Arhiv vol. 67 no. 2

ISSN 0004-1254

ARHIV ZA HIGIJENU RADA I TOKSIKOLOGIJU

ARCHIVES OF INDUSTRIAL HYGIENE AND TOXICOLOGY

Arh Hig Rada Toksikol • Vol. 67 • No. 2 • pp. 83-168 • ZAGREB, CROATIA 2016

CONTENTS Reviews Epidemiological trends of hormone-related cancers in Slovenia

Vesna Zadnik and Mateja Krajc

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Simona Gaberšček and Katja Zaletel

93

Lucija Perharič, Tanja Fatur, and Jernej Drofenik

99

Mini-review European Union’s strategy on endocrine disrupting chemicals and the current position of Slovenia

Anita Klančar, Jurij Trontelj, Albin Kristl, Maja Zupančič Justin, and Robert Roškar

106

Original articles Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study

Rafael Valencia-Quintana, Sandra Gómez-Arroyo, Juana Sánchez-Alarcón, Mirta Milić, José Luis Gómez Olivares, Stefan M. Waliszewski, Josefina Cortés-Eslava, Rafael Villalobos-Pietrini, and María Elena Calderón-Segura

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Assessment of genotoxicity of Lannate-90® and its plant and animal metabolites in human lymphocyte cultures

Syed M. Nurulain, Mohamed Shafiullah, Javed Yasin, Abdu Adem, Juma Al Kaabi, Saeed Tariq, Ernest Adeghate, and Shreesh Ojha

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Terbufos-sulfone exacerbates cardiac lesions in diabetic rats: a sub-acute toxicity study

Angel M. Dzhambov and Donka D. Dimitrova

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Exposure-response relationship between traffic noise and the risk of stroke: a systematic review with meta-analysis

Irena Knežević, Ljiljana Gregov, and Ana Šimunić

152

Salience and conflict of work and family roles among employed men and women

Darinka Purg, Andrej Markota, Damjan Grenc, and Andreja Sinkovič

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Sayed Mahdi Marashi and Zeynab Nasri-Nasrabadi

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Letter to the Editor Is there a role for sildenafil in the management of paraquat-induced lung fibrosis?

A12 A13 A14 A15

Obituary (in English) Report (in English) Reports (in Croatian) Announcement (in Croatian)

Epidemiological trends of iodine-related thyroid disorders: an example from Slovenia

Case report Low-dose intravenous lipid emulsion for the treatment of severe quetiapine and citalopram poisoning

Cover page: Photography by Linda Poščić Borovac.

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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Zadnik V, Krajc M. Epidemiological trends of hormone-related cancers in Slovenia Arh Hig Rada Toksikol 2016;67:83-92

Review

DOI: 10.1515/aiht-2016-67-2731

Epidemiological trends of hormone-related cancers in Slovenia Vesna Zadnik and Mateja Krajc Epidemiology and Cancer Registry, Institute of Oncology Ljubljana, Slovenia [Received in October 2015; CrossChecked in October 2015; Accepted in April 2016] The incidence of hormone-related cancers tends to be higher in the developed world than in other countries. In Slovenia, six hormone-related cancers (breast, ovarian, endometrial, prostate, testicular, and thyroid) account for a quarter of all cancers. Their incidence goes up each year, breast and prostate cancer in particular. The age at diagnosis is not decreasing for any of the analysed cancer types. The risk of breast cancer is higher in the western part of the country, but no differences in geographical distribution have been observed for other hormone-related cancers. Furthermore, areas polluted with endocrine-disrupting chemicals that affect hormone balance such as PCBs, dioxins, heavy metals, and pesticides, do not seem to involve a greater cancer risk. We know little about how many cancers can be associated with endocrine disruptors, as there are too few reliable exposure studies to support an association.

KEY WORDS: attributable fraction; cancer burden; endocrine disruptor; hormone-related cancer Sex hormones have been associated with breast, ovarian and, endometrium cancers, while their role in prostate and testicular cancers is not as clear (1). The thyroid axis hormones and oestrogens may also take part in the development of thyroid cancer (2). These six cancer types are usually referred to as hormone-related cancers (HRCs) (1-3) because they share the same mechanism of carcinogensis. In addition, there is accumulating evidence that sex hormones also play a role in the aetiology of colorectal (4) and liver cancer (5). Relatively high concentrations of oestrogen receptor are also present in the spleen, while low levels have been detected in the kidney, thymus, skin, and lung (6). However, these cancer types are generally (and in our evaluation) not considered as HRCs (1).

Epidemiological evidence of the association between endogenous and exogenous hormones with cancer comes from a variety of sources: from geographical differences in cancer risk, from migration studies, from time trend analyses of incidence and mortality data and from analytical studies comparing populations being exposed and not being exposed to a selected factor (1-3). Global rates of HRCs have been increasing over the past 40-50 years (3) and are the highest in industrialised countries (7). There is an indication that age at exposure is especially important, as there are periods of greater vulnerability during development (such as foetal and prepubertal exposure) (8).

Endocrine disruptors that are most commonly associated with HRCs are polychlorinated biphenyls (PCBs), dioxins, Correspondence to: Vesna Zadnik, Epidemiology and Cancer Registry, Institute of Oncology Ljubljana, Zaloska 2, SI-1000 Ljubljana, Slovenia; E-mail: vzadnik@onko-i.si The subject of this paper has partly been presented at the 2nd Congress of the Slovenian Society of Toxicology “Endocrine disrupting chemicals – from molecule to man� held in Ljubljana, Slovenia, from 23 to 24 April 2015.

some heavy metals, and pesticides (such as DDT and DDE).

Table 1 lists the groups of endocrine disruptors associated with HRCs. Many of these association studies are inconclusive because they typically suffer from methodological limitations such as measurement of exposure at wrong time or disregard of combined exposure (2). For example, several organochlorine and organophosphate pesticides have been linked with an increased risk of prostate cancer, especially in occupational studies, but only two pesticides [inorganic arsenic compounds and dioxin TCDD (tetrachlorodibenzo-pdioxin)] had strong enough evidence to be classified as carcinogenic to humans (IARC Group 1) (1). Five organophosphate pesticides have recently been moved from the IARC Group 3 (not classifiable) to Group 2 (probably carcinogenic to humans), because there is enough evidence that they are carcinogenic in animals. However, evidence from human studies is scarce and inadequate to classify these pesticides as IARC Group 1 (9).

We focused our study on the overall, geographical, and age-specific incidence of six HRCs in Slovenia over the last 50 years. In addition, we put the national statistics in an international context and looked for the reasons for changes in epidemiological indices, paying particular attention to the potential influence of exogenous hormones and hormone-like substances.

Latest research in Slovenia (original data) Our latest research included 77,271 HRC cases diagnosed in Slovenia between 1961 and 2011, as reported by the Slovenian population-based cancer registry.

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Zadnik V, Krajc M. Epidemiological trends of hormone-related cancers in Slovenia Arh Hig Rada Toksikol 2016;67:83-92

Table 1 Groups of chemicals associated with hormone-related cancers (adopted from ref. 2)

Breast

Endometrium

Ovary

Prostate

Testis

Thyroid

Polychlorinated biphenyls, dioxins, furans

x

x

x

x

Polybrominated diphenyl ethers

x

DDT/DDE

x

x

x

x

Pesticides

x

x

x

x

x

Heavy metals

x

x

x

Bisphenol A, parabens

x

x

x

Phthalates

x

Pharmaceutical estrogens

x

x

x

x

x

x

Phytoestrogens

x

x

x

Cancer Registry of the Republic of Slovenia (CRS) is one of the oldest population-based cancer registries in the world. Reporting cancer has been mandatory in Slovenia since the CRS’s foundation in 1950. Cancer data collection complies with all international standards and covers 100 % of the population (10). Data quality (reliability and completeness) is regularly monitored and published in CRS’s annual reports (11). The ratio between mortality and incidence has been stable over the years. The proportion of cases registered based on death certificate only has not exceeded 2 % for several decades. More than 90 % of the registered cases have been confirmed by microscopy. To verify personal information and monitor the vital status of patients, CRS has access to the national population registry, while it updates patient residence data from the national Register of Spatial Units (maintained by the Surveying and Mapping Authority of Slovenia). Population demographics, stratified by gender and five-year age groups, is updated regularly from the Statistical Office of the Republic of Slovenia.

CRS provides information on the incidence, prevalence, and survival of cancer patients. Cancer sites are coded according to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD10). Our study has included incidence data on the following HRCs: breast (ICD10: C50; analysed for women only), endometrial (ICD10: C54), ovarian (ICD10: C56 and D39.1 encoding ovarian tumours of borderline malignancy), prostate (ICD10: C61), testicular (ICD10: C62), and thyroid cancer (ICD10: C73). Ovarian tumours of borderline malignancy (D39.1) have been classified separately at the CRS from the year 2001 on, but we’ve merged both types of ovarian tumour for this analysis.

Incidence rates were age-standardised (ASR) to the European Standard Population using the following formula:

where N is the number of cases in the observed population, P is the number of persons in the observed population, S is the number of persons in the standard population and i is a five-year age group. ASRs are presented by the calendar year of diagnosis. The time trends of ASRs for each examined cancer site were analysed using log-linear joinpoint regression (12). The trends were characterised in terms of average annual percent change (APC), assuming a constant rate of the previous year. In addition, a 95 % confidence interval (CI) of APC is reported. To check if the age at cancer onset is decreasing, we calculated age-specific incidence rates for four age groups: 15-39 years, 40-54 years, 55-69 years, and 70 or more years. In addition, we determined the time trend of the median age at diagnosis for all patients younger than 55 at diagnosis.

Review of the latest research results and earlier research in Slovenia The six HRCs account for almost a quarter of all cancers in the developed countries (including non-melanoma skin cancers). In Europe, their annual age-standardised incidence is 80.4 per 100,000 men and 110.4 per 100,000 women (7).

The annual number of new cases and ASR rates for Slovenia is reported in Table 2. With more than 20 % of all cancer cases, breast and prostate cancers are the most common cancers diagnosed in women and men, respectively.

Endometrial and ovarian cancers rank 5th to 10th in incidence among women. Testicular cancer is generally rare but is the most common cancer among young and middle-aged men.

Thyroid cancer is also relatively uncommon but is approximately four times more frequent in women.

Incidence trends Figure 1 shows that the incidence of prostate, breast, endometrial, testicular, and thyroid cancers have been on the rise, whereas ovarian cancer has been stable over the investigated 50 years. Average annual percent changes in Table 2 give a better view of the increases in incidence from 1992 to 2011 for each investigated HRC. Prostate cancer clearly leads, followed by thyroid and testicular cancer.

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Zadnik V, Krajc M. Epidemiological trends of hormone-related cancers in Slovenia Arh Hig Rada Toksikol 2016;67:83-92

Figure 1 Age-standardised incidence rates for six hormone-related cancers by calendar year, Slovenia 1961-2011

Breast cancer incidence, in turn, was the most prominent between 1992 and 1999 (APC: 3.8, CI 2.1;5.5), since which time it has stabilised (APC: 0.5, CI -0.2;1.2). Endometrial cancer incidence has not changed over the last twenty years, and ovarian cancer has shown a rise of only 0.7Â % per year.

Age at diagnosis With the exception of testicular cancer, where the agespecific incidence rates are the highest between 25 and 35 years, all other HRCs develop at a later age, most frequently after the age of 60. Our time-trend analysis of the age at diagnosis did not indicate any earlier cancer onset in recent

years. Figure 2 shows that the incidence of breast, endometrial, and prostate cancers is rising in the population aged 55 and over. Ovarian cancer is dropping in women older than 40, but the trend seems stable in women of younger ages. Thyroid cancer, in turn, shows a comparable increase in age groups up to 69 and a plateau from 70 years up.

For any of the analysed HRCs the median age at diagnosis in patients diagnosed before the age of 55 has not decreased in the last twenty years. On the contrary, it has risen by one year for prostate, testicular, and ovarian cancers (to 51, 33, and 48 years, respectively). As for breast cancer,

Table 2 Average annual number of new cases (incidence) and age-standardized incidence rates (ASR) for six hormone-related cancers in Slovenia between 2009 and 2011. Average annual percent change of ASR for the six HRCs between 1992 and 2011 Cancer

Average incidence (2009-2011)

Average ASR (2009-2011)

Annual percent change (95Â % CI) (1992-2011)

Breast

1190

88.0

1.5 (1.1;2.0)

Endometrium

305

21.0

0.1 (-0.5;0.7)

Ovary

158

16.3

0.7 (0.1;1.3)

Prostate

1426

117.6

6.4 (5.7;7.1)

Testis

100

9.1

3.4 (2.3;4.5)

Thyroid 150 6.4 ASR: age standardized incidence rate per 100,000 person years (European standard) CI: confidence interval

5.2 (4.2;6.1)

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Zadnik V, Krajc M. Epidemiological trends of hormone-related cancers in Slovenia Arh Hig Rada Toksikol 2016;67:83-92

Figure 2 Age-specific incidence rates for six hormone-related cancers by calendar year, Slovenia 1961-2011

the median age at diagnosis for patients diagnosed before 55 has been 47 for the last twenty years. In the younger age group, it also has not changed: the median for the groups diagnosed before 35 and before 25 has been 33 and 23 years, respectively.

Geographical distribution The GLOBOCAN 2012 statistics (7) indicate that the incidence of all HRCs varies significantly worldwide. As a rule, their incidence is higher in the industrial world and remains low in many parts of Asia and Africa. The incidence of prostate, testicular, endometrial, ovarian, and thyroid cancer varies by more than 25-fold across the world’s regions. For prostate cancer this variation primarily reflects the differences in the availability of prostate-specific antigen (PSA) screening, but for other cancers the variation may reflect differences in lifestyle and genetic factors, as

described in more detail in Discussion. Variations in breast cancer incidence are smaller (nearly fourfold), with rates ranging from 27 per 100,000 in Middle Africa and Eastern Asia to 96 in Western Europe.

HRC incidences do not vary as much between European regions. According to the EUCAN database (13), the variation is the highest for thyroid cancers (over tenfold), with the highest incidence in Austria, Italy, and Lithuania (12-15 per 100,000) and the lowest in Albania, Bosnia and Hercegovina, and Greece (1 per 100,000) (13). Cancer mapping of the Alpine regions (14) shows no geographical variation for endometrial and ovarian cancers. The highest prostate cancer incidence is reported in Austrian communities where PSA testing is extensive, whereas breast cancer incidence is the highest in Italian communities. In an earlier research (15), Zadnik observed variations in breast cancer incidence between Slovenian west and east (highest to

Zadnik V, Krajc M. Epidemiological trends of hormone-related cancers in Slovenia Arh Hig Rada Toksikol 2016;67:83-92

lowest, respectively) (Figure 3). As for other HRCs, we observed no geographical variations.

Recently, we investigated the relationship between cancer burden, environmental pollution and carcinogenic endocrine disruptors (16, 17). The analyses of cancer incidence in Bela krajina, which is heavily polluted with PCBs (16) and of the Celje region, polluted with heavy metals (17) have not established any significant increase in the risk of any HRCs among the population living in these areas.

Risk factors for the development of HRCs include reproductive characteristics associated with oestrogen and other hormones, pharmaceutical hormones, and lifestyle, such as alcohol use and lack of exercise. Below we are going to discuss the reasons that may have led to the rise in HRC burden established in this study, paying particular attention to the potential influence of exogenous hormones and hormone-like substances.

Review of risk factors for HRC development Female breast cancer The first to report high incidence of breast cancer in nuns more than three hundred years ago was Bernardo Ramazzini in his De Morbis Artificum Diatriba (in chapter “De Nutricum Morbis”), pointing to the association between reproduction and cancer risk. Today we know that breast cancer risk is twice as high in nulliparous women as in the parous women. It is the lowest in women who have given birth at an early age (full term pregnancy) and rises with age (18). In fact, women who give birth at the age 30 or later run a higher risk than nulliparous women.

Furthermore, breast cancer risk drops with breastfeeding.

Women who have given birth and breastfed several children over long time run the lowest risk (19, 20).

Menstrual factors are also very important in predicting breast cancer risk. Menarche at an early age and menopause at a later age are associated with the highest risk, presumably due to ovulation activity and changes in endogenous hormonal profiles (21, 22).

Physical activity and obesity may also be related to breast cancer risk. Obesity is associated with higher risk of post-menopausal breast cancer. In premenopausal women obesity-related anovulation seems to reduce the risk, and the conversion of androgens to oestrogens in adipose tissue after menopause seems to increase the risk (1, 21, 23).

Physical activity has been associated with a 23-30 % decrease in breast cancer risk. Some studies have focused on investigating the biological mechanisms mediating this association (23). Physical activity decreases endogenous oestrogens, fat-related insulin resistance, inflammation, and improves adipokine production (leptin and adiponectin) by reducing fat tissue, which are all independently associated with increased breast cancer risk (23, 24). Physical activity also seems to reduce oxidative stress and genomic

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instability; in postmenopausal women exercise decreases circulating oestrogen levels through weight loss (23-25).

Menopausal hormone replacement therapy has been associated with increased breast cancer risk among postmenopausal women, especially in thin women (26).

However, the major risk predictor is the type of hormones used; oestrogen plus progestogen seem to be associated with the highest risk.

Oral contraceptives are associated with increased risk of breast cancer especially among young women, but it falls to baseline after 10 years of discontinuation and is the same as in non-users (26).

According to Barnes et al. (27), the populationattributable risk for non-modifiable breast cancer risk factors (age at menarche, age at menopause, parity, benign breast disease and family history of breast cancer) is 37.2 % for all invasive tumours considered. Among the modifiable risk factors, hormone therapy and physical inactivity contribute the most 19.4 % and 12.8 %, respectively.

Only 3-5 % of breast cancer is due to hereditary predisposition. BRCA1 and BRCA2 genes have been identified to be responsible for the majority of hereditary breast cancer. Carriers of germline mutations in BRCA1/2 genes are at high risk of developing breast and/or ovarian cancer (28, 29). Several moderate and low penetrant genes have also been identified, and their role is still under investigation (29).

Endometrial cancer Hormones play an important role in the aetiology of endometrial cancer, which is believed to arise from oestrogen stimulation that is unopposed by progestins (1).

More than 80 % of cases of endometrial cancers are oestrogen-related. Obesity and menopausal hormone therapy are strong risk predictors, as well as early age at menarche, late age at menopause, and nulliparity. Oral contraceptives are associated with a long-lasting decrease in endometrial cancer risk only if they contain progestogen in addition to oestrogen (30). Unopposed oestrogen use in hormone replacement therapy is associated with a remarkably high risk (two to tenfold increase) which depends on the duration of use and the woman’s body mass (higher in thin women) (31). Obesity is associated with annovulatory cycles in premenopausal women, so the endometrial tissue receives continuous stimulation. In postmenopausal women, the concentration of endogenous oestrogens increases in obese women. Endogenous oestrogens are mainly the product of androgen aromatisation in the fat tissue. Furthermore, excess weight is associated with insulin resistance and chronically elevated insulin concentration and increased concentrations of sex steroids (32) - all these factors are associated with higher endometrial cancer risk. Higher risk is also observed in patients with type 1 and type 2 diabetes. Other clinical features are also associated with higher risk such as polycystic ovary

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Zadnik V, Krajc M. Epidemiological trends of hormone-related cancers in Slovenia Arh Hig Rada Toksikol 2016;67:83-92

Figure 3 Relative risks of breast cancer by municipalities, Slovenia 1995-2002 (15)

syndrome (associated with increased blood androgen levels), infertility, amenorrhea, hirsutism, and tamoxifen therapy (twofold increase compared to non-users) (1).

Two to five percent of endometrial cancers are associated with hereditary syndromes such as Lynch (most frequent), Muir-Torre, Cowden, and the BRCA1 syndrome.

Patients with Lynch syndrome who carry germline mutations in mismatch repair genes (mostly MLH1, MSH2, MSH6) face a 40-60 % lifetime risk of endometrial cancer (1).

Ovarian cancer Nulliparity and infertility are well-known risk factors for ovarian cancers. Another predictor of some types of ovarian cancers (clear cell and endometroid cancers) is endometriosis (33). Beside hormonal factors (elevated gonadotropin levels), some irritants (talc, asbestos) seem to increase the risk of ovarian cancer.

The risk, however, drops with suppressed ovulation (by pregnancy or oral contraceptives; both lower pituitary gonadotropins) (34, 35). Substantially reduced risks have been observed in women who had simple hysterectomy or tubal ligation. While reduced ovulation is known to diminish the risk, it does not explain all of the identified risk factors.

Latest research points to hormonal and immunological factors (36).

A very important risk factor is family history, since it accounts for about 10 % of cases. The risk increases threefold when two or more first-degree relatives have been diagnosed with ovarian cancer. Furthermore, women who carry germline mutations in BRCA1/2 genes and women with Lynch syndrome have a 30-70 % lifetime risk of developing ovarian cancer (37, 38).

Prostate cancer Epidemiological studies, numerous as they are, seem to have failed to pinpoint all the causes of prostate cancer.

We know that the risk increases with age, and is race and family history related (1). About 25 % of men with prostate cancer have a known family history of this cancer (39).

Furthermore, in a twin cohort study (40) 42 % of prostate cancer cases were inheritable.

For a number of other probable exogenous risk factors such as alcohol, smoking, vasectomy, body mass index, exposure to certain chemicals in the workplace, the connection to prostate cancer has been evaluated but not confirmed. Agricultural workers have been identified as having a higher risk of prostate cancers, most probably because of their exposure to pesticides (1).

Among protective factors stand out specific diets such as those rich in carotenoid lycopene (41) or phytoestrogens that have been associated with lower incidence of prostate cancer in Asia (42).

Sex hormones are undoubtedly involved in the aetiology of prostate cancer, but the mechanism is still unclear.

Epidemiological studies have ruled out the association with serum testosterone (1). It is most likely that changes in the metabolism or transfer of hormones on the cellular level are involved in prostate cancer development.

Pharmacological inhibitors of 5-alpha reductase lower the risk (43) whereas insulin-like growth factor seems to increase it (1).

The enormous increase in the incidence of prostate cancer in the developed countries in the last twenty years has mainly been attributed to the intensive use of screening with the PSA. PSA screening has increased the detection of all prostate cancers, including indolent prostate cancers.

It has nearly doubled the diagnosis of prostate cancer but has also reduced its mortality by 20 % (44). However, the

Zadnik V, Krajc M. Epidemiological trends of hormone-related cancers in Slovenia Arh Hig Rada Toksikol 2016;67:83-92

question is not whether it is effective in reducing mortality, but whether it does more harm than good. Compared to breast cancer screening in women PSA screening involves a significantly greater risk of overdiagnosis, unnecessary treatment, and eventually, lower quality of life in overdiagnosed patients.

Testicular cancer Testicular cancer is, in most cases, a germ cell neoplasm.

The incidence of testicular cancer rises with adolescence, and hormonal factors clearly play a role in its aetiology.

The diagnosis peaks in the twenties and thirties. There is a variety of other factors that increase cancer risk, including height, subfertility, and exposure to endocrine disruptors.

Some risk factors also involve exposure to hormones in utero (cryptorchidism, hypospadias, inguinal hernia, low birth weight, short gestational age, and being a twin) or to endogenous hormones (45). Established risk factors for testicular germ cell tumours also include a prior testicular germ cell tumour, a family history of germ cell tumour and various inter-sex syndromes (46, 47). There are very interesting migrant studies that suggest environmental and dietary factors. For example, the incidence of testicular cancer in Finland is half that in Sweden. Furthermore, the second generation Finnish migrants to Sweden face the same testicular cancer incidence as the Swedish population (48).

A small minority of testicular cancers, as in other cancers, appear to be familiar, with approximately 2 % of patients having an affected family member. Men with a first-degree relative with testicular cancer run a three to ten times higher risk of being diagnosed with the disease (46).

Thyroid cancer Thyroid cancer is not as common, but its incidence has been increasing worldwide. This trend may be associated with improved screening and detection, the detection of papillary microcarcinoma in particular (49, 50).

Exposure to ionising radiation, especially in childhood (as it predisposes for papillary thyroid cancer) is among the well-known high risk factors for thyroid cancer. About 5 % of patients develop thyroid cancer due to radiation exposure (1, 50).

The distribution between genders is strikingly uneven, as the cancer affects women three to times more often than men (50) for reasons that may be related to hormones but are far from clear. An association between female hormonal and reproductive factors as drivers for thyroid cancer development has been shown in vitro but has not been confirmed in population-based analyses (51, 52).

According to population-based studies, thyroid cancer might also be moderately associated with height and body mass index in both genders (51). A rare clinical condition - acromegaly (caused by an overproduction of growth hormone) is also associated with increased risk of thyroid

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cancer. It is also known that thyroid cancer development is strongly associated with a history of benign nodules/ adenoma or goitre. Furthermore, iodine deficiency may induce benign thyroid conditions that predispose for thyroid cancer, but it is still unclear to what extent dietary iodine intake might serve as a risk predictor (51).

