cell line culture condition The mouse odontoblast cell line (MDPC-23) was kindly provided by Dr Jacques Eduardo (Dept. Cariology, Restorative Sciences, Endodontics; University of Michigan School of Dentistry, USA). MDPC-23 cells were cultured in DMEM medium (Biowhittaker, Italy) supplemented with 10 % foetal bovine serum (FBS), 2 % glutamine, 2 % sodium pyruvate, 1 % amphotericin, and 1 % (w/v) streptomycin/penicillin at 37 °C in 5 % CO2 atmosphere. The cells were routinely detached using a trypsin-EDTA solution for 2 minutes at 37 °C, and resuspended in DMEM medium. For the cytotoxicity tests, MDPC-23 cells were deposited in the lower chamber of the 24-well culture plate and left for 4 hours at 37 °C before any experiment. Cytotoxicity tests Cytotoxicity tests were performed with the Transwell insert (Sigma-Aldrich, St. Louis, MO, USA) methodology (25). The cytotoxicity of the seven pulp-capping materials was assessed with MDPC-23 cells grown in the lower
chamber of a 24-mm diameter Transwell plate with a 0.3 mm pore size polycarbonate membrane. Each pulp-capping material (300 µL) was mixed following manufacturer’s instructions onto the Transwell membrane of the inner chamber. The Transwell membrane of the inner chamber containing the pulp capping materials was then placed into the lower chamber of the 24-well culture plate containing at the bottom 5x104 cells/well and incubated at 37 °C in 5 % CO2 atmosphere for 24, 48, and 72 h, respectively. Some wells were incubated with only tissue culture media (negative control) whereas others with a 10 % dilution of 30 % H2O2 for 72 h (positive control). At the end of each incubation time, the cell viability was performed with Alamar blue test at 24, 48, and 72 h. The results were presented as percentage of cell viability referred at cells incubated in the absence of pulp capping materials set at 100 %. The MDPC-23 treated with H2O2 did not show cell viability (data not shown). Five replicates for each pulp cupping material were used for each experiment performed in duplicate. The vitality test to Alamar blue reagent acts as an indicator of cell health, determining the reducing power in order to quantitatively measure the proliferative capacity; the reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3-4
Table 1 Characteristic of tested materials Material
Components
LOT
Manufacturer
Two-paste system made of a base paste (1,3-butylene glycol disalicylate, zinc oxide, calcium phosphate, calcium tungstate, iron oxide pigments) and a catalyst paste (calcium hydroxide, N-ethyl-o/p-toluene sulphonamide, zinc oxide, titanium oxide, zinc stearate, iron oxide pigments)
120717
Dentsply Tulsa Dental, Johnson City, TN, USA
Water-based calcium dihydroxide paste
1246209
Voco GmbH, Cuxhaven, Germany
Calcimol LC
Light-curing radiopaque one-component material containing urethane dimethacrylate resin, calcium dihydroxide, dimethylaminoethyl-methacrylate, TEGDMA
1244494
Voco GmbH, Cuxhaven, Germany
Theracal LC®
Light-curing, resin-modified calcium silicate filled liner single paste containing CaO, calcium silicate particles (type III Portland cement), Sr glass, fumed silica, barium sulphate, barium zirconate and resin containing Bis-GMA and PEGDMA
1200012524
Bisco Inc, Schamburg, IL, USA
ProRoot MTA®
Powder containing calcium phosphate, calcium oxide, silica, bismuth oxide.
12001879
Dentsply Tulsa Dental, Johnson City, TN, USA
MTA-Angelus®
Powder containing type Portland cement, bismuth oxide, tricalcium silicate, dicalcium silicate, tricalcium aluminate tetracalcium aluminoferrite
24120
Angelus, Londrina, PR, Brazil
Powder containing tricalcium silicate, calcium carbonate and zirconium oxide. Liquid containing water, calcium chloride (accelerator) and modified polycarboxylate
B06562
Septodont, Saint-Maurdes-Fosses, France
Dycal®
Calcicur®
®
Biodentine®
Poggio C, et al. In vitro cytotoxicity evaluation of different pulp capping materials Arh Hig Rada Toksikol 2015;66:181-188
hours at 37 °C. The degree of fluorescence and the relative values of absorbance were then acquired by reading in a spectrophotometer (BioRad Laboratories, Hercules, CA, USA) at a wavelength of 595 nm. For further control, the percentage of murine odontoblast vitality, at 72 hours, was also assessed with the MTT assay [(3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide)] (Sigma-Aldrich, St. Louis, MO, USA). The MTT test is a standard colorimetric assay for measuring the activity of enzymes that reduce MTT to formazan (a salt blue) in the mitochondria, giving the substance a blue/purple colour. This reaction is assessed and measured by the spectrophotometric reading of the sample, at a wavelength of 570 nm by a microplate reader (BioRad Laboratories, Hercules, CA, USA). Confocal Laser Scanning Microscope (CLSM) Once performed, the cytotoxicity test of the different materials, the Transwell inserts was removed and the land was eliminated from the culture plate. After washing the slides with Buffer-TES, 250 mL of 10 mmol L-1 solution of the fluorescent dye PSVue480™ was added per well, in order to detect the presence of apoptotic cells present in culture. The loss of plasma membrane asymmetry is an early event in apoptosis, independent of cell type, resulting in the exposure of phosphatidylserine (PS) residues at the outer plasma membrane leaflet (26). PSVue reagents are a family of fluorescent probes containing a bis (zinc2+dipicolylamine) group (Zn-DPA), a motif that has been found to bind with high affinity to surfaces enriched with anionic phospholipids, especially phosphatidylserine (PS) exposed on cell membranes. After 2 hours, the solution of PSVue was eliminated and the washing of the plate was carried out with abundant Buffer-TES. The next step involved the addition of the dye Hoechst 33342, affine to DNA for viable cells. After 15 minutes, the images were acquired using CLSM (Carl Zeiss AG, Jena, Germany). Statistical analysis For each pulp capping material, the mean and standard deviation were calculated. Due to the fact that the variance in the population is unknown and the number of observations per material is less than 30 we applied the T-test, after assuming the equality of the variances. The null hypothesis was that there were no differences in the number of vital cells among the pulp capping materials tested for ∝=0.05. The analysis was applied for the data obtained at each time. T test for paired data was conducted to investigate the differences in the number of vital cells after 72 hours for each material. The base assumption to build the analysis was that the vitality should become lower in time; and this is the alternative hypothesis which guided this analysis for ∝=0.05. H0:μd≥0 & H1:μd<0, where μd represents the mean of the differences as shown in Table 2. The analysis was
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conducted with Stata/SE 12.0 for Mac (StataCorp, College Station, TX, USA).
RESULTS Cytotoxicity tests Alamar blue test and MTT assay results are reported in Figure 1. With regard to Alamar blue test (A), Biodentine® showed the highest percentage of cell biocompatibility if compared to the other pulp capping materials. Biodentine® did not show difference in cell viability at the three incubation times whereas MTA-Angelus® and ProRoot MTA® (70 %) were less biocompatible at 72 h; Calcicur® showed a discrete cell biocompatibility (50-60 %) whereas Calcimol LC® and TheraCal LC® were quite cytotoxic but only at the longest incubation time (72 h). Dycal® showed the highest cytotoxic effect (10 % cell viability) among the pulp capping materials independently of the culture times. MTT test (B) confirmed the percentage ratios between the various materials and between the materials and the positive/negative controls determined with the Alamar blue test. MTA-Angelus® showed the best percentage of vitality of all. In general, even though the relationships between the various materials were similar, there was a slight increase in the mean number of cells. CLSM In Figure 2, the CLSM images representative of MDPC23 cells are reported. As clearly shown, in the negative control (A) we can see the nuclei stained in blue with Hoechst, which is used to stain live cells. H2O2 is very cytotoxic and the cells were stained in green fluorescent with PSVue480™ reagent (B). The CLSM images obtained after incubation with different pulp capping materials confirmed the cytotoxicity results: Biodentine® (I), MTAAngelus® (H) and ProRoot MTA® (G) were not cytotoxic, whereas Calcicur® (D) showed some cells fluorescent in green; Calcimol LC® (E) and TheraCal LC® (F) were very slightly cytotoxic if compared to the negative control (A); a few cells were observed in the presence of Dycal®, indicating a high level of cell cytotoxicity (C). Statistical analysis Results regarding statistical analysis are reported in Tables 2-4. As for Alamar blue test (Table 2 and 3), after 24 hours there were no differences in the number of vital cells among ProRoot MTA®, MTA-Angelus®, Biodentine® and the negative control (P>0.05). These results changed uniformly after 48 hours and maintained no significant differences among the materials. After 72 hours, all three materials differed from the negative control, in particular ProRoot MTA® and MTA-Angelus® gained a similar and lower number of vital cells than the control, respectively,
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Poggio C, et al. In vitro cytotoxicity evaluation of different pulp capping materials Arh Hig Rada Toksikol 2015;66:181-188
Figure 1 MDPC-23 cells viability of the different pulp capping materials by using Transwell method. The cell viability was assessed with Alamar blue (A) and MMT (B) tests. Y-axis-percentage of cell viability referred at cells incubated in the absence of pulp capping materials set at 100 %
Figure 2 CLSM images of apoptosis assay. MDPC-23 cells were cultured in the absence (A) or in the presence of: H2O2 (B), Dycal® (C), Calcicur® (D), Calcimol LC® (E), TheraCal LC® (F), ProRoot MTA® (G), MTA-Angelus® (H), and Biodentine® (I)
185
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Table 2 Mean±standard deviation of the number of vital cells for each material tested with Alamar blue Material
24 h
Dycal
48 h
43000±2326
®
Calcicur® Calcimol LC
®
ProRoot MTA
30000±1868n
i
215000±16257b
300000±40580g
262000±7454m
195000±27777
165000±14107
33000±2152n
b
TheraCal LC®
72 h
22000±5740
c
h
208000±20529b
48000±8196j
35000±1193n
475000±53675
f
465000±55629
333000±33157k
MTA-Angelus®
468000±72158a
522000±56089f
333000±59216k
Biodentine
533000±60897
592000±20182
533000±42179l
500000±0a
522000±0f
466000±0o
®
®
a
a
Negative control
f
Positive control* 37000±2738e 25000±1850i Means with the same superscript letters are not significantly different (P>0.05) * cells exposed to a 10 % dilution of 30 % H2O2 for 72 hours
while Biodentine® showed no decrease, as confirmed by the analysis for paired data. Calcicur®, Calcimol LC® and TheraCal LC ® showed similar results after 24 hours (P>0.05); however, the number of vital cells varied significantly after 48 hours for Calcicur® and for TheraCal LC®. The analysis for paired data showed similar results after 72 hours for Calcimol LC® and TheraCal LC®, while Calcicur® maintained a higher number of vital cells. After 24 and 48 hours no significant differences were maintained between Dycal® and the positive control (P>0.05), while after 72 hours a lower biocompatibility was registered for the positive control (P<0.05). When the MTT test (Table 4) was applied no significant differences were recorded among ProRoot MTA®, MTA-Angelus®, Biodentine®, and the negative control (P>0.05). Lower values in the percentage of vitality were obtained with the remaining materials; in particular Calcimol LC® and TheraCal LC® maintained similar results (P>0.05) lower than Calcicur® (P<0.05) and higher than Dycal® (P<0.05).
18000±1332p
DISCUSSION In the last decade, many experimental and clinical studies have been carried out to develop and test new dental materials endowed with safe biocompatibility and antiinfective properties (26-28). The material used should provide an appropriate host response. This means that the tissues that come in contact with the materials should not show any toxic, irritating, inflammatory, allergic, genotoxic or carcinogenic response (29). Pulp capping materials should act as a barrier that protects the vitality of the entire pulp tissue by covering the minimal exposed tissue and by preventing further endodontic treatments. This study evaluated the cytocompatibility of the assayed materials towards a murine odontoblast cell line (MDPC-23). Although a recent study by Moodley et al. (30) reported that human pulp cell-lines showed higher sensitivity than murine 3T3 cell-lines, many authors have recently used murine odontoblasts for cytocompatibility evaluations of dental materials (31, 32). Studies using odontoblast-like
Table 3 Differences between the number of vital cells after 72 hours (μ72) and the number of vital cells after 24 hours (μ24) calculated for each material. μd represents the mean of the differences. Means with the same superscript letters are not significantly different (P>0.05) Material Dycal
24 h
72 h
μd=μ72-μ24
43000±2326
30000±1868
-13000
215000±16257b
262000±7454m
47000
Calcimol LC
195000±27777
33000±2152
-162000
TheraCal LC®
208000±20529b
35000±1193n
®
c
Calcicur® ®
ProRoot MTA
n
b
n
-173000
475000±53675
333000±33157
-142000
MTA-Angelus®
468000±72158a
333000±59216k
-135000
Biodentine
533000±60897
533000±42179
0
Negative control
500000±0
466000±0
-34000
Positive control
37000±2738e
18000±1332p
-19000
®
®
a
a
a
μd μd=μ72-μ24 * cells exposed to a 10 % dilution of 30 % H2O2 for 72 hours
k
1
o
-80111
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Poggio C, et al. In vitro cytotoxicity evaluation of different pulp capping materials Arh Hig Rada Toksikol 2015;66:181-188
Table 4 Percentage of vitality registered for each material with the MTT test after 72 hours. Percentages with the same superscript letters are not significantly different (P>0.05) Material
MTT (%)
Dycal
9.15s
Calcicur®
21.56t
Calcimol LC®
15.04r
TheraCal LC®
14.41r
ProRoot MTA®
28.15q
MTA-Angelus®
35.88q
Biodentine
33.27q
Negative control
26.82q
®
cells are important because odontoblasts make up the layer of cells that line the periphery of the pulp and are the first cells affected by substances that reach the pulp chamber via transdentinal diffusion (33). In this in vitro study, cell viability was recorded at three different times (24, 48, and 72 h) using the Transwell insert methodology by Alamar blue test. In accordance with our previous studies and for further control, the percentage of cell vitality was also assessed with the MTT assay at the end of the incubation period (72 hours) (34). The Transwell insert methodology is a non-direct contact test. The advantage of using a nondirect contact test for the evaluation of dental material cytotoxicity has to do with the fact that cells and materials are usually separated (25). Even though all of the materials that we tested are already available on the market and have passed safety tests, our purpose was to evaluate which material may be preferred in terms of cytotoxic effects in clinical practice use. Our results indicate certain considerable negative effects after the application of each of the materials tested, except Biodentine®, on the culture plate. As shown in Figure 1, the decrease in the number of cells in the culture plate is sizable for calcium hydroxide-based materials. Dycal® demonstrate lower rates of vitality and a strong cytotoxic capability. The remnant calcium hydroxide-based materials (Calcicur®, Calcimol LC®) showed a reduction in the percentage of cell viability after 72 h, suggesting a different rate of biocompatibility long-term. Although Cavalcanti et al. (35) reported lesser cytotoxic effects for calcium hydroxide compared to adhesive pulp-capping materials our results confirm the conclusions of other authors (36-38) regarding the non-complete biocompatibility of calcium hydroxide-based materials. Calcium hydroxide is important in protecting the pulp from thermal, mechanical, and microbiological stimuli (4) because of its antibacterial action and its property of stimulating sclerotic and reparative dentin formation. However, multiple tunnel defects and cell inclusions in bridges following pulp capping with calcium hydroxide have been demonstrated (36). This may lead to leakage and bacteria penetration into pulp tissue. Furthermore, it has equally been demonstrated that,
due to the alkalinity of its pH, calcium hydroxide induces the formation of a layer of coagulation necrosis, when it is in direct contact with the dental pulp (6). TheraCal LC® showed a dramatic decline in the percentage of cell viability at 72 h, so as to be comparable to calcium hydroxide-based materials. This result is quite surprising as the main constituent of TheraCal LC® is Portland cement, whose biocompatibility has been demonstrated in several studies (39, 40). Very different results were obtained from the analysis of the MTA-based materials (ProRoot MTA® and MTA-Angelus®). Both materials reported an excellent percentage of vitality with Alamar blue test and MTT assay after 72 h. Compared to calcium hydroxide, MTA has the ability to induce the formation of a bridge of hard tissue of greater thickness, also managing to cause less inflammation with limited pulp tissue necrosis (less caustic effect) shortly after its application (41, 42). In this study, the in vitro analysis regarding biocompatibility showed the best percentage of for Biodentine®’s cell viability. During the 72 hours of application of Biodentine® on the culture plate, the modifications that occurred underlined the positive trend of mitochondrial activity. Considering the interface between dentin and Biodentine® with confocal microscopy, an increased content of carbonate and the creation of a hybrid layer was demonstrated (43, 44). Furthermore, Biodentine® showed the ability to induce the differentiation of odontoblasts starting from pulp progenitor cells, forming a mineralizing matrix with the characteristics of dentin (45). In accordance with Novicka et al. (46) and Zhou et al. (47), the present study underlined that Biodentine® caused a cytotoxic effect similar to that by MTA and that it may be considered an alternative to MTA in pulp-capping treatment, differing from calcium hydroxide-based materials, which exhibit higher cytotoxicity. Studies such as this are important since they are designed to find, among the available dental materials, those that show the highest degree of biocompatibility. This in vitro study reported significant differences in the biocompatibility effects of the seven tested materials. In particular, calcium-hydroxide based ones demonstrated important cytotoxic activity on murine odontoblasts. Although these findings are limited to an in vitro model system and cannot be directly extrapolated to in vivo situations, they point to the necessity of taking precautions in applying such materials in clinical practice, as they might cause potential irritations and cellular damage. Conflict of interest statement The authors deny any conflict of interest. REFERENCES 1. European Society of Endodontology. Quality guidelines for endodontic treatment: consensus report of the European
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33. Costa CAS, Vaerten MA, Edwards CA, Hanks CT. Cytotoxic effects of current dental adhesive systems on immortalized odontoblast cell line MDPC-23. Dent Mater 1999;15:434-41. doi: 10.1016/S0109-5641(99)00071-8 34. Ceci M, Beltrami R, Chiesa M, Colombo M, Poggio C. Biological and chemical-physical properties of root-end filling materials: A comparative study. J Conserv Dent 2015;18:94-9. doi: 10.4103/0972-0707.153058 35. Cavalcanti BN, Rode SM, Marques MM. Cytotoxicity of substances leached or dissolved from pulp capping materials. Int Endod J 2005;38:505-9. PMID: 16011767 36. Holland R, Souza V, Mello W, Nery MJ, Bernabé PF, Otoboni JA Filho. Permeability of the hard tissue bridge formed after pulpotomy with calcium hydroxide: a histologic study. J Am Dent Assoc 1979;99:472-5. PMID: 112134 37. Goldberg F, Massone EJ, Spielberg C. Evaluation of the dentinal bridge after pulpotomy and calcium hydroxide dressing. J Endod 1984;10:318-20. doi: 10.1016/S00992399(84)80186-7 38. Asgary, S, Eghbal MJ, Parirokh M, Ghanavati F, Rahimi H. A comparative study of histologic response to different pulp capping materials and a novel endodontic cement. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:609-14. doi: 10.1016/j.tripleo.2008.06.006 39. Min KS, Kim HI, Park HJ, Pi SH, Hong CU, Kim EC. Human pulp cells response to Portland cement in vitro. J Endod 2007;33:163-6. doi: 10.1016/j.joen.2006.07.022 40. Zeferino EG, Bueno CE, Oyama LM, Ribeiro DA. Ex vivo assessment of genotoxicity and cytotoxicity in murine fibroblasts exposed to white MTA or white Portland cement with 15% bismuth oxide. Int Endod J 2010;43:843-8. doi: 10.1111/j.1365-2591.2010.01747.x
41. Tabarsi B, Parirokh M, Eghbal MJ, Haghdoost AA, Torabzadeh H, Asgary S. A comparative study of dental pulp response to several pulpotomy agents. Int Endod J 2010;43:565-71. doi: 10.1111/j.1365-2591.2010.01711.x 42. Eskandarizadeh A, Shahpasandzadeh MH, Shahpasandzadeh M, Torabi M, Parirokh M. A comparative study on dental pulp response to calcium hydroxide, white and grey mineral trioxide aggregate as pulp capping agents. J Conserv Dent 2011;14:351-5. doi: 10.4103/0972-0707.87196 43. Atmeh AR, Chong EZ, Richard G, Festy F, Watson TF. Dentin-cement interfacial interaction: calcium silicates and polyalkenoates. J Dent Res 2012;91:454-9. doi: 10.1177/0022034512443068 44. Grech L, Mallia B, Camilleri J. Characterization of set Intermediate Restorative Material, Biodentine, Bioaggregate and a prototype calcium silicate cement for use as root-end filling materials. Int Endod J 2013,46:632-41. doi: 10.1111/ iej.12039 45. About I. Dentin regeneration in vitro: the pivotal role of supportive cells. Adv Dent Res 2011;23:320-4. doi: 10.1177/0022034511405324 46. Novicka A, Lipski M, Parafiniuk M, Sporniak-Tutak K, Lichota D, Kosierkiewicz A, Kaczmarek W, BuczkowskaRadlińska J. Response of human dental pulp capped with biodentine and mineral trioxide aggregate. J Endod 2013;39:743-7. doi: 10.1016/j.joen.2013.01.005 47. Zhou HM, Shen Y, Wang ZJ, Li L, Zheng YF, Häkkinen L, Haapasalo M. In vitro cytotoxicity evaluation of a novel root repair material. J Endod 2013;39:478-83. doi: 10.1016/j. joen.2012.11.026
Procjena citotoksičnosti materijala za prekrivanje zubne pulpe u uvjetima in vitro: usporedno istraživanje Direktnim prekrivanjem zubne pulpe štiti se njena izložena površina kako bi se zadržala vitalnost te očuvala funkcionalna i biološka aktivnost. Cilj ovoga istraživanja bio je usporediti biokompatibilnost sedam različitih materijala za prekrivanje zubne pulpe u uvjetima in vitro: Dycal®, Calcicur®, Calcimol LC®, TheraCal LC®, ProRoot MTA®, MTA-Angelus® i Biodentine®. Primjenom metode Transwell insert® i testa preživljenja stanica s bojom Alamar Blue® procijenili smo citokompatibilnost navedenih materijala na staničnim kulturama mišjih odontoblasta (MDPC-23) izlaganima ispitivanim materijalima u tri različita vremena (24, 48 i 72 h). Radi dodatne kontrole, nakon 72 sata proveden je i MTT-test za procjenu vijabilnosti stanica. Morfološka analiza mišjih odontoblasta provedena je konfokalnom laserskom skenirajućom mikroskopijom. Rezultati upućuju na značajno različitu biokompatibilnost među materijalima drugačijeg sastava. Materijal Biodentine® te proizvodi zasnovani na mineral trioksid-agregatu (MTA) pokazali su nižu citotoksičnost, bitno se razlikujući od materijala zasnovanih na kalcij hidroksidu, koji su bili više citotoksični. Iako je naše istraživanje ograničeno in vitro uvjetima u kojima je provedeno, naš pronalazak da je Biodentine® uzrokovao citotoksičan učinak sličan MTA-u upućuje na to da bi se mogao koristiti kao alternativa u prekrivanju zubne pulpe, jer je poznato kako materijali zasnovani na kalcij hidroksidu iskazuju veće citotoksične učinke. KLJUČNE RIJEČI: biokompatibilnost; mišji odontoblasti; MTT-test; test preživljenja stanica s bojom Alamar Blue®; zubne ispune
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Gürkan M, Hayretdağ S. Acute toxicity of maneb in the tadpoles of common and green toad Arh Hig Rada Toksikol 2015;66:189-195
Original article
DOI: 10.1515/aiht-2015-66-2642
Acute toxicity of maneb in the tadpoles of common and green toad Mert Gürkan and Sibel Hayretdağ Department of Biology, Zoology Section, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey [Received in March 2015; CrossChecked in March 2015; Accepted in September 2015] Pesticides used in agriculture can have hazardous effects on aquatic organisms, and amphibians are even more threatened than other aquatic vertebrates. Maneb is widely used to control fungal diseases on crops, fruits, and vegetables. The aim of this study was to investigate the acute toxic effects of maneb on the common (Bufo bufo) and green toad (Pseudepidalea viridis) tadpoles. Tadpoles at the development stage 21 were exposed to maneb (0-5 mg L-1) for 120 h. Maneb LC50 values at hour 120 were 1.966 mg L-1 for B. bufo and 0.332 mg L-1 for P. viridis. To the best of our knowledge, these are the first published LC50 findings for the two species. Visceral oedema and tail deformations were observed in both species. We also observed liver necrosis, pronephric tubule deformations, somite deteriorations, and visceral oedema at maneb concentrations ≥0.1 mg L-1 for B. bufo and ≥0.05 mg L-1 for P. viridis. Our results show that B. bufo tadpoles have a much higher resilience to maneb than P. viridis tadpoles. This resilience seems to be related to the larger size of the B. bufo tadpoles and their ability to metamorphose faster in adverse conditions. Future research should look into the mechanisms of toxic action of maneb in anurans. KEY WORDS: Bufo bufo; fungicide; histopathology; morphology; Pseudepidalea viridis Pesticides used in agriculture often cause damage and death in non-target organisms and their presence in the environment is gradually increasing as a result of unconscious and/or uncontrolled use (1). One of the most affected organisms among aquatic vertebrates are amphibians, as they use shallow, lentic, and seasonal puddles for breeding and spend their entire larval life in water (2, 3). The decline in the populations of some amphibian species, which is reported on the global scale, has urged toxicologists to intensify their studies in these species (4-6). Maneb (manganese ethylene-bis-dithiocarbamate, CAS Number: 12427-38-2) is a dithiocarbamate pesticide that disturbs endocrine function (7). In agriculture it is often used as a fungicide to treat fruits, vegetables, wheat, and shelled crops such as walnut, hazelnut, and pistachio (8) and is quite common in Turkey (9). The Bufonidae are one of the largest families of anurans and its genera Bufo and Pseudepidalea are widely distributed in Turkey. Widespread in Europe and northern Eurasia, the common toad (Bufo bufo) is also common in our country, especially in north-western Anatolia, Thrace, the Aegean region, and along the Black Sea and Mediterranean shores, whereas the green toad (Pseudepidalea viridis) is widespread in eastern Europe, Russia, and Thrace (10). Correspondence to: Mert Gürkan, Department of Biology, Zoology Section, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Turkey; E-mail: mertgurkan@comu.edu.tr; gurkanmert@hotmail.com
To the best of our knowledge there are only two studies to have studied the toxic effects of maneb in amphibians (11, 12). Considering how common B. bufo and P. viridis are in Turkey, we decided to be the first determine the lethal concentrations of maneb in tadpoles of these two species and to see which morphological and histological anomalies were likely to occur at their most sensitive developmental stages (13, 14).