There are several rare inherited conditions (familial adenomatous polyposis syndrome, Cowden syndrome, Werner syndrome) that are associated with different types of thyroid cancer (53). When investigating medullary thyroid cancers only, about 25 % occur as part of the multiple endocrine neoplasia type 2 (MEN2) syndrome (54). MEN2 syndrome is caused by germline mutation in the RET protooncogene and is involved in phaeochromocytoma, hyperparathyroidism, and medullary thyroid carcinoma. RET protooncogene mutation analysis therefore enables predictive testing and genotype-based tailored prophylactic treatment and serves as a role model of personalised medicine, since medullary thyroid cancers as part of MEN 2 may be prevented by early prophylactic resection of the thyroid gland.

As for non-syndromic thyroid cancer, it is estimated that around 10 % of all non-medullary thyroid cancers are hereditary, since first-degree relatives of patients with thyroid cancer run a ten times higher risk of thyroid cancer than the general population (53).

The influence of risk factors on the burden of HRCs in Slovenia Risk factors for HRC are heterogeneous and are predominantly associated with our lifestyle, reproductive factors, or genetic characteristics. They can have opposite effects in different cancers; oral hormonal contraception increases the risk of breast cancer but decreases the risk of ovarian cancer. Even though the prevalence of HRC risk factors has been poorly investigated in Slovenia, we have enough information to explain, at least in part, the spatial distribution of breast cancer risk (risk is higher in the western part of the country) and the rapid rise in breast and prostate cancer incidence. Several studies of the reproductive factors in Slovenia (55-57) indicate that nulliparity and late first birth (which are both more prevalent in the west of the country) could be responsible for higher breast cancer risk in that part of the country. Reproductive risk factors in Slovenia are also central to the understanding of the increase in breast cancer incidence. The average age at first delivery and the nulliparity are increasing, and Slovenian women today have on average fewer children than earlier generations (15). The dramatic rise in prostate cancer incidence, in turn, is owed to dramatic rise in detection thanks to prostate-specific antigen (PSA) screening, which has been part of regular check-ups since the early 1990s (58).

Although reproductive and genetic factors play a major role in the carcinogenesis of HRCs, the contribution of

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Zadnik V, Krajc M. Epidemiological trends of hormone-related cancers in Slovenia Arh Hig Rada Toksikol 2016;67:83-92

environmental chemicals cannot be dismissed. Endocrine disruptors seem to be involved in carcinogenesis through the same mechanisms as natural hormones. At the moment, however, we do not know how many of the newly diagnosed cancers are associated with endocrine disruptors. Further research should focus on assessing exposure and individual susceptibility, as well as latency and critical exposure windows. In the meantime, public health policies promote a precautionary approach to reasonably reduce exposure to endocrine-disrupting chemicals.

11.

12.

13.

Conflict of interest The authors declare no conflict of interests regarding the publication of this paper.

14.

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Epidemiološki trendi hormonsko odvisnih rakov Spolni hormoni so znan in pomemben nevarnostni dejavnik rakov na dojki, jajčniku in endometriju, medtem ko je njihova vloga pri rakih prostate in mod verjetna, a manj raziskana. Vpletenost hormonov v kancerogenezo se nakazuje tudi pri raku ščitnice. Predvidevamo, da se po enakih mehanizmih kot telesu lastni hormoni v nastanek raka vpletajo tudi hormonski motilci. Rake hormonsko odzivnih tkiv najpogosteje povezujemo z izpostavljenostjo PCB-jem in dioksinom, DDT/DDE ter nekaterim težkim kovinam in pesticidom. Incidenca hormonsko odvisnih rakov je največja v državah razvitega sveta.

V Sloveniji predstavljajo hormonsko odvisni raki četrtino vseh rakov; njihovo število se z izjemo raka jajčnikov vsak leto veča. Tveganje raka dojk je že več desetletji večje na zahodu države, medtem ko pri ostalih lokacijah med območij ne opažamo pomembnih razlik. Hormonskim motilcem pripisljivega deleža rakov trenutno ni mogoče določiti, saj imamo verodostojnih raziskav pri izpostavljenih, predvsem v kritičnih razvojnih obdobjih, premalo.

KLJUČNE BESEDE: dejavniki tveganja; geografska razporeditev; hormonski motilec; incidenčni trendi; pripisljiv delež

Gaberšček S, Zaletel K. Epidemiological trends of iodine-related thyroid disorders: an example from Slovenia Arh Hig Rada Toksikol 2016;67:93-98

Review

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DOI: 10.1515/aiht-2016-67-2725

Epidemiological trends of iodine-related thyroid disorders: an example from Slovenia Simona Gaberšček1,2 and Katja Zaletel1 University Medical Centre Ljubljana, Department of Nuclear Medicine1, University of Ljubljana, Faculty of Medicine2, Ljubljana, Slovenia [Received in October 2015; CrossChecked in October 2015; Accepted in April 2016] The epidemiology of thyroid disorders is significantly associated with iodine supply. In 1999, Slovenia increased iodine content in kitchen salt from 10 mg to 25 mg of potassium iodide per kg of salt. According to the WHO criteria, Slovenia shifted from a mildly iodine-deficient country to a country with adequate iodine intake. Ten years after the increase in iodine intake, the incidence of diffuse goitre and thyroid autonomy decreased. Now patients with diffuse goitre and thyroid autonomy reach older age than the patients before the increase in iodine intake. In addition, patients with thyroid autonomy are less frequently hyperthyroid than ten years ago and iodine-induced hyperthyroidism is less severe. The incidence of highly malignant thyroid carcinoma has also dropped. However, the incidence of Hashimoto’s thyroiditis increased, most probably in genetically predisposed individuals. Over the last ten years, many animal and in vitro studies evaluated the effects of endocrine disrupting chemicals (EDC) on various aspects of the thyroid function. They mostly studied the effects of polychlorinated biphenyls (PCBs) and dioxins, brominated flame retardants, phthalates, bisphenol A, perfluorinated chemicals, and perchlorate. However, human studies on the effects of EDCs on the thyroid function are very scarce, especially the long-term ones. What they do suggest is that PCBs and dioxins interfere with the transport of thyroid hormones and adversely affect the thyroid function. Many authors agree that iodine deficiency predisposes the thyroid gland to harmful effects of EDCs. Therefore the effects of EDCs in iodine-deficient areas could be more severe than in areas with adequate iodine intake.

KEY WORDS: diffuse goitre; dioxins; endocrine disrupting chemicals; Hashimoto’s thyroiditis; iodine supply; iodineinduced hyperthyroidism; polychlorinated biphenyls; thyroid autonomy Beside diabetes, the most frequent disorders of endocrine glands are those affecting the thyroid gland.

Genetic factors largely contribute to the occurrence of various thyroid diseases such as Graves’ disease, Hashimoto’s thyroiditis, or goitre (1). However, their prevalence is also strongly associated with iodine supply in the environment (2), as the thyroid gland requires adequate amounts of iodine to function properly (3). Of all endocrine glands, the thyroid exhibits a unique connection with the environment. It is sensitive to iodine intake and the influence of endocrine disrupting chemicals (EDC) (4).

The aim of this article is to review the effects of environmental factors on the thyroid function and size with the focus on iodine intake and EDCs. Iodine and thyroid hormones Ingested iodine is absorbed as I- in the stomach and upper small intestine. Adult body contains around 15 to 20 mg of iodine, of which 70 to 80 % is in the thyroid gland (5, 6). Iodine concentration within thyroid cells is 20 to 40 Correspondence to: Katja Zaletel, University Medical Centre Ljubljana, Department of Nuclear Medicine, Zaloška 7, 1525 Ljubljana, Slovenia, e-mail: katja.zaletel@kclj.si The subject of this paper has partly been presented at the 2nd Congress of the Slovenian Society of Toxicology “Endocrine disrupting chemicals – from molecule to man” held in Ljubljana, Slovenia, from 23 to 24 April 2015.

times higher than in the blood. The ability of thyroid cells to accumulate iodine against the concentration gradient is enabled by the transport protein called sodium (Na+)-iodide (I-) symporter (NIS). NIS combines the transport of Na+ along the concentration gradient with the transport of Iagainst the concentration gradient using the energy from adenosine triphosphate (ATP) obtained from the activity of Na+-potassium (K+)-ATPase (7). With the help of thyroid peroxidise (TPO), I- is first oxidised, then integrated into glycoprotein thyroglobulin (Tg) as monoiodotyrosine (MIT) and diiodotyrosine (DIT), which are finally coupled into thyroxine (T4) or triiodothyronine (T3) (8). The accumulation of I- and the synthesis of thyroid hormones is regulated by the thyroid-stimulating hormone (TSH, aka thyrotropin), secreted from the pituitary gland (9), as well as by iodine itself (10).

The thyroid gland produces all T4 and only 20 % of T3.

The remaining T3 originates from the conversion of weakly active T4 into very active T3 by enzymes deiodinases, which are present in several tissues (11). To a high degree T4 and T3 bind to transport proteins in serum. Less than 1 % of T4 and T 3 circulate as free thyroxine (fT 4 ) and free triiodothyronine (fT3) (12). However, only free thyroid hormones are active and are transported to different cells,

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Gaberšček S, Zaletel K. Epidemiological trends of iodine-related thyroid disorders: an example from Slovenia Arh Hig Rada Toksikol 2016;67:93-98

where they bind to nuclear, plasma membrane, cytoplasm, and mitochondrial receptors and perform their genomic and non-genomic actions (13, 14). Thyroid hormones regulate metabolism and are especially important for the development of human brain during foetal life and early childhood (15).

In areas with severe iodine deficiency, lack of iodine during pregnancy may cause iodine-deficiency disorders in offspring (16).

Iodine supply In most developed countries the main source of iodine is iodised kitchen salt, since iodine content in soil, and, consequently, in ground water, vegetables, and meat is too low to meet daily needs. The World Health Organization (WHO) recommends iodisation of kitchen salt with 20 to 40 mg of potassium iodide per kg of salt (3). Daily intake of iodine for adults should reach 150 µg. For pregnant and lactating women, a higher daily intake of around 250 µg is recommended. In pregnancy, more thyroid hormones are produced because of increased levels of transport proteins (17). Additionally, iodine is partly transported to the foetus via placenta. During lactation, adequate iodine intake is especially important since a breastfed child depends only on iodine from mother’s milk.

As iodine intake cannot be measured directly, and iodine is largely secreted in urine, its urinary concentration makes a good estimate of the intake. According to the WHO criteria, urinary iodine concentration (UIC) between 50 and 99 µg L-1 suggests mild iodine deficiency, 20 to 49 µg L-1 moderate deficiency, and below 20 µg L-1 severe deficiency (3). A great portion of the world’s population still lives in areas where iodine supply is deficient. Even in Europe, countries without stable and mandatory iodisation of kitchen salt can have areas of mild or moderate iodine deficiency.

Iodine and thyroid disorders Optimal iodine intake lies within a relatively narrow range between 150 and 250 µg a day (3). Too low an intake may lead to iodine deficiency disorders and enlarged thyroid gland, known as goitre. Intake which is too high leads to autoimmune thyroid disorders and hypothyroidism. In addition, any change in iodine supply is associated with a change in the epidemiology of thyroid disorders. One such epidemiological change occurred with the increase in iodine supply in Slovenia in 1999.

From 1953 to 1999, kitchen salt in Slovenia had been iodised with 10 mg of potassium iodide per kg. In 1991 to 1994, we measured UIC in a population of 1740 schoolchildren to be 82.9 µg g-1 of creatinine (18, 19), which suggested a mild iodine deficiency. The average volume of their thyroid gland, measured by ultrasound, was 7.2 mL. In the early 1999, kitchen salt iodisation increased to 25 mg (range: 20 to 30 mg) of potassium iodide per kg of salt. Measurements since 1999 show a daily intake of 9.4 g of kitchen salt in girls and 11.5 g in boys (20) and 9.9 g in

women and 13.0 g in men (21). As soon as 2003, UIC in schoolchildren increased to 148 µg L-1, which suggests adequate iodine intake (19). In pregnant women, it was also high enough - 170.5 µg g-1 of creatinine in the third trimester, and 144 µg g-1 of creatinine after childbirth (22). The volume of the thyroid gland in schoolchildren decreased to 5.8 mL (19).

From 1999 to 2009, the incidence of diffuse goitre dropped over 80 % (19). In 2009, patients with newly diagnosed diffuse goitre were significantly older than those diagnosed with the disease in 1999 (53 vs. 38 years, respectively). This clearly suggests that younger generations benefited from adequate iodine supply. After all, older patients had lived in iodine deficiency longer than the younger subjects.

Thyroid autonomy is caused by various mutations of TSH receptor and/or signalling proteins (23). Autonomous thyroid cells produce thyroid hormones irrespective of regulation by TSH. The incidence of thyroid autonomy has been reported to be higher in iodine-deficient than in iodinesufficient areas (3). From 1999 to 2009, the incidence of thyroid autonomy in Slovenia decreased by 27 % (19).

Furthermore, in 2009, patients with the newly diagnosed thyroid autonomy were older and less frequently hyperthyroid than patients in 1998. This again confirms the effects of iodine deficiency are time-dependent (24).

Literature data regarding iodine supply and thyroid autonomy are scarce and inconclusive. Our data are in accordance with those from Switzerland (25, 26).

Excessive iodine intake is usually related to the antiarrhythmic drug amiodarone or iodinated contrast media in radiology. In predisposed individuals, such as patients with thyroid autonomy, iodine excess most frequently provokes hyperthyroidism. By contrast, in patients with Hashimoto’s thyroiditis, iodine excess may induce hypothyroidism. Andersen et al. (27) reported that in areas with iodine deficiency iodine excess more often causes hyperthyroidism while in areas with adequate iodine intake it may cause hypothyroidism. Ten years after the increase in salt iodisation, our results show a higher incidence of iodine-induced hypothyroidism, a less severe iodineinduced hyperthyroidism, and a shorter duration of treatment of hyperthyroidism, which are all clinical improvements over the earlier period (28).

Literature data about iodine-induced thyroid disorders with respect to iodine supply are very scarce. In Slovenia, we have observed a lower incidence of the most malignant anaplastic thyroid carcinoma between 1998 and 2008 than between 1972 and 1998 (29). After the increase in iodine supply, patients with the newly diagnosed anaplastic thyroid carcinoma were older than before the increase. Again, it seems that younger generations have benefited from adequate iodine supply.

As for Hashimoto’s thyroiditis, its incidence in Slovenia more than doubled after the increase in iodine supply (19).

These findings confirm Iranian reports of up to four-fold

Gaberšček S, Zaletel K. Epidemiological trends of iodine-related thyroid disorders: an example from Slovenia Arh Hig Rada Toksikol 2016;67:93-98

increase in the prevalence of thyroid autoantibodies after the improvement of iodine prophylaxis in iodine-deficient areas (30). Other authors reported the prevalence of thyroid autoantibodies of around 11 % in iodine deficiency (31), 18 % in iodine sufficiency (32), and around 25 % in excessive iodine intake (33, 34). Many tried to elucidate the influence of iodine supply on thyroid autoimmunity.

Some suggest that higher intake of iodine may increase the iodination of thyroglobulin and the level of reactive oxygen species within thyroid cells, thus promoting immunogenicity (35, 36). Others believe that iodine may have a toxic effect on thyroid cells and stimulate the cells of the immune system (1, 37).

Whichever the reason, a change from mild iodine deficiency to iodine sufficiency is associated with many beneficial effects with regard to the incidence and severity of various thyroid disorders.

Endocrine disrupting chemicals Endocrine disrupting chemicals most often affect the NIS, TPO, transport protein transthyretin, deiodinases, and thyroid hormone receptors (4, 38).

Polychlorinated biphenyls (PCBs) and dioxins are persistent environmental pollutants, which accumulate in the food chain and - due to lipophilic properties - in the human body, in spite of the ban from the 1970s. The chemical structure of PCBs and dioxins is similar to the chemical structure of thyroid hormones, as they all have two linked phenyl rings. PCBs displace T4 from transport proteins (39), decrease the serum levels of thyroid hormones and, accordingly, increase the serum levels of TSH (40, 41).

However, Goldey et al. (42) have also reported serum T4 drop without an increase in TSH. Interestingly, hydroxylated PCBs were able to increase the activity of thyroid receptors in rats (43). In vitro, PCBs bind to transport proteins and inhibit the binding of T3 to the receptor (44, 45). In humans, exposure to PCBs was associated with thyroid antibodies and hypothyroidism (1). All in all, it seems that PCBs and dioxins affect the thyroid function adversely.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants and also have chemical resemblance with the thyroid hormones with two halogenated phenyl rings (40). Since PBDEs are lipophilic, they accumulate in several human tissues (46). Human studies with PBDEs are very scarce. In animals, PBDEs decreased the levels of thyroid hormones (47), and affected binding proteins, thyroid hormone receptors, and hepatic clearance (48, 49).

Pesticides are still amply present in the environment.

In animal studies, they adversely affected thyroid hormone levels (50). In vitro, phenol compounds inhibited TPO (51).

Relevant human studies are not available.

In humans as well as in animals, perfluorooctanoate adversely affects fT4 (52, 53). In the National Health and Nutrition Examination Survey carried out in the United

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States (54), individuals with high levels of perfluorooctanoic acid more frequently reported treated thyroid disease.

Human studies with phthalates are very scarce. In children, they negatively correlated with serum T3 (55). A similar correlation was found between phthalates and fT4 in men and pregnant women (56, 57).

In vitro, bisphenol A (BPA) binds to thyroid hormone receptors and therefore disrupts thyroid hormone activity (58). In rats, developmental exposure to BPA caused a condition similar to thyroid resistance syndrome, with increased serum levels of T4 and normal or slightly increased levels of TSH, indicating that BPA could act as a specific antagonist of thyroid hormone receptor β, which is known to be impaired in the thyroid resistance syndrome (59, 60).

Perchlorate in drinking and irrigation waters and in food - a problem particularly familiar to the USA - reduces iodide uptake into the thyroid cells (38, 61). In the general population of women, urinary levels of perchlorate correlated with serum TSH levels (62). In women with urinary iodine below 100 µg L-1 and in women who smoked this correlation was even stronger, since smokers have higher serum levels of thiocyanates than non-smokers, and thiocyanate reduces iodide uptake (63).

For many other EDCs human studies are not available.

In vitro and animal studies indicate significant effects on the thyroid function, but solid data on humans are needed for a more objective view of EDCs. To summarise, potential EDCs are numerous, human studies with EDCs are scarce, exposure to EDCs is universal, and long-term effects are very difficult to study. Therefore, a long-term follow-up of larger groups of exposed individuals would bring valuable information about the effects of EDCs on the thyroid function.

CONCLUSION Changes in iodine supply significantly reflect on the epidemiology of thyroid disorders in an area. Optimum iodine intake - which lies within a relatively narrow range - is the prerequisite for the normal growth and function of the thyroid gland. In contrast, iodine deficiency makes the thyroid gland more sensitive to various adverse effects, including the effects of EDCs. The Slovenian example confirms the importance of a sustained and mandatory iodination programme in abating thyroid disorders.

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Epidemiološki trendi bolezni ščitnice, ki so povezane z jodom: primer Slovenije Epidemiologija bolezni ščitnice je pomembno povezana z jodno preskrbo. V Sloveniji smo leta 1999 zvečali vsebnost joda v kuhinjski soli z 10 mg na 25 mg kalijevega jodida na kg soli. Glede na kriterije SZO smo se iz države z blagim pomanjkanjem joda spremenili v državo z ustreznim vnosom joda. Ugotovili smo, da se je v desetih letih po zvečanju jodne preskrbe zmanjšala pojavnost difuzne golše in avtonomnega tkiva v ščitnici. Bolniki z difuzno golšo in avtonomnim tkivom so sedaj starejši kot pred zvečanjem jodne preskrbe. Poleg tega so bolniki z avtonomnim tkivom redkeje hipertirotični kot pred desetimi leti, hipertiroza, ki je posledica čezmernega vnosa joda, pa je manj izražena. Zmanjšala se je pojavnost najbolj maligne oblike ščitničnega karcinoma. Povečala se je pojavnost Hashimotovega tiroiditisa, verjetno pri genetsko predisponiranih posameznikih. V zadnjem desetletju so v številnih raziskavah na živalih in v raziskavah in vitro ugotavljali vpliv kemijskih povzročiteljev hormonskih motenj (KPHM) na različne vidike ščitnične funkcije. Najpogosteje testirani KPHM so bili poliklorirani bifenili (PCB) in dioksini, bromirani zaviralci gorenja, ftalati, bisfenol A, perfluorirane kemikalije in perklorat. Vendar pa so raziskave o učinkih KPHM na delovanje ščitnice pri ljudeh, zlasti dolgoročne raziskave, zelo redke. Zdi se, da zlasti PCB in dioksini vplivajo na transport ščitničnih hormonov in negativno učinkujejo na delovanje ščitnice. Številni avtorji menijo, da pomanjkanje joda poveča dovzetnost ščitnice za škodljive učinke KPHM. Torej bi lahko bili škodljivi učinki KPMH na področjih pomanjkanja joda resnejši kot na področjih zadostnega vnosa joda.

KLJUČNE BESEDE: avtonomno tkivo ščitnice; difuzna golša; dioksini; Hashimotov tiroiditis; hipertiroza zaradi čezmernega vnosa joda; kemijski povzročitelji hormonskih motenj; poliklorirani bifenili; preskrba z jodom

Perharič L, et al. European Union’s strategy on endocrine disrupting chemicals and the current position of Slovenia Arh Hig Rada Toksikol 2016;67:99-105

Mini-review

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DOI: 10.1515/aiht-2016-67-2728

European Union’s strategy on endocrine disrupting chemicals and the current position of Slovenia Lucija Perharič1, Tanja Fatur1, and Jernej Drofenik2 National Institute of Public Health1, Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection2, Ljubljana, Slovenia [Received in October 2015; CrossChecked in October 2015; Accepted in June 2016] In view of the European Union regulations 1107/2009 and 528/2012, which say that basic substances in plant protection and biocidal products marketed in the European Union (EU) should not have an inherent capacity to cause endocrine disruption, an initiative was started to define scientific criteria for the identification of endocrine disruptors (EDs). The objectives of the EU strategy on EDs are to protect human health and the environment, to assure the functioning of the market, and to provide clear and coherent criteria for the identification of EDs that could have broad application in the EU legislation. Policy issues were to be addressed by the Ad-hoc group of Commission Services, EU Agencies and Member States established in 2010, whereas the scientific issues were to be addressed by the Endocrine Disruptors Expert Advisory Group (ED EAG), established in 2011. The ED EAG adopted the 2002 World Health Organization (WHO) definition of endocrine disruptor and agreed that for its identification it is necessary to produce convincing evidence of a biologically plausible causal link between an adverse effect an endocrine disrupting mode of action. In 2014, the EC proposed four ED identification criteria options and three regulatory options, which are now being assessed for socio-economic, environmental, and health impact. Slovenia supports the establishing of identification criteria and favours option 4, according to which ED identification should be based on the WHO definition with the addition of potency as an element of hazard characterisation. As for regulatory options, Slovenia favours the risk-based rather than hazard-based regulation.

KEY WORDS: endocrine disruptors; EU regulations; hazard characterisation; identification criteria In 1998, the European Parliament adopted a resolution calling upon the European Commission (EC) to improve the regulatory framework for endocrine disruptors and to reinforce related research and communication to the public.

In 1999, the EC proposed activities needed to respond to the public concern, which were based on the precautionary principle. It also proposed a research framework that would elucidate the causes and effects of identified endocrine disturbances. A number of research projects had been carried out since, and the EC had regularly reported on the developments in terms of substances prioritised for further investigation, new test methods, legislation, and further research (1).

Endocrine disruptors and the EU regulations According to the widely accepted 2002 World Health Organization (WHO) definition, An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations. A potential endocrine disruptor is an exogenous substance or mixture Correspondence to: Lucija Perharič, National Institute of Public Health, Zaloška 29, Ljubljana, Slovenia, E-mail: lucija.perharic@nijz.si The subject of this paper has partly been presented at the 2nd Congress of the Slovenian Society of Toxicology “Endocrine disrupting chemicals – from molecule to man” held in Ljubljana, Slovenia, from 23 to 24 April 2015.

that possesses properties that might be expected to lead to adverse health effects in an intact organism, or its progeny, or (sub)populations (2).

The Glossary of Terms in the IPCS Environmental Health Criteria no. 240 define adverse effects as follows: An adverse effect is a change in the morphology, growth, development, reproduction, or lifespan of an organism, system, or (sub)population that results in an impairment of functional capacity, an impairment of the capacity to compensate for additional stress, or an increase in susceptibility to other influences (3).

EC regulations 1107/2009 (4) and 528/2012 (5) stipulate that basic substances, safeners, and synergists in plant protection products and basic substances in biocidal products, respectively, should not have endocrine-disrupting properties that may cause adverse effects if they are to be approved for marketing in the EU. The exceptions for plant protection products are if the exposure of non-target organisms is negligible or the “substance is necessary to control a serious danger to plant health which cannot be contained by other available means including non-chemical method” (4). Similarly, the exceptions for the biocidal products are if the risks are negligible or the substance is “essential to prevent or control a serious danger to human health, animal health or the environment or not approving

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the substance would have “disproportionate negative impacts for society when compared with the risks…” (5).

However, neither regulation defines the criteria for the identification of ED. Until such criteria are adopted, the implementation of the regulations 1107/2009 and 528/2012 relies on the provisions of the classification, labelling and packaging regulation no. 1272/2008 (6) in the sense that substances classified as carcinogenic category 2 and toxic for reproduction category 2 in that regulation “shall be considered to have endocrine disrupting properties. In addition, substances, such as those classified, as toxic for reproduction category 2 and which have toxic effects on the endocrine organs, may be considered to have endocrine disrupting properties” (4). This interim approach, of course, is imprecise, as carcinogenicity, reproductive toxicity, or even endocrine organ toxicity may have little to do with endocrine disruption.

Many other EU regulations are in dire need for clear criteria for identifying EDs (1907/2006, 1223/2009, 93/42/ EEC, 2007/47/EC, and 2000/60/EC) (7-11). Clear criteria will enable their universal application across the regulatory solutions in different settings. The initiative to further develop the EU strategy on EDs has the following objectives: to provide legally clear, predictable, and coherent criteria for the identification of EDs and to enable their universal application across the EU legislation with the ultimate objective of protecting human health and environment and of strengthening the internal EU market (12).

This article presents the latest developments concerning the efforts to come up with these universal, scientific criteria for the identification of EDs as well as the current position of the Republic of Slovenia on this issue.

Development of scientific criteria for the identification of endocrine disruptors In 2010, the EC established an Ad-hoc group of Commission Services, EU Agencies and Member States for policy issues and a year later, a sub-group Endocrine Disruptors Expert Advisory Group (ED EAG) to address scientific issues relevant to endocrine disrupting substances not specific to any regulatory framework, including advice/ orientation on scientific criteria for the identification of EDs. Both groups included representatives of Commission services, EU agencies, member states, industry associations, and non-governmental organisations (NGOs).

The ED EAG was not required to reach consensus and presented differing opinions and options for consideration by the Ad-hoc group (13). It adopted the WHO definition of EDs (2) by analysing each of the definition’s elements.

The starting point for discussion was the state-of-the-art assessment of endocrine disruptors by Kortenkamp et al.

(14). The ED EAG agreed that the elements required for an endocrine disruptor to be identified were the evidence of an adverse effect and its relevance for humans at the

individual and/or offspring level. To quote Kortenkamp, the evidence of an adverse effect requires “a biologically plausible causal link to an endocrine disrupting mode of action and for which disruption of the endocrine system was not a secondary consequence of other non endocrinemediated systemic toxicity” (14). As for the relevance, it should be assumed unless non-relevance can be demonstrated. In relation to wildlife populations, data on all species at the population level are generally considered relevant (13).

Munn and Goumenou (13) give a detailed report about the scientific issues raised by the ED EAG in identifying and characterising EDs. Briefly, potency, severity, irreversibility, and lead toxicity were not considered elements of hazard identification but characterisation. Some experts suggested that these elements could come in handy in setting priorities and ranking the EDs, and/or differentiating EDs into classes or categories of lower or higher concern based on this information, but the suggestion received divided support. Those who opposed it believed that the information could only be used within a risk assessment context. There was no agreement, however, on how to consider these factors with respect to ED hazard characterisation outside the context of risk assessment (13).

Discussing a basic scheme for considering evidence of endocrine disrupting properties of substances, the group singled out mode of action and adversity and favoured the weight-of-evidence approach that would include human epidemiology data, field data, animal experimental toxicology and ecotoxicology studies, in vitro data, and quantitative structure-activity relationship. Within given time, the group could not fully evaluate the adequacy of current assays for specific endocrine pathways but suggested that their development “should be informed by emerging human health issues or observed negative impacts on wildlife populations and hypothesised link to endocrinerelated causes” (13). In a separate report, Munn and Goumenou (15) present issues, such as “effect-thresholds, the non-monotonous dose-response relationship, effects of mixtures, exposure during the critical windows of susceptibility, inadequacy of testing methods for the identification of outcomes at low doses and at the relevant developmental stages”.

In the meantime, the EC also gave a mandate to the European Food Safety Authority (EFSA) Scientific Committee to give their opinion on ED hazard assessment.

The Committee proposed a distinction between an endocrine active substance (EAS) and ED. EAS was defined as “any chemical that was able to interact directly or indirectly with the endocrine system resulting in effect on the endocrine system, target organs and tissues” (16). This interaction, however, does not necessarily result in an adverse effect. In contrast, an ED should be defined by three criteria: an adverse effect in an intact organism or a (sub) population; an endocrine activity; and a plausible causal relationship between the two. Similar to ED EAG, the

Perharič L, et al. European Union’s strategy on endocrine disrupting chemicals and the current position of Slovenia Arh Hig Rada Toksikol 2016;67:99-105

Committee considered critical effect, severity, (ir) reversibility, and potency as elements of ED hazard characterisation (16).

Criteria for identification In June 2014, the EC published the Roadmap of the initiative to define criteria for identifying EDs, in which it proposes four options for identification and three for regulatory decision making (12), as follows: Identification options Option 1: No policy change. No criteria are specified.

The interim criteria set in the plant protection and biocidal products regulations continue to apply.

Option 2: The identification of EDs is based on the WHO definition. This option lists the required evidence and step-by-step procedure for identification.

Option 3: As option 2, but includes categories based on the strength of evidence for fulfilling the WHO definition: Category I - endocrine disruptors; Category II - suspected endocrine disruptors; Category III - endocrine active substances.

Option 4: As option 2, but includes potency as an element of hazard characterisation.

Decision-making options Option A: No policy change.

Option B: Addition of more risk assessment elements into sectorial legislation, so that marketing decisions are not mainly based on hazard identification.

Option C: Inclusion of socio-economic considerations as well as risk-benefit analysis into sectorial legislation to allow marketing endocrine-disrupting products that are “essential to prevent adverse socio-economic impacts” (12).

The Roadmap (12) also summarises the results of the preliminary impact assessment for each of the options of the two aspects.

In the second half of 2014, a public consultation on defining ED identifying criteria generated over 27,000 responses, most of which came from interest groups such as NGOs and farming sector rather than the general public.

The respondents confirmed the need for the EU to establish definitive criteria for EDs (17). The EU strategy is due before the summer of 2016 (18).

Current position of Slovenia Slovenia has actively been participating in the initiative to establish the ED-identifying criteria. Its current position is largely based on the scientific evidence presented in detail in the reports by Damstra et al. (2), Kortenkamp et al. (14), Munn and Goumenou (13, 15), EFSA (16, 19-20), EC Scientific Committees (21-22), Joint German-British position paper (23), and several other peer reviewed publications (24-61).

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As for the identification criteria, Slovenia supports Option 4, which lists the required evidence and provides a step-by-step identification procedure, plus it includes potency to characterise the hazard. Potency here denotes relative toxicity of an agent in relation to a given or implied standard or reference (62); in other words, it is a measure of its strength in respect to other chemicals.

As for the decision-making options, Slovenia is in favour of Option B, which uses risk assessment as the basis for marketing approvals.

Concerning the effect thresholds and other uncertainties, the position of Slovenia is that these should be determined for each case separately, taking into account the weight of evidence for a particular chemical. Depending on the quantity and the quality of available data, either the threshold (42, 51, 54) or the non-threshold (36, 59) approach should be used. Slovenia also favours the use of semiquantitative decision trees for regulatory purposes. In view of uncertainties and the complexity of the endocrine system, Slovenia opts for a higher safety (uncertainty) factor, depending on the quality and quantity of data. It still remains to clearly define which is the sufficient quantity and sufficient quality of data. Considering the trends to minimise the use of animals in toxicological experiments and the ban on animal testing in cosmetics (8), it is unlikely that sufficient data will be generated on the effects in intact organisms for a number of chemicals in everyday use. For those structurally related to “threshold EDs”, it may be appropriate to reconsider using the Threshold of Toxicological Concern approach (63). Slovenia favours creating priority lists for regulation, based on potency, severity of effects, irreversibility, and lead toxicity, as well as the expected magnitude of exposure to a particular ED. Although these are the elements of hazard characterisation and risk assessment, Slovenia believes that ED regulation ought to be based on risk rather than hazard, provided there is sufficient information to assess the risk.

Instead of a conclusion Until the identification criteria are set and EDs regulated across the EU legislation, we believe that it is important to continue raising awareness about EDs through media and events such as the recent conference organised by The Slovenian Society of Toxicology: Endocrine disrupting chemicals - from molecule to man (64).

In view of numerous controversies and uncertainties related to EDs, we believe that it is sensible to reduce exposure to natural and synthetic chemicals by changing our behaviour, regardless of current regulations. The following recommendations to reduce ED exposure are based on the national public health and chemical safety policies (65, 66) as well as common sense: Maintain healthy lifestyle with balanced low-salt, lowsugar, low-fat diet, regular moderate physical activity, and sufficient rest to reduce the risk of illness (and therefore the

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need to take medication). Also avoid alcohol, tobacco, and caffeine.

Closely observe manufacturer’s instructions when using biocidal, chemical, medicinal, plant protection, and consumer products.

Use as few biocidal, chemical, medicinal, plant protection, and consumer products as possible.

Grow and prepare your own food.

Drink tap water.

Store food and water in clear glass containers and at appropriate temperature.

Wash hands before eating.

Minimise the number of consumer products at home and workplace, remove dust, and air rooms regularly.

Recycle and reuse products.

Produce as little waste as possible.

Reduce the use of electrical and electronic appliances.

Cut down on motorised travelling.

Reduce the use of cosmetics and personal hygiene products.

Acknowledgement Lucija Perharič gratefully acknowledges the nomination to the Ad-hoc group of Commission Services, EU Agencies and Member States and the ED EAG by the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection.

Conflicts of interest The authors have no conflict of interest to declare.

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Kemični povzročitelji hormonskih motenj - strategija Evropske unije in stališče Slovenije Uredbi Evropske unije 1107/2009 in 528/2012 navajata, da osnovne snovi fitofarmacevtskih in biocidnih sredstev odobrene za uporabo v Evropski uniji (EU), same po sebi ne povzročajo hormonskih motenj. Zato je bilo potrebno definirati znanstvene kriterije za identifikacijo kemičnih povzročiteljev hormonskih motenj (KPHM). Cilji strategije EU na področju KPHM so varovanje zdravja ljudi in okolja, zagotavljanje delovanja trga ter jasnih in skladnih kriterijev za identifikacijo KPHM, ki bodo omogočali široko uporabo teh kriterijev v zakonodaji. Za obravnavo politik je bila leta 2010 ustanovljena Ad-hoc skupina predstavnikov Evropske komisije, EU agencij in držav članic; leta 2011 pa še ekspertna svetovalna skupina (ESS), ki je obravnavala znanstvene vidike. ESS je privzela definicijo KPHM Svetovne zdravstvene organizacije (SZO) iz leta 2002. Člani ESS so soglašali, da so za identifikacijo KPHM potrebni prepričljivi dokazi biološko verjetne vzročne povezave med škodljivim učinkom in hormonskim načinom delovanja. Evropska komisija je 2014 predlagala 4 možnosti kriterijev za identifikacijo KPHM in 3 možnih usmeritev za nadzor. Začela se je tudi poglobljena ocena socioekonomskih, okoljskih in zdravstvenih vplivov predlaganih možnosti. Slovenija podpira uvedbo 4. možnosti, v skladu s katero kriteriji za identifikacijo KPHM temeljijo na definiciji SZO ob upoštevanju moči kot elementa karakterizacije nevarnosti. Pri nadzornih usmeritvah daje Slovenija prednost nadzoru, ki temelji na oceni tveganja in ne zgolj na oceni nevarnosti.

KLJUČNE BESEDE: EU zakonodaja; karakterizacija nevarnosti; identifikacijski kriteriji

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Klančar A, et al. Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study Arh Hig Rada Toksikol 2016;67:106-115

Original article

DOI: 10.1515/aiht-2016-67-2727

Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study Anita Klančar1, Jurij Trontelj1, Albin Kristl1, Maja Zupančič Justin2, and Robert Roškar1 Faculty of Pharmacy, University of Ljubljana1, Arhel d.o.o.2, Ljubljana, Slovenia [Received in October 2015; CrossChecked in October 2015; Accepted in May 2016] Pharmaceuticals in wastewater have clearly raised concern and a broad range of analytical methods has been used to assess the risk as accurately as possible. The aim of our study was to measure and compare the concentrations of atorvastatin, bisoprolol, carbamazepine, ciprofloxacin, clofibric acid, diclofenac, fluoxetine, metoprolol, and sertraline in wastewater samples taken from one municipal and one hospital wastewater treatment plant in Slovenia and to predict the potential environmental burden using the risk quotient. In both effluents only clofibric acid and fluoxetine were not detected. The measured concentrations of the remaining seven pharmaceuticals varied between the ng L-1 and the µg L-1 range. Hospital effluent showed higher concentrations, except for diclofenac and carbamazepine. However, high risk quotient was found only for ciprofloxacin and diclofenac in both municipal and hospital effluent. In conclusion, our method can provide a useful tool for systematic monitoring of pharmaceuticals commonly found in wastewater, which will enable a reliable assessment of the risks for the aquatic biota and humans. Knowing the risks will help to plan wastewater treatment and preserve our environment.

KEY WORDS: atorvastatin; bisoprolol; carbamazepine; ciprofloxacin; clofibric acid; diclofenac, fluoxetine; hospital; LC-MS/MS; metoprolol; municipal; risk assessment; sertraline Pharmaceuticals in wastewater clearly pose a health risk to humans and aquatic life. First, they add to the growing problem of antimicrobial resistance caused by high and uncontrolled consumption of antibiotics and their continuous presence in the environment (1). Second, pharmaceutical residues in drinking water might disrupt the endocrine system. Endocrine disruptors may affect sperm count or cause breast and testicular cancer. One should bear in mind, however, that all evidence of direct adverse effects on human health is weak and inconclusive (2). On the other hand, the evidence of adverse effects on aquatic biota is much more convincing; long-term exposure to diclofenac impairs renal and gill function (3, 4), exposure to carbamazepine growth retardation (5), and exposure to sertraline reproductive disorders in a variety of fish species (6). Even though drug concentrations found in the environment are much lower than the therapeutic ones, some drugs, such as endocrine disruptors, can affect organisms even at extremely low concentrations. They may interfere with the endocrine system and affect vital functions such as development, reproduction, neurology, and immunity. Examples abound: intersex fish, synthesis of vitellogenin in males, feminisation, and absence of pregnancies (7, 8). Furthermore, lipophilic drugs and their metabolites tend to bioaccumulate (9).

Correspondence to: Robert Roškar, Faculty of Pharmacy, University of Ljubljana, Slovenia, E-mail: robert.roskar@ffa.uni-lj.si The subject of this paper has partly been presented at the 2nd Congress of the Slovenian Society of Toxicology “Endocrine disrupting chemicals – from molecule to man” held in Ljubljana, Slovenia, from 23 to 24 April 2015.

A great many studies have introduced various monitoring methods in order to assess exposure and risk in non-target organisms as accurately as possible (9).

Considering the increasing use of pharmaceuticals worldwide, their concentrations in the environment might reach toxicologically significant levels. There are two main approaches to evaluating environmental levels of a drug.

The theoretical approach is based on factors such as consumption, metabolism, and excretion of these drugs.

Estimating national drug consumption may be challenging, as the consumption of over-the-counter drugs is not as meticulously tracked as that of prescription drugs (10).

Moreover, ingested drugs undergo biochemical reactions and their metabolites and degradation products could exhibit either the same or modified activity as the unaltered parent compound. In other words, evaluations may fail to reflect the true environmental load (11, 12). This is why it is imperative to apply the second, experimental approach, which employs sensitive analytical methods to accurately measure the concentrations of targeted compounds. For compounds present in the environment in trace concentrations an array of methods is available such as liquid or gas chromatography coupled with mass spectrometry (13-15) or enzyme-linked immunosorbent assay (ELISA) (15). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in combination with a sample pre-concentration step seems to be the method of choice for most analytes (16). LC-MS/MS can determine a broad range of pharmaceuticals thanks to its high

Klančar A, et al. Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study Arh Hig Rada Toksikol 2016;67:106-115

sensitivity and selectivity in complex matrices such as wastewaters as well as better precision than with other analytical methods due to a less complicated sample preparation (e. g. no derivatisation is needed) (17).

One of the main reasons for pharmaceuticals entering bodies of water is low removal rate in wastewater treatment plants (WWTPs) (18-20). Because municipal WWTPs are considered the main route of pharmaceutical release into the aquatic environment, hospital effluents are frequently overlooked in exposure estimates. With this study we tried to address this oversight using the state-of-the art method.

Our aim was (i) to measure the concentrations of eight common drugs and one metabolite from different classes that are released in Slovene wastewaters, (ii) to compare these concentrations between municipal and hospital wastewaters, and (iii) to assess environmental burden using the risk quotient.

MATERIALS AND METHODS Our analysis targeted pharmaceuticals that are commonly used and well covered by literature data, namely atorvastatin, bisoprolol, carbamazepine, ciprofloxacin, clofibric acid, diclofenac, fluoxetine, metoprolol, and sertraline. Furthermore, their occurrence in wastewater has already been evidenced (21-24). Carbamazepine and clofibric acid were included due to their long history of use and persistence (25). Beta-blockers and NSAIDs were selected because of their widespread use (26). Diclofenac and bisoprolol are the 3rd and the 5th most prescribed drugs in Slovenia, respectively (27). In terms of ecotoxicity, carbamazepine, diclofenac, and metoprolol have been evidenced to cause growth retardation and heart abnormalities in fish embryos (28). Fluoxetine seems to cause endocrine-mediated reproduction dysfunction and developmental abnormalities, also in fish embryos (29, 30).

Antibiotic residues in aquatic systems have clearly showed a potential to induce resistance in bacterial strains and become a serious threat for public health (31, 32).

Chemicals and reagents Standards for carbamazepine (CAR), clofibric acid (CLO), diclofenac (DIC), fluoxetine (FLU), metoprolol (MET), and sertraline (SER) were purchased from SigmaAldrich (Steinheim, Germany). Atorvastatin (ATOR) and bisoprolol fumarate (BIS) were purchased from Sequoia Researcher Products (Pangbourne, UK) and ciprofloxacin (CIP) from AppliChem GmbH (Darmstadt, Germany). The reagents used for standard and sample preparation including acetonitrile (ACN), acetic acid (98 %), formic acid (98100 %), methanol (MeOH), 2-propanol (iPrOH) and potassium dihydrogen phosphate were provided by Merck (Darmstadt, Germany). Ultra-pure water was produced by a Millipore Milli-Q water purification system A10 Advantage (Millipore Corporation, Billerica, MA, USA).

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Solvents for LC-MS/MS analyses were LC-MS-grade acetonitrile ChromasolV® (Sigma-Aldrich, Steinheim, Germany), Milli-Q water and formic acid (98-100 %) Suprapur® (Merck, Darmstadt, Germany).

Sample collection The samples were collected from two WWTPs in central Slovenia: one urban municipal WWTP with a capacity of 9900 population equivalents and daily flow of approximately 2200 m3 and the other WWTP serving a medium-sized hospital with 220 beds with a capacity of 1000 population equivalents and daily flow of approximately 40 m3. It also receives a negligible portion of wastewater from a nearby settlement. Both WWTPs employ a similar treatment: primary mechanical processing followed by biological treatment. Samples were collected both before the treatment point (influent) and after the final treatment (effluent). We obtained three litres of grab wastewater samples (influent or effluent) from each WWTP in accordance with the ISO/ IEC-17025 standard. Samples were stored at 4 °C and analysed within 24 h. From the municipal WWTP influent and effluent wastewater samples were taken in March and June 2015, and from the hospital WWTP five times at regular weekly intervals in May 2015. All wastewater samples had pH around 7.

Sample extraction and analysis Standard stock solutions of each analyte were prepared by dissolving 5 mg of accurately weighed standard in methanol to obtain the concentration of 1 mg mL-1. The final concentration of the working solution of 1 mg L-1 was prepared by mixing the corresponding volumes of the stock solutions of each analyte and by diluting them further with ultra-pure water (standard mixture of pharmaceuticals).

To evaluate the concentration of pharmaceuticals in wastewater we employed the method of standard addition (33). The wastewater samples (250 mL) were first diluted with 250 mL of 250 mmol L-1 potassium dihydrogen phosphate buffer at pH 3. One aliquot of diluted sample was extracted without any additions in order to determine the original concentrations of the analytes. To other aliquots we added the appropriate amounts of the standard mixture of pharmaceuticals before extraction to obtain the final concentrations of 200, 500, 1000, and 2000 ng L-1. All samples were prepared in triplicate. Analyte concentrations were calculated using the linear regression curve.

All samples were extracted using a semi-automated sample preparation system SPE-DEX (Horizon Technology, Salem, NH, USA) with HLB Horizon discs. Rough samples were sequentially treated using a pre-developed method programmed in the EnvisionTM platform Controller software (Horizon Technology). Of all tested rinse solvents, a mixture of ACN (50 %), MeOH (25 %), and iPrOH (25 %) provided the best results.

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Klančar A, et al. Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study Arh Hig Rada Toksikol 2016;67:106-115

The obtained samples were analysed with the Agilent 1290 Infinity LC coupled to Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). One microlitre of the sample was injected onto a 100×3.0 mm, 2.7 µm Poroshell EC-C18 column (Agilent Technologies, Palo Alto, CA, USA) at 50 °C and eluted with mobile phase A (0.1 % formic acid in water) and B (ACN) using the following linear gradient (time min; % B; flow-rate mL min-1): (0;5;0.35), (0.5;5;0.35), (1.1;9;0.35), (1.2;40;0.65), (1.3;50;0.65), (2.0;60;0.65), (2.5;60; 0.65).

Run time was 3.2 min. After each injection, the sampling needle was washed with a washing solvent (MeOH:H2O=80:20 v/v). For MS, a JetStream® (Agilent Technologies, Santa Clara, CA, USA) electrospray source was used. Instrument parameters were set as follows: drying gas temperature 275 °C, drying gas flow 5 L min-1, nebuliser 45 psi, sheath gas temperature 320 °C, sheath gas flow 11 L min-1, capillary entrance voltage 4000 V, nozzle voltage 1000 V. Quadrupoles Q1 and Q3 were set at a wide mass resolution (1.2 amu). Instrument control, data acquisition, and quantification were performed by MassHunter Workstation software (Agilent Technologies, Santa Clara, CA, USA). The MRM transitions and other quantification settings for analytes are presented in Table 1.

Quality control The analytical method was validated using five calibration standards (in triplicate) in the concentration range between 0.01-10 µg L-1. Process efficiency was assessed on samples of ultra-pure water and the June samples of the municipal WWTP influent and effluent wastewater spiked with the standard mixture of pharmaceuticals at different concentrations. The values were estimated by comparison of the peak area obtained from extracted sample and standard solution at the same final concentration (Eq. 1). The imprecision was expressed in terms of the relative standard deviation (RSD) of six replicates. The limit of quantification was defined as the concentration level with a signal to noise ratio of at least 10:1. The influence of the sample matrix on analyte response was determined as an absolute matrix effect (ME) according

to equation 2, based on Matuszewski et al. (34). The rationale of the equation will be described in a separate publication (manuscript in preparation). Values >0 indicate ionisation enhancement and <0 ion suppression.

[Eq. 1]

[Eq. 2]

[Eq. 3] Environmental risk assessment Environmental risk was assessed in terms of each compound's RQ, by calculating the ratio between measured environmental concentration (MEC) and the predicted noeffect concentration (PNEC) according to Eq. 3. PNEC values were based on previously reported acute toxicity data for the most sensitive species (Table 4). A commonly accepted risk ranking criterion was used: RQ<0.1 means minimal risk to aquatic organisms, 0.1≤RQ<1 moderate risk, and RQ≥1 high risk (35, 36).