MATERIALS AND METHODS Animals The B. bufo and P. viridis tadpoles were obtained from adult specimens collected in Çanakkale, Turkey in February 2010. The adult specimens were carefully collected by hand from areas near seasonal puddles, generally after the sunset, and brought to the laboratory in wet cloth bags. In order for spawning to take place, the specimens in amplexus were placed in plastic containers (259×476×209 mm) with 5-cmdeep tap water and tree branches. The adult specimens were removed after the fertilisation and the fertilised B. bufo and P. viridis eggs were divided into four equal parts and transferred to 350×350×250 mm glass aquaria with 10-cmdeep tap water. Water temperature, dissolved oxygen, and pH were controlled using a Thermo Orion 3 Star portable ecological kit (Thermo Fisher Scientific, Waltham, MA, USA) and ranged from 15 to 17 °C, 8.2 to 8.5 mg L -1, and
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Gürkan M, Hayretdağ S. Acute toxicity of maneb in the tadpoles of common and green toad Arh Hig Rada Toksikol 2015;66:189-195
7.5 to 7.7, respectively. At the early stages of development, the tadpoles were fed vegetable feed for fish, and at later stages boiled lettuce and spinach. The light/dark cycle corresponded to natural seasonal conditions at this stage of tadpole development. Experimental design The tadpoles were divided in six groups of about 20 per species (in triplicate), of which one was control (unexposed) and the remaining five were exposed to 98 % maneb (SigmaAldrich, Saint Louis, MO, USA) in one of the following concentrations: 0.01, 0.05, 0.1, 1, and 5 mg L-1. Exposure started at their developmental stage 21, when the external gills are fully developed, and lasted 120 h. The stock solution was prepared afresh at the start of exposure and applied to the groups by thinning it out with soft water to the concentrations mentioned above. Our maneb concentrations for the acute toxicity study are based on the reported maximum solubility in water at 25 °C, which is 6 mg L-1 (15). Dead tadpoles were recorded and removed together with faeces every 24 hours.
Statistical analysis For statistical analysis we used the SPSS 17.0 program (IBM, New York, NY, USA). Lethal concentrations (LC10, LC50, and LC90) were calculated with Finney’s probit analysis. With this method we created a concentrationsurvival curve showing the percentages of surviving tadpoles in relation to maneb concentrations. Tadpole survival in each group was also estimated using the KaplanMeier estimate, and the differences between the groups were assessed with the log-rank (Mantel-Cox) test for pairwise comparisons. Morphological measurement data were tested for normality with the Kolmogorov-Smirnov test, and differences in measurements (wet weight, total body length, tail length, and width) between the groups with one-way analysis of variance (ANOVA). Differences <0.05 were considered statistically significant.
RESULTS
Morphological study The tadpoles were measured for wet weight (g), total body length (mm), tail length (mm), and width (mm) at the beginning and the end of the experiment. Wet weight was measured on a scale sensitive to 0.0001 g. The tadpoles were placed onto a slide of determined weight and excess water was dried with paper. To measure total body length, tail length, and width we used a digital compass sensitive to 0.01 mm. These measurements were made on 10 tadpoles from each group. For detailed morphological exam we used an Olympus SZ binocular microscope (Olympus, New York, NY, USA) with bottom illumination. We looked for anomalies such as visceral oedema, tail deformations, and spinal curvature, and took microscopic photographs (at 8x and 40x magnification). Histopathological study At the end of the experiment (hour 120), all surviving tadpoles were evaluated for histopathological changes by fixing them in Bouin’s solution for 8 h and then preserving them in 70 % ethyl alcohol (Sigma-Aldrich) until the preparation day. On the preparation day, the tadpoles were passed through 80 %, 90 %, 96 %, and absolute ethyl alcohol series and later through xylene (Sigma-Aldrich) and paraffin series to be embedded in paraffin blocks. We made 8-µmthick cross sections and stained them with haematoxylin & eosin (H&E; Sigma-Aldrich) for histopathological examination with an Olympus CX21 light microscope. We also took photographs with a camera adapted to the Olympus BX51 light microscope and analysed them with Olympus Analysis LS software.
Lethal concentrations and survival Table 1 shows the LC10, LC50, and LC90 values of maneb in B. bufo and P. viridis tadpoles after 120 h of exposure. To the best of our knowledge, these are the first LC findings in these two species. Table 1 The 120-hour lethal concentrations (LC10, LC50, and LC90) of maneb in B. bufo and P. viridis tadpoles Lethal concentrations (mg L-1) Species
LC10 (95 % CI)
LC50 (95 % CI)
LC90 (95 % CI)
B. bufo
0.700 (0.071-1.341)
1.966 (0.818-3.313)
5.518 (3.280-23.457)
P. viridis
0.066 (0.011-0.015)
0.332 (0.145-0.626)
1.664 (0.857-5.446)
Figure 1 shows the survival rates in B. bufo and P. viridis tadpoles from the beginning (0 h) to the end (120 h) of the experiment. Morphological findings Table 2 shows the number of morphological anomalies in the ten analysed B. bufo and ten P. viridis tadpoles. B. bufo At the end of the experiment, no morphological anomaly was found in control tadpoles (Figure 2a), 95 % of which survived, completed their operculum development, and reached stages 25 to 27, at which the hind limb starts to
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Gürkan M, Hayretdağ S. Acute toxicity of maneb in the tadpoles of common and green toad Arh Hig Rada Toksikol 2015;66:189-195
Table 2 Morphological anomalies in B. bufo and P. viridis tadpoles (N=10 each) Maneb (mg L-1) Morphological anomalies
Control
0.01
0.05
0.1
1
5
B. bufo
P. viridis
B. bufo
P. viridis
B. bufo
P. viridis
B. bufo
P. viridis
B. bufo
P. viridis
B. bufo
P. viridis
Visceral oedema
0
0
0
1
1
3
3
3
5
6
8
9
Tail deformation
0
0
0
1
1
2
4
3
5
7
7
8
develop. Maneb concentrations of 0.01 and 0.05 mg L-1 caused no significant anomaly except for slight tail deformations. In addition, no significant developmental delay was detected in these groups. In the tadpoles exposed to maneb concentrations of 0.1 and 1 mg L-1 the most significant anomaly was visceral oedema. Tadpoles exposed to maneb at 0.1 mg L-1 were between developmental stages 23 and 26 (Figure 2b), whereas tadpoles exposed to 1 mg L-1 were between stages 22 and 24 (Figure 2c). The concentration-dependent effects were also visible in tail deformations (Figure 3), which were the most prominent in tadpoles exposed to maneb at 1 mg L-1. Figure 4 shows average width, tail length, and total length at the end of the experiment and Figure 5 differences in weight gain (0-120 h) between the groups. With only two surviving tadpoles, the group exposed to 5 mg L-1 was not included in morphological evaluation. P. viridis Similar to B. bufo, no morphological anomaly was found in the P. viridis control tadpoles, which reached stages 26 to 28, and developed their hind limbs (Figure 6a). Tadpoles exposed to 0.01 mg L-1 maneb also showed no anomaly and reached stages 26 to 28. Maneb started to suppress development at 0.05 mg L -1 ; this group reached developmental stages 23 to 26. In addition, it exhibited a few tail deformations. Groups exposed to maneb
concentrations 0.1 and 1 mg L-1 had not completed their operculum development and reached stages 23 to 25 and 23 to 24, respectively (Figure 6b, 6c). They also exhibited severe visceral oedema. All tadpoles exposed to maneb at 5 mg L-1 died by the end of the experiment and were not included in the morphological study. Figure 7 shows average width, tail length, and total length at the end of the experiment and Figure 8 differences in weight gain (0-120 h) between the groups. Histopathological findings Table 3 shows the number histopathological changes observed in ten B. bufo and ten P. viridis tadpoles. B. bufo Severe visceral oedema started to appear with maneb concentrations of 0.1, 1, and 5 mg L-1 (Figure 9). Liver necrosis was observed at 0.1, 1, and 5 mg L-1 and was concentration-dependent (Figure 10). Deformation of pronephric tubule epithelial cells was observed at maneb concentrations of 1 and 5 mg L-1 (Figure 11). P. viridis This species was more sensitive to maneb than B. bufo. Mild visceral oedema was already observed at 0.01 and
Figure 1 Kaplan-Meier 120-hour survival curves at different maneb concentrations in: a) B. bufo and b) P. viridis tadpoles
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Gürkan M, Hayretdağ S. Acute toxicity of maneb in the tadpoles of common and green toad Arh Hig Rada Toksikol 2015;66:189-195
Figure 2 Visceral oedema (e) in B. bufo tadpoles exposed to maneb: a) control (stage 27); b) 0.1 mg L-1 (stage 23); c) 1 mg L-1 maneb (stage 22)
0.05 mg L-1 and became severe at 0.1 and 1 mg L-1 (Figure 12). Advanced liver necrosis was observed at 1 mg L-1 (Figure 13). Whereas tubular deformation started to appear at 0.01 mg L-1, and it was obviously observed at 0.1 and 1 mg L-1 of maneb (Figure 14).
DISCUSSION To the best of our knowledge this acute toxicity study is the first to report lethal maneb concentrations in B. bufo and P. viridis tadpoles. Maneb was highly toxic to either of the species even at lower concentrations. Like all adverse effects observed in our study, tail deformations were concentration-dependent. Deformed tails severely limit the survival of a larva. A similar finding to ours was reported by Bancroft et al. (11) in Xenopus laevis larvae exposed to maneb. Furthermore, maneb slowed down the development of either species in a concentration-dependent manner. Others
Figure 3 Tail deformations in B. bufo tadpoles exposed to maneb: a) control (stage 27); b) 0.05 mg L-1 (stage 27); c) 0.1 mg L-1 (stage 23); d) 1 mg L-1 (stage 22)
Figure 4 Width, tail length, and total length of B. bufo tadpoles exposed to maneb. Bars represent mean values (±SEM). Different letters denote significant differences between bars (ANOVA, p<0.05)
have also reported that exposure to maneb suppresses growth and development (16, 17). The organs where histopathological changes are first observed in organisms exposed to a pollutant are the liver and the kidneys. These changes directly affect the life of an organism. Our necrosis and pronephric tubule deformation findings show that maneb affects these organs severely. Similar histopathological findings were reported in albino mice exposed to maneb and zineb (18). However, judging by the survival rates and adverse morphological, histopathological, and developmental effects, B. bufo is more resilient to maneb than P. viridis. This difference can partly be related to the fact that the B. bufo tadpoles were significantly bigger than the P. viridis tadpoles. According to Geng et al. (19), the larger the body size, the stronger the resilience to pesticide effects. Resilience is also related to growth rate and the time needed
Figure 5 Mean (±SEM) wet weight in B. bufo tadpoles at the beginning and the end of the experiment. Different letters denote significant differences between bars (ANOVA, p<0.05)
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Gürkan M, Hayretdağ S. Acute toxicity of maneb in the tadpoles of common and green toad Arh Hig Rada Toksikol 2015;66:189-195
Table 3 Histopathological findings in B. bufo and P. viridis tadpoles (N=10 each) Maneb (mg L-1) Histopathological findings
Control
0.01
0.05
0.1
1
5
B. bufo
P. viridis
B. bufo
P. viridis
B. bufo
P. viridis
B. bufo
P. viridis
B. bufo
P. viridis
B. bufo
P. viridis
Epithelial cell deformation
0
0
0
1
0
3
2
4
4
8
7
8
Liver necrosis
0
0
0
1
0
1
3
3
6
6
8
9
Visceral oedema
0
0
0
1
0
4
3
4
6
6
8
8
Tubular deformation
0
0
0
1
0
3
1
5
3
8
7
9
Figure 6 Visceral oedema (arrow) in P. viridis tadpoles exposed to maneb: a) control (stage 26); b) 0.1 mg L-1 (stage 23); c) 1 mg L-1 (stage 23)
Figure 8 Mean (±SEM) wet weight in P. viridis tadpoles at the beginning and the end of the experiment. Different letters denote significant differences between bars (ANOVA, p<0.05)
Figure 7 Width, tail length and total length of P. viridis tadpoles exposed to maneb. Bars represent mean values (±SEM). Different letters denote significant differences between bars (ANOVA, p<0.05)
Figure 9 Histopathological findings of visceral oedema (blue and yellow star) in B. bufo tadpoles exposed to maneb: a) control; b) 0.1 mg L-1, c) 1 mg L-1; d) 5 mg L-1. (Mo: medulla oblongata; Pt: pronephric tubule; Nt: notochorda; L: liver; I: intestine)
194
Gürkan M, Hayretdağ S. Acute toxicity of maneb in the tadpoles of common and green toad Arh Hig Rada Toksikol 2015;66:189-195
Figure 10 Liver necrosis (blue and yellow ellipse) in B. bufo tadpoles exposed to maneb: a) control; b) 0.1 mg L-1; c) 1 mg L-1; d) 5 mg L-1. (L: liver)
Figure 11 Pronephric tubule epithelial cell deformations (blue arrow) in B. bufo tadpoles exposed to maneb: a) control group; b) 1 mg L-1; c) 5 mg L-1. (Pt: pronephric tubule)
Figure 13 Liver necrosis (blue and yellow ellipse) in P. viridis tadpoles exposed to 1 mg L-1 maneb. (L: liver) Figure 12 Histopathological findings of visceral oedema (blue and yellow star) in P. viridis tadpoles exposed to maneb: a) control; b) 0.05 mg L-1; c) 0.1 mg L-1; d) 1 mg L-1. (Mo: medulla oblongata; Pt: pronephric tubule; Nt: notochorda; L: liver; I: intestine)
Figure 14 Pronephric tubule epithelial cell deformations(blue arrow) in P. viridis tadpoles exposed to maneb: a) 0.1 mg L-1 and b) 1 mg L-1. (Pt: pronephric tubule)
Gürkan M, Hayretdağ S. Acute toxicity of maneb in the tadpoles of common and green toad Arh Hig Rada Toksikol 2015;66:189-195
for a larva to metamorphose. Nunes et al. (20) reported earlier that B. bufo tadpoles were able to shorten the time to metamorphosis to cope better with adverse conditions. While our study may have established the toxic effects of maneb in the two species, it has not addressed the biological mechanisms of its action, and we hope that future studies with different concentrations and frog and toad species will be able to answer this question.
9. 10. 11.
Acknowledgements This study was part of a PhD thesis and was approved by the Çanakkale Onsekiz Mart University Scientific Research Projects Commission (BAP 2010/02).
12.