RESULTS AND DISCUSSION Analytical method validation Table 2 shows the process efficiencies, imprecision, and matrix effects, quantified in ultrapure and/or in influent and effluent wastewater. Process efficiencies were 81-106 %, 68-102 %, and 73-99 % for ultrapure, influent, and effluent wastewater, respectively. Lower recoveries in wastewater

Table 1 Quantification settings Analyte

MRM (m/z)

CE (eV)

Fr (V)

P

atorvastatin

559.3

>

440.2

17

180

+

bisoprolol

326.2

>

116.1

9

144

+

carbamazepine

237.1

>

194.1

13

100

+

ciprofloxacin

332.1

>

314.1

16

134

+

clofibric acid

213.0

>

127.0

10

80

-

diclofenac

296.0

>

214.0

15

55

+

fluoxetine

310.1

>

148.1

1

100

+

metoprolol

268.2

>

116.0

12

96

+

sertraline 306.1 > 158.9 24 MRM: multiple-reaction monitoring; CE: collision energy; Fr: fragmentor; P: polarity

60

+

0.3 0.9997 1.2 1.2 9.7 10.4 2.0 73.2 68.2 SER 87.6 *at the concentration of 0.5 µg L-1

0.9994 1.3 1.2 0.9 3.8 2.0 96.6 80.5 MET

99.2

40.0

2.0 0.9994

0.9993 2.4

1.2 1.1

2.0 4.1

7.0 8.2

12.0 6.7

2.0 80.2

88.3

86.6 FLU

79.5

105.3 DIC

69.3

7.0 0.9990 1.1 1.0 1.0 4.2 5.0 92.8 89.4 CLO

89.7

5.0 0.9991 6.9 7.8 7.1 12.1 7.0 99.4 105.9 CIP

85.1

0.2

2.5 0.9996

0.9996 1.7

1.8 1.6

1.6 1.0

3.6 9.0

5.2 1.0

2.0 96.8

82.4 88.6

CAR

102.1

87.2

105.9

BIS

0.9996 1.2 1.2 4.7 10.3 4.0 81.3 80.8 84.1 ATOR

Ultrapure water

Influent wastewater

Effluent wastewater

Ultrapure water

Influent wastewater

Effluent wastewater

Influent wastewater

Effluent wastewater

Determination coefficient (range 0.01-10 µg L-1) Matrix effect (%)* Process efficiency (%)* Analyte

Table 2 Validation parameters

Imprecision (% RSD)*

Pharmaceutical concentrations in municipal wastewater To the best of our knowledge, this the first study reporting pharmaceuticals in Slovene municipal and hospital wastewaters, but their concentrations in tap and river water have already been reported (37). Table 3 shows municipal wastewater influent and effluent concentrations of the selected compounds and detection frequencies. Six out of nine pharmaceuticals were detected at every sampling and one (atorvastatin) was found in 75 % of the samples.

Clofibric acid, an active metabolite of the lipid-modifying drug clofibrate, and fluoxetine were not detected in any of the samples. This does not surprise, since both drugs have become quite obsolete and have been replaced by safer alternatives such as HMG CoA reductase inhibitors (statins) or new-generation selective serotonin reuptake inhibitors (sertraline, escitalopram, and paroxetine). Instead of clofibrate we found its alternative atorvastatin and instead of fluoxetine we found sertraline in both influent and effluent wastewater.

The concentrations of the remaining pharmaceuticals were mostly similar with other reports (38). Higher ciprofloxacin concentrations were reported in Italy (630 ng L-1) (10) and Spain (2200 ng L-1) (39). High concentrations of the nonsteroidal anti-inflammatory drug diclofenac in our study seem to confirm its ranking as the third most prescribed medicine in Slovenia (27). Another reason for its high concentration may be poor elimination efficiency via activated sludge, as reported earlier (21, 40, 41). The highest measured absolute concentration reported in literature is 11 µg L-1 (9), while generally, its concentrations vary from a few ng L-1 up to 5.5 µg L-1, depending on the country and wastewater treatment technology (42-44).

The comparison between influent and effluent concentrations in our study clearly demonstrates insufficient removal rates for all monitored compounds. Another curiosity worth noting is that diclofenac effluent concentrations (that is to say, after treatment) were higher than influent concentrations in both March and June samples. This phenomenon may be closely related to diclofenac metabolism (45). Several authors suggest that

2.0

LOQ (ng L-1)

samples are probably related to sample complexity.

Imprecision expressed as RSD (%) of six samples spiked at 0.5 µg L-1 in all types of water was not higher than 12 % and, not surprisingly, the most complex water sample, influent wastewater had the highest RSD. Calibration curves were linear with determination coefficients greater than 0.999 for all compounds. The linearity using the standard addition method was also confirmed for all analytes. The matrix effects were below 2.5 %, except for ciprofloxacin. Limits of quantification (LOQ) for the investigated compounds were in the low ng L-1 range. Based on the validation data we concluded that the applied method was appropriate for further investigation of pharmaceuticals in wastewater.

0.5

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Klančar A, et al. Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study Arh Hig Rada Toksikol 2016;67:106-115

110

KlanÄ?ar A, et al. Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study Arh Hig Rada Toksikol 2016;67:106-115

Figure 1 Concentrations of pharmaceuticals detected in the hospital WWTP effluent wastewater over five consecutive weeks in May 2015. CIP and DIC are shown on the separate numeric scale. ATOR: atorvastatin; BIS: bisoprolol; CAR: carbamazepine; CIP: ciprofloxacin; DIC: diclofenac; MET: metoprolol; SER: sertraline Table 3 Concentrations (ng L-1) and frequency (%) of pharmaceuticals in the municipal WWTP samples Analyte

Freq. (%)

Influent concentration (ng L-1)

Effluent concentration (ng L-1)

March

June

March

June

ATOR

75

< LOQ

23.3

< LOQ

nd

BIS

100

130.1

77.5

216.4

36.0

CAR

100

269.6

193.8

482.4

340.5

CIP

100

135.4

165.8

101.9

< LOQ

CLO

0

nd

nd

nd

nd

DIC

100

881.1

4056.9

1155.8

4760.0

FLU

0

nd

nd

nd

nd

MET

100

12.5

18.8

67.4

25.0

49.6

29.1

24.9

SER 100 48.9 nd: not detected; <LOQ: below the limit of quantification

Figure 2 Comparison between the mean concentrations of pharmaceuticals detected in municipal WWTP (A) and hospital WWTP (B) effluents. ATOR: atorvastatin; BIS: bisoprolol; CAR: carbamazepine; CIP: ciprofloxacin; DIC: diclofenac; MET: metoprolol; SER: sertraline

Klančar A, et al. Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study Arh Hig Rada Toksikol 2016;67:106-115

glucuronide and sulphate conjugates of diclofenac may be cleaved by enzymes during wastewater treatment and convert to their parent compound (42, 46). Moreover, the results from the evaluation of the matrix effect in influent and effluent wastewater (Table 2) exclude the potential analytical error (47).

Pharmaceutical concentrations in hospital wastewater and the comparison between municipal and hospital concentrations In order to avoid daily bias for hospital wastewater composition we measured them weekly over entire May 2015 (five samplings). Figure 1 shows their concentrations.

Again, clofibric acid and fluoxetine were not detected, quite likely because of the same reason as for the municipal wastewater: these two obsolete drugs have been replaced by new ones. Concentrations of atorvastatin, metoprolol, and sertraline were roughly consistent across all five samplings, while bisoprolol and carbamazepine showed a higher variability. The highest and the most variable concentrations were observed for ciprofloxacin (0.73.6 µg L-1) and diclofenac (1.1-3.2 µg L-1), yet ciprofloxacin concentrations in our study were slightly lower than in some other hospital wastewater studies (35, 48). Diclofenac concentrations, in turn, were between the ranges reported

by Oliveira et al. (0.03-0.2 µg L-1) (37) and Lin and Tsai (up to 70 µg L-1) (49). All in all, our findings are in line with other published hospital wastewater studies (35), and confirm differences in the loading patterns for specific pharmaceuticals, depending on several factors reported earlier (37, 50). We believe that further investigation should look into these loading patterns.

The comparison between the municipal and hospital wastewater shows that hospital effluents contained pharmaceuticals in 1.5 to 24 times higher concentrations, except for diclofenac and carbamazepine (Figure 2). The greatest difference concerns the antibiotic ciprofloxacin, which is not surprising, due to far more intense use of antibiotics among hospitalised patients. In contrast, diclofenac concentrations were 1.6 times higher in municipal wastewater effluents. One of the reasons could be that the nonsteroidal anti-inflammatory drug diclofenac is not as common among hospitalised patients as in outpatients and the ageing population with musculoskeletal disorders.

In terms of environmental threat one should bear in mind that effluent concentrations were measured directly at the outlet and that wastewater gets significantly diluted before it reaches surface waters. Therefore, we expect that even the highest concentrations measured in our study should drop below the predicted critical downstream concentrations reported by Fick et al. (51). This does not mean that we should neglect the potential environmental threat of ciprofloxacin, diclofenac, or carbamazepine.

111

Environmental risk assessment Our findings do not provide grounds for a precise risk assessment due to the low number of samples and the nature of grab sampling. However, they may give a general idea of the risk. We calculated the risk quotient for the lowest and highest measured concentrations and expressed it as a range. To do that we used previously reported PNECs for the most sensitive species (52-57) and combined them with the highest MECs to establish the worst-case scenario. Table 4 shows high risk for only two compounds: ciprofloxacin and diclofenac. Both belong to therapeutic classes (antibiotics and NSAIDs, respectively) categorised as environmental hazards (58). When the risk quotient is higher than 1 (as is the case with these two drugs) the threat to exposed organisms is serious. Permanent presence of antibiotics in the environment leads to antimicrobial resistance (36). Even the WWTP sludge that retains about 70 % of the influent concentration is known to be used as a fertiliser and can seriously affect the human food chain (59). Global studies have demonstrated that almost all bacterial species have become resistant to fluoroquinolone.

Neisseria gonorrhoeae has rapidly developed fluoroquinolone resistance, which is now as high as 100 % in Asia and 10-30 % in Europe and North America (1).

While ciprofloxacin poses a risk in terms of long-term exposure, diclofenac's toxicity is both acute and chronic in Daphnia. Long term exposure, in turn, leads to renal and gill function impairment in several fish species (3, 4).

Furthermore, diclofenac residues have virtually destroyed the Pakistani vulture population (Gyps bengalensis) due to renal failure and visceral gout, as it fed on dead domestic livestock treated with diclofenac (60).

The risk quotient for carbamazepine and sertraline ranged between 0.1 and 1 in our study, which implies moderate environmental risk. Exposure to carbamazepine in an early life stage can slow down growth in Danio rerio (61). Sertraline as one of the most active selective serotonin re-uptake inhibitors may affect the nervous and/or hormonal systems of non-target organisms, such as the ones reported in Ceriodaphnia dubia (6). While our findings should not raise immediate concern if we look separately at the levels of each drug, the mixture of these pharmaceuticals may pose a far greater risk than either compound individually (52, 61).

CONCLUSION This leads us to the shortcomings of our research. In addition to suboptimal sampling method and the modest number of samples mentioned above, it lacks experimental toxicology data about the exposed organisms and information about downstream (diluted) concentrations measured in surface water. Even so, our findings do confirm that antibiotics and NSAIDs are the most hazardous drug classes regardless of the source of wastewater, as reported

112

Klančar A, et al. Levels of pharmaceuticals in Slovene municipal and hospital wastewaters: a preliminary study Arh Hig Rada Toksikol 2016;67:106-115

Table 4 PNEC (for the most sensitive species) and the range of risk quotients for the selected pharmaceuticals Compound

PNEC (ng L-1)

Species assayed

RQ (municipal WWTP)

RQ (hospital WWTP)

atorvastatin 160 0.0 0.1-0.4 (P. subcapitata) (53) bisoprolol na carbamazepine 2500 0.1-0.2 0.1-0.2 (C. dubia) (54) ciprofloxacin 50 0.7-2.0 14.7-71.2 (M. aeroginosa) (55) clofibric acid 64 no risk no risk (C. dubia) (54) diclofenac 100 11.6-55.6 11.0-32.5 (C. dubia) (54) fluoxetine 50 no risk no risk (P. subcapitata) (56) metoprolol 7900 0.0 0.0 (D. subspicatus) (52) sertraline 90 0.3-0.3 0.4-0.7 (C. dubia) (57) PNEC: predicted no-effect concentration; na: not available in literature; RQ: risk quotient; WWTP: wastewater treatment plant

earlier (35). Our method can serve as a preliminary tool for monitoring pharmaceutical compounds in wastewater, as it allows expanding from a small to a large number of compounds. We believe that with a substantial number of samples this method can provide enough information for a relevant and reliable risk assessment.

However, in light of these findings, we believe that wastewater treatment should include new technologies of hazardous compound removal. For instance, diclofenac is significantly better eliminated by oxidative treatment with ozone or hydrogen peroxide (62), and replacing activated sludge with attached-growth biomass has also shown promising effects (40).

6.

7.

8.

9.

Acknowledgements This study was supported by the LIFE PharmDegrade project »Degradation of pharmaceuticals in wastewaters from nursing homes and hospitals« (LIFE 13 ENV/ SI/000466).

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Koncentracije zdravilnih učinkovin v slovenskih komunalnih in bolnišničnih odpadnih vodah: preliminarna raziskava Pojavljanje ostankov zdravilnih učinkovin v odpadnih vodah postaja vedno bolj aktualna tematika in posledično se širi nabor analiznih metod, ki omogočajo natančno ugotavljanje njihove prisotnosti in služijo kot orodje za napovedovanje tveganja teh onesnažil v vodnem okolju. Namen naše raziskave je bil kvantitativno ovrednotiti prisotnost izbranih zdravilnih učinkovin (atorvastatin, bisoprolol, ciprofloksacin, diklofenak, fluoksetin karbamazepin, klofibrinska kislina, metoprolol in sertralin) na iztoku ene komunalne in ene bolnišnične čistilne naprave. Na osnovi meritev koncentracij smo z uporabo količnika tveganja ocenili okoljsko breme vključenih spojin. Ugotovili smo prisotnost sedmih zdravilnih učinkovin, medtem ko klofibrinske kisline in fluoksetina nismo zaznali v nobenem vzorcu. Izmerjene koncentracije so bile v širokem koncentracijskem območju (od ng L-1 do µg L-1), praviloma višje v bolnišnični odpadni vodi, z izjemo diklofenaka in karbamazepina. Izračunan količnik tveganja nakazuje na visoko tveganje za ciprofloksacin in diklofenak v vseh analiziranih vzorcih odpadnih voda. Raziskava je pokazala, da je razvita metoda primerno orodje za nadaljnje študije, ki bodo na podlagi sistematičnega spremljanja teh novodobnih onesnažil v odpadnih vodah omogočile zanesljivejšo oceno tveganja za izpostavljene vodne organizme in tudi za zdravje ljudi. Poznavanje teh tveganj bo prispevalo k načrtovanju ustrezne tehnologije čiščenja odpadnih voda in posledično k ohranjanju čistega in zdravega okolja.

KLJUČNE BESEDE: atorvastatin; bisoprolol; bolnišnična čistilna naprava; ciprofloksacin; diklofenak; fluoksetin; karbamazepin; klofibrinska kislina; komunalna čistilna naprava; LC-MS/MS; metoprolol; ocena tveganja; sertralin

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Valencia-Quintana R, et al. Assessment of genotoxicity of Lannate-90® and its plant and animal metabolites in human lymphocyte culture Arh Hig Rada Toksikol 2016;67:116-125

Original article

DOI: 10.1515/aiht-2016-67-2763

Assessment of genotoxicity of Lannate-90® and its plant and animal metabolites in human lymphocyte cultures Rafael Valencia-Quintana1, Sandra Gómez-Arroyo2, Juana Sánchez-Alarcón1, Mirta Milić6, José Luis Gómez Olivares4, Stefan M. Waliszewski5, Josefina Cortés-Eslava2, Rafael VillalobosPietrini3, and María Elena Calderón-Segura2 Laboratorio “Rafael Villalobos-Pietrini” de Toxicología Genómica y Química Ambiental, Facultad de Agrobiología, Universidad Autónoma de Tlaxcala, Tlaxcala1, Laboratorio de Genotoxicología Ambiental2, Laboratorio de Mutagénesis Ambiental3, Centro de Ciencias de la Atmósfera, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa4, Centro de Investigaciones Biomédicas, Universidad Veracruzana, Veracruz5, Institute for Medical Research and Occupational Health, Mutagenesis Unit, Zagreb, Croatia6 [Received in December 2015; CrossChecked in December 2015; Accepted in May 2016] This study evaluated direct and metabolic genotoxic effects caused by Lannate-90®, a methomyl-based formulation (90 % active ingredient), in human lymphocyte cultures using sister chromatid exchange assay (SCE). Two processes were used for the plant promutagens evaluation: in vivo activation, applying the insecticide systemically in plants for 4 h and subsequently adding plant metabolites containing extracts to lymphocyte cultures; and in vitro activation, where the insecticide was incubated with Vicia faba S10 mix plus human lymphocyte culture. Direct treatment with the insecticide significantly increased SCE frequency in human lymphocytes (250-750 mg L-1), with cellular death observed at 1000 mg L-1 concentration. Using the extracts of Vicia faba treated with Lannate-90® to treat human lymphocytes, a dose-response relationship was observed. In lymphocyte cultures treated directly with the insecticide for 2 h, a negative response was obtained. When S10 mix was added, SCE frequency did not change significantly. Meanwhile, a mixture of S9 mammalian metabolic mix and Lannate-90® increased the SCE frequency, with an observed concentration-dependent response.

Although Lannate-90® induced cellular death at the highest concentrations, it did not cause a delay in cell proliferation in any of the treatments, confirming its genotoxic action. This study is one of the first to evaluate and compare the direct effect of Lannate-90® in two bioassays, animal and vegetal, and the effect of plant and animal metabolism on its genotoxic potential.

KEY WORDS: animal metabolism; carbamate insecticides; cellular proliferation kinetics; plant metabolism; replication index; sister chromatid exchange Despite the beneficial effects associated with the use of agrochemicals in agriculture and households, many of these products could be potentially hazardous because of their extensive use (1). Among various types of agrochemicals, large quantities of carbamates are particularly applied to different crops. These pesticides have produced inconclusive results in mutagenicity tests according to different cytogenetic end-points evaluated or the pesticide assessed (2).

Lannate-90® is a methyl carbamate insecticide in which the active ingredient is methomyl. Methomyl (S-methylN-(methylcarbamoyloxy)thioacetimidate; CAS 16752-775; Fig. 1) is an oxime carbamate insecticide that controls a broad spectrum of arthropods. This contact insecticide exerts rapid knockdown effects on insects and acts as an ovicide, larvicide and adulticide. Methomyl was first Correspondence to: Sandra Gómez-Arroyo, Laboratorio de Genotoxicología Ambiental, Centro de Ciencias de la Atmósfera, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, 04510 México D.F. México, E-mail: slga@atmosfera.unam.mx

introduced by E.I. du Pont de Nemours in 1966 (3). In 1978, the US Environmental Protection Agency classified it as a restricted-use pesticide (4). Like other carbamates, its main action is through the inhibition of acetylcholinesterase (AChE) activity, resulting in nerve and/or tissue failure and possible cell death. Due to these findings, methomyl was classified as ''highly hazardous'' (class 1B) according to the WHO (5) classification.

In relation to genotoxicity, methomyl has been evaluated with different biological tests. Negative results have been observed in the majority of these tests, particularly in those detecting gene mutation, as in bacteria with or without metabolic activation (6-10), Drosophila (11-13), and cultured cells (6, 14). However, positive results have been reported in yeast (15). Additionally, methomyl induced micronuclei formation in mice (16), and elevated levels of sister chromatid exchange (SCE) and chromosomal aberrations (CA) in mice and cultured cells (17-20). As for plants, Vicia faba root tips also demonstrated elevated levels of CA (21, 22). Drosophila S2, HeLa, and HEK293 cells

Valencia-Quintana R, et al. Assessment of genotoxicity of Lannate-90® and its plant and animal metabolites in human lymphocyte culture Arh Hig Rada Toksikol 2016;67:116-125

Figure 1 Methomyl structure

showed significantly higher DNA damage and apoptosis after treatment (3). Methomyl did not induce carcinogenic activity in rats (23) or dogs (24). Teratogenic effects were also not observed in pregnant rats or rabbits (24).

Besides the concentration of insecticides and time period in which cells and their genetic material are exposed to the insecticide action (3), genotoxic and carcinogenic risks of insecticides also depend on the complexity of insecticide compounds, since sometimes they can stimulate a toxic effect with their presence or during/after biotransformation in a plant/animal organism. Also, the metabolites of pesticides produced by plants and animals can act as promutagens, which is why it is necessary to evaluate not only the chemical itself but also the plant/ animal metabolites produced after exposure to insecticides in order to determine their genotoxic risks to human health.

When working on human lymphocyte cultures, it is necessary to accompany these with S9 rat liver enzymatic fraction in order to obtain data on those metabolites that would normally be found after biotransformation of a chemical in the human body. In case of plant exposure, Vicia faba is considered to be a good plant system for mutagenic studies since it can metabolise the studied pesticide and form metabolically active compounds. As such, it is a sensitive and effective plant to study the effects of pesticide metabolites on genome stability measured by the SCE assay either in the plant or on other cell lines with promutagen extracts (25-27).

Although the genotoxic effect of methomyl alone is rather known, genotoxicity of Lannate-90® as a commercial methomyl-based formulation needs to be explained in more detail, since it is one of the carbamate pesticides most widely used in Mexico due to its broad spectrum of action in all three stages of pest development.

One of the assays commonly used not only as an indicator of genotoxic effects in cells but also as a biomarker of exposure and repair is the SCE assay. SCE involves reciprocal exchanges of chromatid segments that occur at low levels in untreated cells, but their frequency increases substantially following exposure to DNA-damaging agents.

SCEs are primarily formed through homologous recombination, an error-free type of DNA repair. Due to the differential labelling of SCEs, achieved by the incorporation of base analog 5′-bromodeoxyuridine (BrdU) in a cell culture during semiconservative DNA replication, these exchanges can be visualised in second-division metaphase cells whose further division was stopped by spindle inhibitor (e.g. colchicine) in order to accumulate

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cells in that phase. SCE can be measured both in mammals and non-mammalian systems. When cells are exposed to a test substance, it is recommended to use appropriate metabolic activation systems together with the test, since metabolites can cause a higher frequency of SCE (28).

The aim of this study was to examine a possible genotoxic effect of Lannate-90 ® (measured as SCE frequency) caused directly to human lymphocyte cultures.

Moreover, we wanted to examine the influence of the in vivo and in vitro transformation metabolites of Lannate-90® retrieved from the treated Vicia faba plant in a lymphocyte culture. Likewise, plant and animal metabolites were induced in vitro by S10 mix and S9 metabolic lymphocyte activation to estimate their influence on genotoxic effects, cell-cycle progression and proliferation measured as a proportion of the first (M1), second (M2), and third (M3) metaphases as well as the replication index ratio (RI).

MATERIALS AND METHODS Chemicals RPMI medium 1640 with L-glutamine and phytohemagglutinin were purchased from Gibco, of Mexico; Lannate-90®-WD (methomyl CAS number 1675277-5) was provided by Dupont of Mexico. The following chemicals were purchased from Sigma Chemical, St. Louis MO, USA: mitomycin C (MMC, CAS number 50-07-7), cyclophosphamide (CAS number 6055-19-2), bromodeoxyuridine (BrdU, CAS number 59-14-3), Giemsa (CAS number 51811-82), ethanol (purity 99.2 %, CAS number 6415-5), methanol (purity 99.8 %, CAS number 6756-1), acetic acid (purity 99.7 %, CAS number 5323-2645), sodium phosphate monobasic (NaH2PO4, CAS number 7558-80-7), sodium phosphate dibasic (Na2HPO4, CAS number 7758-79-4) and sodium chloride (NaCl, CAS number 7647-14-5). Colchicine (CAS number 64-86-8) and potassium chloride (KCl, CAS number 222425) were purchased from Merck of Mexico.

Direct treatments, without the metabolic activation of human lymphocyte cultures Whole blood samples were used separately in different experiments. Samples were taken from three healthy donors, male non-smokers and non-drinkers, with an average age of 30 years. Donors did not use any medications in the last 6 months before sampling, and did not undergo any radiation/diagnostic treatment in the same period. This study was performed in accordance with the principles stated in the Declaration of Helsinki. All participants were acquainted with the content of the study, accepted the idea of taking part in it, and they subsequently signed an informed consent form.

Since the SCE baseline frequencies among donors were not significantly different, the influence of inter-individual

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Valencia-Quintana R, et al. Assessment of genotoxicity of Lannate-90® and its plant and animal metabolites in human lymphocyte culture Arh Hig Rada Toksikol 2016;67:116-125

variability was negligible in response to the mitogens, culture conditions or blood samples themselves, as suggested by Morgan and Crossen (29), and Speit et al.

(30).

Two experiments were carried out for each treatment using 3 mL of RPMI medium 1640 with L-glutamine and 0.2 mL of phytohemagglutinin added to 0.5 mL of a healthy donor's blood in a culture flask.

The cultures were incubated at 37 °C for 72 h. After 24 h, BrdU was added to the culture medium at a final concentration of 5 μg mL-1. In the experiments without metabolic activation, the cultures were directly exposed to 50, 100, 250, 500, 750, and 1000 mg L-1 of Lannate-90®.

Mitomycin C (400 ng mL-1) was used as a positive control. Lannate-90® was dissolved in sterilised distilled water. The negative control was handled under the same experimental conditions, but lymphocytes were treated with the same amount of distilled water. Preliminary experiments were used to identify optimal concentrations of Lannate-90® that did not cause cellular death.

The cultures were incubated for additional 48 h at 37 °C, and 0.1 mL of colchicine (5 µM) was applied to the cultures 2 h before harvesting. Metaphase cells were harvested by centrifugation, treated with 0.075 M KCl for 20 min, and fixed in methanol-acetic acid (3:1).

Slides were stained using the fluorescence-plus-Giemsa technique (31). In addition to the study of SCE, a BrdU differential staining technique was used to estimate the effect of Lannate-90® on cell replication. According to the OECD guideline, it is enough to analyse 25 well-spread metaphases per culture and treatment.

For the evaluation of cytokinetics, the proportion of the M 1, M 2, and M 3 metaphases was obtained from 100 consecutive mitotic cells for each treatment, and the RI was calculated as follows: RI = 1M1 + 2M2 + 3M3/100 (32). To avoid bias, the slides were scored blindly.

Treatments with metabolic activation In vivo activation. Treatment of human lymphocyte cultures with extracts of Vicia faba roots exposed to Lannate-90® Vicia faba (var. minor) seeds were germinated between two cotton layers soaked in tap water. When primary roots reached a length of 4-6 cm, they were immersed in Lannate-90® at 500, 1000, 1500, 2000, and 2500 mg L-1 in a water solution at 20 °C in the dark. The concentrations were chosen based on preliminary experiments that examined the phytotoxicity of the insecticide. The positive control roots were exposed to 3600 mg L-1 of ethanol (0.1 M) for 4 h at 20 °C. Ethanol is a promutagen in Vicia faba and increases the SCE frequency (27, 33-35). The negative control was handled under the same experimental conditions, but the roots of Vicia faba were immersed in distilled water alone to obtain the S10 fraction.

After the treatment, the roots were rinsed three times with distilled water and cut at approximately 2 cm from the primary root tips. The roots were macerated and homogenised at 4 °C in 0.1 M sodium-phosphate buffer, pH 7.4. The ratio of the buffer solution in millilitres (2.0-2.5) to the fresh weight of root cuttings in grams (2.0-2.5) was 1:1 (26).

Homogenised roots were centrifuged for 15 min at 10000 x g at 4 °C. The supernatant was sterilised using Millipore filters (0.45 μm pore size) and immediately used to treat human lymphocytes in culture. The total protein concentration in these extracts was determined using the Bio-Rad method (35). Protein concentration was fairly constant from one experiment to the other, with values between 5.0-5.9 μg μL-1.

In vitro activation. Treatment using in vitro promutagen activation by Vicia faba S10 mix Lymphocytes cultured for 48 h were exposed to Lannate-90® at 500, 1000, 1500, 2000, and 2500 mg L-1 (concentrations chosen in preliminary experiments) for 2 h in the dark at 37 °C with and without the S10 mix (cultures were stationary for one hour and were then gently shaken for another hour). After the treatment, the cells were rinsed twice in 0.9 % sodium chloride and incubated for 24 h in RPMI medium containing BrdU at a final concentration of 5 μg mL-1. Colchicine (0.1 mL, 5 μM) was added 70 h after the start of the culture, and the same harvesting and staining procedures were performed as described above.

The metabolic activation system with the S10 mixture was prepared from the microsomal S10 fraction at a 1:9 ratio (v/v) with following compounds: 8 mM MgCl2, 3.3 mM KCl, 5 M glucose-6 phosphate, 4 mM NADP and NAD, and 0.1 M Na2HPO4-NaH2PO4 at pH 7.4. The 48 h cultures were incubated for 2 h at 37 °C with 500 μL of the activation system and several concentrations of the insecticide. 3600 mg L-1 ethanol (0.1 M) was used as a positive control (26-27, 33, 35).

In vitro activation. Treatment using in vitro promutagen activation mammalian S9 mix The commercial form (Mol Tox, from Molecular Toxicology Inc USA) of the rat liver microsomal S9 fraction was prepared according to Ames et al. (36) and Frantz and Malling (37). The S9 mixture was prepared with 1 mL of S9 extract and NADPH generator system containing sterile cofactors of 0.2 mL (0.4 M) MgCl 2, 5 mL of pH 7.4 phosphate buffer (0.2 M Na2HPO4 and NaH2PO4), 3.35 mL of distilled water, 0.4 mL of NADP (0.1 M) and 0.05 mL of glucose-6-phosphate (1 M); all of the reagents were freshly prepared. Similarly to direct treatments, BrdU at a final concentration of 5 μg mL-1 was added after 24 h, and 500 µL of the S9 mixture and Lannate-90® were added 48 h later and cultured for 2 h (cultures were stationary for one hour and were then gently shaken for another hour). After each treatment, cell cultures were washed twice with 0.9 %

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sodium chloride and incubated for 24 h with RPMI medium containing 100 µL of BrdU. The positive control was 50 µL (40 µM) cyclophosphamide (CP) with S9, and the negative control was 50 µL CP (40 µM) without S9.

As expected, significant differences were observed in the SCE frequency, cell kinetics and RI with the positive control MMC (Table 1).

Treatments with metabolic activation

Statistical analysis To quantify the SCE frequencies, twenty five metaphase cells at the second division were scored for each concentration. Two experiments were performed, and the results obtained were compared using the Student's t-test.

SCEs were statistically analysed using the ANOVA assay to determine significant differences among the treated groups. When a significant F value (p<0.0001) was found, a Newman-Keuls multiple comparison test was applied to identify groups with significant differences at p<0.001 when compared with controls. A chi squared (c2) test was used for RI. The parts of decomposed c2 were used to compare the values of M1, M2, and M3.

RESULTS Direct treatments without the metabolic activation of human lymphocyte cultures Table 1 shows the averages of two experiments in which Lannate-90® was added directly to human lymphocyte cultures. Low concentrations of the insecticide did not induce significant differences between the SCE frequencies and the negative control. Meanwhile, a concentrationdependent relationship was observed starting at 250 mg L-1 and up to 750 mg mL-1; 1000 mg L-1 caused cell death. Cell kinetics and the RI are also listed in Table 1, no significant differences were found.

In vivo activation. Treatment of human lymphocyte cultures with extracts of Vicia faba roots exposed to Lannate-90® The extracts from untreated roots (negative control) did not increase the SCE frequency (Table 2).

When ethanol was added directly to lymphocyte cultures, the SCE frequency was not significantly affected (negative control); however, significant differences were obtained with root extracts treated with the same concentration of ethanol (positive control).

The in vivo activation by S10 Vicia faba is shown in Table 2. When root extracts were first treated for 4 h with the insecticide, and were then applied to lymphocyte cultures for 48 h, an increase in the SCE frequency was observed for all concentrations of Lannate-90® metabolites.

The transformed Lannate-90®, contained in the Vicia faba root extracts that were applied to lymphocyte cultures, did not produce significant effects on either cell kinetics or RI (Table 2).

In vitro activation. Lannate-90® treatment with in vitro promutagen activation by mammalian S9 mix and Vicia faba S10 mix As shown in Table 3, when Lannate-90® was applied directly to lymphocyte cultures for 2 h, non-significantly different SCE frequencies compared to control values were obtained. Meanwhile, when the S9 metabolic fraction was added, a positive concentration-dependent relationship was observed. However, non-significantly different higher SCE frequencies were also obtained when the S10 metabolic

Table 1 Sister chromatid exchanges induction and effects on cell kinetics (M1, M2 and M3 cells) and replication index (RI) by direct treatments of Lannate-90® in human lymphocyte culturesa X̄ ±S.E. M M M % RIb 1

2

3

Negative control

4.79±0.35

26

38

36

2.10

MMC (400 ng mL-1) (positive control)

15.36±0.65*

51

31

18**

1.67**

50

6.36±0.34

31

46

23

1.92

100

6.45±0.35

25

33

42

2.17

250

8.94±0.53*

24

37

39

2.15

500

11.18±0.54*

20

39

41

2.21

750

13.44±0.74*

25

41

34

2.09

Lymphocyte cultures directly treated with Lannate-90® (mg L-1)

1000 Cellular deathc a n=50 metaphase cells in two experiments. b Replication index, n=100 consecutive metaphases. c Stimulated cells were not observed. *Significant differences among controls and each treated group were obtained by analysis of variance F=57.83, p value is <0.0001, and therefore a Newman-Keuls multiple comparison test was applied p<0.001. **Significant with c2, p<0.05

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fraction was added. Significant differences in the SCE frequency were observed in the positive controls, which were cyclophosphamide (CP) addition to the S9 mammalian metabolic fraction and ethanol addition to the S10 Vicia faba metabolic fraction. Again, no significant effect was seen in either cell kinetics or RI changes, whether after direct treatment or treatment together with S9 or S10 mix.

DISCUSSION Carbamates are part of a large group of synthetic pesticides generally used in agriculture as insecticides, fungicides, herbicides, nematicides or sprout inhibitors (39).

Many carbamates cause genotoxicity (3). Up to now, their genotoxicity, including DNA damage, micronucleus (MN) frequency, elevated levels of CA, SCE, and effects on plant systems and mammalian cells have been evaluated under in vitro and in vivo conditions (20, 22, 40, 41).

On the other hand, diverse studies about genotoxic and/ or cytotoxic effects obtained with pure and commercial formulations of diverse pesticides indicate that commercial formulations may contain additional unsafe xenobiotics supporting the importance of evaluating not only the active principle but also the commercial formulation, which in fact constitutes the real hazard from agrochemicals (42-51).

Methomyl is a carbamate insecticide that has shown genotoxic effects under in vitro and in vivo conditions; it has induced elevated MN frequency in human blood lymphocytes (52) and CA in agricultural (53) and greenhouse exposed workers (54). Its commercial formulation, Lannate®, has also demonstrated genotoxicity in previous studies. Lannate-20® induced a significant increase in the number of sex-linked recessive lethal (55)

and CA in mice germ cells including polyploidy and sex univalent (56). Lannate-25® had a higher clastogenic activity than pure methomyl in vivo (16) and in vitro (18).

With Lannate-90®, elevated levels of CA and SCE were found in Vicia faba root tips (22, 40).

Based on all these studies, genotoxicity of Lannate-90® insecticide has already been demonstrated, but not much is known about its metabolic compounds and their influence on plant/animal organisms. The aim of this study was to examine the influence of Lannate-90®, a formulated carbamate insecticide (90 % methomyl), on a possible genotoxic effect (measured as SCE frequency) caused in human lymphocyte cultures at a wide range of concentrations, chosen on the basis of no cell-toxicity induction. Although the evaluated concentrations of Lannate-90® in this study are expected to be rare in the environment, perhaps only observed when specific events occur (e.g. direct application), we cannot rule out the possibility that organisms and occupationally exposed human workers could be exposed accidentally to such high concentrations. We also need to point out that plants are exposed to those concentrations, and in Mexico the registered uses of methomyl based on labels or label translations provided by the manufacturer go up to 1500-12000 mg L-1 for wheat, barley, maize, sorghum, alfalfa, pea, asparagus, potato, onion, cauliflower, broccoli, beans, lettuce, pepper, cotton, cucumber, cabbage, peanut, apple, grape, citrus, melon, watermelon, strawberry, tomato (57). We also need to underscore that methomyl at these concentrations is not used only in Mexico, but it is registered for use as a pesticide to control a large variety of chewing and sucking insects on a wide range of crops in many countries.

Table 2 Sister chromatid exchanges induction and effects on cell kinetics (M1, M2 and M3 cells) and replication index (RI) by Lannate-90® with in vivo metabolic activation by Vicia faba in human lymphocyte culturesa X̄ ±S.E. M M M % RIb Negative control Lymphocyte cultures + V. faba extracts, untreated (negative control)

1

2

3

4.79±0.35

26

38

36

2.10

4.57±0.35

20

37

43

2.23

Lymphocyte cultures + ethanol 3600 mg L-1 without metabolic activation (negative control)

4.35±0.21

28

39

38

2.20

Lymphocyte cultures + V. faba extracts from the treatment with ethanol 3600 mg L-1 (positive control)

8.96±0.53*

17

37

46

2.29

Lymphocyte cultures + V faba extracts from the treatment with Lannate-90® (mg L-1)

500

7.54±0.42*

20

28

52

2.32

1000

7.59±0.51*

22

30

53

2.41

1500

7.88±0.35*

23

30

47

2.24

2000

8.38±0.52*

20

27

53

2.33

2500

9.29±0.56*

25

27

48

2.39

3000 Cellular deathc a n=50 metaphase cells in two experiments. b Replication index, n=100 consecutive metaphases. c Stimulated cells were not observed. *Significant differences among controls and each treated group were obtained by analysis of variance F=19.84, p value is <0.0001, and therefore a Newman-Keuls multiple comparison test was applied p<0.001.

2500 8.52±0.37 43 38 19 1.75 16.75±2.21* 49 31 20 1.71 6.78±0.81 50 36 14 1.64 n=50 metaphase cells in two experiments; bReplication index, n=100 consecutive metaphases *Significant differences among controls and each treated group were obtained by analysis of variance; F=94.37, p value is <0.0001, and therefore a Newman-Keuls multiple comparison test was applied p<0.001; **Significant with c2, p<0.05 a

1.78 46 30 24 5.96±0.28 1.73 46 36 19 14.07±0.98* 1.77 6.50±0.39 2000

43 37 20

1.76

1.66 50 34 16

46 32 22 5.46±0.29

6.94±0.38 1.67

1.74 45 36 19

47 39 14 9.82±0.40

13.78±0.39* 1.82

1.86

6.67±0.34

40 38 22

7.20±0.48

1500

Lanate-90® (mg L-1)

1000

39 36 25

1.76

1.77 44 35 17

46 32 22 7.16±0.38

9.10±0.25* 1.67

1.87 40 33 27

48 37 15 7.96±0.47

8.41±0.54 1.74

1.67 47 39 14

42 42 16 6.94±0.44

6.01±0.35 Ethanol

500

1.67

1.66 48 38 14

50 33 17 7.40±0.34

4.84±0.31 1.80

1.80 40 40 20

41 38 21 5.86±0.34

41.19±2.14* 1.75

1.77 42 39 19

43 39 18 6.59±0.34

Control

Concentration

Cyclophosphamide

RIb Metaphases M1 M2 M3 SCE/metaphase X̄ ±S.E. RIb Metaphases M1 M2 M3 Metaphases M1 M2 M3

RIb

SCE / metaphase X̄ ±S.E.

With S10 plant metabolic activation With S9 animal metabolic activation Without metabolic activation

SCE/ Metaphase X̄ ±S.E. 4.98±0.30

Table 3 Sister chromatid exchanges induction and effects on cell kinetics (M1, M2 and M3 cells) and replication index (RI) by Lannate-90® without and with in vitro animal and plant metabolic activation in human lymphocyte culturesa

Valencia-Quintana R, et al. Assessment of genotoxicity of Lannate-90® and its plant and animal metabolites in human lymphocyte culture Arh Hig Rada Toksikol 2016;67:116-125

121

Another interest of the study was to examine the behaviour of the in vivo-obtained plant metabolites of Lannate-90® retrieved from the treated Vicia faba plant towards lymphocytes and of the in vitro-induced plant and animal metabolites in respect of the same end point: human lymphocytes using S10 mix from Vicia faba and S9 fraction for metabolic activation. The objective was to estimate their influence on genotoxic effects, cell-cycle progression and proliferation measured as a proportion of the M1, M2, and M3 metaphases as well as the RI ratio.

When meristematic cells of Vicia faba were treated with Lannate-90®, chromatid type aberrations were observed (22). Regarding the type of aberrations produced, Lannate-90® can be considered an S-dependent agent that produces clastogenic effects mainly due to its alkylating groups (22). This is in agreement with the findings of Gómez-Arroyo et al. (58) who concluded that the carbamate herbicide molinate in Vicia faba is an S-dependent agent.

Since SCE is an S-dependent event (59, 60), this type of agent is expected to be an efficient SCE inducer. This fact was confirmed in other studies since treatments of meristematic root cells of Vicia faba with Lannate-90® significantly increased SCE frequency, with a concentrationdependent effect (40).

Bolognesi et al. (16) have observed that in vivo methomyl treatments caused indirect damage of DNA through the formation of active oxygen species. The same authors observed a dose-dependent increase in DNA adducts with Lannate-25® commercial formulation, while negative results were obtained with pure methomyl. The induction of OH8dG by methomyl suggests that DNA oxidative damage could account for the induction of single strand breaks and implies that this insecticide can act indirectly through the formation of hydroxyl radicals, responsible for DNA strand breaks (61). The formation of OH 8dG is considered to be a relevant factor for DNA damage and potential genotoxic and carcinogenic effects due to reactive oxygen species (62). The negative results generally obtained with gene mutation assays, together with the positive results from the test of chromosome damage, as in this study, are also consistent with an indirect mode of action of Lannate-90®.

Bonatti et al. (18), found a dose-related increase in MN frequency with both methomyl and Lannate-25®. However, a higher proportion of MN was induced by Lannate-25, indicating a relatively stronger clastogenic activity of the technical product. A major difference between the pure and the technical product was that Lannate-25 ®, but not methomyl, was able to induce DNA damage in the alkaline elution assay and oxidative damage in the HPLC-EC assay.

Direct treatments with Lannate-90® in lymphocyte cultures for 48 h induced a significant increase in SCE frequency starting at 250 mg L-1, and a concentrationdependent response was observed. These results are in agreement with the data obtained with Lannate-25® (18) in a study on CA in lymphocyte cultures where authors

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observed a dose-dependent increase of chromatid-type and total aberrations. However, our results were not in agreement with the SCE results from that study since we have found a significant increase only at the highest concentrations tested.

No significant differences were found in cell cycle kinetics and the RI in cells treated with Lannate-90®. Our results were in agreement with those of Wei et al. (19) who observed that the rate of cell proliferation in CHO cells treated with methomyl was not significantly altered.

When the extracts obtained from the Vicia faba roots treated for 4 h with different concentrations of the insecticide were applied to lymphocyte cultures, a positive response was obtained in all cases. The products gained from the in vivo transformation by Vicia faba of Lannate-90® applied to the lymphocyte cultures were capable of increasing the SCE frequency, which means that this compound acted directly and indirectly. Additionally, the toxicity diminished with plant metabolism, indicating the possibility that some detoxification mechanisms were involved. Direct treatment with 1000 mg L-1 of Lannate-90® induced cell death, but much higher concentrations (3000 mg L-1) of the insecticide were required to cause cell death when cells were treated with Vicia faba extracts. This is in agreement with Gómez-Arroyo et al. (63) and CortésEslava et al. (64).

Comparing both plant activation systems, in vivo and in vitro, significant SCE frequencies were only observed under in vivo conditions, and these differences could be related to the exposure time of lymphocytes and/or to active metabolites. In the in vivo activation experiments, the treatments lasted for 48 h, while the exposure lasted only for 2 h for the in vitro activation. The latter time period may not have been sufficient for the cells to metabolise the insecticides or this may have been due to the enzyme inactivation because direct contact of the insecticides with the S10 fraction could have inhibited the insecticides' metabolism.

In plant metabolism, peroxidases are among the most important enzymes involved in the oxidative transformation of xenobiotics (65). Peroxidases catalyse two categories of oxidative reactions in plant cells; the peroxidative reaction requiring H2O2 and the oxidative reaction using molecular oxygen (66). Calderón-Segura et al. (34) described the phytotoxic effect of the thiocarbamic herbicide butylate in Vicia faba.

Furthermore, 2 h of treatment with Lannate-90® and the mammalian S9 fraction activated the insecticide. A significant concentration-dependent relationship of SCE frequencies was obtained at concentrations starting at 1500 mg L-1, but cell cycle kinetics and the RI were not affected. Direct treatment for 2 h showed no significant results, and cell kinetics and the RI were not altered.

The effects induced by a commercial formulation of any pesticide cannot account for the active component of the mixture alone and it has been suggested that a genotoxic

agent(s) might be present in the technical formulate (18).

The possibility of oxidative damage as a causative factor for genotoxicity of Lannate-90® has some support in the experimental evidence of the enzyme-inhibiting activities of different methyl carbamate pesticides. Enzymes involved in the defence against harmful oxygen species, such as superoxide dismutase, catalase, and glutathione transferase, are inhibited by these pesticides (67).

Finally, our results highlight that rounded knowledge of the toxic effect/s of the active ingredient of a pesticide is not enough in biomonitoring studies and agrochemical/s' toxic effect/s should be evaluated according to its/their commercial formulation available on the market.

Furthermore, the deleterious effect/s of the excipient/s present within the commercial formulation should neither be discarded nor underestimated. The components of pesticide formulations used as wettable powders usually consist of the carrier, mineral clays, surface active agents, and other ingredients such as stabilisers and dyers. The presence of surface-active agents could increase the genotoxic activity of the active ingredient, favouring the amount of active metabolites at critical cellular targets (16).

Metabolic transformation has been shown to have a great impact on pesticide genotoxicity. Therefore, pesticide constituents and metabolites should also be investigated for genotoxic effects in further studies on active ingredients.

CONCLUSIONS Many carbamate pesticides produce genotoxic effects and long-term exposure of humans to this chemical could produce genetic damage. This study indicates Lannate-90®'s ability to induce genotoxic damage by increasing SCE frequency in human lymphocytes in direct treatments. This highlights the environmental risk of this commercial formulation. Our observations were in agreement with the classification proposed by the WHO for a potential deleterious effect of methomyl.

This study is one of the first to evaluate and compare not only the direct effect of Lannate-90® but also the effect of plant and animal drug metabolism on its genotoxic potential.

The results also confirmed that Lannate-90® is able to exert genotoxic and/or cytotoxic effects on human lymphocytes in a culture, with or without the presence of microsomal metabolic S9 or S10 fractions during cultivation. Thus, the non-consistent results reported so far by different research groups about the deleterious effects of methomyl could be attributed to the ability to convert this type of pesticide into its carbamate derivatives by different cellular systems employed. Then, it could be assumed that the deleterious effect induced by this insecticide on these bioassays is committed to the pesticide itself or to any metabolite/s or any other sub-products generated during the treatment period.

Valencia-Quintana R, et al. Assessment of genotoxicity of Lannate-90® and its plant and animal metabolites in human lymphocyte culture Arh Hig Rada Toksikol 2016;67:116-125

Finally, the study clearly demonstrated that Lannate-90® exerts both genotoxicity and cytotoxicity in human lymphocytes. The hypothesis that coformulants and impurities may play a role in technical grade methomylinduced genotoxicity should be taken into account.

Acknowledgements The authors would like to thank Ana Rosa Flores Márquez for technical assistance, Pietro Villalobos for editing the tables, Claudio Amescua assistance in editing, and Rafael A. Valencia-Sánchez for language editing.

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Procjena genotoksičnosti insekticida Lannate-90® i njegovih biljnih i životinjskih metabolita u kulturi ljudskih limfocita Korištenjem testa izmjena sestrinskih kromatida (eng. Sister Chromatide Exchange Assay – SCE) u kulturama ljudskih limfocita ispitivani su izravni i metabolički genotoksični učinci insekticida Lannate-90®, formulacije koja se temelji na metomilu (90 % aktivni sastojak). Za procjenu biljnih promutagena provedena su dva postupka: in vivo aktivacija, kod koje se insekticid četiri sata sustavno primjenjivao na biljci, a potom su kulturama limfocita dodani biljni metaboliti s ekstraktom, i aktivacija in vitro, kod koje je insekticid inkubiran mješavinom S10 biljke Vicia faba i kulturom ljudskih limfocita. Izravno tretiranje insekticidom značajno je povećalo učestalost SCE-a u ljudskim limfocitima (250-750 mg L-1), a stanična smrt uočena je pri koncentraciji od 1000 mg L-1. Nakon tretiranja ljudskih limfocita ekstraktima biljke Vicia faba koji su tretirani insekticidom Lannate-90®, primijećen je odnos između doze i učinka. Kod kultura limfocita koje su dva sata bile izravno tretirane insekticidom primijećen je negativan odgovor. Kada je dodana S10 mješavina za metaboličku aktivaciju, učestalost SCE-a nije se značajnije promijenila. Naspram tomu, metabolička mješavina S9 za kultivirane stanice sisavaca i Lannate-90® povećali su učestalost SCE-a, uz zamijećen koncentracijski ovisan odgovor. Premda je Lannate-90® inducirao staničnu smrt pri najvišim koncentracijama, nije uzrokovao zastoj stanične proliferacije ni u jednom postupku, čime se potvrđuje njegovo genotoksično djelovanje. Ovo je ispitivanje među prvima kojim se procjenjivao i uspoređivao izravan učinak insekticida Lannate-90® u dvama biološkim testovima, životinjskom i biljnom, te učinak biljnog i životinjskog metabolizma na njegov genotoksični potencijal.