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1. Garcia-Munoz E, Guerrero F, Parra G. Effects of copper sulfate on growth, development and escape behavior in Epidalea calamita embryos and larvae. Arch Environ Contam Toxicol 2009;56:557-65. doi: 10.1007/s00244-0089201-y 2. Tyler MJ. Australian Frogs. A Natural History. 1st ed. Ithaca (NY): Cornell University Press; 1997. 3. Hopkins WA. Amphibians as models for studying environmental changes. ILAR J 2007;48:270-7. doi: 10.1093/ilar.48.3.270 4. Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, Waller RW. Status and trends of amphibian declines and extinctions worldwide. Science 2004;306:17836. doi: 10.1126/science.1103538 5. Beebee TJC, Griffiths RA. The amphibian decline crisis: A watershed for conservation biology? Biol Conserv 2005;125:271-85. doi: 10.1016/j.biocon.2005.04.009 6. Wyman RL. What’s happening to the amphibians. Conserv Biol 1990;4:350-2. doi: 10.1111/j.1523-1739.1990.tb00307.x 7. Edwards IR, Ferry DG, Temples WA. Fungicides and related compounds. In: Hayes WI, Laws ER, editors. Handbook of pesticide toxicology. New York (NY): Academic Press; 1991. p. 1410-5. 8. Kapoor J, Kumar A, Gupta U, Rao ALJ. Spectrophotometric determination of maneb by ternary complex formation with
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PAR and CTAB. Talanta 1994;41:2061-5. doi: 10.1016/00399140(94)00179-0 Tezcan H. Arılara dost fungusit kullanımı [Usıng bee-frıendly fungıcıdes, in Turkish]. Uludağ Arıcılık Dergisi 2009;9:328. Özeti N, Yılmaz Y. Türkiye Amfibileri [The Amphibia of Turkey, in Turkish]. Serisi. No: 151. Izmir: Ege Üniversitesi Fen Fakültesi Kitaplar; 1994. Bancroft R, Prahlad KV. Effect of ethylenebis (dithiocarbamic acid) disodium salt (nabam) and ethylenebis (dithiocarbamato) manganese (maneb) on Xenopus laevis development. Teratology 1973;7:143-50. doi: 10.1002/tera.1420070205 Zavanella T, Zaffaroni NP, Arias E. Abnormal limb regeneration in adult newts exposed to the fungucide maneb 80. A histological study. J Toxicol Environ Health 1984;13:743-5. doi: 10.1080/15287398409530535 Gosner KL. A Simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 1960; 16: 183-190 Herkovits J, Oerez-Coll CS. Stage-dependent susceptibility of Bufo arenarum embryos to cadmium. Environ Contam Toxicol. 1993; 50: 608-611 Kidd H, James DR. The agrochemicals handbook, Third edition. Royal society of chemistry information services. Cambridge, UK. 1991; 1-56 Beck SL. Prenatal and postnatal assessment of maneb exposed CD-1 mice. Reprod Toxicol 1990; 4: 283-290. doi: 10.1016/0890-6238(90)90040-3 Nemec MD, Arce GT, Holson JF, Piccirillo VJ. Assessment of the effect of orally administered maneb technical on prenatal development in the rat. Teratology 1993; 47: 430 Özbay G, Barlas N, Kolankaya D. Histopathological effects of the residual maneb and zineb in the lettuces on the liver and kidney of albino mice. J Islam Acad Sci 1991; 4: 336-339 Geng BR, Yao D, Xue QQ. Acute toxicity of the pesticide dichlorvos and the herbicide butachlor to tadpoles of four anuran species. Bull Environ Contam Toxicol 2005; 75: 343-349. doi: 10.1007/s00128-005-0759-z Nunes AL, Orizaola G, Laurila A, Rebelo R. Morphological and life-history responses of anurans to predation by an invasive crayfish: an integrative approach. Ecol Evol 2014; 4: 1491-1503. doi: 10.1002/ece3.979
Akutna toksičnost maneba u punoglavaca smeđe i zelene krastače Pesticidi u poljoprivrednoj uporabi mogu biti opasni za vodene organizme, a najugroženiji među vodenim kralježnjacima su vodozemci. Maneb se rabi za suzbijanje gljivičnih bolesti usjeva, voća i povrća. Cilj ovog istraživanja bio je utvrditi akutnu toksičnost maneba u punoglavaca smeđe (Bufo bufo) i zelene krastače (Pseudepidalea viridis). U tu smo svrhu punoglavce u 21. stadiju razvoja izložili manebu (0-5 mg L-1). Po svršetku izloženosti (120 h), LC50 maneba iznosio je 1,966 mg L-1 u B. bufo te 0,332 mg L-1 u P. viridis. Koliko znamo, ove vrijednosti dosada nisu objavljene za ove dvije vrste. Osim letalne koncentracije, pri koncentracijama maneba ≥0.1 mg L-1 za B. bufo odnosno ≥0.05 mg L-1 za P. viridis utvrdili smo i nekrozu jetara, deformacije predbubrežnoga kanalića, propadanje somita te visceralni edem. Naši rezultati pokazuju da su punoglavci smeđe krastače značajno otporniji na maneb od punoglavaca zelene krastače. KLJUČNE RIJEČI: Bufo bufo; fungicid; histopatologija; morfologija; Pseudepidalea viridis
Jurica K, et al. Quantitative analysis of arbutin and hydroquinone in strawberry tree leaves by GC-MS Arh Hig Rada Toksikol 2015;66:197-202
Original article
197
DOI: 10.1515/aiht-2015-66-2696
Quantitative analysis of arbutin and hydroquinone in strawberry tree (Arbutus unedo L., Ericaceae) leaves by gas chromatography-mass spectrometry Karlo Jurica1,*, Irena Brčić Karačonji2,*, Sandra Šegan4, Dušanka Milojković Opsenica5, and Dario Kremer3 Special Security Operations Directorate, Ministry of the Interior1, Analytical Toxicology and Mineral Metabolism Unit, Institute for Medical Research and Occupational Health2, Faculty of Pharmacy and Biochemistry, University of Zagreb3, Zagreb, Croatia, Institute of Chemistry, Technology and Metallurgy4, Faculty of Chemistry5, University of Belgrade, Belgrade, Serbia [Received in July 2015; CrossChecked in August 2015; Accepted in September 2015] The phenolic glycoside arbutin and its metabolite with uroantiseptic activity hydroquinone occur naturally in the leaves of various medicinal plants and spices. In this study, an extraction procedure coupled with gas chromatography-mass spectrometry (GC-MS) was developed to determine arbutin and hydroquinone content in strawberry tree (Arbutus unedo L., Ericaceae) leaves. The method showed good linearity (R2>0.9987) in the tested concentration range (0.5-200 µg mL-1), as well as good precision (RSD<5 %), analytical recovery (96.2-98 %), and sensitivity (limit of detection=0.009 and 0.004 µg mL-1 for arbutin and hydroquinone, respectively). The results obtained by the validated GC-MS method corresponded well to those obtained by high performance liquid chromatography (HPLC) method. The proposed method was then applied for determining arbutin and hydroquinone content in methanolic leaf extracts. The amount of arbutin in the leaves collected on the island of Koločep (6.82 mg g-1 dry weight) was found to be higher (tpaired=43.57, tc=2.92) in comparison to the amount of arbutin in the leaves collected on the island of Mali Lošinj (2.75 mg g-1 dry weight). Hydroquinone was not detected in any of the samples. The analytical features of the proposed GC-MS method demonstrated that arbutin and hydroquinone could be determined alternatively by gas chromatography. Due to its wide concentration range, the method could also be suitable for arbutin and hydroquinone analysis in leaves of other plant families (Rosaceae, Lamiaceae, etc.). KEY WORDS: Ericaceae; food safety; GC-MS; HPLC; solvent extraction Arbutin is a hydroquinone-β-D-glucopyranoside present in the leaves of various plant species, especially in Ericaceae [Arbutus unedo L. (strawberry tree), Arctostaphylos uvaursi (L.) Spreng. (bearberry), Vaccinium vitis-idaea L. (lingonberry)], while less often in some other species such as marjoram or Origanum majorana L. from the Lamiaceae family (1, 2). This phenolic glycoside is a secondary plant metabolite that forms as a plant’s response against infectious diseases and environmental stress conditions (e.g. extremely low or high temperatures) (3, 4). Arbutin is used as an antiseptic for treating urinary infections as well as a skin whitening agent in cosmetics due to its inhibitory effect on tyrosinase activity (5, 6). Its bactericidal activity is probably brought by the hydroquinone released from arbutin by the action of β-glucosidase (3, 7). After ingestion, arbutin is absorbed from the gastrointestinal tract and hydrolysed by intestinal flora to form the aglycone hydroquinone (not to be confused with the naturally-occuring hydroquinone found in certain plants following release from arbutin upon plant β-glucosidase activity) (3, 6, 8). In herbal preparations Correspondence to: Karlo Jurica, MS, Ministry of the Interior, Vukovarska 33, 10000 Zagreb, Croatia, E-mail: juricakarlo@gmail.com
Equally contributed
*
used to fight pathogenic microorganisms, hydroquinone is recognized as an active substance at the site of action (urinary tract) and is crucial for therapeutic activity. Since hydroquinone appears to have hepatotoxic, nephrotoxic, and genotoxic potential and has been detected not only in glycolysated form as arbutin, but also in free form in the leaves of several plant families (9), its application in the treatment of human urinary infections should be controlled through food safety systems such as by monitoring the dietary intake of both hydroquinone and its glycoside derivative arbutin. A. unedo, an evergreen wild shrub that grows in the Mediterranean region (10), contains many pharmacologically relevant polyphenols and one of its main phenolic compounds is arbutin (1, 11). A. unedo leaf infusions are known to have diuretic, astringent, and uroantiseptic properties and are used in folk medicine for treatment of hypertension, diabetes, and inflammation (12-15). As phenolic compounds have powerful antioxidative activity, the identification of individual compounds in leaves responsible for pharmacological activity has been the focus of interest in recent years.
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Jurica K, et al. Quantitative analysis of arbutin and hydroquinone in strawberry tree leaves by GC-MS Arh Hig Rada Toksikol 2015;66:197-202
Several methods have been reported for arbutin and/or hydroquinone quantification in leaves, including high performance liquid chromatography (HPLC) with diode array detection (DAD) (2, 11, 16), UV detection (17, 18), mass spectrometry (MS) detection (19), as well as gas chromatography-mass spectrometry (GC-MS) (20), nuclear magnetic resonance (NMR) spectroscopy (1), and densitometry (21). To the best of our knowledge, only one GC-MS method has been reported for arbutin quantification in plant leaf extracts (20) and no validated GC-MS method has been developed for the simultaneous quantification of arbutin and hydroquinone in leaves. The aims of this study were as follows: compare the effectiveness of different solvents for extracting arbutin and hydroquinone from A. unedo leaves, validate the GC-MS method for determining arbutin and hydroquinone in leaf extracts, and compare the results with those obtained by HPLC analysis. Finally, the proposed method was used to determine arbutin and hydroquinone content in A. unedo plant leaves growing at two different geographical locations.
MATERIALS AND METHODS Chemicals and reagents Standards of arbutin and hydroquinone were purchased from Sigma-Aldrich (Steinheim, Germany). Methanol, dichloromethane, ethyl acetate, acetonitrile (all of HPLC grade) and formic acid (p.a.) were purchased from Merck (Darmstadt, Germany), while acetic acid (MS grade) was purchased from Sigma-Aldrich (Steinheim, Germany). N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS) and N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA) with 1 % TMCS were obtained from Restek (Bellefonte, PA, USA). Ultrapure water (MicroPure water purification system, 0.055 µS cm-1, TKA; Thermo Fisher Scientific, Niederelbert, Germany) was used to prepare standard solutions for HPLC analysis. Plant material A. unedo leaves were collected in May 2013 on the Croatian islands of Mali Lošinj (GPS coordinates: 44°31′50″ N; 14°28′06″ E; 14 m a.s.l.) and Koločep (GPS coordinates: 42°40’34” N; 18°00’35” E; 34 m a.s.l.). The leaves of 25 randomly selected mature plants were harvested on a dry day and mixed to obtain randomly selected samples. The leaves were air-dried for twenty days in a wellventilated room at 60 % air humidity and room temperature (22 °C), single-layered and protected from direct sunlight. The dried leaves were then milled in a laboratory mill and stored in plastic containers in a dark place at room temperature.
Preparation of sample extracts and standard solutions Following literature data (4) for the evaluation of extraction efficiency, two extraction methods (sonication and vortexing) and two different volumes (5 and 15 mL) of four different solvents were tested: methanol, 80 % methanol, dichloromethane, and ethyl acetate. Three different sample weights (25, 50, and 200 mg) and two derivatisation reagents (BSTFA+1 % TMCS and MSTFA+1 % TMCS) were also considered. All of the experiments were carried out in triplicate with homogeneous plant material collected from the same site. In the final optimised procedure, 25 mg of dried leaf sample was mixed with 5 mL of methanol in an ultrasonic bath for 30 minutes at room temperature. The mixture was vortexed after 15 minutes in order to additionally facilitate the extraction process. Extracts were filtered through a 0.45-μm Whatman filter paper and aliquots of 1 mL were used for further GC-MS analyses. For the comparison of GC-MS and HPLC, 3 g of dried leaf sample was extracted with methanol using procedure described above. The obtained extracts were frozen and lyophilised in a vacuum freeze dryer Hetosic (Heto, Denmark). The lyophilisate was grounded in a laboratory mill and stored in a desiccator due to its hygroscopic characteristics. For GC-MS analysis, 25 mg of lyophilisate were mixed with 5 mL of methanol by vortexing for 10 min and an aliquot of 1 mL was used for further analysis. An aliquot of 1 mL of extract (dried leaves and lyophilisate) was evaporated to dryness under a stream of nitrogen at room temperature. The dried extract was dissolved in 100 µL BSTFA with 1 % TMCS. The mixture was heated at 65 °C for 30 min, and 1 µL of sample cooled to room temperature was injected into the GC-MS system. Each sample was analysed in triplicate and arbutin and hydroquinone content were expressed on dried leaf weight. Stock standard solution (0.5 mg mL-1) was prepared by dissolving arbutin and hydroquinone in methanol. The working standard solutions were obtained by using appropriate aliquots of stock standard solution diluted with methanol. Standard solutions were stored at –20 °C in a freezer. One-mL aliquots of a set of working standard solutions ranging from 0.5 to 200 µg mL-1 for arbutin and hydroquinone were evaporated to dryness, derivatised, and analysed in the same way as leaf extracts. GC-MS analysis The analyses of the derivatised samples and standard solutions were carried out using a Trace 1300 gas chromatograph (Thermo Scientific, Milan, Italy) coupled to a ITQ 700 ion trap mass spectrometer (Thermo Scientific, Austin, TX, USA). The analytes were separated on a TG5MS capillary column (30 m×0.25 mm ID, 0.25 µm film thickness) from Thermo Scientific (Runcorn, UK). Helium was used as the carrier gas at a flow rate of 1 mL min-1. The
Jurica K, et al. Quantitative analysis of arbutin and hydroquinone in strawberry tree leaves by GC-MS Arh Hig Rada Toksikol 2015;66:197-202
oven temperature programme was: 120 °C for 1 min, then increased to 280 °C at 20 °C min−1 and held for 3 min. Both injector and transfer line temperatures were set at 280 °C. Samples (1 µL) were injected in split mode with a split ratio of 10:1. The MS detector operated in electron impact (EI) ionisation mode. The ion source temperature was set at 220 °C. Quantification was done in selected ion monitoring (SIM) using m/z 254 for arbutin and m/z 239 for hydroquinone. External standard calibration was used: peak area of each target analyte was plotted against its concentration. The analytes were identified by matching the retention times and mass spectral data with the calibration standards. Validation of GC-MS method The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using a signal-tonoise ratio of 3 and 10, respectively. Precision was expressed as relative standard deviation (RSD) using six replicates of standard solution at concentrations of 20 and 100 µg mL-1. The recovery of the method was evaluated by analysing samples of methanolic leaf extracts of known analyte concentrations (N=6) spiked with arbutin and hydroquinone at concentrations of 20 and 100 µg mL-1. HPLC-DAD analysis Five mg of lyophilisate was dissolved in 1.5 mL of ultrapure water, and centrifuged at 13,000×g at room temperature. Clear supernatant was decanted, mixed with a standard solution of arbutin (80 µg mL-1) in 1:1 (v/v) ratio, filtered through a syringe filter (13 mm, polytetrafluoroethylene (PTFE) membrane 0.45 μm; Supelco, Bellefonte, PA, USA) and 20 μL of extract was injected into the HPLC system. An Agilent 1260 Infinity HPLC system was consisted of a 1260 Quat Pump (G1311B), 1260 ALS Autosampler (G1329B), 1260 TCC Column heater (G1316A), and DAD 1260 VL+ (G1315C) detector (Agilent Technologies, Santa Clara, CA, USA). Separations were performed using a Zorbax Eclipse Plus C18 column, 4.6×150 mm in size, with a 1.8 µm particle diameter (Agilent, Technologies, Santa
199
Clara, CA, USA). The mobile phase was (A) 0.1 % formic acid and (B) methanol. The gradient program was as follows: 0–2 min 95 % A, 2–6 min 95 % A to 85 % A, 6-8 min 85 % A to 75 % A, 8-12 min 75 % A to 30 % A, 1214 min 30 % A to 5 % A, 14-20 min 5 % A, 20–25 min 5 % A to 95 % A. The flow rate was 0.5 mL min-1. Arbutin was detected and quantified at 290 nm. Quantification was performed using the calibration curve of the arbutin standard, concentration range 20-150 µg mL-1. Data analysis Excalibur software (ver. 2.1, Thermo Scientific, San Jose, CA, USA) was used for data acquisition and chromatographic data analysis. Microsoft Office Excel 2007 was used for statistical calculations. Comparison of the amount of arbutin in the samples collected on two localities (the islands of Koločep and Mali Lošinj) was performed by one-tailed paired t-test. Linear regression analysis using the least squares method was used to evaluate the calibration curve of analytes. Data are expressed as mean±SD (standard deviation of average value).
RESULTS AND DISCUSSION Optimisation of the extraction procedure Organic solvent extraction is the most common method for extracting phenolics (22). In order to establish the most effective extraction of arbutin and hydroquinone in dried leaf material, variables such as type and volume of solvent and extraction method were optimised. Since hydroquinone was not detected in the leaf samples, a standard solution of hydroquinone in methanol was added to samples to contain 1 mg g-1 dried weight to optimise the extraction procedure. This concentration was chosen according to the literature data regarding hydroquinone content in the leaves of Arctostaphylos uva-ursi, Vaccinium vitis-idaea, and Origanum majorana (17, 18). Arbutin and hydroquinone were extracted from samples with 5 or 15 mL of methanol, 80 % methanol, ethyl acetate, and dichloromethane using
Figure 1 Effect of solvent (methanol, 80 % methanol, ethyl acetate, and dichloromethane) and extraction type (sonication and vortexing) on the extraction efficiency of arbutin naturally present in Arbutus unedo leaf samples (2.80 mg g-1) (A) and extraction efficiency of hydroquinone added to A. unedo leaf samples at 1 mg g-1 (B)
200
Jurica K, et al. Quantitative analysis of arbutin and hydroquinone in strawberry tree leaves by GC-MS Arh Hig Rada Toksikol 2015;66:197-202
Figure 2 Selected ion chromatograms for arbutin (m/z=254) and hydroquinone (m/z=239) standard solution (20 µg mL-1) (a and b) and for a leaf extract sample obtained by the proposed GC-MS method (c and d). Peaks: 1=hydroquinone bis(trimethylsilyl ether); 2=arbutin penta(trimethylsilyl ether)
vortexing for 10 min or an ultrasonic bath for 30 min. Room temperature was used during extraction to prevent the degradation of the analytes due to the increased temperature (22). Figure 1 shows the extraction efficiency for different solvents and extraction types. Ultrasound-assisted extraction with methanol gave the highest recovery for both arbutin and hydroquinone. The volume of solvent did not affect the extraction efficiency, so 5 mL were used as the volume for all of the solvents in further procedures. As mentioned, sonication has been shown to be more effective than conventional vortexing. Most relevant studies performed arbutin and/or hydroquinone extraction from leaves using ultrasound assisted extraction (2, 17, 20). As for the amount of sample, 25 mg of sample showed better method precision, less baseline noise, and much better peak shape and resolution in the chromatogram compared to 50 or 200 mg. According to this experiment, the most suitable extraction procedure for arbutin and hydroquinone was ultrasound extraction with 5 mL of methanol for 30 min using 25 mg of dried leaf sample.
Optimisation of the derivatisation reaction Due to the low volatility of the GC analysis target compounds, a derivatisation step using a silylation reagent was necessary to convert arbutin and hydroquinone to more volatile derivatives (4). BSTFA and MSTFA, both containing 1 % TMCS as the catalyst, were compared in this study. The temperature (60 °C) and reaction time (30 min) were chosen according to the preliminary experiment (data not shown) and recommendations from previous studies (4, 23). The type of derivatisation reagent did not affect the arbutin and hydroquinone peak shape and peak area. Since the BSTFA reaction produces corrosive by-products that can damage metal syringe needles and plungers as well as the capillary GC column, MSTFA+1 % TMCS was used as a derivatisation reagent in further analyses. The MSTFA+1 % TMCS volume sufficient to complete a derivatisation reaction was tested by dissolving dry extract (from leaf samples and arbutin and hydroquinone standard in methanol at 100 µg mL-1) in 100 and 200 µL of
Table 1 Analytical features of the proposed GC-MS method Analyte Arbutin
LOD (µg mL-¹)
LOQ (µg mL-¹)
0.009
0.029
Precisiona (RSD %)
Recoveryb (%)
20 µg mL-¹
100 µg mL-¹
20 µg mL-¹
100 µg mL-¹
4.3
3.8
96.2
97.5
Hydroquinone 0.004 0.017 3.9 2.8 97.3 LOD–limit of detection; LOQ–limit of quantification a refers to standard solution of arbutin and hydroquinone at two concentrations (N=6) b refers to the sample spiked with standard solution of arbutin and hydroquinone at two concentrations (N=6)
98.0
Jurica K, et al. Quantitative analysis of arbutin and hydroquinone in strawberry tree leaves by GC-MS Arh Hig Rada Toksikol 2015;66:197-202
derivatisation reagent. The results showed that a volume of 100 µL was sufficient to complete a derivatisation reaction. The optimised conditions for derivatisation were dissolving dry methanolic extract in 100 µL of MSTFA+1 % TMCS and heating the mixture at 60 °C for 30 min in a tightly capped glass tube. Analytical features of the proposed GC-MS method The proposed GC-MS conditions allowed a good separation of arbutin and hydroquinone with no interfering peaks. Figure 2 shows selected ion chromatograms for the standard solution of arbutin and hydroquinone (20 µg mL-1) and for a sample containing 2.80 mg of arbutin per g of dried leaf weight. The calibration curves were linear (R2>0.9987) over the tested concentration range (0.5200 µg mL-1). Table 1 shows the LOD, LOQ, and precision of the developed method as well as the analytical recovery of the studied compounds. The precision of the method (RSD<5 %) and analytical recovery (>96 %) were comparable to the previously developed HPLC methods for arbutin and hydroquinone quantification in leaf extracts (17, 18) or arbutin quantification in medicinal plant extracts (24) as well as to the GC-MS method for arbutin quantification in leaf extracts (20). The LOD for arbutin (0.009 µg mL-1) and for hydroquinone (0.004 µg mL-1) obtained in this study showed an improvement in method sensitivity over the HPLC methods reported by Fecka and Tu r e k ( 1 7 ) a n d Ry c h l i n s k a a n d N o w a k ( 1 8 ) (LODs>0.49 µg mL-1) and the GC-MS method reported by Lamien-Meda et al. (20) (LOD for arbutin=0.13 µg mL-1). An LOD for arbutin similar to the LOD achieved in our study was reported only for the HPLC method developed by Thongchai et al. (24) (LOD=0.005 µg mL-1). GC-MS and HPLC-DAD method comparison For a comparison of the GC and HPLC methods, lyophilisate was analysed in triplicate and results were expressed as mg of arbutin per g of dried leaf. The GC result was 2.85 mg g-1, while the HPLC analysis of arbutin content yielded 2.95 mg g-1, which indicated that these results were in good agreement. The slightly lower level of arbutin obtained by the GC method could be explained by the loss of a certain amount of arbutin during the evaporation and derivatisation process; in HPLC analysis, the procedure is applied directly to the lyophilisate. Sample analysis The determination of arbutin and hydroquinone content in leaf samples is of great importance for estimating dosages of herbal products and evaluating the toxicity of hydroquinone to insure proper uroantiseptic activity. The proposed method was applied for determining arbutin and hydroquinone levels in A. unedo leaves from two geographical locations. A. unedo leaves collected on the island of Koločep showed statistically significant
201
(tpaired=43.57, tc(one-tailed)=2.92, p≤2.63x10-4) higher arbutin content (6.82±0.12 mg g-1) compared to the leaves collected on the island of Mali Lošinj (2.75±0.06 mg g-1). The differences in arbutin content may have been related to climate and soil characteristics of the sites where the plants grow. Our results for arbutin levels in A. unedo leaves are quite similar to those reported in other studies, ranging from 0.6 mg g-1 (1) to 12.4 mg g-1 (25). Hydroquinone was not detected in any of A. unedo leaf samples, which is in accordance with results reported by Pavlović et al. (16).
CONCLUSION To the best of our knowledge, this is the first GC-MS method reported for the simultaneous analysis of arbutin and hydroquinone in leaf samples. In this study, methanol has been shown to be the most effective solvent for arbutin and hydroquinone extraction. The developed GC-MS method was shown to be sensitive, precise, and accurate. It is a good alternative for laboratories without liquid chromatographs and enables an unambiguous confirmation of the structure of the tested compounds. The analytical features of this method allow for arbutin and hydroquinone to be determined in a wide concentration range, which is why this method could also be used to analyse leaves of other plants used for therapeutic purposes. Acknowledgements This work was supported by the Mali Lošinj Tourist Board. The authors wish to thank the football club GNK Dinamo and the city of Zagreb for their generous financial support in purchasing the GC-MS instrument as well as Dr Jurasović for valuable technical support. Conflicts of interest The authors declare no conflict of interest. REFERENCES 1. Fiorentino A, Castaldi S, D’Abrosca B, Natale A, Carfora A, Messere A, Monaco P. Polyphenols from the hydroalcoholic extract of Arbutus unedo living in a monospecific Mediterranean woodland. Biochem Syst Ecol 2007;35:80911. doi: 10.1016/j.bse.2007.04.005 2. Lukas B, Schmiderer C, Mitteregger U, Novak J. Arbutin in marjoram and oregano. Food Chem 2010;121:185–90. doi: 10.1016/j.foodchem.2009.12.028 3. Blaut M, Braune A, Wunderlich S, Sauer P, Schneider H, Glatt H. Mutagenicity of arbutin in mammalian cells after activation by human intestinal bacteria. Food Chem Toxicol 2006;44:1940-7. doi: 10.1016/j.fct.2006.06.015 4. Stalikas CD. Extraction, separation, and detection methods for phenolic acids and flavonoids. J Sep Sci 2007;30:326895. doi: 10.1002/jssc.200700261 5. Chisvert A, Sisternes J, Balaguer Á, Salvador A. A gas chromatography-mass spectrometric method to determine
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skin-whitening agents in cosmetic products. Talanta 2010;81:530-6. doi: 10.1016/j.talanta.2009.12.037 European Medicines Agency. Assessment report on Arctostaphylos uva-ursi (L.) Spreng., folium [displayed 14 September 2015]. Available at http://www.ema.europa.eu/ docs/en_GB/document_library/Herbal_-_HMPC_ assessment_report/2011/07/WC500108750.pdf Hildebrand DC, Powell CC Jr, Schroth SM. Fire blight resistance in Pyrus: localization of arbutin and betaglucosidase. Phytopathology 1969;59:1534-9. PMID: 5374248 Schindler G, Patzak U, Brinkhaus B, von Niecieck A, Wittig J, Krähmer N, Glöckl I, Veit M. Urinary excretion and metabolism of arbutin after oral administration of Arctostaphylos uvae ursi extract as film-coated tablets and aqueous solution in healthy humans. J Clin Pharmacol 2002;42:920-7. doi: 10.1177/009127002401102740 de Arriba SG, Naser B, Nolte K-U. Risk assessment of free hydroquinone derived from Arctostaphylos uva-ursi folium herbal preparations. Int J Toxicol 2014;32:442-53. doi: 10.1177/1091581813507721 Šilić Č. Atlas dendroflore (drveće i grmlje) Bosne i Hercegovine. Čitluk: Matica Hrvatska Čitluk; 2005. Parejo I, Viladomat F, Bastida J, Codina C. A single extraction step in the quantitative analysis of arbutin in bearberry (Arctostaphylos uva-ursi) leaves by high-performance liquid chromatography. Phytochem Anal 2001;12:336-9. doi: 10.1002/pca.602 Afkir S, Nguelefack TB, Aziz M, Zoheir J, Cuisinaud G, Bnouham M, Mekhfi H, Legssyer A, Lahlou S, Ziyyat A. Arbutus unedo prevents cardiovascular and morphological alterations in L-NAME-induced hypertensive rats Part I: cardiovascular and renal hemodynamic effects of Arbutus unedo in L-NAME-induced hypertensive rats. J Ethnopharmacol 2008;116:28895. doi: 10.1016/j. jep.2007.11.029 Mariotto S, Esposito E, Di Paola R, Ciampa A, Mazzon E, de Prati AC, Darra E, Vincenzi S, Cucinotta G, Caminiti R, Suzuki H, Cuzzocrea S. Protective effect of Arbutus unedo aqueous extract in carrageenan-induced lung inflammation in mice. Pharmacol Res 2008;57:110-24. doi: 10.1016/j. phrs.2007.12.005 Oliveira I, Baptista P, Bento A, Pereira JA. Arbutus unedo L. and its benefits on human health. J Food Nutr Res 2011;50:73-85.
15. Amel B. Traditional treatment of high blood pressure and diabetes in Souk Ahras District. J Pharmacogn Phytotherpy 2013;5:12-20. doi: 10.5897/JPP11.065 16. Pavlović RD, Lakušić B, Došlov-Kokoruš Z, Kovačević N. Arbutin content and antioxidant activity of some Ericaceae species. Pharmazie 2009;64:656-9. doi: 10.1691/ph.2009. 9551 17. Fecka I, Turek S. Determination of polyphenolic compounds in commercial herbal drugs and spices from Lamiaceae: thyme, wild thyme and sweet marjoram by chromatographic techniques. Food Chem 2008;108:1039-53. doi: 10.1016/j. foodchem.2007.11.035 18. Rychlińska I, Nowak S. Quantitative determination of arbutin and hydroquinone in different plant materials by HPLC. Not Bot Horti Agrobo 2012;40:109-13. 19. Pop C, Vlase L, Tamas M. Natural resources containing arbutin. Determination of arbutin in the leaves of Bergenia crassifolia (L.) Fritsch. acclimated in Romania. Not Bot Horti Agrobo 2009;37:129-32. 20. Lamien-Meda A, Lukas B, Schmiderer C, Franz C, Novak J. Validation of a quantitative assay of arbutin using gas chromatography in Origanum majorana and Arctostaphylos uva-ursi extracts. Phytochem Anal 2009;20:416–20. doi:10.1002/pca.1142. 21. Pyka A, Bober K, Stolarczyk A. Densitometric determination of arbutin in cowberry leaves (Vaccinium vitis idaea). Acta Pol Pharm 2007;64:395-400. PMID: 18540157 22. Khoddami A, Wilkes MA, Roberts TH. Techniques for analysis of plant phenolic compounds. Molecules 2013;18:2328-75. doi: 10.3390/molecules18022328 23. Robbins RJ. Phenolic acids in foods: an overview of analytical methodology. J Agric Food Chem 2003;51:286687. doi: 10.1021/jf026182t 24. Thongchai W, Liawruangrath B, Liawruangrath S. Highperformance liquid chromatographic determination of arbutin in skin-whitening creams and medicinal plant extracts. Int J Cosmet Sci 2007;29:488. doi: 10.1111/j. 14682494.2007.00391_4.x 25. Pavlović DR, Branković S, Kovačević N, Kitić D, Veljković S. Comparative study of spasmolytic properties, antioxidant activity and phenolic content of Arbutus unedo from Montenegro and Greece. Phyther Res 2011;25:749-54. doi: 10.1002/ptr.3460
Kvantitativna analiza arbutina i hidrokinona u listovima planike (Arbutus unedo L., Ericaceae) plinskokromatografskom metodom uz detekciju masenim spektrometrom Fenolni glikozid arbutin i hidrokinon, njegov metabolit s uroantiseptičkim djelovanjem, prirodni su sastojci lišća različitih biljaka koje se koriste u terapeutske svrhe te začinskih biljaka. U ovom radu optimiran je postupak ekstrakcije te je razvijena i validirana plinskokromatografska metoda uz detekciju masenim spektrometrom (GC-MS) za određivanje sadržaja arbutina i hidrokinona u lišću obične planike (Arbutus unedo L., Ericaceae). Metoda je pokazala dobru linearnost (R2>0,9987) u ispitivanom koncentracijskom rasponu (0,5-200 µg mL-1), kao i dobru preciznost (RSD<5 %), analitički povrat (96,2-98 %) i osjetljivost (granica detekcije=0,009 µg mL-1 za arbutin i 0,004 µg mL-1 za hidrokinon). Rezultati dobiveni validiranom GC-MS metodom u dobroj su suglasnosti s rezultatima dobivenim metodom tekućinske kromatografije visoke učinkovitosti (HPLC). Predložena metoda je primijenjena za određivanje sadržaja arbutina i hidrokinona u metanolnom ekstraktu lišća. Sadržaj arbutina određen u lišću obične planike prikupljenom na otoku Koločepu (6,82 mg g-1 suhe mase) bio je veći u usporedbi sa sadržajem arbutina u lišću prikupljenom na otoku Malom Lošinju (2,75 mg g-1 suhe mase). Hidrokinon nije detektiran ni u jednom uzorku. Analitičke značajke predložene GC-MS metode pokazale su da se arbutin i hidrokinon mogu alternativno određivati plinskom kromatografijom. S obzirom na široki koncentracijski raspon, metoda je pogodna za analizu arbutina i hidrokinona, ne samo u lišću obične planike, nego također i u lišću biljaka drugih porodica (Rosaceae, Lamiaceae, itd.). KLJUČNE RIJEČI: ekstrakcija otapalom; Ericaceae; GC-MS; HPLC; sigurnost hrane
Viegas C, et al. Assessment of exposure to the Penicillium glabrum complex in cork industry using complementing methods Arh Hig Rada Toksikol 2015;66:203-207
Original article
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DOI: 10.1515/aiht-2015-66-2614
Assessment of exposure to the Penicillium glabrum complex in cork industry using complementing methods Carla Viegas1, Raquel Sabino1,2, Daniel Botelho1, Mateus dos Santos1, and Anita Quintal Gomes1,3 Environment and Health RG, Lisbon School of Health Technology, Polytechnic Institute of Lisbon1, Mycology Laboratory, National Institute of Health Dr Ricardo Jorge2, Institute of Molecular Medicine, Faculty of Medicine of Lisbon3, Lisbon, Portugal [Received in January 2015; CrossChecked in January 2015; Accepted in July 2015] Cork oak is the second most dominant forest species in Portugal and makes this country the world leader in cork export. Occupational exposure to Chrysonilia sitophila and the Penicillium glabrum complex in cork industry is common, and the latter fungus is associated with suberosis. However, as conventional methods seem to underestimate its presence in occupational environments, the aim of our study was to see whether information obtained by polymerase chain reaction (PCR), a molecular-based method, can complement conventional findings and give a better insight into occupational exposure of cork industry workers. We assessed fungal contamination with the P. glabrum complex in three cork manufacturing plants in the outskirts of Lisbon using both conventional and molecular methods. Conventional culturing failed to detect the fungus at six sampling sites in which PCR did detect it. This confirms our assumption that the use of complementing methods can provide information for a more accurate assessment of occupational exposure to the P. glabrum complex in cork industry. KEY WORDS: fungal exposure; molecular methods; PCR; Portugal; suberosis Cork is produced from the bark of the cork oak (Quercus suber L), which grows in the Mediterranean (1). Worldwide, cork oak plantations occupy close to 2.2 million hectares, 33 % of which in Portugal and 23 % in Spain (2). In 2012, 64.7 % of all cork was produced in Portugal, followed by Spain with 16.0 % (3). Portugal’s cork industry employs about ten thousands workers (4). This type of industry has already been associated with exposure to several fungal species, most commonly with the Penicillium glabrum complex and Chrysonilia sitophila (5-13). The presence of the P. glabrum complex in this industry involves the risk of respiratory diseases such as suberosis, a type of hypersensitivity pneumonitis that is one of the most prevalent diseases among cork workers (8-15). Conventional methods may underestimate this fungal burden, as the detection of clinically relevant species with lower growth rates such as the P. glabrum complex may be hampered in samples with fast-growing fungi such as C. sitophila (9, 16-19). We therefore decided to complement conventional methods with real-time polymerase chain reaction (PCR) in assessing fungal contamination in three cork plants in suburban Lisbon, hoping that the higher sensitivity of PCR compared to the conventional methods will give us a clearer idea of the potential health risk posed by this fungus. Correspondence to: Carla Viegas, Environment and Health RG, Lisbon School of Health Technology, Polytechnic Institute of Lisbon, Lisbon, Portugal, E-mail: carla.viegas@estesl.ipl.pt
MATERIALS AND METHODS Plants assessed This pilot cross-sectional study included three cork plants. Plant A employs 26 workers and produces cork boards for further processing by other industries. Plant B employs 93 workers and mainly produces natural bottle corks. Plant C employs 150 workers and specialises in several cork-derived articles such as cork tiles, paper, and textile. All three plants provide respiratory protection devices to their workers, but they do not use them on a regular basis. All the plants work five days a week in two eight-hour shifts. Samples were taken from the workplaces where workers spend most of their time. Sample collection Samples for both conventional and PCR methods were collected in January and February 2014 as shown in Table 1. Conventional sampling methods Air samples (50-100 L) were collected from 10 indoor sections (agglomeration, sanding, cutting, selecting, baking, tracing, grinding, pressing, mixing, and sawing) using an impactor with a flow rate of 140 L min-1 (Millipore air Tester, Millipore - Billerica, Massachusetts, USA) onto malt
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Table 1 Number of samples collected Plants
Conventional Methods
Molecular Biology
Indoor Air Samples
Surface Samples
Indoor Air Samples
Surface Samples
Plant A
5
5
4
NP
Plant B
3
3
3
NP
5
5
5
NP
Plant C NP-not performed
extract agar supplemented with chloramphenicol (0.05 %). Samplers were placed at the breathing level of 0.6-1.5 m above the floor and as close as possible to workers. An outdoor sample was also collected for reference. For a proper fungal characterisation from the assessed occupational environments, surface samples were also collected by swabbing the same indoor sites with a 10 by 10 cm square stencil, disinfected with 70 % alcohol solution between samplings, in line with the ISO 18593 requirements (20). The obtained swabs were then plated onto malt extract agar. All the collected samples were incubated at 27 °C for 5 to 7 days. The fungal species were quantified (CFU m-3 for air samples and CFU m-2 for surface samples) and identified as described by Hoog et al. (21). Sampling for PCR For PCR identification we collected 250-litre air samples through impinger method using a Coriolis µ air sampler (Bertin Technologies, Montigny-le-Bretonneux, France) at the airflow rate of 300 L min-1. Samples were collected onto 10-mL sterile phosphate-buffered saline with 0.05 % Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). The collection liquid was used for DNA extraction with the ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. Five millilitres of the liquid were centrifuged at 2500 g at room temperature for 10 min, supernatant was removed, and DNA extracted. The P. glabrum complex was identified with the realtime polymerase chain reaction (qPCR) using the RotorGene 6000 qPCR Detection System (Corbett, Quiagen, Valencia, Califórnia, USA). Reactions included 1× iQ Supermix (Bio-Rad, Berkeley, California, USA), 0.5 µM of each primer (Table 2), and 0.375 µM of TaqMan probe (Table 3) in a total volume of 20 µL. Amplification followed three steps: 40 cycles with denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s.
A non-template control was used in every PCR reaction. For positive control we used DNA samples obtained from the reference strain provided by the Mycology Laboratory of the National Institute of Health Dr Ricardo Jorge. We sequenced the internal transcribed spacers (ITS) from rDNA as well as the genes that codify the calmodulin and beta tubulin proteins. The obtained sequences matched 100 % the sequence of the reference P. glabrum complex.
RESULTS Fungal identification and load determined with conventional methods The conventional identification of fungal species in air confirmed the presence of countless colonies of C. sitophila in Plant A. In Plant B C. sitophila was also prevalent, whereas Plant C presented a larger diversity of fungal species, among which the most prevalent were Penicillium genus (76.5 %) and Geotrichum (11.8 %) (Table 3). In addition to these two genera, Aureobasidium sp., Chrysonilia sp., Cladosporium sp. and species from Aspergillus niger complex were also identified. The distribution of fungal species in the surface samples of Plants A and B was similar, with isolates from the A. fumigatus complex being the only ones found in addition to C. sitophila. In Plant C the most prevalent genera were Trichoderma and Penicillium (52.9 %; 29.4 %). Aureobasidum sp. and species belonging to A. fumigatus complex were also isolated. All three plants had higher fungal load indoors than outdoors. Fungal detection using PCR Real-time PCR identified the P. glabrum complex in 10 out of the 12 air samples, that is, in six more sampling sites than the conventional method did (Table 4). The fact that the DNA was amplified at quite high quantitation cycles
Table 2 Sequence of primers and TaqMan probes used for Real Time PCR (from http://www.epa.gov/microbes/moldtech.htm#penicillium) P. glabrum
Sequence
Primer Forward Primer Reverse Prob
5'-CATTACTGAGTGAGGGCCCTCT-3' 5'-CGTGAGGCGGGAGCA-3' 5'-CCAACCTCCCACCCGTG-3'
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Viegas C, et al. Assessment of exposure to the Penicillium glabrum complex in cork industry using complementing methods Arh Hig Rada Toksikol 2015;66:203-207
Table 3 Most prevalent fungal distribution in three cork production plants Plant A
Plant B
Plant C
Fungi species
Air (CFU m-3)
Surfaces (CFU m-2)
Air (CFU m-3)
Surfaces (CFU m-2)
C. sitophila
Air (CFU m-3)
Surfaces (CFU m-2)
Countless
Countless
Countless
Countless
40
ND
Penicillium sp.
ND
ND
ND
ND
780
5x104
A. fumigatus complex
ND
2x104
10
20x103
ND
1x104
Geotrichum sp.
ND
ND
ND
ND
120
ND
Trichoderma sp. ND-not detected
ND
ND
ND
ND
ND
9x104
(Cq) suggests that the fungal load was low. It is important to note that these values are above the limit of detection and are specific, as no amplification occurs when samples from other fungi (e.g. A. fumigatus complex and A. flavus complex) are amplified with this pair of primers. The fact that the Cq does not reflect colony-forming units might indicate that qPCR detects fungal particles that have not germinated under the growth conditions used. For example, in the air sample collected from the raw material grinding section in Plant B we did not detect any Penicillium colonies (Table 4), but the Cq value obtained by qPCR was the lowest, suggesting a higher DNA content. In other words, we might have detected a sporulated form of the fungus that did not germinate in our media.