KLJUČNE RIJEČI: izmjena sestrinskih kromatida; karbamatni insekticidi; kinetika stanične diobe; metabolizam biljaka; metabolizam životinja; replikacijski indeks

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Nurulain SM, et al. Terbufos sulfone exaggerates cardiac lesions in diabetic rats: a sub-acute study Arh Hig Rada Toksikol 2016;67:126-135

Original article

DOI: 10.1515/aiht-2016-67-2776

Terbufos-sulfone exacerbates cardiac lesions in diabetic rats: a sub-acute toxicity study Syed M. Nurulain1,2, Mohamed Shafiullah2, Javed Yasin3, Abdu Adem2, Juma Al Kaabi3, Saeed Tariq4, Ernest Adeghate4, and Shreesh Ojha2 Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan1, Department of Pharmacology and Therapeutics2, Department of Internal Medicine3, Department of Anatomy4, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE [Received in January 2016; CrossChecked in January 2016; Accepted in May 2016] Organophosphorus compounds (OPCs) have a wide range of applications, from agriculture to warfare. Exposure to these brings forward a varied kind of health issues globally. Terbufos is one of the leading OPCs used worldwide. The present study investigates the cardiac effect of no observable dose of a metabolite of terbufos, terbufos-sulfone (TS), under nondiabetic and streptozotocin-induced diabetic condition. 100 nmol rat-1 (1/20 of LD50) was administered intraperitoneally to adult male Wister rats daily for fifteen days. The left ventricle was collected for ultrastructural changes by transmission electron microscopy. The blood samples were collected for biochemical tests including RBC acetylcholinesterase, creatinine kinase (CK), lactate dehydrogenase (LDH), cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides, ALT, AST, and GGT. The study revealed about 10 % inhibition of RBC-AChE in two weeks of TS treatment in non-diabetic rats whereas RBC-AChE activity was significantly decreased in diabetic TS treated rats. CK, LDH, and triglycerides were significantly higher in diabetic TS treated rats. Electron microscopy of the heart showed derangement and lesions of the mitochondria of cardiomyocytes in the TS treated groups. The present study concludes that a non-lethal dose of TS causes cardiac lesions which exacerbate under diabetic condition. Biochemical tests confirmed the ultrastructural changes. It is concluded that a non-lethal dose of TS may be a risk factor for a cardiovascular disease, which may be fatal under diabetic condition.

KEY WORDS: Acetylcholinesterase; cardiac lesion; cardiotoxicity; diabetes mellitus; lipid profile; myocyte injury markers; organophosphates Organophosphorus compounds (OPCs) are used in agriculture as insecticides, pesticides, helminthicides, acaricides, nematocides, and herbicides. Furthermore, they are used as a warfare agent and in controlling disease vectors for public health purposes. Over the years, the use of OPCs has increased dramatically with new applications still being developed. To date, there are over 100 different OPCs available, with a similar generalised chemical structure.

Wide accessibility, along with indiscriminate and inadvertent use or exposure, may result in intentional poisoning or unintentional toxicity and it therefore poses a risk to the users and workers involved in their production and application. The acute toxicity of different OPCs ranges from that of an extremely toxic nerve gas to less than that of table salt. Toxicity of all OPCs is due to irreversible inhibition of the neurotransmitter enzyme acetylcholinesterase (AChE).

A significant association between OPC exposure and toxic effects on different organs has been reported in Correspondence to: Dr Shreesh Ojha, Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, UAE E-mail: shreeshojha@uaeu.ac.ae

numerous epidemiological studies (1-3). Among several toxic manifestations, cardiotoxicity is believed to be one of the unrecognised risk factors of OPCs. The effects of OPCs on the cardiovascular system are speculative or based on individual clinical case reports (4-7). The risk of exposure to sub-acute low levels of OPCs for the heart has been scarcely addressed and somehow did not garner much attention from researchers. Albeit, only electrophysiological alterations have been reported but morphological maladies have barely been touched upon. The nature of their toxic effects is variable ranging from transient to permanent (8).

It is noteworthy that cardiotoxicity with OPCs may be independent of AChE inhibition. For instance, dimethoate toxicity in guinea pigs has been reported to cause cardiac failure without any effect on AChE inhibition (9). Likewise, studies also suggest that toxicity of OPCs causes mitochondrial dysfunction mediating a non-cholinergic mechanism (10).

Simultaneously, cardiovascular diseases are increasing significantly globally, as is the prevalence and incidences of diabetes mellitus (DM), one of the important risk factors for cardiovascular diseases. Taken all together, diabetes and cardiovascular diseases are predicted to increase even further over the next decades. Hence, it is reasonable to

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institutional animal ethics committee for the care and use of laboratory animals (Ethical Approval #A2-13).

Induction of experimental diabetes mellitus

Figure 1 Structural formula and some chemical properties of terbufos- sulfone Molecular weight: 320.43 g/mol; empirical formula: C9H21O4PS3 Log P: 2.64; C Log P: 2.58; XLogP3: 2.5 Chemical name/synonyms: Phosphorodithioic acid, S-((1,1dimethylethyl)sulfonylmethyl) O,O-diethyl ester, Dithiophosphoric acid O,O-diethyl S-[(tert-butylsulfonyl)methyl] ester; S-[(tertbutylsulfonyl)Methyl] O,O-diethyl phosphorodithioate, S-tertbutylthiomethyl O,O-diethyl phosphorodithioate

predict that exposure to OPCs may have a significant impact on the manifestations of cardiotoxicity in patients with diabetes and prediabetes much faster than in normal healthy individuals. It may be noted that the effect of OPCs under diabetic pathological condition is scarcely reported (11, 12).

Therefore, the present study was carried out to investigate the effect of sub-acute exposure to terbufos sulfone (TS), a highly toxic OPC as per the World Health Organization (WHO) classification of insecticides and pesticides. Terbufos is registered in more than 20 countries; however, its usage has been banned in Europe since 2006.

The present study was designed to demonstrate whether TS relevant to no-observable adverse effect level (NOAEL) dose could exacerbate cardiac injury in non-diabetic and streptozotocin-induced (STZ) diabetic rats. This study will have significant implications because sub-acute exposure to TS or to any other OPC has inadequately been addressed in the literature.

MATERIALS AND METHODS Experimental animals The original stock of Wistar rats were purchased from Harlan Laboratories (Harlan Laboratories, Oxon, England).

The animals used in the actual experiments were bred at our own animal facility from the original stock. Male adult rats, weighing 200-250 g were used in the present study.

The animals were housed in polypropylene cages (43x22.5x20.5 cm; six rats per cage) in climate and access controlled rooms maintained under standard laboratory conditions (temperature: 23±1 °C; humidity: 50±4 %; day/ night cycle: 12h/12h). The rodent pellet diet was procured from the Emirates Feed Factory, Abu Dhabi, UAE and water was available ad libitum. All the experimental procedures were in strict compliance with the protocol approved by the

To induce type I diabetes, after an overnight fast, the rats were administered a single intraperitoneal (i.p.) injection of streptozotocin (60 mg kg-1 body weight) prepared freshly in citrate buffer (0.1 mol L-1 pH 4.5) solution. The induction of diabetes in rats was confirmed measuring random blood glucose levels using a One-Touch Glucometer (Lifescan Inc., Milpitas, CA, USA). Animals with blood glucose above 235 mg dL-1 were considered diabetic and were used further for the experiments.

Experimental design There were four experimental groups, each containing 6 rats. Terbufos sulfone (TS) was administered i.p. at a dose of 100 nmol per rat (1/20 of LD50) following our previous laboratory studies (13). The animals were treated daily for two weeks.

Group 1 (G1): Non-diabetic control (saline-treated nondiabetic rats) Group 2 (G2): Diabetic control group (No treatment with TS) Group 3 (G3): Non-diabetic, TS 100 nmol rat-1 treatment Group 4 (G4): Diabetic group treated with 100 nmol rat-1 of TS Chemicals The TS stock solution (100 mmol L-1) was prepared in dry acetone. The working solution for the i.p. injection was prepared extempore by diluting the stock solution with physiological saline. The chemicals, TS and streptozotocin, were purchased from Sigma-Aldrich Chemie (SigmaAldrich Chemie GmbH, Steinheim, Germany).

Blood glucose measurement Random blood glucose was checked on day 8 of each week before the injection of TS. Blood samples were collected from the tail vein.

RBC-AChE activity The blood sample for RBC-AChE measurement was collected from the tail vein of the animal. The RBC-AChE activity was measured in diluted whole blood samples in the presence of a selective butyrylcholinesterase inhibitor, ethoproprazine, as described previously (14).

Blood collection for biochemical tests The blood samples were collected from rats before decapitation and were subsequently centrifuged at 2555 g for 10 minutes. The obtained serum was then stored frozen at -80 °C. The markers for cardiac function viz. myocyte

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injury marker enzymes such as lactate dehydrogenase (LDH) and creatinine kinase (CK), for lipid profile such as cholesterol, triglycerides, high density lipoprotein (HDL), low density lipoprotein (LDL), and liver function enzymes such as alanine transaminase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) were measured by an autoanalyser (COBAS INTEGRA 400 PLUS from Roche Diagnostics, Germany).

significance in comparison to control values, the MannWhitney order rank test was used. The limit of statistical significance was set at p<0.05.

Transmission electron microscopy

Figure 2 shows the change in body weight of experimental animals after the first and second weeks of saline (controls) and TS treatment. The non-diabetic animals increased body weight over time irrespective of whether treated with saline or TS. The diabetic animal groups decreased their body weight. This decrease was more conspicuous in the TS treated than in the saline treated group. However, the change was not found statistically significant compared to the pre-treatment or first-week body weight.

The left ventricle from the heart of animals of each experimental group was cut into small pieces and fixed for 5 hours in 2.5 % glutaraldehyde in Karnovsky’s solution (15). The samples were washed overnight in cacodylate buffer (40 °C) and post fixed with 1 % osmium tetroxide for 1 hour. The samples were further washed six times in cacodylate buffer and dehydrated in ethyl alcohol at graded concentrations. They were later treated with propylene oxide; two changes of 15 minutes and were then immersed in propylene oxide and resin (1:1) for 1 hour followed by pure resin overnight. Afterwards, the samples were embedded and polymerised at 60 °C for 24 hours. Semi and ultrathin sections were cut using a diatome knife procured from Agar Scientific, Essex, England. The semi thin sections were placed on glass slides and stained with toluidine blue.

The ultrathin sections were mounted on 3.05 mm 200 mesh copper grids and contrast stained first with saturated aqueous uranyl acetate for 30 min and then with Renold’s lead citrate for 5 minutes. The ultrathin sections were viewed under a Philips CM10 transmission electron microscope (Eindhoven, The Netherlands).

Statistical analysis Statistical analysis was performed for all the parameters obtained from the experimental groups using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). To determine the

Figure 2 Changes in body weight of different experimental groups

RESULTS Body weight

Blood glucose Random blood glucose levels across G1 to G4, over a period of two weeks, are depicted in Figure 3. The average two-week blood glucose levels in G1 and G2 (control groups) were 100±9 mg dL -1 and 473±72 mg dL -1, respectively. The average two-week blood glucose level in G3 was 100±8 mg dL-1. The mean blood glucose level for diabetic rats treated with TS100 (G4) was 511±29 mg dL-1 over the two-week period. This increase was statistically significant (p<0.05) compared to the pre-treatment value (432±29 mg dL-1).

RBC-Acetylcholinesterase The activity of RBC-AChE enzyme was determined as a percentage of baseline and is depicted in Figure 4. The

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Nurulain SM, et al. Terbufos sulfone exaggerates cardiac lesions in diabetic rats: a sub-acute study Arh Hig Rada Toksikol 2016;67:126-135

Figure 3 Changes in blood glucose level over weeks in different experimental groups

* *

Figure 4 Changes in RBC-AChE over weeks in control and TS treated groups *Statistically significant at p≤0.05; Mann-Whitney order rank test

Table 1 Serum levels of myocyte injury markers; CK and LDH in different experimental groups. *p≤0.05; Mann-Whitney order rank test.

Groups

Non-diabetic control

Non-diabetic TS treated

Diabetic control

Diabetic TS treated

LDH (IU/L)

Mean±SD 95% CI

937±365 (484-1390)

1354±559 (660-2049)

989±301 (615-1362)

1910±179* (1688-2132)

CK (IU/L)

Mean±SD 95% CI

343±117 (198-487)

644±226* (363-924)

404±140 (230-578)

871±88* (762-980)

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intraperitoneal injection of 100 nmol of TS administered to non-diabetic rats (G2) did not produce a significant change in the RBC-AChE activity. The same dose in STZinduced diabetes (G4) produced a sharp and statistically significant decrease (p<0.05) in week 1 (38 %±18) and week 2 (11 %±3). The diabetic control group (G3) showed a moderate decrease which was statistically not significant.

CK and LDH

The ultrastructure of the heart (transmission electron microscopy)

Lipid profile

The ultrastructure of the heart by transmission electron microscopy is represented in Figure 5. A representative image of the ultrastructure of the heart of non-diabetic control rats is represented in Figure 5A. Myofibrils were observed intact with thick and definite architecture and linearly arranged mitochondria. Figure 5C shows the ultrastructure of the heart of non-diabetic TS treated rats.

A distorted architecture of myofibrils, large intermyofibrillar spaces and swollen mitochondria are conspicuous and are the effect of TS treatment. Figure 5B shows the diabetic control group. Thin myofibril, deranged mitochondria, and inter-myofibrillar spaces are a characteristic pathological effect of diabesity. The mitochondria (m) of the cardio myocytes of the heart of diabetic rats treated with TS (Figure 5D) contain deranged mitochondria (m) and large muscle inter-fibre spaces.

Mitochondrial lesions were more conspicuous in diabetic rats (Figure 5D) when compared to those of the non-diabetic and diabetic control groups (Figure 5A & B), and TS treated non-diabetic (Figure 5C) rats. A damaged architecture of myofibrils was clearly observable between the TS treated and non-treated groups though diabetes itself damages the heart structure.

Table 1 shows the creatine kinase (CK) and lactate dehydrogenase (LDH) levels in the control and TS treated groups after two weeks of treatment. CK and LDH levels were found significantly elevated (p<0.05) in the diabetic TS treated groups, indicating a cardiac manifestation of TS treatment.

Table 2 shows the measurement of cholesterol, HDL, LDL, triglycerides, ALT, AST, and GGT in the serum of the treated and non-treated groups. The non-diabetic TS treated groups did not show statistical significance (Table 3) in the alteration of the tested parameters when compared to their corresponding non-diabetic saline control except for AST, which decreased significantly. However, diabetic rats treated with TS showed marked and statistically significant (p<0.05) changes in all the tested parameters except for ALT, which was found to be non-significantly different than in the diabetic saline control group. Among all the parameters of the lipid profile, triglycerides markedly surged in the diabetic TS treated group (mean±SD: 1.61±0.79) compared to the diabetic and non-diabetic controls (mean±SD: 0.60±0.12, 0.90±0.40 respectively).

DISCUSSION The present study demonstrates that even a relatively non-lethal dose of terbufos sulfone and sub-acute exposure to it causes toxic effects to the heart of non-diabetic and STZ-induced diabetic rats. Terbufos belongs to an extremely toxic group of OPCs as per the WHO’s classification of hazardous chemicals (16) and its metabolites are also

Table 2 Blood lipid profile in different experimental groups. *p≤0.05; Mann-Whitney order rank test. *Statistically significant when compared to non-diabetic saline control (p<0.05). **Statistically significant when compared to diabetic saline control (p<0.05) Experimental groups

Non-diabetic saline control

Non-diabetic TS treated

Diabetic saline control

Diabetic TS treated

Cholesterol (mmol L-1)

Mean±SD 95 % CI

1.26±0.23 0.97-1.55

1.22±0.17 1.04-1.40

1.60±0.27 1.32-1.88

1.20±0.21** 0.98-1.42

HDL (mmol L-1)

Mean±SD 95 % CI

0.84±0.08 0.74-0.94

0.77±0.31 0.44-1.09

0.96±0.13 0.82-1.09

0.58±0.26** 0.32-0.86

LDL (mmol L-1)

Mean±SD 95 % CI

0.11±0.02 0.08-0.14

0.16±0.06 0.09-0.23

0.30±0.10 0.19-0.41

0.07±0.04** 0.03-0.11

Triglyceride (mmol L-1)

Mean±SD 95 % CI

0.90±0.40 0.39-1.40

0.79±0.08 0.71-0.87

0.60±0.12 0.48-0.72

1.61±0.79** 0.78-2.43

ALT (IU L-1)

Mean±SD 95 % CI

40.20±4.44 34.69-45.71

37.67±13.90 23.08-52.25

135.83±62.97 69.75-201.92

96.00±34.29 60.02-131.98

AST (IU L-1)

Mean±SD 95 % CI

97.60±11.63 83.16-112.04

159.83-31.49* 126.78-192.88

711.17±258.65 484.46-937.84

224.50±71.71** 149.24-299.76

GGT (IU L-1)

Mean±SD 95 % CI

5.00±0.71 4.12-5.88

5.50±0.55 4.93-6.07

1.50±0.55 0.93-2.07

4.67±2.50** 2.04-7.29

131

Nurulain SM, et al. Terbufos sulfone exaggerates cardiac lesions in diabetic rats: a sub-acute study Arh Hig Rada Toksikol 2016;67:126-135

Table 3 p-values of multiple comparison of Table 2.

Non-diabetics saline control Non-diabetic -saline control Non-diabetic 0.925 TS treated Cholesterol Diabetics saline control 0.701

HDL

LDL

Triglyceride

ALT

AST

GGT

Non-diabetic TS treated

Diabetics saline control

Diabetic TS treated

0.925

0.701

0.062

--

0.019

0.019

0.019

--

0.023

0.062

0.019

0.023

--

--

0.715

0.008

0.144

0.715

--

0.199

0.199

0.008

0.199

--

0.004

0.144

0.199

0.004

--

--

0.142

0.117

0.006

0.142

--

0.020

0.020

0.117

0.020

--

0.004

0.006

0.020

0.004

--

--

0.715

0.045

0.068

0.715

--

0.016

0.016

0.045

0.016

--

0.006

0.068

0.016

0.006

--

--

0.647

0.010

0.035

0.647

--

0.030

0.030

0.010

0.030

--

0.150

0.035

0.030

0.150

--

--

0.011

0.006

0.006

0.011

--

0.006

0.006

0.006

0.006

--

0.016

0.006

0.006

0.016

--

--

0.219

0.402

0.005

0.219

--

0.003

0.003

Diabetics saline control

0.402

0.003

--

0.007

Diabetic TS treated

0.005

0.003

0.007

--

Diabetic TS treated Non-diabetics saline control Non-diabetic TS treated Diabetics saline control Diabetic TS treated Non-diabetics saline control Non-diabetic TS treated Diabetics saline control Diabetic TS treated Non-diabetics saline control Non-diabetic TS treated Diabetics saline control Diabetic TS treated Non-diabetics saline control Non-diabetic TS treated Diabetics saline control Diabetic TS treated Non-diabetics saline control Non-diabetic TS treated Diabetics saline control Diabetic TS treated Non-diabetics saline control Non-diabetic TS treated

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Nurulain SM, et al. Terbufos sulfone exaggerates cardiac lesions in diabetic rats: a sub-acute study Arh Hig Rada Toksikol 2016;67:126-135

B

Figure 5 Electron microscopic changes in the heart tissue. Scale bar=0.5Â Âľm.

A=non-diabetic control; B=diabetic control; C=non-diabetic TS treated, and D=diabetic rat treated with TS; m=mitochondrion

Nurulain SM, et al. Terbufos sulfone exaggerates cardiac lesions in diabetic rats: a sub-acute study Arh Hig Rada Toksikol 2016;67:126-135

persistent in the environment (http://www.abcbirds.org/ abcprograms/policy/toxins/profiles/terbufos.html). TS is the active metabolite of terbufos, the fourth most frequently used pesticide in the USA, reported for its use in fish kill incidents, which is also suggestive of its presence in the food chain (17). To the best of our knowledge and based on the available literature, no data on the cardiovascular condition following sub-acute exposure to this compound has been reported. Hundreds of different OPCs share a common mechanism of action but are toxicologically different one from the other, ranging from deadly poison nerve agents to those with virtually no toxic potential (18).

According to the website Scorecard (19), the list of more than 500 cardiovascular toxins causing different types of cardiovascular complications contains only few OPCs. This list reflects the paucity of studies with OPCs. Asymptomatic low-level exposure to OPCs has been reported to cause various health effects including cardiac lesions (8, 20, 21).

Different individual toxicokinetics of OPCs necessitates investigating the effect on the heart under normal and diabetic conditions (22). The cardiovascular manifestation of OPC poisoning is mainly attributed to acute poisoning (23–27), which comprises bradycardia or tachycardia or impaired heart rate. There are reports showing an increased level of creatine kinases and lactate dehydrogenases, besides myocardial necrosis (28). The results of the present study reveal that TS causes a leakage of myocyte injury marker enzymes into the serum, as evidenced by the elevated levels of serum CK and LDH. The structural change in the heart was further correlated with the myocyte injury marker enzymes’ leakage from cardiomyocytes to the serum. Abdou et al. (29) reported increased LDH and histologically large areas of degenerating muscle fibres with an evident loss of transverse striations and wide inter-fascicular spaces when treated with diazinon, an OPC. Velmurugan (30) used 1/50th of LD50 dosage of monochrotophos against Wister rats and found elevated LDH and CK-MB in the blood plasma along with signs of nonspecific inflammatory changes and oedema between muscle fibres of the heart.

The present study also investigated ultrastructural changes following the administration of a non-lethal dose of TS to non-diabetic and STZ-induced diabetic rats.

Ultrastructural changes after acute OPC poisoning have been reported by numerous clinical case studies (31–34).

We also observed similar cardiac lesions in diabetic rats with sub-acute and apparently non-lethal exposure to TS. The injurious effect on the heart in diabetic rats was further confirmed by low HDL and high triglycerides. These observations are in line with other previous studies which report that exposure to repeated doses of profenfos, an OPC, significantly increased LDH and CK enzymes (35). The probable reason for this leakage could be cell death as Razavi et al. (36) reported with sub-chronic exposure to diazinon, an organophosphate. In the present study, we also observed that TS administration led to a structural impairment of the mitochondrion in non-diabetic TS treated

133

rats, but the lipid profile was not significantly affected. This observation was supported by a previous study wherein a higher but non-lethal dose of 20 mg kg-1 body weight of diazinon administered for four weeks did not reveal a change in the cholesterol and triglyceride levels (37).

OPC elicits its toxicity by inhibiting acetylcholinesterase.

The severity of poisoning depends upon the extent of AChE inhibition and consequent cholinergic crises. This may be correlated with the observation that inhibition of AChE was severe in diabetic rats at the end of the second week and it resembled acute toxicity. The effect of anticholinesterases on the heart is mainly attributed to acute toxicity, especially the delayed or late phase effect (5). Different mechanisms and pathways have been reported to be involved in the cardiotoxicity of acute OPC poisoning. One of the studies suggests that morphologic damage may be instigated by uninterrupted vagus nerve stimulation of the accumulated acetylcholine (5). Herein, the magnitude of myocardial damage correlated with the duration of exposure. It has also been found that cardiac damage may be independent of the extent of AChE inhibition contrary to the high level inhibition of AChE in acute poisoning. Since acetylcholine receptors have a high binding capacity for calcium ions (38), the disorder of cellular morphology may be ascribed to a massive influx of Ca2+ into the cytoplasm. The results also signify that TS even after two weeks has a potential to raise triglyceride levels, cause myocyte injury and subsequent myocardial injury. Interestingly, the changes observed were irrespective and independent of the degree of AChE inhibition, as it was observed to be high in diabetes and low in non-diabetic rats. This suggests that occupational exposure to TS, even at low doses, may pose a risk in those who are prone to develop diabetes and can worsen cardiotoxic manifestations in diabetics. In addition, ultra-structural studies may be a vital tool in identifying the non-lethal dose associated cardiac effect, since biochemical changes may be not evident at such dose or can manifest after very long exposure. Considering the high rise of diabetes and cardiovascular diseases, the exposure to TS may impact cardiovascular health, worsen the disease condition and accelerate progression.

CONCLUSION TS at a relatively non-lethal dose may be associated with cardiovascular complications including myofibril degeneration, wide inter-fascicular spaces, and mitochondria mutilation. The impact may become exacerbated under diabetic condition, which may lead to mortality and morbidity. The effect may be independent of ACh inhibition as observed in non-diabetic rats or might be related to ACh inhibition, as was seen in diabetic rats.