DISCUSSION The studied plants were all microbiologically contaminated but with different levels of fungal load. All air samples with countless colonies can safely be regarded as exceeding threshold values for occupational exposure proposed by ACGIH and the World Health Organization (WHO) (22-24). Since it was impossible to assess real contamination, for health protection purposes it would be
wise to assume the most critical contamination scenario (25). Furthermore, fungi found in the surfaces samples sites can also be aerosolised (26). The Trichoderma species were isolated only in the surface samples, highlighting the importance of analysing surfaces for a more accurate assessment of fungal contamination (27). The A. fumigatus complex was present in all the plants. This saprophytic fungus is very common and may increase occupational health risk (28, 29) posed by the P. glabrum complex, since it has also been implicated in the development of suberosis (30, 31). The Penicillium genus was identified only in plant C, and the same is true for the Geotrichum and Trichoderma genera. This is probably because countless colonies of C. sitophila could have obstructed an effective counting of other colonies in the collected samples (16, 18, 19). Thanks to qPCR, however, we were able to detect the P. glabrum complex in all three plants. The use of the two types of analytical methods in this study, culture analysis and PCRbased detection, has given a more comprehensive idea of fungal contamination in cork industry. On the one hand, with the culture analysis we were able to identify and quantify organisms posing a higher occupational risk from inhalation and compare their levels with legal and scientific
Table 4 Conventional quantification of isolates belonging to Penicillium genera and molecular detection of P. glabrum Air (CFU/m3)
Surfaces (CFU/m2)
Real Time PCR (Air) (Cq– Cycle threshold)
Tracing - Plant A
ND
ND
-
Cutting - Plant A
ND
ND
+ (37.07)
Baking - Plant A
ND
ND
+ (35.07)
Selecting - Plant A
ND
ND
+ (35.30)
Rectification - Plant B
ND
ND
+ (36.45)
Grinding of raw materials - Plant B
ND
ND
+ (34.71)
Grinding in mills - Plant B
ND
ND
Agglomeration - Plant C
20
2x10
Mixing - Plant C
60
ND
-
Sawing - Plant C
660
ND
+ (36.51)
40
ND
+ (38.73)
ND
3x104
+ (35.58)
Sampling sites
Pressing - Plant C Sanding - Plant C +-detected; ND-not detected
+ (35.76) 4
+ (35.21)
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guidelines. On the other hand, this conventional method is limited by several factors, including incubation conditions and competition between species (32), which might be the reason for not identifying Penicillium species in plants A and B. These limitations were overcome by the use of qPCR. This method is based on the amplification of genomic regions specific to certain fungal species, which increases sensitivity and removes interference by other species present in the sample (32). Although the primers and probes used in this study may amplify DNA of P. lividum, P. pupurescens, P. spinulosum, and P. thomii, none of these species has ever been described in the cork industry, and it is safe to assume that our detection is limited to the P. glabrum complex. In addition, unlike PCR, the conventional methods can hardly distinguish between the Penicillium species. We therefore believe that these two methods should be used in parallel, as they complement each other to provide useful information for the assessment of occupational exposure to fungi (18, 19). REFERENCES 1. Green Cork. Montado de Sobro e o Sobreiral [displayed July 2 0 1 5 ] . Av a i l a b l e a t h t t p : / / w w w. g r e e n c o r k . org/a-floresta-a-cortica-e-a-rolha/montado-de-sobro-e-osobreiral/. 2. Pestana M, Tinoco I. A indústria e o comércio da cortiça em Portugal durante o século XX [Industry and the trade of cork in Portugal during the 20th century, in Portuguese]. Silva Lusitana 2009;17:1-26. 3. The Portuguese Cork Association (APCOR). [Estudo de caracterização setorial 2013, in Portuguese]. 4. Ministério do Trabalho e da Solidariedade Social (MTSS). [Boletim do Trabalho e Emprego 2009, in Portuguese]. 5. Danesh P, Caldas F, Marques J, San-Romão M. Mycobiota in Portuguese “normal” and “green” cork throughout the manufacturing process of stoppers. J Appl Microbiol 1997;82:689-94. doi: 10.1046/j.1365-2672.1997.00188.x 6. Oliveira A, Peres CM, Correia Pires JM, Silva Pereira C, Vitorino S, Figueiredo Marques JJ, Barreto Crespo MT, San Romão MV. Cork stoppers industry: defining appropriate mould colonization. Microbiol Res 2003;158:117-24. doi: 10.1111/j.1574-6941.2012.01419.x 7. Santos M, Bragança M, Casimiro P. Microrganismos associados à cortiça em diferentes fases da sua fileira [Cork oak associated microorganisms throughout cork manufacture process, in Portuguese]. Silva Lusitana 2005;13:75-93. 8. Winck JC, Delgado L, Murta R, Lopez M, Marques J. Antigen characterization of major cork moulds in Suberosis (cork worker’s pneumonitis) by immunoblotting. Allergy 2004;59:739-45. PMID: 15180761 9. Pereira R, Pires A, Valle MJ, Boas LV, Marques JJF, Romão MVS. Role of Chrysonilia sitophila in the quality of cork stoppers for sealing wine bottles. J Ind Microbiol Biotechnol 2000;24:256-61. doi: 10.1038/sj.jim.2900815 10. Serra R, Peterson S, Venâncio A. Multilocus sequence identification of Penicillium species in cork bark during plank
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preparation for the manufacture of stoppers. Res Mycrobiol 2008;159:178-86. doi: 10.1016/j.resmic.2007.12.009 Basílio M, Gaspar R, Pereira C, Romão M. Penicillium glabrum cork colonising isolates-preliminar analysis of their genomic similarity. Rev Iberoam Micol 2006;23:151-4. PMID: 17196021 Cruz J. [Doença Respiratória dos Trabalhadores da Indústria da Cortiça, in Portuguese]. [PhD thesis]. 2003; Porto: Faculdade de Medicina da Universidade do Porto; 2003. Oliveira B. Identification and fingerprinting of cork fungi: a phenetic approach [PhD thesis] Lisboa: Faculdade de Ciências da Universidade de Lisboa; 2011. Pimentel JC, Avila R. Respiratory disease in cork workers (“suberosis”). Thorax 1973;28:409-23. doi: 10.1136/ thx.28.4.409 Villar A, Munõz X, Cruz MJ, Morell F. Hypersensitivity pneumonitis caused by Mucor species in a cork worker. Arch Bronconeumol 2009;45:405-7. doi: 10.1016/S15792129(09)72939-8 Francuz B, Year H, Geraut L, Bensefa-Colas L, Nghiem ZH, Choudat D. Occupational asthma induced by Chrysonilia sitophila in a worker exposed to coffee grounds. Clin Vaccine Immunol 2010;17:1645-6. doi: 10.1128/CVI.00134-10 Malta-Vacas J, Viegas S, Sabino R, Viegas C. Fungal and microbial volatile organic compounds exposure assessment in a waste sorting plant. J Toxicol Environ Health A 2012;75:1410-7. doi: 10.1080/15287394.2012.721175 Viegas C, Malta-Vacas J, Sabino R. Molecular biology versus conventional methods - Complementary methodologies to understand occupational exposure to fungi. In: Arezes P, Baptista JS, Barroso MP, Carneiro P, Cordeiro P, Costa N, Melo R, Miguel AS, Perestrelo GP, editors. Proceedings of the International Symposium on Occupational Safety and Hygiene SHO - 2012; 9-10 February 2012; Guimarães, Portugal. Guimarães: SPOSHO; 2012. p. 643-7. Viegas C, Malta-Vacas J, Sabino R, Viegas S, Veríssimo C. Accessing indoor fungal contamination using conventional and molecular methods in Portuguese poultries. Environ Monit Assess 2014;186:1951-9. doi: 10.1007/s10661-0133509-4 International Organization for Standardization – ISO 18593:2004 : microbiology of food and animal feeding stuffs: horizontal methods for sampling techniques from surfaces using contact plates and swabs. Geneva: ISO, 2004. Hoog DC, Guarro J, Gené G, Figueras M. Atlas of Clinical Fungi. 2nd ed. Utrecht: Centraalbureau voor Schimmelcultures; 2002. ACGIH. (1989). Guidelines for the assessment of bioaerosols in the indoor environment. Committee on Bioaerosols, American Conference of Governmental Industrial Hygienists (ACGIH). Cincinnati, OH. WHO guidelines for indoor air quality. Dampness and mould. Geneva: World Health Organization. 2009. Goyer N, Lavoie J, Lazure L, Marchand G. Bioaerosols in the Workplace: Evaluation, Control and Prevention Guide. Montréal: Bibliothètec national du Québec; 2001. Kromhout, H. Design of measurement strategies for workplace exposures. Occup Environ Med 2002;59:349-54. doi: 10.1136/oem.59.5.349 Roussel S, Reboux G, Bellanger AP, Sornin S, Grenouillet F, Dalphin JC, Piarroux R, Millon L. Characteristics of
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dwellings contaminated by mould. J Environ Monit 2008;10:724-9. doi: 10.1039/b718909e 27. Viegas C, Alves C, Carolino E, Pinheiro C, Rosado L, SilvaSantos C. Assessment of fungal contamination in a group of Lisbon´s gymnasiums with a swimming pool. Ital J Occup Environ Hyg 2011;2:15-20. 28. Dagenais TRT, Keller NP. Pathogenesis of Aspergillus fumigatus in invasive Aspergillosis. Clin Microbiol Rev 2009;22:447-65. doi: 10.1128/CMR.00055-08 29. Gutarowska B, Skóra J, Stepien L, Twaruzek M, BlajetKosicka A, Otlewska A, Grajewski J. Estimation of fungal contamination and mycotoxin production at workplaces in composting plants, tanneries, archives and libraries. World Mycotox J 2014;7:345-55. doi: 10.3920/WMJ2013.1640
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30. Morell F, Roger A, Cruz MJ, Muñoz X, Rodrigo MJ. Clinical study and new etiologic agents in a series of eight patients. Occup Environ Lung Dis 2003;124:1145-52. doi: 10.1378/ chest.124.3.1145 31. Knob A, Beitel SM, Fortkamp D, Terrasan CRF, de Almeida AF. Production, purification, and characterization of a major Penicillium glabrum xylanase using Brewer’s spent grain as substrate. Biomed Res Int 2013;2013:728735. doi: 10.1155/2013/728735 32. Méheust D, Le Cann P, Reboux G, Millon L, Gangneux J. Indoor fungal contamination: Health risks and measurements methods in hospitals, homes and workplaces. Crit Rev Microbiol 2014;40:248-60. doi: 10.3109/1040841X.2013. 777687
Ocjena izloženosti kompleksu Penicillium glabrum u proizvodnji pluta s pomoću komplementarnih metoda Hrast plutnjak druga je vrsta po učestalosti u portugalskim šumama, zbog čega je ta zemlja najveći izvoznik pluta na svijetu. Profesionalna je izloženost plijesnima Chrysonilia sitophila i Penicillium glabrum česta u proizvodnji pluta, a potonja vrsta povezana je s plućnom bolesti suberozom. Međutim, prilikom procjene izloženosti konvencionalnim se metodama često podcjenjuje prisutnost te vrste u radnom okolišu. Stoga je cilj ovog istraživanja bio provjeriti pretpostavku da polimerazna lančana reakcija (PCR) kao molekulska metoda dopunjuje nalaze konvencionalnih metoda i time daje bolji uvid u profesionalnu izloženost radnika u proizvodnji pluta. U tu smo svrhu istražili onečišćenje kompleksom P. glabrum u trima tvornicama pluta u okolici Lisabona oslanjajući se na konvencionalne i molekulske metode. PCR je otkrio prisutnost plijesni u šest uzoraka, u kojih konvencionalne metode nisu otkrile njihovu prisutnost. To potvrđuje našu pretpostavku da se primjenom komplementarnih metoda može steći bolji uvid i napraviti točnija procjena profesionalne izloženosti kompleksu P. glabrum u proizvodnji pluta. KLJUČNE RIJEČI: izloženost plijesnima; molekulske metode; PCR; Portugal; suberoza
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Mason HJ, et al. Levels of house dust mite allergen in cars Arh Hig Rada Toksikol 2015;66:209-212
Original article
DOI: 10.1515/aiht-2015-66-2684
Levels of house dust mite allergen in cars Howard J. Mason1, Ian Smith1, Siti Marwanis Anua2,3, Nargiz Tagiyeva2, Sean Semple2, and Graham Devereux2 Health & Safety Laboratory, Harpur Hill, Buxton1, Respiratory Group, Division of Applied Health Sciences, School of Medicine and Dentistry, University of Aberdeen, Foresterhill, Aberdeen2, UK, School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia3 [Received in June 2015; CrossChecked in June 2015; Accepted in July 2015] This small study investigated house dust mite (HDM) allergen levels in cars and their owners’ homes in north-east Scotland. Dust samples from twelve households and cars were collected in a standardised manner. The dust samples were extracted and measured for the Dermatophagoides group 2 allergens (Der p 2 and Der f 2) and total soluble protein. Allergen levels at homes tended to be higher than in the cars, but not significantly. However, they significantly correlated with paired car dust samples expressed either per unit weight of dust or soluble protein (rho=0.657; p=0.02 and 0.769; p=0.003, respectively). This points to house-to-car allergen transfer, with the car allergen levels largely reflecting levels in the owner’s home. Car HDM allergen levels were lower than those reported in Brazil and the USA. Twenty-five percent of the houses and none of the cars had allergen levels in dust greater than 2000 ng g-1. This value is often quoted as a threshold for the risk of sensitisation, although a number of studies report increased risk of sensitisation at lower levels. This small study does not allow for characterisation of the distribution of HDM allergen in vehicles in this geographic area, or of the likely levels in other warmer and more humid areas of the UK. Cars and other vehicles are an under-investigated micro-environment for exposure to allergenic material. KEY WORDS: Der f 2; Dermatophagoides; Der p 2; Scotland; vehicles
House dust mites (HDM) are almost ubiquitous in houses. Centrally heated, well-insulated homes with pets means that allergenic material related to HDM and cats and dogs is common (1, 2). Estimates from the Pet Food Manufacturers Association are that approximately 20 % of UK households keep dogs or cats. Sensitisation and subsequent allergic symptoms may be a relatively common outcome from such domiciliary indoor exposure scenarios. However, there may be other exposure scenarios for such allergens. In Great Britain, over 77 % of households own one or more cars (3) and recent data suggest that every person in Great Britain, as a passenger or driver, travels on average about 14 miles and spends 35 minutes every day in a car (4). Employee car-sharing schemes and shared school-runs for children have become increasingly common in the UK. In contrast to the house indoor environment, we can find little data, and none from Europe (5, 6) on the levels of common allergenic material in cars. In a recent study investigating why bakers’ children are more likely to have asthma we clearly demonstrated workto-home transfer of bakery allergens (7, 8). Subsequently we became interested in whether we could find evidence Correspondence to: Howard J. Mason, Health & Safety Laboratory, Harpur Hill, Buxton, SK17 9JN, UK, E-mail: howard.mason@hsl.gsi.gov.uk
of transfer of indoor household allergens, such as HDM, from houses to vehicles as an alternative exposure scenario for such allergens, and whether the levels found in vehicles could be of health concern. Based on samples collected as part of the bakery-home study, we report the levels of HDM allergenic material using a Dermatophagoides species group 2 allergen assay in dust samples collected from a small number of matched homes and associated cars.
MATERIALS AND METHODS The data reported here form part of an investigation in north-east Scotland on the transfer of allergens from bakeries to workers’ homes (7). Dust samples had been collected from 34 houses and 13 cars. Twenty-four of the 34 house samples were collected in bakery workers’ homes and 10 were control samples taken from the homes of university staff and students. The thirteen vehicles sampled belonged to bakery workers. The focus of this short report is the level of Der group 2 allergens in dust samples from the 12 matched households and cars. These matched dust samples had been collected by vacuuming using Dustream collectors (Indoor Biotechnologies, Cardiff, UK) from twelve households in Scotland and their associated cars. Description of the
210 sampling techniques has been fully reported (7), but essentially all household samples were obtained by vacuuming the seat of a well-used lounge chair and nearby floor area for approximately 5 minutes. Car dust samples were obtained by vacuuming the driver’s seat and foot-well. Dust samples were weighed and extracted for 2 hours by mixing them with 0.1 % Tween 20 (Sigma-Aldrich, Poole, UK) in phosphate buffered saline at 10 % weight/ volume ratio. Extracts were centrifuged and the supernatant analysed for Dermatophagoides species group 2 allergens (Der p 2 + Der f 2) using the ELISA kit (Indoor Biotechnology, Cardiff, UK) and total soluble protein using the standard bicinchoninic acid colorimetric assay (Sigma-Aldrich, Poole, UK). The batch-to-batch method imprecision for the allergen and protein methods are 14 % and 10 % respectively. The lower limit of quantification calculated for the allergen assay using ProQuant software (QIVX Inc. Fort Collins, CO, USA) was 2 ng g-1 dust. We measured Der group 2 allergens rather than the more usual Der p 1, as the latter can be underestimated in the presence of bakery/ wheat components (9).
RESULTS AND DISCUSSION The levels of HDM in the 12 bakery households matching the cars were not different from the larger data set of 24 bakery homes. Measurable levels of the Der group 2 allergens were detected in all dust samples collected from the twelve houses and the matching cars. Their medians (and ranges) were 55 (5-12,863) ng g-1 and 23 (3-454) ng g-1 dust, respectively, or 6.48 (0.28-740.1) and 2.93 (0.14-65.11) when expressed as ng mg-1 soluble protein (Figure 1). Although allergen levels were higher in house samples
Mason HJ, et al. Levels of house dust mite allergen in cars Arh Hig Rada Toksikol 2015;66:209-212
expressed either way, these differences did not achieve statistical significance (Friedman test; p=0.082 and 0.586, respectively). However, allergen levels correlated significantly between the paired dust samples from cars and houses; Spearman’s coefficients of correlation (rho) were 0.657, p=0.02 for the results expressed per gram of dust and 0.769, p=0.0034 when expressed per mg of soluble protein (Figure 2). For the reason noted earlier, we measured Der group 2 rather than the more usual Der p 1. Attempting to convert our results to likely Der p 1 levels can only yield approximate values. Custovic (10) showed good correlation in UK field samples between Der p 1 and p 2, whereby 1 ng of Der p 2 was equivalent to 1.9 ng of Der p 1. Our group 2 measurement also theoretically includes any Der f 2 present, but in the UK D. pteronyssinus is predominant over D farina. Thus we assume that likely Der p 1 levels are somewhere between the same as, or require doubling of, our measurements. Justino et al. (6) measured Der group 1 allergens in car dust samples from Brazilian university staff and students, reporting a geometric mean [Der p 1 +Der f 1] of 540 ng g-1 dust. Car allergen levels in our study are lower than these. Neal et al. (5) conducted a study of paired vehicles and homes in Ohio, USA. Their overall means of Der group 1 in houses and vehicles were 25,600 and 1,300 ng g-1 dust, respectively. While Neal et al. (5) presents arithmetic means without any indication of the distribution of allergen data, which were very non-normal in both Justino’s and our study, their results suggest much higher vehicle and household levels of HDM allergens than we report. Interestingly, Neal et al. (5) also found a positive association between house and vehicle allergen levels.
Figure 1 Levels of Der group 2 (Der p 2 + Der f 2) found in houses and cars
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Figure 2 Correlation between car and house levels of HDM allergens The value of 2000 ng g-1 of Der p 1 in settled dust is often quoted as the threshold for the risk of sensitisation (11). Our data suggest that 25 % of the matched households would exceed this. No allergen levels in the cars exceed it, and therefore suggest no risk of sensitisation. However, a number of studies (12-14) show that exposure to allergen levels of less than 2000 ng g-1 can still be a significant sensitisation risk. Dose-response relationships for other outcomes (rhinitis, upper and lower respiratory symptoms) in sensitised individuals are unclear. Therefore, while the health risk in this small vehicle group is probably small, their HDM levels deserve further investigation in a larger UK cohort, particularly if focussed on households that are likely to have high HDM levels. What also deserves further investigation is the use of allergen levels in settled dust as the inhalation exposure metrics for health outcomes (15). Such metrics have been employed for HDM allergens, as air measurements have often failed to detect airborne levels of Der p 1 unless there is significant dust disturbance or human activity. This possibly relates to the Der p 1 particle size. Analysis of airborne HDM allergens after normal human activity in both the house and the car may help clarify exposure-risk relationships. Our household allergen levels are comparable with those reported in a Dutch study of living room carpets (16). The group 2 measurement reflects mite body proteins and is more heat-stable than the Der p 1 allergen associated with a faecal protein (11), and Der p 2 has been shown to correlate with mite numbers indoors (17). However, we do not know whether the vehicle results reflect the transfer of the living mites from home or only the transfer of the allergenic protein. The significant association between the allergen levels in matched household and car dust samples suggests transfer from home to car. We have already established transfer of
allergens between workplaces and homes through contaminated clothes, hair, skin, with increased bakery allergen levels found in bakers’ cars used for commuting (7). This study also suggests that the car could be a relatively neglected micro-environment for secondary exposure to indoor and occupational allergenic material. Levels of house dust allergen in a car may reflect the levels of the allergen in the house and the extent of houseto-car transfer. While HDM allergen levels measured in this study in north-east Scotland indicate low risk (18), the small sample size can hardly serve to predict the distribution of HDM allergen, especially the levels that might constitute a health risk. Further investigations of HDM levels (including Der p 1 measurements) in UK households and vehicle dust are warranted, especially where high household HDM levels are likely to be found (i.e. higher ambient temperatures and humidity). Given that the major health risks are due to inhalation rather than indirectly from settled dust content and that airborne HDM allergen derives largely from human activity and disturbance of settled dust, we suggest measuring vehicle airborne allergen levels during real-life use of vehicles. We also hope this study will raise further interest in exposure to allergens and their possible health risks in all forms of transport. REFERENCES 1. Randall A, Hillier A, Cole L, Kwochka KW, Needham G, Wassom DL. Quantitation of house dust mite allergens (Der f 1 and group 2) on the skin and hair of dogs Am J Vet Res 2005;66:143-9. PMID: 15691050 2. Fahlbusch B, Heinrich J, Gross I, Jäger L, Richter K, Wichmann H. Allergens in house-dust samples in Germany: results of an East-West German comparison. Allergy. 1999;54:1215-22. doi: 10.1034/j.1398-9995.1999.00196.x
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3. Leibling D. Car Ownership in Great Britain. London: RAC Foundation for Motoring; 2008. 4. Department for Transport. National Travel Survey: 2010 [displayed 14 July 2015]. Available at https://www.gov.uk/ government/uploads/system/uploads/attachment_data/ file/8932/nts2010-01.pdf 5. Neal J, Arlian L, Morgan M. Relationship among house-dust mites, Der 1, Fel d 1, Can f 1 on clothing and automobile seats with respect to densities in houses. Ann Allergy Asthma Immunol 2002;88:410-5. PMID: 11991559 6. Justino C, Segundo G, Pereira F, Silva D, Sopelete M, Sung SS, Taketomi EA. Mite and pet allergen exposure in Brazilian private cars. Ann Allergy Asthma Immunol 2005;94:658-61. PMID: 15984598 7. Tagiyeva N, Anua S, Semple S, Dick F, Devereux G. The ‘take home’ burden of workplace sensitizers: Flour contamintaion in bakers’ families. Environ Int 2012;46:44-9. doi: 10.1016/j.envint.2012.04.014. 8. Tagiyeva N, Devereux G, Semple S, Sherrif A, Henderson J, Elias P,Ayres JG. Parental occupation is a risk factor for childhood wheeze and asthma. Eur Respir J 2010;35:987-93. doi: 10.1183/09031936.00050009 9. Barber D, Pernas M, Chamorro M, Carreira J, Artega C, Sanchez-Monge R, Polo F, Salced G. Specific depletion of the house dust mite allergen Der p 1 by cereal flour prolamins. J Allergy Clin Immunol 1996;97:963-5. doi: 10.1016/S00916749(96)80071-6 10. Custovic C, Taggart S, Francis H, Chapman M, Woodcock A. Exposure to house dust mite allergens and the clinical activity of asthma. J Allergy Clin Immunol 1996;98:64-72. doi: 10.1016/S0091-6749(96)70227-0 11. Platts-Mills T, Vervloet D, Thomas W, Aalberse R, Chapman M. Indoor allergens and asthma: report of the Third
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International Workshop J Allergy Clin Immunol 1997;100(6 Pt 1):S1-S24. doi: 10.1016/S0091-6749(97)70292-6 Cullinan P, MacNeill S, Harris J, Moffat S, White C, Mills P, Newman Taylor AJ. Early allergen exposure, skin prick responses, and atopic wheeze at age 5 in English children: a cohort study. Thorax. 2004;59:855-61. doi:10.1136/ thx.2003.019877 Huss K, Adkinson NF Jr, Eggleston PA, Dawson C, Van Natta ML, Hamilton RG. House dust mite and cockroach exposure are strong risk factore for positive allergy skin test responses in the Childhold Asthma Management Program. J Allergy Clin Immunol 2000;107:48-54. PMID: 11149990 Wahn U, Lau S, Bergman R, Kulig M, Forster J, Bergmann K, Bauer CP, Guggenmoos-Holzmann I. Indoor allergen exposure is a risk factor for sensitisation during the first three years of life. J Allergy Clin Immunol 1997;99:763-9. doi: http://dx.doi.org/10.1016/S0091-6749(97)80009-7 Paufler P, Gebel T, Dunkelberg H. Quantitation of house dust mite allergens in ambient air. Rev Environ Health 2001;16:65-80. 10.1515/REVEH.2001.16.1.65 Meijer G, Van der Heide S, Postma D, de Reus D, Koeter G, van Aalderen W. House dust mite exposure in asthmatic and healthy children: the difference is carpeting. Pediatr Allergy Immunol 1995;6:187-91. doi: 10.1111/j.1399-3038.1995. tb00283.x Liao E-C, Lin Y-H, Tsai J-J. Detection of group 2 Dermatophagoides pteronyssinus allergen for environmental monitoring of dust mite infestation. BioScience Trends 2013;7(2):82-8. doi: 10.5582/bst.2013.v7.2.82 Macan J, Kanceljak-Macan B, Milković-Kraus S. Pyroglyphid mites as a source of work-related allergens. Arh Hig Rada Toksikol 2012;63:57-66. doi: 10.2478/10004-1254-63-20122133
Razine alergena prašinskih grinja u automobilima U ovome smo preliminarnom istraživanju izmjerili razine alergena prašinskih grinja u automobilima i domovima njihovih vlasnika u sjevernoistočnoj Škotskoj. Uzorci prašine uzeti su na standardizirani način iz dvanaest domova i dvanaest odgovarajućih automobila. Nakon ekstrakcije izmjerene su razine 2. skupine alergena grinja roda Dermatophagoides (Der p 2 i Der f 2) te njihove ukupne topljive bjelančevine. Razine alergena u kućama bile su mahom više nego u automobilima, ali ne značajno. Uočena je međutim značajna korelacija njihovih razina s razinama u automobilima, bez obzira na to jesu li iskazane udjelom težine po gramu prašine ili topljivim bjelančevinama (rho=0,657; p=0,02 odnosno 0,769; p=0,003). To upućuje na prijenos alergena iz kuće u auto tj. pokazuje da razine alergena u autima odražavaju razine u domovima njihovih vlasnika. Razine alergena kućnih prašinskih grinja u automobilima u Škotskoj bile su niže od onih izmjerenih u Brazilu i SAD-u. U 25 % domova razina alergena bila je viša od 2000 ng g-1, a ni u jednom uzorku nije izmjerena razina viša od te. Ta se vrijednost često smatra graničnom za rizik od senzitizacije, premda neka istraživanja govore o povišenom riziku od senzitizacije i pri nižim razinama alergena. Rezultati ovog istraživanja ne daju uvida u stvarnu prisutnost alergena kućnih prašinskih grinja u vozilima u sjeveroistočnoj Škotskoj niti upućuju na to kolike bi mogle biti njihove razine u toplijim i vlažnijim krajevima Ujedinjenog Kraljevstva. Automobili i ostala vozila zanemareni su kao izvori izloženosti alergenima. KLJUČNE RIJEČI: Der f 2; Dermatophagoides; Der p 2; Škotska; vozila
Manfredo I. Accidental discovery of asbestos-related occupational pleural disease in unemployed carpenter Arh Hig Rada Toksikol 2015;66:213-215
Case report
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DOI: 10.1515/aiht-2015-66-2682
Accidental discovery of asbestos-related occupational pleural disease in unemployed carpenter: a healthcare safety net that needs mending Irena Manfredo Medicina dela, prometa in ĹĄporta Ltd., Zagorje, Slovenia [Received in June 2015; CrossChecked in June 2015; Accepted in August 2015] Unemployed persons are often on the margins of the healthcare system and under the radar of safety and health organisations, as no systematic records are kept of occupational diseases caused by exposure at previous work place. Law in Slovenia requires that asbestos-related occupational diseases are verified by establishing the causal relationship between exposure at work and its effect on the worker. This report describes a case of verifying occupational pleural disease in an unemployed carpenter who was referred for consultation with occupational health specialist as part of the regular procedure for the unemployed registered at the Employment Service of Slovenia. At the consultation it turned out that the carpenter had been exposed to asbestos when he worked as a teenage apprentice. The diagnosis of the bilateral pleural disease and asbestosis was confirmed by X-ray and high-resolution computed tomography. Because he had no record of exposure in that period, we analysed his past working environment for minerals and found chrysotile in all asbestos board samples. The case was presented to an interdisciplinary committee, which verified his disease as occupational. This case points to the need of adopting guidelines for occupational health specialists providing counsel to the national employment service so that the number of unrecorded occupational diseases is minimised and their treatment is covered by the state. KEY WORDS: apprentice; asbestos boards; legislation; occupational disease; pleural plaques The Slovenian employment agency (Employment Service of Slovenia, ESS) works with occupational health specialists, who review medical records of unemployed persons in order to assess their work ability and advise social workers about the most appropriate job for them. As the consultation does not involve medical exam, this is where the occupational health physicianâ&#x20AC;&#x2122;s role stops. The unemployed are often left on the margins of the healthcare system and dwell outside the scope of work safety and health organisations or trade unions. As safety at work strategies are mostly designed to meet current needs, they do not target the unemployed or the retired, and occupational diseases caused by exposure at a previous work place are seldom diagnosed and verified. Diseases that can result from occupational asbestos exposure are asbestosis, pleural disease, pulmonary cancer, malignant mesothelioma of the pleura and peritoneum, as well as cancer in other sites (1). The EU resolution of 14 March 2013 on risks related to asbestos exposure emphasises the role of medical staff in recognising the professional origin of asbestos-related diseases, especially due to a very long period of latency (2). According to this resolution, the burden of proof should not be on the patient. Correspondence to: Irena Manfredo, MD, MSc, occupational health specialist, Medicina dela, prometa in ĹĄporta Ltd., Cesta zmage 1, 1410 Zagorje, Slovenia, E-mail: irena.manfredo@siol.net
However, in Slovenia, this burden of proof still lies with the patient. Slovenian laws and regulations clearly define who the rightful claimants are, types of asbestos and asbestos products, and the diseases that can result from asbestos exposure (3, 4), but it is left to patients with asbestos-related diseases to start the process of proving the occupational origin of their disease. Once they do, occupational health specialists take over. They collect evidence of asbestos exposure at workplace, assess the risk, diagnose the disease and submit their expertise to an interdisciplinary committee for verification. If the disease is verified as occupational, the patient has the right to claim compensation. This report presents one outstanding case of an unemployed patient from the margins of the healthcare system who, unlike many others, was lucky enough to chance upon occupational health specialist who helped him prove occupational asbestos-related pleural disease and claim his rights. Case description In 2014, a 60-year-old carpenter, who had been unemployed for 10 years, was referred by the ESS to occupational health specialist for the assessment of fitness for work. The patient had already had reduced ability to work because of ventral hernia waiting for the seventh surgery. Unable to shift and lift heavy loads the patient had
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Manfredo I. Accidental discovery of asbestos-related occupational pleural disease in unemployed carpenter Arh Hig Rada Toksikol 2015;66:213-215
been laid off and could not find another employment for over ten years. Answering the question about his health, he said that he felt well but that “there was something in his lungs supposedly due to asbestos”. He had smoked about 15 packs years of cigarettes for 35 years and quit in 2010. From July 1971 to August 1974 he had worked as apprentice in a carpenter’s workshop and continued to work as carpenter in the same workshop until August 1975. At that time, apprenticeship consisted of six months at school and six months of practice in a workshop over a school year. During the practice, apprentices worked over eight hours a day for six days a week. Instead of working with wood, our patient worked on installing asbestos insulating boards (1250x2050 mm, 6-15 mm thick) of Belgian make (Masal, Fasal or Glasal) at different sites in Slovenia and former Yugoslavia. The boards were cut, sawed, and screwed on walls without masks, ventilation, or other safety measures. The workers knew that the boards were made of asbestos, but had no idea of its harmful effects. They usually had their lunch breaks at the workplace. Our patient had not been exposed to asbestos ever since, but continued to work with wood as carpenter proper. In 2008, surgery of his right shoulder pointed to pulmonary embolism, leading to a follow up lung X-ray once a year. Between 2008 and 2011, the follow up revealed no suspicious changes, but in 2012 it showed extensive pleural plaques and atypical condensation. The radiologist suspected those plaques were the result of asbestos exposure. The diagnosis of bilateral pleural disease was confirmed by X-ray at the Clinic of Pulmonary Diseases in Zagorje (Figure 1) and by high-resolution computed tomography at the University Clinic of Pulmonary and Allergic Diseases Golnik in 2014. We at the occupational health office made a list of buildings and places where our carpenter worked. There was no paper evidence, no contracts, no environmental
measurements, and no risk assessment of the working conditions involving asbestos. The master-carpenter died, and the apprentice’s working diaries were burnt. The only evidence was a signed statement of the master-carpenter’s collaborator that our patient had worked with asbestos boards. It took 41 years from the first exposure in 1971 to the diagnosis of pleural plaques in 2012. We submitted our expertise to the interdisciplinary committee with a confirmed diagnosis of asbestos disease and the statement of the collaborator confirming the patient’s work history. The expertise was rejected as insufficient. As asbestos products have been banned from the EU since 2005 (5), we could not track down boards of the same make to any of the stores. The only possibility to find a proof was to get a sample of the insulating board from one of the patient’s old workplaces. We found it in an indoor swimming pool in Zasavje, where the carpenter used to work (Figure 2). The building manager allowed us to take a few samples. Mineral analysis, done at the Laboratory for Concrete, Stone, and Recycled Materials of the Slovenian
Figure 2 Asbestos boards on the ceiling of the Zasavje swimming pool facility where the patient worked in the 1970s
National Building and Civil Engineering Institute using scanning electron microscopy (Figure 3), and electronic dispersion spectroscopy on apparatus JEOL 5500 LV confirmed the presence of white asbestos-chrysotile in all of the samples. We resubmitted the amended expertise, and this time the committee verified asbestos exposure. Since there were experts on the committee, the diagnosis was even extended from bilateral pleural disease to asbestosis. Figure 1 X-ray of the patient’s lungs taken in April 2014 to verify findings from 2012. It shows bilateral calcified pleural plaques, mostly in the peripheral lung diaphragm areas, and a nodular condensation in the left apex
CONCLUSION Our case illustrates that at the moment, the unemployed are on the margins of the system, as no procedure has been
Manfredo I. Accidental discovery of asbestos-related occupational pleural disease in unemployed carpenter Arh Hig Rada Toksikol 2015;66:213-215
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REFERENCES
Figure 3 Chrysotile fibres on electronic microscope (magnification 120x)
established to diagnose and keep record of occupational diseases contracted at their earlier workplaces. However, it may also give some guidelines for improvement. It should start with extending the role of occupational health counsellors working for the ESS and defining activities to pursue suspicions of past occupational exposure. The burden of proof should lie with the system and not the individual. This will require a change in the mind-sets of current decision makers, and we hope our case will provide an incentive in this direction. Acknowledgments I would like to thank my patient VB for consenting to the publication of his case and Ana Mladenović, PhD, Head of the Laboratory for Concrete, Stone, and Recycled Materials, who kindly welcomed my request to analyse the boards.
1. Dodič-Fikfak M, Črnivec R, Mandelc Grom M. Occupational diseases due to asbestos exposure. In: Dodič-Fikfak M, Črnivec R, editors. Verification of occupational diseases in the Republic of Slovenia I. Ljubljana: Univerzitetni klinični center Ljubljana, Klinični inštitut za medicino dela, prometa in športa; 2009. p. 1-11. 2. European Parliament. European Parliament resolution of 14 March 2013 on asbestos related occupational health threats and prospects for abolishing all existing asbestos (2012/2065 (INI)) [displayed 14 August 2015]. Available at http://www. europarl.europa.eu/sides/getDoc.do?pubRef=-//EP// TEXT+TA+P7-TA-2013-0093+0+DOC+XML+V0//EN 3. Ministry of Health. Pravilnik o spremembah in dopolnitvah Pravilnika o pogojih za določitev bolezni zaradi izpostavljenosti azbestu in merilih za določitev višine odškodnine [Rules Amending the Rules on the conditions for the establishment of disease from exposure to asbestos and the criteria for fixing the amount of compensation, in Slovenian] [displayed 30 September 2008]. Available at http://www.pisrs.si/Pis.web/pregledPredpisa?id=PRAV9058 4. National Assembly. Zakon o spremembah in dopolnitvah zakona o odpravljanju posledic dela z azbestom (ZOPDA-A) [Act Amending the Act Concerning Remedying the Consequences of Work with Asbestos, in Slovenian] [displayed 19 June 2009]. Available at http://www.pisrs.si/ Pis.web/pregledPredpisa?id=ZAKO4853 5. EUR-Lex. Commission Directive of 26 July 1999 adapting to technical progress Annex I to Council Directive 76/769/ EEC on the approximation of the laws, regulations and administrative provisions of the Member States relating to restrictions on the marketing and use of certain dangerous substances and preparations (asbestos) [displayed 6 August 1999]. Available at http://eur-lex.europa.eu/legal-content/ EN/TXT/?uri=CELEX:31999L0077
Poklicna bolezen plevre zaradi izpostavljenosti azbestu pri mizarju Nezaposleni so pogosto na robu zdravstvenega sistema in izven dosega varnosti in zdravja pri delu. Poklicnih bolezni zaradi izpostavljenosti na prejšnjih delovnih mestih v Sloveniji ne spremljamo. Zakonodaja predpisuje upravičence, vrste azbesta in azbestnih izdelkov, bolezni, ki se štejejo kot bolezni zaradi izpostavljenosti azbestu in postopek priznanja poklicne bolezni zaradi izpostavljenosti azbestu. Pri dokazovanju poklicnega izvora bolezni je potrebno objektivizirali vzročno zvezo med izpostavljenostjo azbestu na delovnem mestu in reaktivnostjo delavca. Opisan je primer verifikacije poklicne bolezni zaradi izpostavljenosti azbestu pri nezaposlenem mizarju, ki je bil predstavljen zdravnici- svetovalki na zavodu za zaposlovanje v sklopu zaposlitvenega svetovanja. Azbestu je bil izpostavljen kot vajenec v mizarski delavnici. Diagnoza bilateralnih plevralnih plakov je bila postavljena z RTG p.c. in HR-CT. Ker ni bilo nobenih pisnih dokumentov iz obdobja izpostavljenosti azbestu, smo opravili natančno delovno anamnezo, izvedli mineraloško analizo na enem od delovišč in dokazali krizotil v vseh odvzetih vzorcih. Interdisciplinarna komisija je potrdila poklicno bolezen zaradi izpostavljenosti azbestu. Primer opozarja na potrebo po sprejetju smernic za delo zdravnikov-svetovalcev, s čimer bi se zmanjšala verjetnost spregledanih poklicnih bolezni. V primeru suma na poklicno bolezen pri nezaposlenih bi morala stroške dokazovanja prevzeti država. KLJUČNE BESEDE: azbestno-cementne plošče; plevralni plaki; poklicna bolezen; vajenec; zakonodaja
Editorial Foreword Arh Hig Rada Toksikol 2015;66:217
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Editorial foreword to the Letters to the Editor The following two letters to the editor follow up on the article by Marashi et al. “Can sodium bicarbonate really help in treating metabolic acidosis caused by aluminium phosphide poisoning?” published in the Archives issue no. 1, volume 66, 2015 (pp. 83-84). The first is the reaction of Professor Zvonko Rumboldt to this article, and the second is the authors’ response. We decided to publish both, as some of our readers may find aluminium phosphide poisoning treatment an interesting topic, but wish you to note that we endorse neither of the opinions. Instead, we hope that this little confrontation of the views may start a broader discussion on the topic. Archives Editorial Board
Rumboldt Z. Hydroxyethyl starch should not be used to manage severe aluminium phosphide poisoning Arh Hig Rada Toksikol 2015;66:219
Letter to the Editor
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DOI: 10.1515/aiht-2015-66-2647
Hydroxyethyl starch should not be used to manage severe aluminium phosphide poisoning Zvonko Rumboldt Split University School of Medicine, Split, Croatia
In the Archives January issue (1), I have read with interest and surprise a letter by Marashi and Nasrabadi concerning the use of hydroxyethyl starch (HES, hetastarch; best known under the registered name Plasmasteril) to treat severe aluminium phosphide (AlP) poisoning. At the current level of scientific evidence such a claim seems unacceptable. Aluminium phosphide poisoning (the compound is commercially available as a fumigant for stored cereals) is believed to be caused by phosphine (PH3), as it induces mitochondrial damage and severe multi-organ failure (2). The poisoning is mostly reported from the Indian subcontinent, and is largely unknown in Europe. The management is exclusively supportive (gastric lavage with KMnO4 solution, treatment of shock, lactic acidosis, etc.), since there is no specific antidote (2, 3). Some traditional (e.g. coconut oil) or “modern” interventions (e.g. haemodialysis) have not been adequately assessed, and rest instead on pathophysiological assumptions, anecdotal reports, and wishful thinking (2, 3). Lactic acidosis type A, a form of high anion gap metabolic acidosis, is just one of the many aspects of AlP poisoning with fatal prognosis. Treatment with NaHCO3 in this situation is highly controversial, as the authors (1) appropriately underscore. However, the suggested administration of colloid solutions is potentially even more dangerous. The authors corroborate their proposal by quoting just one hypothetic paper of their own on the role of HES in acute AlP poisoning (4) with no further experimental evidence. It is not clear whether they are advocating starch solution administration at arterial pH >7 (as stated in the letter) or <7, or what the dosage of 5001000 mL of HES really means (i.e. isotonic, 6 g L-1, or hypertonic, 9 g L-1 solution)? Contrary to their claim (1), colloidal solutions such as dextrans, pentastarch, polygeline, and HES in particular may worsen metabolic acidosis and shock, possibly due to
Correspondence to: Zvonko Rumboldt, Professor emeritus, Split University School of Medicine, 21000 Split, Croatia, E-mail: zr@mefst.hr
the substance’s deposition in the tissue and its interaction with fibrinogen, leading to even more severe coagulopathy and organ failure (5). Crystalloid solutions are better tolerated and equally effective plasma expanders (6). Beware of ill-founded therapeutic enthusiasm! REFERENCES 1. Marashi SM, Nasri-Nasrabadi Z. Can sodium bicarbonate really help in treating metabolic acidosis caused by aluminium phosphide poisoning? Arh Hig Rada Toksikol 2015;66:83-4. doi: 10.1515/aiht-2015-66-2637 2. Agrawal VK, Bansal A, Singh RK, Kumawat BL, Mahajan P. Aluminium phosphide poisoning: possible role of supportive measures in the absence of specific antidote. Indian J Crit Care Med 2015;19:109-12. doi: 10.4103/09725229.151019 3. Gurjar M, Baronia AK, Azim A, Sharma K. Managing aluminium phosphide poisoning. J Emerg Trauma Shock 2011;4:378-84. doi: 10.4103/0974-2700.83868 4. Marashi SM, Arefi M, Behnoush B, Nasrabad MG, Nasri Nasrabadi Z. Could hydrohyethyl starch be a therapeutic option in the management of acute aluminium phosphide toxicity. Med Hypotheses 2011;76:596-8. doi: 10.1016/j. mehy.2011.01.009 5. Hartog CS, Natanson C, Sun J, Klein HG, Reinhart K. Concerns over use of hydroxyethyl starch solutions. BMJ 2014;349:g5891. doi: 10.1136/bmj.g5981 6. The European Medicines Agency’s Pharmacovigilance Risk Assessment Committee. Hydroxyethyl-starch solutions (HES) should no longer be used in patients with sepsis or burn injuries or in critically ill patients [displayed 14 July 2015]. Available at: http://www.ema.europa.eu/ema/index. jsp?curl=pages/medicines/human/referrals/Hydroxyethyl_ starch-containing_solutions/human_referral_prac_000012. jsp&mid=WC0b01ac0580 5c516f
Marashi SM, Nasri-Nasrabadi Z. Response to Professor Rumboldt’s reaction to our letter on hydroxyethyl starch in AlP poisoning Arh Hig Rada Toksikol 2015;66:221-223
Letter to the Editor
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DOI: 10.1515/aiht-2015-66-2649
Response to Professor Rumboldt’s reaction to our letter on hydroxyethyl starch use in managing aluminium phosphide poisoning Sayed Mahdi Marashi1 and Zeynab Nasri-Nasrabadi2 Trauma Research Center, Shiraz University of Medical Sciences, Shiraz1, Department of Pediatrics, Children’s Medical Center, Pediatric Center of Excellence, Tehran University of Medical Sciences, Tehran2, Iran We appreciate Professor Rumboldt’s interest in our article and his caring comments (1). As he said, most published articles propose mitochondrial damage as the mechanism of phosphine toxicity; in fact, this hypothesis has been repeated in almost every medical article on aluminium phosphide (AlP) poisoning since the 1990s (24). To our knowledge, it has not been backed up by any studies in humans. Moreover, in vitro studies indicate incomplete inhibition of the mitochondrial function (5). As we know, the main problem with severe AlP poisoning is refractory hypotension, which is almost always associated with severe refractory metabolic acidosis and fatal prognosis (6). There is a general consensus that the only possible management is supportive. However, despite advanced intensive care strategies, mortality rates vary between 24 and 66 % within the first few days (7, 8). This urged us to ask ourselves the following: 1. What really happens when phosphine is adsorbed by the gastric or pulmonary epithelium? 2. Is inhibition of cytochrome c oxidase really addressing the toxicity of AlP? 3. Considering the high mortality rate, are the current treatment protocols justified? To answer these questions let us review the related pathological events. In our experience, accumulation of serous fluid in the pleural and peritoneal cavities is common. Literature reports congestion of the vital organs as the most prominent finding in autopsy examinations (9, 10). In our opinion, this points to the disruption of vascular integrity as soon as the absorbed phosphine enters the bloodstream. This condition can explain the rapid progress of hypotension, which is usually refractory to treatment with crystalloid solutions and vasopressors. Refractory hypotension leads to circulatory failure and consequently Correspondence to: Zeynab Nasri-Nasrabadi, Department of Paediatrics, Tehran University of Medical Sciences, 16 Azar Avenue, Tehran, Iran, E-mail: drmr44@yahoo.com
metabolic acidosis. In this condition, cell metabolism will decrease to enhance the chance of cell survival (11). Even though our hypothesis has not yet been tested, it explains every fact about AlP poisoning. One such fact is that current treatment protocols are not addressing the problem. Almost all of them repeat the ancient errors and often fail to alleviate poisoning symptoms (12-14). We recently evaluated misconceptions such as using of charcoal, gastric lavage with KMnO4 solution, and cardioactive steroids in managing AlP poisoning-induced heart failure (12-14) and decided not to follow these protocols in our patients. Jaiswal et al. (6) report that 12 of the 14 patients presenting with hypotension died despite intensive care. In our experience, successful treatment of hypotension is usually associated with the improvement of lactic acidosis and patient survival. We propose Voluven® administration (6 % hydroxyethyl starch 130/0.4 in 0.9 % sodium chloride) at doses from 10 to 15 mL kg-1 body weight, without concomitant administration of additional doses of bicarbonate at arterial pH>7. However, we can not speak about “no need for administration of additional bicarbonate” at arterial pH <7, as none of our patients had an arterial pH below seven on admission. We would like to make clear that in addition to Voluven we administered normal saline in dosages as high as 4000-5000 mL over the first 24 hours to resuscitate the volume as well as other acceptable treatments such as magnesium sulphate, calcium gluconate, and N-acetyl cysteine. We would also like to make clear that when we as a referral hospital receive our patients, most of them have already received considerable doses of sodium bicarbonate and even vasopressors before admission. We are agree with Professor Rumboldt that crystalloid solutions are better tolerated than colloidal, but disagree that “they are equally effective plasma expanders” in AlP poisoning treatment. In fact, considering that compromised vascular integrity is the main mechanism of phosphineinduced haemodynamic failure, only high molecular weight plasma expanders can remain in blood vessels.