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Nurulain SM, et al. Terbufos sulfone exaggerates cardiac lesions in diabetic rats: a sub-acute study Arh Hig Rada Toksikol 2016;67:126-135

Acknowledgement The research work in part was supported by research grants of Dr. Shreesh Ojha, College of Medicine and Health Sciences, United Arab Emirates University, UAE. The authors would also like to acknowledge Mahmoud Hag Ali, Animal Research Facility controller for his help in animal care and welfare.

Authors’ contribution All the authors (SMN, MS, JY, AA, JA, ST, EA, SO) provided an important intellectual content, reviewed the content, and approved the final version of the manuscript.

SMN, AA, JA, EA, SO contributed significantly, read and approved the manuscript. SMN,JA, EA, SO conceived and designed the experiments. SMN and MS performed the animal experiments. SMN and JY performed laboratory investigation. SMN and ST performed electron microscopy.

SMN, EA and SO analysed the data. SMN, JA, EA and SO contributed with reagents/materials/analysis tools. SMN, EA and SO wrote the paper.

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Conflict of interest The authors declare no conflict of interest. There are no patents, products in development or marketed products to declare. The research grant funding organisations had no role in the study design, data collection and analysis, decision to publish or the preparation of the manuscript.

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Jaacks LM, Staimez LR. Association of persistent organic pollutants and non-persistent pesticides with diabetes and diabetes-related health outcomes in Asia: A systematic review. Environ Int 2015;76:57-70. doi: 10.1016/j. envint.2014.12.001 Nurulain SM, Petroianu G, Shafiullah M, Kalász H, Oz M, Saeed T, Adem A, Adeghate E. Sub-chronic exposure to paraoxon neither induces nor exacerbates diabetes mellitus in Wistar rat. J Appl Toxicol 2013;33:1036-43. doi: 10.1002/ jat.2794 Lorke DE, Nurulain SM, Hasan MY, Kuča K, Petroianu GA. Prophylactic administration of non-organophosphate cholinesterase inhibitors before acute exposure to organophosphates: assessment using terbufos sulfone. J Appl Toxicol 2014;34:1096-103. doi: 10.1002/jat.2939 Worek F, Mast U, Kiderlen D, Diepold C, Eyer P. Improved determination of acetylcholinesterase activity in human whole blood. Clin Chim Acta 1999;288:73-90. doi: 10.1016/ S0009-8981(99)00144-8 Karnovsky M. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 1965;27:137A-8A. The WHO recommended classification of pesticides by hazard and guidelines to classification 2009 [displayed 29 May 2015]. Available at http://apps.who.int//iris/ handle/10665/44271 Bonner MR, Williams BA, Rusiecki JA, Blair A, Freeman LEB, Hoppin JA, Dosemeci M, Lubin J, Sandler DP, Alavanja MC. Occupational exposure to terbufos and the incidence of cancer in the Agricultural Health Study. Cancer Causes Control 2014;21:871-7. doi: 10.1007/s10552-010-9514-9 Nurulain SM. Different approaches to acute organophosphorus poison treatment. J Pak Med Assoc 2012;62:712-7. PMID: 23866522 Cardiovascular Toxicants. Suspected Cardiovascular Or Blood Toxicants [displayed 23 May 2015]. Available at http:// scorecard.goodguide.com/health-effects/chemicals-2. tcl?short_hazard_name=cardio&all_p=t Eskenazi B, Kogut K, Huen K, Harley KG, Bouchard M, Bradman A, Boyd-Barr D, Johnson C, Holland N. Organophosphate pesticide exposure, PON1, and neurodevelopment in school-age children from the CHAMACOS study. Environ Res 2014;134:149-57. doi: 10.1016/j.envres.2014.07.001

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Terbufos sulfon pogoršava srčane lezije u štakora koji boluju od dijabetesa: studija subakutne toksičnosti Organofosforni spojevi (eng. Organophosphorous Compounds - OPCs) imaju široku primjenu, od one u poljoprivredi do one u vojne svrhe. Izlaganje takvim spojevima izaziva niz različitih zdravstvenih problema od globalnog značaja.

Terbufos je jedan od vodećih OPC-a koji se koriste diljem svijeta. U ovom je istraživanju na modelu štakora bez dijabetesa i sa streptozotocinom izazvanim dijabetesom ispitivan metabolit terbufos-sulfon (TS) u najvišoj dozi koja ne izaziva učinak te njezin utjecaj na srce. Odrasli mužjaci štakora soja Wistar dobivali su petnaest dana dnevnu dozu od 100 nmol štakor-1 (1/20 LD50) intraperitonealno. Transmisijskim elektronskim mikroskopom istražene su ultrastrukturne promjene lijeve klijetke. Na krvnim uzorcima provedeni su biokemijski testovi, uključujući aktivnost acetilkolinesteraze u crvenim krvnim stanicama, razinu kreatinin kinaze (CK), laktat dehidrogenaze (LDH), kolesterola, lipoproteina visoke gustoće (HDL), lipoproteina niske gustoće (LDL), triglicerida, ALT, AST i GGT. Istraživanjem je otkriveno oko 10 % inhibicije AChE-a u crvenim krvnim stanicama nakon dva tjedna izlaganja štakora bez dijabetesa TS-u, dok je u štakora s dijabetesom aktivnost AChE-a bila značajno smanjena. Razine CK, LDH i triglicerida bile su značajno više u TS tretiranim štakorima s dijabetesom. Elektronsko-mikroskopska analiza srca upućuje na narušenu strukturu i lezije u mitohondrijima u kardiomiocitima skupina štakora koji su tretirani TS-om. Zaključuje se da nesmrtonosna doza TS-a uzrokuje srčane lezije koje se pogoršavaju u prisutnosti dijabetesa. Biokemijski testovi potvrdili su ultrastrukturne promjene. Navedena doza TS-a može biti rizični čimbenik za kardiovaskularne bolesti, koje se mogu pokazati smrtonosnima uz istovremeno postojanje dijabetesa.

KLJUČNE RIJEČI: acetilkolinesteraza; diabetes mellitus; kardiotoksičnost; lipidni profil; marker oštećenja miocita; organofosfati; srčana lezija

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Dzhambov AM, Dimitrova DD. Exposure-response relationship between traffic noise and stroke: a systematic review with meta-analysis Arh Hig Rada Toksikol 2016;67:136-151

Original article

DOI: 10.1515/aiht-2016-67-2751

Exposure-response relationship between traffic noise and the risk of stroke: a systematic review with meta-analysis Angel M. Dzhambov1 and Donka D. Dimitrova2 Department of Hygiene and Ecomedicine1, Department of Health Management and Health Economics2, Faculty of Public Health, Medical University of Plovdiv, Plovdiv, Bulgaria [Received in November 2015; CrossChecked in November 2015; Accepted in May 2016] Traffic noise is an established risk factor for some cardiovascular diseases such as hypertension and ischaemic heart disease, but the evidence regarding stroke is still limited. In this study we aimed to systematically review the related epidemiological data and make a meta-analysis of the risk of stroke morbidity associated with road and air traffic noise exposure. We searched articles in English, Spanish, and Russian indexed in MEDLINE, EMBASE, and Google Scholar on 24 November 2015. Qualitative synthesis was made for 13 studies, and 11 studies were included in quality effects meta-analyses. Overall, they were of high quality. Based on six studies (n≈8,790,671 participants) for road traffic noise, we found a pooled relative risk (RR) of stroke per 10 dB to be 1.01 (95 % CI: 0.96, 1.06). In the 70-75 dB noise range (versus <55 dB) RR increased to 1.29 (95 % CI: 0.74, 2.24). For air traffic noise we pooled five studies (n≈16,132,075 participants) and the RR per 10 dB was1.01 (95 % CI: 1.00, 1.02). Road traffic group showed high heterogeneity whereas the air traffic group had none. Both groups showed evidence of publication bias. In conclusion, we have established a small but elevated risk of stroke to be associated with both road and air traffic noise exposure, but the association was statistically significant only with the latter. The effect of road traffic noise followed a non-linear trend.

KEY WORDS: aircraft; cerebrovascular disease; cardiovascular disease; noise exposure; road traffic; transportation Traffic noise is already a recognised risk factor for cardiovascular disease (1). It acts as a general environmental stressor, adversely affecting the neuroendocrine system and sleep architecture, which ultimately leads to increased levels of plasma catecholamine and cortisol, impaired carbohydrate and lipid metabolism, and increase in blood pressure and vascular reactivity (2). Meta-analyses have found that the pooled risk of ischaemic heart disease is 1.04 (95 % CI: 1.00, 1.10) per 10 dB increase in road traffic day-eveningnight sound level (Lden) and 1.06 (95 % CI: 1.04, 1.08) per 10 dB increase in air traffic Lden (3). With respect to the air traffic noise, the odds ratio (OR) for the exposed population to develop hypertension is 1.63 (95 % CI: 1.14, 2.33) (4).

Burden-of-disease analyses have shown that traffic-noiseattributed myocardial infarction is associated with 61 000 disability-adjusted life-years in Western Europe (5).

Stroke can be defined as central nervous system infarction or cell death attributed to ischaemia or intracerebral/subarachnoid haemorrhage (6). It is a leading mortality cause worldwide associated with considerable social and economic costs (7, 8). The number of stroke victims has increased in the past twenty years, with most of the burden in middle-to-low income countries and an alarming rise in stroke morbidity and mortality in young

people and even children (9). However, little is known about the effects of traffic noise on the risk of stroke (2). This would be particularly important, given that some studies point towards an increased risk (10, 11), whereas others show little or no effect (12, 13). Quantitative risk data are necessary not only to delineate the effect of noise on this specific cardiovascular outcome but also to assess the burden of disease associated with it. As far as we are aware, there has been only one ad-hoc attempt to quantitatively synthetize the results from primary studies (14), and it estimated a pooled relative risk (RR) per 10 dB of 1.04 (95 % CI: 1.00, 1.09) (14). However, the validity of this finding is uncertain.

Therefore, in this study we aimed to systematically review the epidemiological data and to run a meta-analysis on the risk of stroke associated with exposure to noise from road and air traffic.

Correspondence to: Angel M. Dzhambov, Department of Hygiene and Ecomedicine, Faculty of Public Health, Medical University of Plovdiv, No. 15-A Vassil Aprilov Boulevard, 4002 Plovdiv, Bulgaria e-mail: angelleloti@gmail.com

Search strategy

MATERIALS AND METHODS Systematic review

Our systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and

Dzhambov AM, Dimitrova DD. Exposure-response relationship between traffic noise and stroke: a systematic review with meta-analysis Arh Hig Rada Toksikol 2016;67:136-151

Meta-Analyses (PRISMA) guidelines (15) and the Metaanalysis of Observational Studies in Epidemiology (MOOSE) statement (16). The research question was defined as the exposure-response relationship between traffic noise exposure (road and air traffic) and the risk of stroke. Railway noise was not considered for this review.

We developed an a priori review protocol and data extraction forms.

Two independent electronic searches were carried out in MEDLINE (PubMed), EMBASE (ScienceDirect), and the Internet (Google Scholar) on 24 November 2015. The search string included the following free-term keywords in different combinations: stroke, cerebrovascular, road traffic noise, aircraft noise. Language restrictions were English, Spanish, and Russian. Articles were screened on three levels: titles, abstracts, and full-texts.

Duplicate publications were excluded. Only peerreviewed epidemiological studies exploring the risk of stroke/cerebrovascular disease in adults associated with objectively measured traffic noise exposure were eligible.

We excluded experimental studies, studies using selfreported exposure, and reviews. Authors and experts in the field were contacted in order to identify additional records.

Hand-searching of the reference lists of included articles complemented the search.

Data extraction Different estimates of the relative risk (e.g. hazard ratio, odds ratio, incidence rate ratio, mortality ratio) were considered for the meta-analysis, assuming that they were empirically similar enough to be pooled together (17, 18).

The outcome of interest was stroke or cerebrovascular disease and was assessed either objectively (ICD codes) or it was self-reported in a questionnaire. If possible, we extracted morbidity estimates but, for studies reporting only mortality estimates, those were taken as a conservative proxy for risk of morbidity. Studies analysing a summary health outcome (stroke + other cardiovascular disease) were not included in the meta-analysis. Studies using combined traffic noise exposure indicator were also excluded from it.

Risks were generally extracted for the fully adjusted models.

In some cases educated choice was done; for example from the Evrard et al. study (19) we took the risk adjusted for NO2 instead of PM2.5 (to ensure comparability with other studies). From cohort studies we extracted data for the longest follow-up. When results had been stratified by age, such as in Sørensen et al. (20), we included both age groups in the meta-analysis, if no summary result for all age groups had been reported. The selected noise indicator was Lden, but when it was not available we extracted data associated with daytime noise exposure to achieve uniformity across studies. The only extracted estimate associated with nighttime noise exposure (Lnight) was that from Hoffmann et al.

(21), who did not report results for daytime noise, but, given the high correlation between daytime and night-time traffic

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flow and noise, this is not a problem in linear trend estimation (12).

Quality assessment The methodological quality of each study included in the qualitative synthesis was rated according to the a priori criteria listed in Appendix 1. They were developed based on a previously used protocol (22) and expert discussions.

Meta-analysis Studies selected for quantitative synthesis were allocated into two groups: road and air traffic exposure. In the road traffic group two types of risks were used and meta-analysed: linear trend of risk per 10 dB increase in noise exposure and categorical risk in the range 50-75 dB (reference <55 dB). In the air traffic group we pooled only the trend of risk per 10 dB. For the trend meta-analysis we either used already reported estimates per 10 dB or transformed the categorical risks. When the number of stroke cases and the size of the total population in each exposure group were reported in addition to the risk estimates (10, 23), we used the generalised least squares (STATA “glst”) (24), as previously done by Babisch (1) and Vienneau et al. (3). When only the risk estimates for each category were available, we instead used the variance-weighted least squares command (STATA “vwls”) (24), employed by Vienneau et al. (3). To estimate the trend per 10 dB for Hoffmann et al. (21), we used the exponential approximation proposed by van Kempen et al.

(25), assuming that the difference in compared exposure categories was 26 dB. Command lines for “glst” and “vwls” are shown below: Trend estimation without correction for covariance of risk estimates (“vwls”) gen double logRR=log(RR) gen double loglci=log(lci) gen double loguci=log(uci) gen double logse=((loguci - loglci)/(2*invnorm(.975))) vwls logRR Noise, noconstant sd(logse) lincom Noise*10, eform where “log” is natural logarithm; “RR” is the risk estimate in exposure category; “lci” is the lower limit of the confidence interval of the risk estimate; “uci” is the upper limit of the confidence interval of the risk estimate; “logse” is the standard error of the log-transformed risk estimate; and “Noise” is the midpoint of exposure category. Note that the reference exposure level was omitted from the trend estimation because it had a standard error of zero!

Trend estimation with correction for covariance of risk estimates (“glst”) gen double logRR=log(RR) gen double loglci=log(lci) gen double loguci=log(uci) gen double logse=((loguci - loglci)/(2*invnorm(.975))) glst logRR Noise, se (logse) cov (N Cases) ci

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lincom Noise*10, eform where “N” is the total number of cases in the exposure category and “Cases” is the number of stroke cases. The reference exposure level was set to 0 dB to comply with the algorithm!

For dichotomous comparisons, we used the following exponential approximation formula (25): RR per 10 dB=log(RR)*(10/ΔdB), logse=(log(uci)-log(lci)/3.92)*(10/ΔdB), 95 % CI for RR per 10 dB=exp(logRR±1.96*logse) where “ΔdB” is the difference in noise levels between the exposed and the reference group.

The range of open-ended categories was assumed to be the same as that of the adjacent category, and the exposure level in them was fixed at the midpoint when applying transformations (e.g., 55-60 dB and >60 dB → 55-60 dB and 62.5 dB). Noise indicators were entered in the analyses after conversion to Lden using the following approximations: LAeq,16h+2 dB, Ldn+0.3 dB (1, 3). However, when the effect was already reported per 10 dB, noise indicators were not transformed to Lden because it would not affect the risk estimate.

Even so, transforming categorical to linear trend estimates loses some information. This is why we also conducted a categorical meta-analysis using only high quality studies (with fewer sources of bias) reporting categorical risks. Categories for the pooled results were delineated as follows: <55 dB (reference), 55-60 dB, 6065 dB, 65-70 dB, and 70-75 dB. After converting the exposure levels from each study to the Lden metric, we pooled together the estimates for each of the five exposure categories. Finally, we plotted the pooled risks from each category against its midpoint (e.g. 55-60 dB → 57.5 dB) and compared linear, quadratic, and cubic polynomials through the data points to select the best fitting function.

For the reference category (<55 dB), a value of 52.5 dB was assigned.

For the meta-analysis we preferred the quality-effects model (26, 27) over the random-effects model, as the latter underestimates the error of the effects and may produce spuriously significant results (28). The quality-effects model uses an artificial quality index (Qi) to correct for studyspecific information about its methodological rigor and thus give more weight to studies with higher quality (fewer sources of bias). We used the quality score for each study as input to generate the Qi. For comparison, we also report results from the random-effect model. Doi plots were used to check for possible publication bias, as they are more sensitive than the funnel plots (29). A symmetrical mountain-like plot with Luis Furuya-Kanamori (LFK) index <|1| indicates no asymmetry; LFK index between |1| and |2| suggests minor asymmetry; and LFK index >|2| suggests major asymmetry (29). Statistical heterogeneity can be suspected when Cochran’s Q is significant at p<0.1, I2 is >30 %, and/or tau-squared is >1.

For the meta-analyses we used MetaXL v. 3.1 (EpiGear International Pty Ltd, Sunrise Beach, Queensland, Australia).

RESULTS Literature search results We identified 18 records in PubMed, 182 in ScienceDirect, 3,040 in Google Scholar, and three from the reference lists of the reviews we consulted. The study of Babisch et al. (23) was identified through Google Scholar and missed by the other search engines. Having removed duplicates and applied stroke and traffic noise exposure filters, we screened the titles of the remaining 3,062 records.

Twenty-three of the 43 abstracts were excluded. Twenty full-texts were read and seven excluded because they were either irrelevant to the research question or contained no useful data regarding the relationship between traffic noise and stroke. No additional articles were retrieved after handsearching the reference lists of already included articles.

Figure 1 shows the flow diagram of the searches.

Road traffic noise Qualitative synthesis All the seven studies included in this group (See Table 1) were conducted in European countries with a high socioeconomic standard. The study of Floud et al. (30) was, in fact, an international survey across six countries. Except for Babisch et al. (23) and Beelen et al. (12), the collected data span after the year 2000 [Sørensen et al. (31) spanned 1993-2009 and Kluizenaar et al. (32)-1991-2004]. Four studies were cohort (12, 21, 31, 32), two were crosssectional (23, 30), and one was ecological (using aggregate data) (10). The average follow-up in the cohort studies was around 10 years.

Sample sizes ranged from 2,512 (23) to 8.61 million (10) people and from 35 (23) to 62,513 stroke cases (10).

All samples included both men and women from the general population, except for Babisch et al. (23), which included only men. The participants were middle aged in Babisch et al. (23), Beelen et al. (12), Hoffmann et al. (21) and Sørensen et al. (31). Halonen et al. (10) reported separate estimates for those aged ≥25 years and those aged ≥75 years.

The definition of the outcome varied across studies.

Three studies looked at the ICD-code-defined stroke (10, 21, 31), while Beelen et al. (12) used ICD codes for the more general cerebrovascular disease. (See Table 1) Babisch et al. (23) and Floud et al. (30) relied on self-reported stroke.

Sørensen et al. (31) studied different subtypes of stroke and reported the main results for ischemic stroke. Kluizenaar et al. (32) used combined outcome (ischemic heart disease + cerebrovascular disease). Beelen et al. (12), in fact,

Total/cases=4 350/71; M (47.9 %) + F; 45-74 yrs.

Cohort; Heinz Nixdorf Recall study (2000-2009); Germany

Cross-sectional; Caerphilly study; UK

Hoffmann et al.

(21); Quality score=34

Babisch et al.

(23); Quality score=22

Total/cases= 2 512/35; M; 45-59 yrs.

Total/cases=51 569/1 999; M (47 %) + F; 56.2 yrs. (50.7-64.2)

Cohort; Danish Diet, Cancer and Health cohort (1993-2009)

Sørensen et al.

(31); Quality score=38

Stroke morbidityICD-10: I61, I63, I64; Hospital admissions and death certificates

Total=8.61 million; Morbidity: cases= 62 513; M + F; ≥25 yrs.

Total/cases=4 712/62; M (49.6 %) + F; 45-70 yrs.

Cross-sectional; Hypertension and Environmental Noise near Airports study (2004-2006); six countries

Ecological; London, UK (2003-2010)

Self-reported doctordiagnosis (morbidity)

Total/ cases=117 528/1 175; M (47.2 %) + F; 55-69 yrs.

Noise assessment

Self-reported stroke morbidity

Stroke morbidityICD-10: I61, I63, I64; Medical records

Ischemic stroke morbidity-ICD-10: I63; Medical records + expert validation

Cerebrovascular disease (mortality)-ICD-9: 430438, ICD-10: I60-I69; Death certificates

Measurements; LAeq,6-22h (LAeq,16h) at 10 m from the center of the road

Modeling (END); Lnight

Modelling (SoundPLAN); Lden

Modelling (TRANEX); LAeq,16h

Modelling (INM)-10 m grid; LAeq,24h

Modelling (EMPARA)-25 m grid; Indicator similar to Lden

ROAD TRAFFIC GROUP

Cohort; Netherlands Cohort Study on Diet and Cancer (19871996)

Outcome

Population

Design

Halonen et al.

(10)-adults; Quality score=33

Floud et al. (30)road traffic; Quality score=32

Beelen et al.

(12); Quality score=33

Study

Table 1 Characteristics of studies included in the systematic review

n/a

Average of 7.9 yrs. (± 1.5)

10 yrs.

n/a

n/a

9 yrs.

Follow-up

RR calculated from raw data

Cox regression (HR)

Cox regression (IRR)

Poisson regression (RR)

Logistic regression (OR)

Cox regression (HR)

Analysis

None

A, G, recruitment year, MS, E, Em, S, SES, PA, BMI, Al

A, G, E, SES, S, D, Al, PA, BMI, calendar-year, NO2

A, G, SES, Eth, S, PM2.5

A, G, BMI, E, Eth, aircraft noise

A, G, S, SES, TI, BS

Adjustments

51-55, 56-60, 61-65, 66-70

Trend per 10 dB

Trend per 10 dB

<55, 55-60, >60

Trend per 10 dB

<50, 50-55, 55-60, 60-65, >65

Noise categories (dB)

Dzhambov AM, Dimitrova DD. Exposure-response relationship between traffic noise and stroke: a systematic review with meta-analysis Arh Hig Rada Toksikol 2016;67:136-151

139

Cohort; GLOBE study; Netherlands (1991-2004)

Cohort; Danish Diet, Cancer and Health cohort (1993-2006)

Cohort; Danish Diet, Cancer and Health cohort (1993-2006)

Ecological; France (2007-2010)

Cross-sectional; Hypertension and Environmental Noise near Airports study (2004-2006); six countries

**Sørensen et al. (20)-<64.5 yrs.; Quality score=36

**Sørensen et al. (20)->64.5 yrs.; Quality score=36

Evrard et al.

(19); Quality score=37

Floud et al.

(30)-aircraft; Quality score=32

Design

*Kluizenaar et al. (32); Quality score=35

Study

Total/cases=4 712/62; M (49.6 %) + F; 45-70 yrs.

Total=1.9 million; M+F

Total/cases=51 485/929; M + F; ≥64.5 yrs.

Total/cases=51 485/952; M + F; <64.5 yrs.

Total/ cases=18 213/1 547; M (47 % and 63.9 %) + F; 46.4 yrs. (± 15.9) and 59.3 yrs. (± 9.1)

Population

Modelling (SoundPLAN); Lden

Modelling (SoundPLAN); Lden

Modelling (SKM2); Lden

Noise assessment

Self-reported doctordiagnosis (morbidity)

Stroke mortalityICD-10: I60-I64, excluding I63.6; Mortality register

Modelling (INM)-10 m grid; LAeq,16h

Modelling (INM); Lden

AIR TRAFFIC GROUP

Ischemic heart disease (ICD-9: 410-414) + cerebrovascular disease (ICD-9: 430-438) morbidity; Hospital admission registry First hospitalization for stroke (morbidity)ICD-8: 431.0, 431.9, 432.0, 432.9, 433.09, 433.99, 434.09, 434.99, 436.0, 436.9 and ICD10: I61, I63, I64; Hospital discharge register First hospitalization for stroke (morbidity)ICD-8: 431.0, 431.9, 432.0, 432.9, 433.09, 433.99, 434.09, 434.99, 436.0, 436.9 and ICD10: I61, I63, I64; Hospital discharge register

Outcome

n/a

n/a

Average of 10.1 yrs. among cohort members and 6 yrs. among cases Average of 10.1 yrs. among cohort members and 6 yrs. among cases

13 yrs.