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Marashi SM, Nasri-Nasrabadi Z. Response to Professor Rumboldt’s reaction to our letter on hydroxyethyl starch in AlP poisoning Arh Hig Rada Toksikol 2015;66:221-223
In his letter Professor Rumboldt has expressed concern about worsening the patients’ prognosis, referring to the article by Hartog et al. “Concerns over use of hydroxyethyl starch solutions” (15). To address his concern, we read this and other articles arguing against the use of HES in critically ill patients (16-20). Most of the concerns raised refer to coagulopathy, acute kidney injury, and increased mortality. Jonville-Bera et al. (16) suggest that HES administration of more than 33 mL kg-1 day-1, or the cumulative dose more than 80 mL kg-1 may be induce coagulopathy. In our patients, generally lower doses sufficed to overcome refractory hypotension. Therefore, this may not be an important issue. Rioux et al. (17) showed that administration of more than 14 mL kg-1 was associated with acute kidney injury in 10 % of patients treated with a 10 % Pentastarch solution (250 kDa/0.45) following cardiac surgery. Considering the high mortality of acute AlP poisoning, we believe that an even higher acute kidney injury rate may be acceptable if this treatment strategy saves the patient’s life. Other studies compare 90-day mortality in severe sepsis patients receiving more than 2500 mL of HES vs. crystalloid solutions in the first four days of treatment. Myburgh et al. (18) found no significant difference in mortality rates between the treatments; Perner et al. (19) did; whereas Guidet et al. (20) reported that HES product needed less volume (generally more than 1000 mL) and stabilised the haemodynamics more rapidly than normal saline in severe sepsis patients at similar mortality rates. In contrast, administration of 500-1000 mL of Voluven within the first 6-12 hours sufficed to stabilise our patients. It is only fair to admit, however, that our successful experience with HES solutions in AlP poisoning is limited to only five cases. Considering the poor prognosis of AlP; we believe that more remains to be learned from future randomised trials that would show the specific properties HES. If it proves successful, we could introduce this remedy as an antidote, even if its other uses are contraindicated. REFERENCES 1. Marashi SM, Nasri-Nasrabadi Z. Can sodium bicarbonate really help in treating metabolic acidosis caused by aluminium phosphide poisoning? Arh Hig Rada Toksikol 2015;66:83-4. doi: 10.1515/aiht-2015-66-2637 2. Siwach SB, Jagdish K, Katyal VK, Dhall A, Bhardwaj G. Prognostic indices in aluminium phosphide poisoning: Observations on acidosis and central venous pressure. J Assoc Physicians India 1997;45:693-5. 3. Siddaiah LM, Adhyapak SM, Jaydev SM, Shetty GG, Varghese K, Patil CB, Iyengar SS. Intra-aortic balloon pump in toxic myocarditis due to aluminum phosphide poisoning. J Med Toxicol 2009;5:80-3. doi: 10.1007/BF03161093 4. Agrawal VK, Bansal A, Singh RK, Kumawat BL, Mahajan P. Aluminium phopsphide poisoning: possible role of supportive measures in the absence of specific antidote. Indian J Crit Care Med 2015;19:109-12. doi: 10.4103/09725229.151019
5. Chefurka W, Kashi KP, Bond EJ. The effect of phosphine on electron transport in mitochondria. Pestic Biochem Physiol 1976;6:65-84. doi:10.1016/0048-3575(76)90010-9 6. Jaiswal S, Verma RK, Tewari N. Aluminum phosphide poisoning: Effect of correction of severe metabolic acidosis on patient outcome. Indian J Crit Care Med 2009;13:21-4. doi: 10.4103/0972-5229.53111 7. Soltaninejad K, Nelson LS, Bahreini SA, Shadnia S. Fatal aluminum phosphide poisoning in Tehran-Iran from 2007 to 2010. Indian J Med Sci 2012;66:66-70. doi: 10.4103/00195359.110909 8. Shadnia S, Mehrpour O, Soltaninejad K. A simplified acute physiology score in the prediction of acute aluminum phosphide poisoning outcome. Indian J Med Sci 2010;64:5329. doi: 10.4103/0019-5359.75928 9. Jain AK, Nigam M, Garg SD, Dubey BP, Arora A. Aluminium phosphide poisoning autopsy findings. J Indian Acad Forensic Med (JIAFM) 2005;27:35-9. 10. Mehrpour O, Dolati M, Soltaninejad K, Shadnia S, Nazparvar B. Evaluation of histopathological changes in fatal aluminum phosphid e poisoning. Indian J Forensic Med Toxicol 2008;2:34-6 [displayed 4 March 2015]. Available from: http://www.indmedica.com/journals.php?journalid=11& issueid=131&articleid=1739& action=article 11. Marashi SM, Arefi M, Behnoush B, Nasrabad MG, Nasri Nasrabadi Z. Could hydroxyethyl starch be a therapeutic option in management of acute aluminum phosphide toxicity? Med Hypotheses 2011;76:596-8. doi: 10.1016/j. mehy.2011.01.009 12. Marashi SM, Majidi M, Raji Asadabadi H, Nasri Nasrabadi Z. A common misconception in the management of aluminium phosphide poisoning. Arh Hig Rada Toksikol 2013;64:475-6. doi: 10.2478/10004-1254-64-2013-2404 13. Nasri Nasrabadi Z, Marashi SM. Comments on “A systematic review of aluminium phosphide poisoning”. Arh Hig Rada Toksikol 2012;63:551. doi: 10.2478/10004-1254-63-20122321 14. Marashi SM, Majidi M, Sadeghian M, Ahmadi S, Raji Asadabadi H, Nasri Nasrabadi Z. Is the use of cardioactive steroids appropriate in managing aluminium phosphide poisoning-induced heart failure? Arh Hig Rada Toksikol 2013;64:477-8. doi: 10.2478/10004-1254-64-2013-2439 15. Hartog CS, Natanson C, Sun J, Klein HG, Reinhart K. Concerns over use of hydroxyethyl starch solutions. BMJ 2014;349:g5981. doi: 10.1136/bmj.g5981 16. Jonville-Bera AP, Autret-Leca E, Gruel Y. Acquired type I von Willebrand’s disease associated with highly substituted hydroxyethyl starch. N Engl J Med 2001;345:622-3. doi: 10.1056/NEJM200108233450818 17. Rioux JP, Lessard M, De Bortoli B, Roy P, Albert M, Verdant C, Madore F, Troyanov S. Pentastarch 10% (250 kDa/0.45) is an independent risk factor of acute kidney injury following cardiac surgery. Crit Care Med 2009;37:1293-8. doi: 10.1097/CCM.0b013e31819cc1a0 18. Myburgh JA, Finfer S, Bellomo R, Billot L, Cass A, Gattas D, Glass P, Lipman J, Liu B, McArthur C, McGuinness S, Rajbhandari D, Taylor CB, Webb SA; CHEST Investigators; Australian and New Zealand Intensive Care Society Clinical Trials Group. Hydroxyethyl starch or saline for fluid resuscitation in intensive care. N Engl J Med 2012;367:190111. doi: 10.1056/NEJMoa1209759
Marashi SM, Nasri-Nasrabadi Z. Response to Professor Rumboldt’s reaction to our letter on hydroxyethyl starch in AlP poisoning Arh Hig Rada Toksikol 2015;66:221-223
19. Perner A, Haase N, Guttormsen AB, Tenhunen J, Klemenzson G, Åneman A, Madsen KR, Møller MH, Elkjær JM, Poulsen LM, Bendtsen A, Winding R, Steensen M, Berezowicz P, Søe-Jensen P, Bestle M, Strand K, Wiis J, White JO, Thornberg KJ, Quist L, Nielsen J, Andersen LH, Holst LB, Thormar K, Kjældgaard AL, Fabritius ML, Mondrup F, Pott FC, Møller TP, Winkel P, Wetterslev J; 6S Trial Group; Scandinavian Critical Care Trials Group. Hydroxyethyl
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starch 130/0.42 versus Ringer’s acetate in severe sepsis. N Engl J Med 2012;367:124-34. doi: 10.1056/NEJMoa1204242 20. Guidet B, Martinet O, Boulain T, Philippart F, Poussel JF, Maizel J, Forceville X, Feissel M, Hasselmann M, Heininger A, Van Aken H. Assessment of hemodynamic efficacy and safety of 6% hydroxyethyl starch 130/0.4 vs. 0.9% NaCl fluid replacement in patients with severe sepsis: The CRYSTMAS study. Crit Care 2012;16:R94. doi: 10.1186/ cc11358
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The 2nd Croatian Symposium on Membrane Transporters: membrane transporters in toxicological and pharmacological research Arh Hig Rada Toksikol 2015;66:225-227
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DOI: 10.1515/aiht-2015-66-2712
Note
The 2nd Croatian Symposium on Membrane Transporters (2. hrvatski simpozij o transporterima): Membrane Transporters in Toxicological and Pharmacological Research Ivan SaboliÄ&#x2021;1 and Tvrtko Smital2 Unit of Molecular Toxicology, Institute for Medical Research and Occupational Health1, Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute2, Zagreb, Croatia Numerous studies in the past 25 years have shown that most compounds, generated in metabolic processes (metabolic intermediates and products; endogenous compounds) or introduced orally or parenterally (nutritive compounds, medicaments, environmental toxins and other xenobiotics), do not enter or exit cells freely, by simple diffusion. Rather, their transmembrane movement is mediated by various membrane-bound proteins (transporters) localized in the cell membrane. Most membrane transporters belong to the SLC (SoLute Carriers) superfamily, which in humans encompass ~400 members grouped in 52 families (1). The functional properties and roles in cell physiology have been described for most of them, but about 40% of all SLCs are still orphans, without known substrates and biological characterization (1-5). The well-characterized members act as secondary- or tertiary-active exchangers, coupled transporters or electrochemically-driven facilitators in mediating the transport of amino acids, sugars, cholesterol, fatty acids, vitamins, inorganic ions, essential and some toxic metals, and various organic anions and cations, including drugs used in human and veterinary medicine. Depending on the electrochemical gradients of their substrates, these transporters can operate in influx or efflux mode, and are especially important in the intestine, liver and kidneys for handling the (re)absorptive and Correspondence to: Dr Ivan SaboliÄ&#x2021;, Unit of Molecular Toxicology, Institute for Medical Research and Occupational Health, Ksaverska c. 2, Zagreb, HR-10000. E-mail: sabolic@imi.hr
secretory processes. Due to their roles in the physiology of various organs, pathophysiology in various human diseases, and pharmacology (drug therapy, drug transport, drug-drug interactions, drug toxicity, drug development), the most characterized are the organic anion (OATs), cation (OCTs) and zwitterion transporters (family SLC22) (2-7), multidrug and toxins extruders (MATE/SLC47) (4, 8), and sodiumindependent (GLUTs/SLC2) and sodium-dependent (SGLTs/SLC5) glucose transporters (9-11). Functional defects or malfunctions of the specific SLC transporters due to either inactive gene, diminished gene expression, or single nucleotide polymorphisms (SNPs)-dependent gene variants have been implicated in various human conditions and diseases, such as primary carnithine deficiency, rheumatoid arthritis, inflammatory bowel disease, cisplatininduced toxicity, urolithiasis, mood-related disorders, diabetes, diminished renal secretion of organic anions and cations, variations in liver handling and renal secretion of organic cations, variations in drug pharmacokinetics and therapeutic efficiency, occurrence of drug-drug interactions and drug-induced organ toxicity, etc. (2, 4-6, 8). Another important superfamily of membrane transporters comprises several families of the primaryactive ABC (ATP Binding Cassette) members that operate as the ATP-driven efflux pumps (12). Most renown of these transporters are multidrug resistance protein 1/P-glycoprotein (MDR1/P-gp/ABCB1), various MRPs (multidrug resistance associated proteins/ABCC family), and breast cancer
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The 2nd Croatian Symposium on Membrane Transporters: membrane transporters in toxicological and pharmacological research Arh Hig Rada Toksikol 2015;66:225-227
resistance protein (BCRP/ABCG2). Their main physiological role is elimination from the cells and body various cationic and some anionic metabolic products which are too large to be substrates for the SLC transporters. In humans, P-gp is located in the intestinal and renal proximal tubule cells (apical membrane), hepatocytes (canalicular membrane), brain capillaries (endothelial cells, being an active component of blood brain barrier), and placental syncytiotrophoblast (apical membrane). As an efflux pump, P-gp exports various xenobiotics from these tissues, thus protecting them from drug toxicity but also causing resistance to various therapeutic drugs. MRPs are more ubiquitous, and also mediate multiple drug resistance to cancer therapy, whereas BCRP is particularly responsible for poor effects of breast cancer chemotherapy. In addition, variable effects of drug therapy, drug-drug interactions and drug-induced organ toxicity in different individuals, as well as some specific genetic diseases may be related to the gene polymorphism of these transporters (12-15). Recent studies have indicated that some transporters of the ABC families (and also from the SLC families) can be therapeutic or diagnostic targets in specific diseases. In vitro studies on identification and detailed molecular characterization of various membrane transporters, followed or complemented by the in vivo evaluation of its physiological and/or defence role in established animal research models, represents an important and wellestablished research area in pharmacology and toxicology. However, although the orthologs of the above-mentioned human membrane transporters have been detected and extensively studied in rodents and other experimental animals, in many aspects their translational relevance to human transporters appear to be questionable due to species differences in their characteristics and expression (16). From the viewpoint of environmental toxicology, however, membrane transporters are important as integral elements of the cellular defence mechanisms involved in processing of endo- and xenobiotics in other animals. For example, aquatic organisms are continuously exposed to a variety of environmental chemicals originating from various anthropogenic sources, and evolutionary conserved cellular defence mechanisms that mediate the overall bioavailability and potential toxicity of xenobiotics have been shown to be similar to those described in mammals. They include the regulation of absorption by uptake (SLC) transporters, the well-documented biotransformation by phase I and phase II enzymes (17), and finally the more recently demonstrated active efflux of xenobiotics by the ABC proteins (18). Furthermore, recent ecotoxicological studies have shown that the expression levels of some membrane transporters in specific organs of wild animals may be relevant indicators of the environmental pollution (17, 19). Finally, it has been demonstrated that the transport activity of ABC transporters can be sensitive to the presence of environmental chemicals which can act as specific inhibitors (18, 20, 21). These chemicals have the potential to block the ABC transportersâ&#x20AC;&#x2122;
mediated active efflux of xenobiotics, causing a significant increase in their intracellular accumulation. From an ecotoxicological viewpoint, the main consequence of this type of inhibition is an increase in sensitivity of aquatic organisms toward the many xenobiotics typically present in aquatic environments. Studies on the role of membrane transporters in ecotoxicological context are rare in comparison to efforts in mainstream toxicology, especially regarding research on SLC transporters. Despite their physiological importance and well documented role in the cellular detoxification in mammals, none of these transporters have been characterized in non-mammalian organisms before recent studies done on zebrafish (22-24), and the knowledge about polyspecific uptake transporters in non-mammalian species remains modest. Consequently, apart from new insights relevant for our fundamental understanding of the evolution, biological role and (eco)toxicological significance of polyspecific membrane transporters, this type of research is a necessary prerequisite for a better understanding and ultimately prediction of toxicity, and should enable a more reliable risk assessment in the context of both human and environmental toxicology. All these findings in the fast-growing field of membrane transporters led us to organize the 1st Croatian symposium on membrane transporters in 2013, which largely dealt with the physiological aspects of transporters in various experimental models performed by Croatian scientists in the Croatian research institutions. The presentations were held in Croatian. The present symposium was designed to be more international, with contributions from a few renown scientists from abroad, with all presentations in English and focused on a few selected transporters with a view toward the increasingly important pharmacological and (eco) toxicological impacts. REFERENCES 1. Bioparadigms [displayed 1 September 2015]. Available at http://www.bioparadigms.org/slc/menu.asp 2. Hediger MA, guest editor. Special issue: The ABCs of solute carriers: physiological, pathological, and therapeutic implications of human membrane transport proteins. PflĂźgers Arch - Eur J Physiol 2004;447. 3. Srimaroeng C, Perry JL, Pritchard JB. Physiology, structure, and regulation of the cloned organic anion transporters. Xenobiotica 2008;38:889-935. doi: 10.1080/ 00498250801927435 4. Fromm MF, Kim RB, editors. Drug Transporters. Handbook of Experimental Pharmacology 201. Berlin, Heidelberg: Springer-Verlag; 2011. 5. Koepsell H. Polyspecific organic cation transporters: their functions and interactions with drugs. Trends Pharmacol Sci 2004;25:375-81. PMID: 15219980 6. Rizwan AN, Burckhardt G. Organic anion transporters of the SLC22 family: biopharmaceutical, physiological, and
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7. 8.
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pathological roles. Pharmaceut Res 2007;24:450-70. doi: 10.1007/s11095-006-9181-4 Koepsell H. The SLC22 family with transporters of organic cations, anions and zwitterions. Mol Aspects Med 2013;34:413-35. doi: 10.1016/j.mam.2012.10.010 Omote H, Hiasa M, Matsumoto T, Otsuka M, Moriyama Y. The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations. Trends Pharmacol Sci 2006;27:587-93. PMID: 16996621 Chen L-Q, Cheung LS, Feng L, Tanner W, Frommer WB. Transport of sugars. Annu Rev Biochem 2015;84:865-94. doi: 10.1146/annurev-biochem-060614-033904 Poulsen SB, Fenton RA, Rieg T. Sodium-glucose cotransport. Curr Opin Nephrol Hypertens 2015;24:463-9. doi: 10.1097/ MNH.0000000000000152 Vrhovac I, Balen Eror D, Klessen D, Burger C, Breljak D, Kraus O, Radović N, Jadrijević S, Aleksic I, Walles T, Sauvant C, Sabolić I, Koepsell H. Locations of Na+-Dglucose cotransporters SGLT1 and SGLT2 in human kidney and of SGLT1 in human small intestine, liver, lung and heart. Pflügers Arch – Eur J Physiol 2015;467:1881-98. doi: 10.1007/s00424-014-1619-7 Stieger B, Higgins CF, guest editors. Twenty years of ABC transporters. Pflügers Arch - Eur J Physiol 2007;453. Endres CJ, Hsiao P, Chung FC, Unadkat JD. The role of transporters in drug interactions. Eur J Pharmaceut Sci 2006;27:501-17. doi: 10.1016/j.ejps.2005.11.002 König J, Müller F, Fromm MF. Transporters and drug-drug interactions: Important determinants of drug disposition and effects. Pharmacol Rev 2013;65:944-66. doi: 10.1124/ pr.113.007518 Ho RH, Kim RB. Transporters and drug therapy: Implications for drug disposition and disease. Clin Pharmacol Therapeut 2005;78:260-77. doi: 10.1016/j.clpt.2005.05.011 Sabolić I, Breljak D, Ljubojević M, Brzica H. Are mice, rats, and rabbits good models for physiological, pharmacological and toxicological studies in humans? Period biol 2011;113:7-16.
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17. van der Oost R, Beyer J, Vermeulen NPE. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ Toxicol Pharmacol 2003;13:57-149. doi: 10.1016/ S1382-6689(02)00126-6 18. Epel D, Stevenson CN, MacManus-Spencer LA, Luckenbach T, Hamdoun A, Smital T. Efflux transporters: newly appreciated roles in protection against pollutants. Environ Sci Technol 2008;42:3914-20. doi: 10.1021/es087187v 19. Luckenbach T, Fischer S, Sturm A. Current advances on ABC drug transporters in fish. Comp Biochem Physiol C - Toxicol Pharmacol 2014;165:28-52. doi: 10.1016/j.cbpc.2014.05.002 20. Smital T, Luckenbach T, Sauerborn R, Hamdoun AM, Vega RL, Epel D. Emerging contaminants - pesticides, PPCPs, microbial degradation products and natural substances as inhibitors of multixenobiotic defense in aquatic organisms. Mut Res 2004;552:101-17. doi: 10.1016/j.mrfmmm. 2004.06.006 21. Žaja R, Terzić S, Senta I, Lončar J, Popović M, Ahel M, Smital T. Identification of P-glycoprotein (P-gp, Abcb1) inhibitors in contaminated freshwater sediments. Environ Sci Technol 2013;47:4813-21. doi: 10.1021/es400334t 22. Popović M, Žaja R, Smital T. Organic anion transporting polypeptides (OATP) in zebrafish (Danio rerio): phylogenetic analysis and tissue distribution. Comp Biochem Physiol A - Mol Integr Physiol 2010;155:327-35. doi: 10.1016/j. cbpa.2009.11.011 23. Popović M, Žaja R, Fent K, Smital T. Molecular characterization of zebrafish Oatp1d1 (Slco1d1), a novel organic anion transporting polypeptide. J Biol Chem 2013;288:33894-911. doi: 10.1074/jbc.M113.518506 24. Popović M, Žaja R, Fent K, Smital T. Interaction of environmental contaminants with zebrafish uptake transporter Oatp1d1 (Slco1d1). Toxicol Appl Pharmacol 2014;280:149-58. doi: 10.1016/j.taap.2014.07.015
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Transporters as mediators of cisplatin effects and side-effects Giuliano Ciarimboli1,2, Anna Hucke1,2, Rita Schröter1,2, Antoinette Am Zehnhoff-Dinnesen2,3, Claudia Lanvers4, Alex Sparreboom5, Jason Sprowl5, and Eberhard Schlatter1,2 Experimental Nephrology, Medical Clinic D1; Interdisciplinary Center for Clinical Research (IZKF)2, Department of Phoniatrics and Pedaudiology3, University Hospital Münster; Department of Pediatric Hematology and Oncology, University Children’s Hospital Münster4, Münster, Germany, Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, USA5 The use of the effective antineoplastic agent cisplatin is hampered by serious side effects, such as nephro-, oto-, and neurotoxicity. In this work, we studied whether organic cation transporters (OCT) mediate the uptake and hence the toxicities of cisplatin in vitro and in vivo. Interaction of cisplatin with the transport of the fluorescent cation 4-(4-(dimethylamino)styril)-methylpyridinium (ASP+) was investigated in HEK293 cells stably transfected with hOCT1 or hOCT2 and in freshly isolated human hepatocyte and human proximal tubules. Cisplatin preferentially inhibited ASP+ transport via hOCT2. Incubation of hOCT2 cells with cisplatin induced apoptosis, which was completely suppressed by co-incubation by the hOCT2 substrate cimetidine. In vivo, the effects of cisplatin treatment on kidney and hearing (auditory brainstem response, ABR) functions were compared in wild type (WT) and OCT1/2 double knock-out (KO) mice. While in WT cisplatin led to reduced ABR and increased renal glucose, water, protein excretion and apoptosis, no sign of ototoxicity and only mild nephrotoxicity was observed after cisplatin treatment of KO mice. Co-medication of WT mice with cisplatin and cimetidine protected from ototoxicity and partly from nephrotoxicity. We also showed that OCT2 is expressed on hair cells of the cochlea and in dorsal root ganglia. Tumour-derived cell lines did not show a significant expression of mRNA for OCT2, suggesting that cisplatin uptake is mediated by other mechanisms here. These findings are very important for establishing chemotherapeutical protocols aimed at maximizing the antineoplastic effect of cisplatin, while reducing the risk of toxicities. Supported by IZKF Münster, Grant Cia2/013/13. KEY WORDS: cisplatin; cochlea; Corti-organ; dorsal root ganglia; kidneys; proximal tubules; side effects; transporters; tumour cells
CFEX (Slc26a6) in rat kidneys, liver, and small intestine in an experimental model of oxalate nephrolitiasis Dean Karaica1, Davorka Breljak1, Hrvoje Brzica1, Jovica Lončar2, Marija Ljubojević1, Carol M HerakKramberger1, Vedran Micek1, Ivana Vrhovac1, Jana Ivković Dupor1, Ivan Mihaljević2, Petra Marić2, Tvrtko Smital2, Birgitta C. Burckhardt3, Gerhard Burckhardt3, and Ivan Sabolić1 Molecular Toxicology, Institute for Medical Research and Occupational Health1, Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute2, Zagreb, Croatia, Physiology & Pathophysiology, University Medical Center, Göttingen, Germany3 CFEX (chloride/formate exchanger; Slc26a6) is an important anion exchanger of chloride, bicarbonate, oxalate (OX), formate, and hydroxyl ions in kidneys, liver, and the small intestine. Studies on CFEX-knockout mice indicated a possible role of CFEX in the development of hyperoxaluria and OX urolithiasis/nephrolithiasis, which in humans is more frequent in men. Here we studied the expression of the CFEX protein and mRNA in the organs of male and female rats, and employed a rat model of ethylene glycol (EG)-induced OX urolithiasis in order to correlate the expression of CFEX with sex-related hyperoxaluria. Rats drank EG in water (0.75 % vol/vol) or water (control) for 30 days. Tissue expressions of the CFEX protein and mRNA were analysed by immunochemical methods and qRT-PCR, respectively. The specificity of an anti-CFEX antibody, used in immunochemical studies, was confirmed in HEK293 cells transiently transfected with CFEX cDNA. In kidneys, the CFEX protein was immunolocalized to the proximal tubule brush-border membrane (BBM) with segmental (S3>>S1~S2) and sex (male>female) differences. Sex-unrelated expression was detected in the BBM of enterocytes (duodenum>jejunum) and in the hepatocyte canalicular membrane. In immunoblots, the CFEX protein band of ~120 kDa in various organs showed an expression pattern comparable to that in immunocytochemistry; however, renal CFEX mRNA expression was not sex-dependent. Compared to controls and EG-treated females, the EG-treated male rats exhibited hyperoxalemia, hyperoxaluria and OX crystaluria, but the expression of CFEX mRNA and protein remained unaffected in the organs of both sexes. Thus, basic CFEX expression in both rat sexes was sufficient for OX handling even upon EG-treatment, indicating that in rats, CFEX plays no major role in generating EG-induced hyperoxaluria and nephrolithiasis. KEY WORDS: ethylene glycol; hyperoxaluria; immunocytochemistry; transporters; proximal tubule; qRT-PCR; urolithiasis; Western blotting
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Inhibitors of glucose transporter SGLT1 in the treatment of diabetes mellitus will not act only in the kidneys; the transporter is also present in other rodent and human organs Ivana Vrhovac1, Davorka Breljak1, Dean Karaica1, Nikola Radović2, Ognjen Kraus3, Stipislav Jadrijević4, Hermann Koepsell5, and Ivan Sabolić1 Molecular Toxicology Unit, Institute for Medical Research and Occupational Health1, Urology, Clinical Hospitals Dubrava2 and Sisters of Mercy3, Department for Surgery of the Digestive Tract, Clinical Hospital Merkur4, Zagreb, Croatia; Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany5 Diabetes mellitus, one of the most common chronic diseases in population, is becoming a major health and economic problem. Current therapies with insulin and metformin, aimed at reducing blood glucose, are often ineffective and/ or problematic due to the induction of hypoglycemia, body weight gain, and occasional death resulting from cardiovascular disease. A novel generation of oral anti-diabetics inhibits sodium-D-glucose cotransporters in the small intestine (SGLT1/SLC5A1), thus diminishing the absorption of glucose from the diet, as well as in the kidneys (SGLT1 and/or SGLT2/SLC5A2), thus decreasing glucose reabsorption along the nephron and enhancing its excretion through urine. Overall, this improves glycemia, reduces body weight, lowers blood pressure, and decreases damage to the cardiovascular system. However, our recent studies showed that in humans, SGLT1 is not expressed only in the intestinal and renal epithelium; it was also detected in the liver (bile duct epithelium), lungs (bronchiolar Clara cells and alveolar type II cells), and heart (blood capillaries). These places represent possible targets for novel SGLT1 inhibitors. SGLT1 or dual (SGLT1+SGLT2) inhibitors may inhibit various SGLTs-related functions, such as fluid absorption in the lungs, energy supply to Clara cells, and glucose release from the heart capillaries, and may thus cause functional disorders. In addition, our novel unpublished data showed that in mice, Sglt1 is localized in the kidneys, small intestine, liver, pancreas, salivary glands, tongue, prostate, seminal vesicles, and uterus. The newly discovered localizations of SGLT1/Sglt1 suggest certain novel functions for this transporter, which could be of great physiological and biomedical importance. KEY WORDS: human organs; immunocytochemistry; mouse organs; qRT-PCR; sodium-D-glucose cotransporters; SGLT1; SGLT2
Genetic variability in organic cation transporters: pharmacology, pathophysiology, and beyond Mladen V. Tzvetkov, Frank Faltraco, Nawar Dalila, Tina Seitz, Robert Stalmann, and Jürgen Brockmöller Institute of Clinical Pharmacology, University Medical Center Göttingen, Göttingen, Germany Naturally occurring genetic variants substantially affect the expression and function of organic cation transporters. Therefore, these variants may lead to inter-individual variations in plasma and organ concentrations of endogenous molecules and drugs and may cause variations in drug pharmacokinetics, efficacy and toxicity, or may confer susceptibility to diseases. Common genetic variants causing loss of OCT1 activity have been shown to affect the hepatic uptake and pharmacokinetics of the drugs morphine, tramadol and tropisetron. On the other hand, genetic variants causing loss of OCT1 activity are suggested to modulate the efficacy of drugs acting in the liver, like metformin. Here we give an overview on the available data about the genetically-determined loss of OCT1 activity and discuss potential applications for personalised drug therapy. Furthermore, specific global patterns of loss of OCT1 activity were observed. We discuss how these patterns may confer inter-ethnical variability of pharmacokinetics and efficacy of clinically relevant drugs and how they may point to selection pressure for losing or maintaining OCT1 activity. In OCT2, the common polymorphism Ala270Ser was repeatedly reported to associate with inter-individual variability in metformin pharmacokinetics. We present meta-analyses of currently available studies showing the inconsistency and limited size of these effects. In contrast, polymorphisms in OCTN1 and OCTN2 were repeatedly reported as risk factors for Crohn’s disease. Meta-analyses of available studies are presented illustrating the high consistency of these observations. Finally we discuss the potential effects of regulatory polymorphisms in the MATE1 and MATE2K genes on the pharmacokinetics and efficacy of metformin. KEY WORDS: Crohn’s disease; genetic polymorphisms; hepatic uptake; metformin; morphine; pharmacokinetics; renal clearance; SNPs; tramadol; tropisetron
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Importance of transporters in the prediction of drug efficacy and adverse events Nada Božina, Mila Lovrić, and Lana Ganoci Department of Laboratory Diagnostics, University Hospital Center Zagreb, Zagreb University School of Medicine, Zagreb, Croatia An important role of drug transporters in modulating pharmacokinetic and pharmacodynamic properties has been documented for many drugs. Many studies have investigated the association between gene polymorphisms of the efflux transporters belonging to the ATP-binding cassette (ABC) superfamily of membrane proteins, as well as of influx transporters of the SLC superfamily, and therapy response and clinical outcomes. Convincing results have been obtained for ABCB1 gene variants and variability of therapy by digoxin, HIV protease inhibitors, some antiepileptics, antidepressants, antipsychotics, immunosuppressants; ABCC2 and mycophenolic acid, anticancer drugs like methotrexate, cisplatin, irinotecan, antibiotics; ABCG2 and anticancer drugs, statins, alopurinol, cimetidin, and lamotrigin. Drug-drug interactions on the level of variable drug metabolism by phase I and phase II enzymes and drug transporters often called for a phase III modulate at many barrier tissues – such as the intestine, liver, blood-brain barrier, kidney, placenta – plasma and cerebrospinal fluid drug concentrations that can lead to non-responsiveness, resistance or serious or even lethal adverse drug reactions. The most prominent case is the development of rhabdomyolysis due to statin therapy in case of variant/ ineffective allele carriers of SLCO1B1 521C>T with an aggravating role of ABCG2 421C>A and some CYP450 gene polymorphisms. Data also emphasize the role of ABCB1 and ABCG2 polymorphisms as promising predictors of the clinical outcome of tyrosine kinase inhibitor (TKI) therapy with drugs like imatinib, nilotinib, and sunitinib. Based on clinical evidence of drug transporter-mediated drug-drug interactions (DDIs) and some functional polymorphisms affecting drug efficacy and safety, both the US Food and Drug Administration and European Medicines Agency recommend preclinical evaluation and, when appropriate, clinical assessment of transporter-mediated DDIs. Although variability of bioavailability and drug response may be attributed to certain polymorphisms in transporter genes, transcriptional regulation or post-transcriptional modification in certain cases seems to be even more critical. KEY WORDS: adverse drug reactions; drug bioavailability; drug-drug interaction; drug transporters; genetic polymorphism
Age and sex differences in expression of P-glycoprotein (P-gp/Mdr1/Abcb1) in rat liver and kidneys Jana Ivković Dupor1, Marija Ljubojević1, Davorka Breljak1, Ivana Vrhovac1, Dean Karaica1, Vedran Micek1, Carol M. Herak-Kramberger1, Roberto Antolović2, and Ivan Sabolić1 Molecular Toxicology Unit, Institute for Medical Research and Occupational Health, Zagreb1, Department of Biotechnology, University of Rijeka, Rijeka2, Croatia P-glycoprotein (P-gp; ABCB1 in humans/Abcb1 in rodents) is an ATP-dependent multidrug efflux transporter in the cell membrane, also known as the multidrug resistant protein 1 (MDR1 in humans/Mdr1 in animals), constitutively expressed in the a) bile canaliculi of hepatocytes, b) brush-border membrane of the renal proximal tubule cells, c) luminal membrane of the intestinal enterocytes, d) blood-brain, blood-testis, and mother-foetus barriers, and e) hematopoietic cells. P-gp mediates the transport of some endogenous compounds and various xenobiotics (drugs, toxins, environmental organic compounds, and their metabolites) out of the cells, thus protecting the cell’s interior from the potentially toxic effects of these compounds. Although P-gp is a highly studied transporter in a variety of (patho)physiological conditions in human and animal organs, its age-dependent expression is still a controversial issue. Here, we investigated the ontogenic pattern of P-gp protein expression in rat liver and kidneys to provide insight into the drug transport capacity in these organs at different ages. P-gp protein expression was studied by immunocytochemistry and Western blotting in organs from neonatal (age, 1 day), prepubertal (age, 3 weeks), adult (age, 3 months), and old (age, 2 years) male and female Wistar rats. In both sexes, the liver P-gp expression pattern was: neonatal (highest) > prepubertal > adult < old. In the kidneys, the P-gp expression exhibited the pattern: neonatal < prepubertal < adult (highest) > old. Better knowledge of the ontogeny of P-gp expression may improve experimental design and the interpretation of results of toxicity studies in juvenile animals as well as the understanding of drug toxicity in different age groups in translational studies. KEY WORDS: age differences; immunocytochemistry; kidney; liver; Mdr1; proximal tubule; P-gp; Western blotting
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Identification of xenobiotic interactors with multidrug and toxin extrusion (MATE/ SLC47) proteins in zebrafish (Danio rerio) Jovica Lončar1, Petra Krznar2,3, Marta Popović1,5, Roko Žaja1,4, and Tvrtko Smital1 Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute1, Department for Biology, Faculty of Science, University of Zagreb2, Zagreb, Croatia; Institute of Molecular Systems Biology, Department of Biology, ETH Zürich, Zürich, Switzerland3; Sir William Dunn School of Pathology4, Department of Oncology5, University of Oxford, Oxford, United Kingdom Multidrug and toxin extrusion (MATE) proteins are involved in the extrusion of endo- and xenobiotics across the plasma membrane, similar to the ATP-binding cassette (ABC) transporters but without the consumption of ATP. MATEs are conserved from bacteria to mammals with a differing number of genes within groups. In humans, three MATEs have been found (MATE1, 2, and 2k), whereas in zebrafish (Danio rerio), we have found six members annotated as DrMate 3-8 which form a distinct cluster separated from the tetrapod MATEs. Tissue expression profiling showed a high expression of zebrafish MATEs in toxicologically important tissues, kidney and liver, as well as in the testes. MATEs transport activity was analysed in transiently overexpressing HEK293 cell system measuring the uptake of model cationic fluorescent dyes. Assay conditions were optimised so that the protein interaction with a battery of toxicologically interesting endo- and xeno-biotics could be evaluated. Basic kinetic parameters together with the type of interaction – whether a compound is transported or if it merely inhibits the transport activity – were determined for over 20 selected physiological and/or xenobiotic interactors of mammalian MATEs, including hormones, bile salts, drugs, and pesticides. KEY WORDS: efflux transporters; MATEs; physiological and xenobiotic interactors; zebrafish
Functional and structural characterization of organic cation transporter 1 (Oct1) in zebrafish (Danio rerio) Ivan Mihaljević1, Marta Popović1, 3, Roko Žaja1, 4, Nikola Maraković2, and Tvrtko Smital1 Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute1, Biochemistry and Organic Analytical Chemistry Unit, Institute for Medical Research and Occupational Health2, Zagreb, Croatia; Department of Oncology3, Sir William Dunn School of Pathology4, University of Oxford, Oxford, United Kingdom Organic cation transporters (OCTs) are members of the SLC22 family within the SLC (Solute Carriers) superfamily of membrane proteins. These transporters are responsible for the uptake of numerous organic cations and neutral molecules, alongside other inorganic ions. There are three OCTs in humans, two of which play crucial roles in ADME (administration, distribution, metabolism and excretion) processes, with high expression of OCT1 in liver and OCT2 in kidneys. In zebrafish, an important vertebrate model organism, there are two Oct members, with Oct1 dominantly expressed in kidneys and livers, where it potentially has the compensatory role of human OCT1 and OCT2, whereas Oct2 showed lower expression in toxicologically less relevant tissues, which indicates its potentially more specific physiological role. Using transiently transfected human embryonic kidney cells (HEK239) as a heterologous expression system, we developed an in vitro tool for the functional analysis of Oct1 by measuring the uptake of five identified fluorescent substrates of Oct1. Functional analysis revealed the interaction of Oct1 with numerous endo- and xenobiotics. Steroid hormones showed potent inhibition of 4-(4-(dimethylamino)styryl)N-methylpyridinium iodide (ASP+) uptake by Oct1, with Ki values in low micromolar range, along with potent interactions with numerous xenobiotics ranging from various pharmaceuticals to deleterious environmental contaminants such as organotin compounds. However, further analysis focused on the type of interaction with the identified interactors was limited due to the complexity of the Oct1 active site, which we characterized in more detail using homology modelling and molecular docking analysis. The structural models of zebrafish Oct1 and human OCT1 and OCT2 revealed a characteristic transmembrane organization of proteins and spacious active regions with several binding sites. Our research offers novel insight into the function and transport mechanism of Oct1 as a potentially crucial factor in steroid hormone homeostasis and toxicological response in zebrafish. KEY WORDS: functional characterization; organic cation transporters; physiological and xenobiotic interactors; zebrafish
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Interactions of secondary metabolites from cyanobacteria and invasive tropical algae with phase 0 membrane transporters in zebrafish (Danio rerio) Petra Marić, Ivan Mihaljević, Jovica Lončar, Jelena Dragojević, and Tvrtko Smital Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute, Zagreb, Croatia One advantageous feature of cyanobacteria and invasive tropical algae from the genus Caulerpa is their ability to produce secondary metabolites with diverse biological activities. Some secondary metabolites are toxic and thus represent a significant threat to the environment and animal/human health, especially during periods of intensive blooms. So far, the understanding of their interaction with membrane transporters involved in the cellular defence mechanism has been poor. Therefore, the main goal our study was to determine the interactions of secondary metabolites from C. racemosa, C. taxifolia and selected cyanobacterial strains, including genera Anabaena, Nostoc, Phormidium and Oscillatoria, with the phase 0 membrane transporters involved in the cellular detoxification mechanism of an important model vertebrate species, the zebrafish (Danio rerio). The toxic inhibitory effects of secondary metabolites from Caulerpa and cyanobacterial strains to the activity of zebrafish anion (DrOatp1d1) and cation (DrOct1) uptake transporter were determined using the human embryonic kidney (HEK293) expression system. In addition, we performed a preliminary identification of the biologically active substances that cause the observed toxic effects using the effects-directed analyses (EDA) approach. Significant toxicity for these complex biological samples towards toxicologically relevant zebrafish uptake transporters DrOatp1d1 and DrOct1 was determined. Caulerpin (CLP) was determined as the major metabolite in C. racemosa while caulerpenyne (CYN) appeared to be the dominant biologically active compound in C. taxifolia. CYN was confirmed as an inhibitor of the DrOatp1d1 anion transporter. Finally, aquatic cyanobacterial strains, especially the Oscillatoria strain, showed the most significant and potentially (eco)toxicologically highly relevant inhibitory effects towards the transport activity of DrOatp1d1 and DrOct1 transporters. KEY WORDS: cellular detoxification; cyanobacteria; invasive tropical algae; phase 0 membrane transporters; secondary metabolites; zebrafish (Danio rerio)
Invasive vs. native bivalves - differences in tolerance to anthropogenic stress Ivana Bošnjak1,3, Ana Bielen1, Martina Jaklič4, Marija Cvitanić2, Kristina Sepčić5, Tatjana Simčič4, Jasna Lajtner2, and Sandra Hudina2 Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology1, Department of Zoology2, Department of Botany3, Faculty of Science, University of Zagreb, Zagreb, Croatia, National Institute of Biology4, Department of Biology, Biotehnical Faculty, University of Ljubljana5, Ljubljana, Slovenia Tolerance towards environmental stress has been frequently considered as one of the key determinants of invasion success. However, empirical evidence supporting the assumption that invasive species endure unfavourable conditions better compared to native species is limited and has even yielded opposing results. We examined tolerance to thermal stress and heavy metal zinc pollution (ZnCl2) in two phylogenetically related and functionally similar freshwater bivalve species; the native Anodonta anatina and the invasive Sinanodonta woodiana. We assessed their response to stress using several cellular response assays: metabolic rates (ETS - electron transport system), efficiency of the multixenobiotic resistance (MXR) mechanism activity, and enzymatic biomarkers (ChE - cholinesterase, GST - glutathione-S-transferase and CAT - catalase). Overall, S. woodiana coped with unfavourable conditions much better. This was evident from (i) a significantly more pronounced MXR mechanism activity; (ii) significantly higher ETS activity, and (iii) lower response of stress-related enzymes (ChE, GST and CAT) under thermal stress and ZnCl2 pollution. The overall better tolerance to thermal extremes is an especially important physiological advantage for the future of invasion success of S. woodiana in European freshwaters, especially in the context of climate change. KEY WORDS: freshwater mussels; heavy metal zinc pollution; invasion success; metabolic rate; MXR mechanism activity; thermal stress
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NEW EDITIONS Hebrang Grgić I, urednica. Hrvatski znanstveni časopisi. Zagreb: Školska knjiga; 2015. 278 str. ISBN 978-9530-61770-4 Urednica Ivana Hebrang Grgić okupila je 22 iskusnih urednika, znanstvenika i stručnjaka i njihove tekstove objedinila u dosad najopsežniji uvid u stanje znanstvenog izdavaštva u Hrvatskoj, ali i u onom dijelu svijeta koji utječe na njega. Toliko korisnih informacija za urednike hrvatskih znanstvenih časopisa i pametnog razmišljanja o budućnosti izdavaštva u domovini dosada nisam imao prilike vidjeti na jednom mjestu. Knjiga je podijeljena u tri cjeline: Organizacija uredničkog rada; Autori: prava, obveze, vještine te Posebnosti znanstvenih područja. Meni je osobno najzanimljivija bila prva cjelina, budući da veoma detaljno razmatra mnoge aspekte uređivanja znanstvenih časopisa i izazove s kojima se svakodnevno suočavamo. Budući da mi je prostor ograničen, usredotočit ću se baš na ovu prvu cjelinu. Ana i Matko Marušić već svojim intrigantnim naslovom “Znanstveni časopisi u Hrvatskoj: teška pitanja na koja nije teško odgovoriti” daju naslutiti da i nije sve tako crno, ali isto tako članak zaključuju predviđanjem da će “dobri postati još bolji, a slabi još slabiji…”. To su oni koji se neće prilagoditi novim tehnologijama. Svakako pročitati. Bojan Macan i Jelka Petrak daju podroban i jasan pregled bibliometrijskih pokazatelja za procjenu kvalitete časopisa i citatnih baza na čijim se podacima mahom temelji takva procjene te govore o njihovim prednostima i nedostacima i alternativama poput altmetrije. Jako mi se svidio (r)evolucionaran pristup Jadranke Stojanovski. U članku naslovljenom “(R)evolucija znanstvenih časopisa” vrlo argumentirano ukazuje na problem hiperprodukcije znanstvenih časopisa (tu mislim i na članke i na časopise). Citiram: “Iako je bit znanstvene komunikacije u dijeljenju, danas najaktualnija tema znanstvene komunikacije nisu novi načini komuniciranja i dijeljenja utemeljeni na novim tehnologijama već je to
ekonomija znanstvenog izdavaštva koja je cijeli sustav dovela u ozbiljnu krizu”. Pokušaji da se odgovori na komercijalizaciju znanosti kao općeg dobra su različiti, više ili manje uspješni, a jedan se pridjev provlači kroz cijeli članak - “otvoreno”: otvoreni pristup, otvoreni formati i mediji, otvoreni podaci, otvorena recenzija, otvoreni doprinos autora. U četvrtom poglavlju autori Mrša i sur. govore kako se u Hrvatskoj financiraju znanstveni časopisi. Ova je tema škakljiva, nekima i bolna, ali kad pročitate članak, u kojem se uspoređujemo i sa susjedima, shvatit ćete da bi moglo biti i gore, a možda i hoće, promijeni li se model financiranja. Poglavlje Franje Pehara i Zorana Velagića mahom se bavi najpoznatijim online sustavom za upravljanje uredničkim postupkom, tzv. OJS-om (Online Journal System), ali ćete tu naći i mnoštvo drugih korisnih informacija. Druga, nama u uredništvu zanimljiva, cjelina govori o autorima, njihovim pravima, čestitosti i informacijskoj pismenosti. Svakako bih istaknuo članak Ksenije Baždarić koji daje korisne naputke uredništvima kako se nositi s autorskom “nečestitosti”. Zahvaljujući članku Sonje Špiranec (“Informacijska pismenost kao oslonac znanstvene komunikacije”) shvatio sam koliko sam zapravo nepismen, a možda i tvrd. U sili koncepata i apstraktnih misli jedino što sam razumio jest da je za informacijsku pismenost važan otvoreni pristup. I tu se vraćamo na meni mnogo jasnije izlaganje Jadranke Stojanovski. U trećem se dijelu govori o posebnostima izdavaštva vezanog uz biotehničke, prirodne, knjižničko-informatičke, društvene i jezične znanosti, kao i njihova vrednovanja. Za svakog ponešto. Meni je za oko zapeo članak Vanje Borša “Neznanstveni odnos prema domaćim znanstvenim časopisima i u njima”, a osobito dio o ulozi i ponašanju uredništva. Kritika mu je prilično utemeljena, premda bih dodao sljedeće: slažem se da autori ne postoje zbog časopisa (str. 238, pri dnu), ali ne i da časopisi postoje zbog autora, već i jedni i drugi postoje zbog čitatelja. Knjiga završava s posebnim dodatkom - Hrvatskom deklaracijom o otvorenom pristupu. Otvoreni pristup je očito nit vodilja koja će imati sve važniju ulogu u našem znanstvenom izdavaštvu. On ga je vjerojatno i spasio. Dado Čakalo