Follow-up

Logistic regression (OR)

Poisson regression (MRR)

Cox regression (IRR)

Cox regression (IRR)

Cox regression (RR)

Analysis

A, G, BMI, E, Eth, road traffic noise

A, G, SES, lung cancer mortality, NO2

A, G, calendaryear, S, D, BMI, PA, Al, E, SES, AN, RN, NOx

A, G, calendaryear, S, D, BMI, PA, Al, E, SES, AN, RN, NOx

A, G, BMI, S, E, PA, MS, Al, SES, employment, PM10

Adjustments

Trend per 10 dB

Trend per 10 dB

Digitized from graph: <55, 55-58, 58-61, 61-64, 64-67, 67-70, 70-73 Digitized from graph: <55, 55-58, 58-61, 61-64, 64-67, 67-70, 70-73

Trend per 10 dB

Noise categories (dB)

140 Dzhambov AM, Dimitrova DD. Exposure-response relationship between traffic noise and stroke: a systematic review with meta-analysis Arh Hig Rada Toksikol 2016;67:136-151

Cohort; Canada (19942002)

*Gan et al.

(34);Quality score = 39

Modelling (INM); Ldn

Stroke mortality-ICD-9: 430-434, 436-438 and ICD-10: I60-I69 excluding I63.6; Death registration database

Modelling (CadnaA)-6-digit postal code area; Lden; Combined road + air & railway traffic noise

4 yrs.

n/a

5 yrs.

n/a

Follow-up

Cox regression (HR)

Poisson regression (RR)

Cox regression (HR)

Poisson regression (RR)

Analysis

A, G, SES, PM2.5, NO2, black carbon, diabetes, COPD

Trend per 10 dB

Trend per 10 dB (digitized from graph)

A, G, MS, Eth, nationality, urban/rural, language region, type of building, SES (nonmovers for >15 yrs.)

A, G, race, SES, Eth, PM2.5, ozone

<45, 45-49, 50-54, 55-59, ≥60

A, G, Eth, SES, lung cancer

Noise categories (dB) ≤ 51, 51-54, 54-57, 57-60, 60-63, >63

Adjustments

M-male, F-female, A-age, G-gender, S-smoking, SES-socio-economic status, BMI-body mass index, TI-traffic intensity, BS-black smoke, E-education, Eth-ethnicity, AN-aircraft noise, PM2.5-fine particulate matter, NO2-nitrogen dioxide, D-diet, PA-physical activity, MS-marital status, Em-employment, Al-alcohol, RN-railway noise, NOx-nitrogen oxide, COPD-chronic obstructive pulmonary disease; *not included in meta-analysis, **included only in categorical meta-analysis

Total/cases = 445 868/1 288; M (45-46%) + F; 45-85 yrs.

Ecological; US (2009) COMBINED TRAFFIC GROUP

Cerebrovascular disease morbidity-ICD-9: 430438; Hospitalization records

Total/cases=6 million/1343.3 (1 092.5-1 652.2); M + F; ≥65 yrs.

Correia et al.

(33); Quality score=31

Modelling-100 m grid; Ldn

Primary or concomitant stroke mortalityICD-10: I60-I64 excluding I63.6; Death certificate

Total/cases=4.6 million/12 102; M + F; ≥30 yrs.

Cohort; Swiss National Cohort (20002005)

Modelling (ANCON)-10 m grid; LAeq,16h

Main reason for hospitalization is stroke (morbidity)-ICD-10: I61, I63-I64; National registry

Huss et al.

(13); Quality score=33

Noise assessment

Outcome

Total/cases=3.6 million/16 983; M+F

Population

Ecological; UK (2001-2005)

Design

Hansell et al.

(11); Quality score=33

Study

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141

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estimated the risk of cerebrovascular disease mortality, whereas the others reported morbidity estimates. Most studies employed noise modelling approaches, while Babisch et al. (23) carried out field measurements. Selected noise indicators differed: three studies used Lden (12, 31, 32), one used LAeq,24h (30), two used LAeq,16h (10, 23), and one used Lnight (21).

All four cohort studies fitted the Cox proportional hazards model to study the risk of stroke (12, 21, 31, 32).

Halonen et al. (10) used Poisson regression, and Floud et al. (30) logistic regression. Adjustment sets included the core demographics (age, gender) and the rest controlled for dietary habits or body mass index, except for two studies (10, 12). Beelen et al. (12), Sørensen et al. (31), Kluizenaar et al. (32), and Halonen et al. (10) adjusted for indicators of air pollution, while Floud et al. (30) adjusted for air traffic noise. Babisch et al. (23) reported unadjusted data. Overall, Beelen et al. (12), Kluizenaar et al. (32), and Hoffmann et al. (21) did not find elevated risk associated with noise exposure in the adjusted models. In Kluizenaar et al. (32) the risk per 10 dB was elevated (RR=1.09, 95 % CI: 0.90, 1.32) only in the subgroup with a history of cardiovascular disease, whereas the relative risk in the whole sample was 1.00 (95 % CI: 0.91, 1.10). Others did find the risk above 1.00, but it was statistically significant only in Sørensen et al. (31) and Halonen et al. (10). There were no risks for haemorrhagic strokes in the study of Sørensen et al. (31), and the risk was higher for those exposed for one year (IRR=1.19, 95 % CI: 1.09, 1.31). In Halonen et al. (10) the elderly (≥75 years) had a slightly higher risk than the whole

Figure 1 Study selection flow-chart

sample of adults (≥25 years). Night-time noise exposure >60 dB was associated with a lower risk than daytime exposure: RR=1.01 (95 % CI: 0.98, 1.05) in those aged ≥25 years and RR=1.02 (95 % CI: 0.97, 1.08) in those aged ≥75 years. The risk estimates in Beelen et al. (12) and Halonen et al. (10) were categorical, in Hoffmann et al. (21) they were reported per one interquartile range increase in Lnight, and Babisch et al. (23) reported only the prevalence of stroke across noise categories, from which we derived a linear trend per 10 dB. The rest reported risks per 10 dB. Overall, the studies were of high quality, with the score of Sørensen et al. (31) being the highest.

We included Hoffmann et al. (21), Halonen et al. (10), Sørensen et al. (31), Floud et al. (30), Beelen et al. (12), and Babisch et al. (23) in the linear trend meta-analysis.

The categorical meta-analysis included Beelen et al. (12), Halonen et al. (10), and two coefficients from another study

by Sørensen et al. (20) based on the same cohort, which reported results from categorical analysis stratified by the age threshold of 64.5 years. Those two subgroups were included separately in the meta-analysis. Kluizenaar et al.

(32) was included in neither of the two quantitative syntheses because the outcome included ischemic heart disease.

Meta-analysis The meta-analysis was based on the most comparable effect sizes. Appendix 2 reports the input data for linear trend estimation. Figure 2 presents a forest plot of the risk

Dzhambov AM, Dimitrova DD. Exposure-response relationship between traffic noise and stroke: a systematic review with meta-analysis Arh Hig Rada Toksikol 2016;67:136-151

of stroke morbidity associated with 10 dB increase in road traffic noise exposure. The pooled risk was 1 % (95 % CI: -4 %, 6 %). The coefficient from Halonen et al. (10) was associated with the highest weight (84.5 %). There was high statistical heterogeneity (tau-squared=0.0008, significant Cochran’s Q at p=0.00, I2=79 %). The Doi plot in Figure 3 was asymmetrical with LFK index >|2|, suggesting gross publication bias. For comparison, under the random effects model the pooled risk was similar (RR=1.01, 95 % CI: 0.98, 1.05), but the weight of Halonen et al. (10) dropped to 41.4 % and that of Beelen et al. (12) increased from 6.6 % to 37.6 %.

Results from sensitivity analysis, shown in Table 2, reveal that, upon exclusion of most studies one-at-a-time, the pooled effect remained unchanged but the study of Halonen et al. (10) was driving the effect, which dropped <1.00 when it was excluded. If only cohort studies were pooled (three studies), the relative risk would be 0.99 (95 % CI: 0.81, 1.20). If the analysis was limited to studies originally reporting linear trend estimates per 10 dB (three studies), the relative risk would be 1.15 (95 % CI: 1.05, 1.25).

Figure 4 shows individual study risks included in the categorical meta-analysis (see Appendix 3 for risk estimates).

The pooled categorical risk is given in Figure 5.

Although it failed in statistical significance, it was elevated in the categories 55-60 dB (RR=1.04, 95 % CI: 0.87, 1.24), 60-65 dB (RR=1.05, 95 % CI: 0.75, 1.46), 65-70 dB (RR=1.21, 95 % CI: 0.81, 1.81) and 70-75 dB (RR=1.29, 95 % CI: 0.74, 2.24). The linear function explained only 89 % of the variance in the risk of stroke. The cubic polynomial did not provide substantial improvement (R2=96 %) to the quadratic function. Therefore, the best fitting and most parsimonious approximation of the risk as a function of Lden in the range <55-75 dB was the following unweighted quadratic polynomial: Risk=0.00066295186571*(Lden) 2-0.06779154098343* (Lden)+2.73847143171073, R2=0.95 Table 2 Sensitivity of road traffic noise estimates (linear trend meta-analysis) Pooled Risk Excluded study estimate I2 % (95 % CI) Hoffmann et al. (21) 1.01 (0.96, 1.07) 83.07 Halonen et al. (10)

0.99 (0.85, 1.15)

74.66

Sørensen et al. (31)

1.01 (0.97, 1.05)

72.56

Floud et al.-road traffic (30)

1.01 (0.96, 1.06)

83.03

Beelen et al. (12)

1.01 (0.91, 1.13)

60.85

Babisch et al. (23)

1.01 (0.96, 1.06)

82.23

143

Because the reference category <55 dB represented a wide range of exposure levels below 55 dB, we tested the function in several scenarios, changing the reference exposure level from 47.5 to 50, and finally to 52.5 dB, and found only a marginal impact on the slope or the coefficient of determination.

Air traffic noise Qualitative synthesis Five studies were included in this group. Three of those were ecological (11, 19, 33), one cross-sectional (30), and one cohort (13). The only non-European study was that of Correia et al. (33), as it was carried out in the United States.

Sample sizes ranged from 4,712 (30) to 6 million (33) people. All samples included men and women, and that of Correia et al. (33) was limited to the elderly (≥65 years).

Only Correia et al. (33) defined the outcome as cerebrovascular events, while the rest specified it as types of stroke. Only Floud et al. (30) used self-reported doctor diagnosis. Two of the studies examined the risk of stroke mortality (13, 19). All studies assessed noise exposure objectively through validated models. Selected indicators were Lden (19), Ldn (13, 33), and LAeq,16h (11, 30). Ecological studies analysed their data with Poisson regression; Huss et al. (13) used Cox proportional hazards model; and Floud et al. (30) used logistic regression. Adjustments were made for key demographics, but some important individual-level factors could not be measured in the ecological studies. All studies were of high quality, with Evrard et al. (19) scoring the top 37 of 42 points. All found some increase in the risk of stroke with increasing exposure, ranging from 1.001 (95% CI: 0.99, 1.01) (13) to 1.08 (95% CI: 0.82, 1.41) (19).

Evrard et al. reported elevated risk in men (MRR=1.10, 95 % CI: 0.90, 1.33) but not in women (MRR=1.00, 95 % CI: 0.85, 1.19) (19). Some authors found lower risk of night-time in comparison to daytime noise (10), whereas others reported higher relative risk (1.29; 95 % CI: 1.14, 1.46) for Lnight >55 dB (11) and higher odds ratio per 10 dB Lnight (1.18; 95 % CI: 0.89, 1.57) (30). All studies were included in quantitative synthesis.

Meta-analysis Figure 6 shows the results of the linear trend metaanalysis. The pooled effect was 1 % (0.2 %, 2 %) when the quality effects model was used. There was no heterogeneity (tau-squared=0.00, non-significant Cochran’s Q at p=0.76, I2=0.00 %), but there was evidence of major asymmetry in the Doi plot (LFK >|2|) (Figure 7). When the random effects model was used, the effect remained virtually the same (RR=1.01; 95 % CI: 1.00, 1.01).

In sensitivity analysis the pooled risk remained unchanged upon exclusion of each study one-at-a-time (Table 3). If only studies reporting stroke morbidity were analysed, the risk would remain unchanged (RR=1.01; 95 %

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CI: 1.00, 1.01). Restriction to studies originally reporting linear trend estimates would raise the relative risk per 10 dB to 1.03 (95 % CI: 0.97, 1.09).

not differentiate between road and air traffic noise, we did not include in our meta-analysis.

DISCUSSION

Combined traffic noise

Main findings

Table 3 Sensitivity of air traffic noise estimates (linear trend meta-analysis) Excluded study

Pooled Risk estimate (95 % CI)

I2 %

Evrard et al. (19)

1.01 (1.00, 1.01)

0.00

Hansell et al. (11)

1.01 (0.99, 1.02)

0.00

Floud et al.-air traffic (30)

1.01 (1.00, 1.02)

0.00

Correia et al. (33)

1.01 (1.00, 1.02)

0.00

Huss et al. (13)

1.01 (1.00, 1.02)

0.00

Qualitative synthesis Only one study was included in this group (34). Gan et al. used linked administrative health insurance databases to assemble this population-based cohort from Vancouver in Canada. People aged 45-85 years were enrolled and followed for four years. Data about stroke were obtained from death registry and cases were defined by ICD codes.

The authors used CadnaA software to model Lden for combined traffic noise (road, air, and railway) (34).

The risk of stroke mortality per 10 dB increase in that study was 1.03 (95% CI: 0.91, 1.16). For ischaemic and haemorrhagic stroke, the risk was 1.02 (95% CI: 0.78, 1.33) and 1.14 (95% CI: 0.91, 1.44), respectively. The analysis relied on Cox regression, adjusted only for some important covariates, since the authors lacked individual-level questionnaire data (34). This study received high-quality score (39 of 42 points), but since the exposure indicator did

With respect to road traffic noise, the risk of stroke increased 1 % (95 % CI: -4 %, 6 %) with every 10 dB increase in Lden. This finding was driven by the study of Halonen et al. (10), because it included all London area residents. In categorical analysis we found non-linearity in the effect, and the pooled risk reached 29 % (-26 %, 24 %) in the category 70-75 dB. Overall, road traffic noise studies were methodologically heterogeneous, which may be expected to lead to differences in the observed effects.

As far as the air traffic noise is concerned, the pooled risk per 10 dB was also 1 % (95 % CI: 0.2 %, 2 %). However, these studies were more consistent in their effects and methodologies.

Even so, both groups showed gross asymmetry in the Doi plots as evidence of publication bias, which may be skewing the pooled results, if primary studies are being published selectively, depending on the effect they find.

For the only study using a combined traffic noise indicator (34), the overall effect was similar to those for road and air traffic noise. Previously Vienneau et al. (3) argued that ignoring the type of noise source might not be much of an issue because the pooled risks of ischemic heart disease in relation to road and air traffic noise were close.

While we also found no difference in the pooled point estimates associated with either type of traffic noise, the precision of the estimates in our meta-analysis was higher for air traffic and there was no heterogeneity in that group.

Therefore, we do not recommend combining studies investigating the effects of different noise sources, as the

Figure 2 Forest plot of studies included in the meta-analysis of the linear trend of stroke risk per 10 dB increase in road traffic noise exposure

Dzhambov AM, Dimitrova DD. Exposure-response relationship between traffic noise and stroke: a systematic review with meta-analysis Arh Hig Rada Toksikol 2016;67:136-151

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Figure 3 Doi plot with Luis Furuya-Kanamori index for the detection of publication bias in the road traffic group (linear trend metaanalysis)

Figure 4 Individual risk estimates for stroke reported in studies included in the categorical meta-analysis

Figure 5 Pooled categorical risk of stroke associated with road traffic noise exposure The solid line represents the pooled linear trend of the risk per 10 dB (based on 6 estimates). The dashed line represents a quadratic polynomial approximation of the categorical risk: 50-55 dB (reference); 55-60 dB is based on 4 estimates; 60-65 dB is based on 4 estimates; 65-70 dB is based on 3 estimates; 70-75 dB is based on 2 estimates

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issues of heterogeneity and precision should not be taken lightly. Based on our findings, we contend that the risk of 4 % (96 % CI: 0 %, 9 %) per 10 dB reported by Houthuijs et al. (14) might be an overestimation.

Limitations The first limitation of our systematic review and metaanalysis is that it should have included more studies to have sufficient power for subgroup analysis and formal publication bias tests. However, our meta-analysis included a greater number of studies than are usually included in this type of meta-analysis (35)and it also included a higher number of participants. After all, the meta-analysis that has served as the basis for ischemic heart disease burden of disease estimation in Europe also included but a few studies (36). As for the environmental noise research, there are not enough studies to begin with, as it has only recently turned its focus to stroke as an endpoint.

Another issue might be pooling together different point estimates. This choice can be justified due to the empirical similarity of those estimates, especially given the small effect sizes and that there was only one study reporting odds ratio (which is the least conservative estimate), while the others employed Cox proportional hazards or Poisson

regression models (17, 18). Therefore those should be reasonable measures of the relative risk.

Taking the risk of stroke mortality as a proxy for the risk of morbidity makes the pooled effect conservative, as it ignores non-fatal cases. On the other hand, we included only one (out of six) mortality estimate in the road traffic group and two (out of five) in the air traffic group, and their exclusion did not affect the pooled risk. The same can be deduced from some studies reporting both morbidity and mortality risks (11); others, however, showed lower risk for noise-attributed stroke mortality than for noise-attributed stroke morbidity (10). The same bias towards the null applies to the cerebrovascular disease when it is used as a proxy for stroke. Therefore the categorical risk, based on Beelen et al. (12), who reported estimates for cerebrovascular disease mortality, is probably underestimated and conservative.

In line with Vienneau et al. (3), we found that studies originally reporting linear trend per 10 dB were associated with higher risk than those for which such a trend was derived by us. Estimating a linear trend per 10 dB from reported categorical risks is associated with information loss. While the generalised least squares approach was specifically designed for this purpose, the assumption of

Figure 6 Forest plot of studies included in meta-analysis of the linear trend of stroke risk per 10 dB increase in air traffic noise exposure

Figure 7 Doi plot with Luis Furuya-Kanamori index for the detection of publication bias in the air traffic noise group (linear trend meta-analysis)

Dzhambov AM, Dimitrova DD. Exposure-response relationship between traffic noise and stroke: a systematic review with meta-analysis Arh Hig Rada Toksikol 2016;67:136-151

the variance-weighted least squares that the log relative risk estimates are independent is never confirmed in practice (24). Even so, when the covariance matrix cannot be specified, the latter is an alternative (3). For studies reporting sufficient information (10, 23) we used generalised least squares but we also estimated the linear trend via variance-weighted least squares and found very similar results.

The categorical approach requires for the included studies to be similar in terms of exposure assessment, noise indicators, and reference levels and to be generally of high quality, because of which few studies are usually fit for this approach (37). This is why we should interpret it with caution. Furthermore, including the two subgroups of Sørensen et al. (20) in the categorical meta-analysis might violate the assumption of statistical independence and, although the subsamples do not share subjects, the fact that the investigators of these effects were the same implies some bias (38). This choice was made only because Sørensen et al. (20) did not report combined categorical risk for the whole sample.

Finally, the high heterogeneity in the road traffic group and the considerable bias associated with some estimates (23) were addressed by using the quality effects model (28, 39) and by excluding Babisch et al. (23) from the categorical meta-analysis.

Future research The results of this meta-analysis can be used for preliminary calculation of disease burden. However, in order to grow and strengthen the evidence of the exposureresponse relationship, additional studies with compatible methodologies need to be reported. Special emphasis should be put on the effect with regards to different types of stroke and comparable ICD definitions of the outcome.

Although ecological studies do provide the opportunity to analyse large samples, they lack control for some important individual and behavioural factors that might be confounding or moderating the effects of noise. Gender, age, and ethnic group stratification is also necessary in order to identify vulnerable subpopulations.

Furthermore, there is no evidence from middle- and low-income countries where both stroke incidence and traffic noise exposure are considerably higher than in Western Europe.

The effect of night-time noise has not been addressed sufficiently either. Studies should report both trend and categorical risk estimates to facilitate data meta-synthesis.

Finally, the issue of the publication bias should be addressed more carefully.

CONCLUSIONS In conclusion, we found a small but elevated risk of stroke associated with both road and air traffic noise

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exposure, but it was statistically significant only for the latter. The effect of road traffic noise followed a non-linear trend. As soon as the results from other high-quality studies are published this exposure-response relationship should be updated and adjusted.

Conflict of interest statement This study was conducted without any relationships that could be construed as potential conflict of interest. We were not financially supported in any way.

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Appendix 1 Quality scoring checklist Design a) b) c) Sample a) b) c) d) Outcome a)

Cohort: yes (3) / no (0) Cross-sectional/case-control: yes (2) / no (0) Ecological: yes (1) / no (0) Representative: yes (3) / no or no information (1) Random: yes (3) / no or no information (1) Sufficient sample size: yes (3) / no or no information (1) Response rate: ≥80 % (3) / 60-79 % (2) / <60 % or no information (1)

Objective assessment (medical records, death certificates, ICD-classification, etc.): yes (3) / self-reported or no information (1) b) Definition: stroke (ischemic/haemorrhagic) (3) / cerebrovascular disease (1) Noise exposure a) Objective assessment (modelling, measurements): yes (3) / no information on the model (1) b) Indicator: Lden (3) / other or no information (1) Analysis a) Adequate: yes (3) / no or no information (1) b) Adjustments: demographics (age, gender, socio-economic status/education/ethnicity) + diet/physical activity/body mass index + smoking + genetics/family history + hypertension/diabetes mellitus/hearth rhythm disorders/kidney disease + blood lipids + other environmental exposures (6) / most of those including age, gender, diet/physical activity/body mass index and smoking (4) / some of those including age and gender (3) / not including age and gender (1) Extracted effect size a) Transformations necessary: no (3) / yes (1) b) Extraction: straightforward (3) / no (1) Maximum=42 points

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Appendix 2 Input data for the estimation of linear trend of stroke risk per 10 dB increase in road traffic and air traffic noise exposure Mid-category noise levels Risk estimates Study (method) (original metric/Lden/for (95 % CI) for trend Risk per 10 dB trend estimation) estimation Beelen et al. (12) ("vwls") 0.97 (0.96, 0.99) 47.5/47.5/omitted 1.00 52.5/52.5/52.5 0.90 (0.78, 1.04) 57.5/57.5/57.5 0.89 (0.76, 1.05) 62.5/62.5/62.5 0.61 (0.44, 0.84) 67.5/67.5/67.5 0.95 (0.55, 1.66) Halonen et al. (10) ("glst")* 1.01 (1.00, 1.01) 47.5/49.5/0 1.00 52.5/54.5/54.5 1.04 (1.02, 1.07) 62.5/64.5/64.5 1.05 (1.02, 1.09) Hoffmann et al. (21) (exponential fit) Reported per 26 dB 0.91 (0.40, 2.06) 0.96 (0.70, 1.32) Floud et al.-road traffic (30) Reported per 10 dB 1.07 (0.75, 1.52) Floud et al.-aircraft (30) Reported per 10 dB 1.08 (0.82, 1.41) Sørensen et al. (31) Reported per 10 dB 1.17 (1.06, 1.29) Babisch et al. (23) ("glst")* 1.07 (0.96, 1.19) 53/55/0 1.00 58/60/60 1.20 (0.36, 3.96) 63/65/65 1.06 (0.37, 3.05) 68/70/70 2.53 (0.88, 7.23) Hansell et al. (11) ("vwls") 1.01 (1.003, 1.02) 49.5/51.5/omitted 1.00 52.5/54.5/54.5 1.03 (0.98, 1.09) 55.5/57.5/57.5 1.04 (0.98, 1.12) 58.5/60.5/60.5 1.04 (0.95, 1.14) 61.5/63.5/63.5 1.10 (0.96, 1.25) 64.5/66.5/66.5 1.24 (1.08, 1.43) Evrard et al. (19) Reported per 10 dB 1.06 (0.93, 1.21) Huss et al. (13) ("vwls") 1.001 (0.99, 1.01) 42/42.3/omitted 1.00 47/47.3/47.3 1.03 (0.92, 1.14) 52/52.3/52.3 1.02 (0.90, 1.15) 57/57.3/57.3 0.96 (0.82, 1.13) 62/62.3/62.3 0.88 (0.58, 1.34) Correia et al. (33) Reported per 10 dB 1.02 (0.95, 1.09) *linear trend estimates generated with “vwls” are similar to the first decimal place; “vwls”-variance-weighted least squares; “glst”generalized least squares

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Appendix 3 Input data for the estimation of categorical risk of stroke associated with road traffic noise exposure Study Sørensen et al. <64.5 yrs. (20)

Sørensen et al. >64.5 yrs. (20)

Beelen et al. (12)

Halonen et al. (10)

Original noise category/trend category (Lden)

Risk estimates (95 % CI)

<55/reference 55-58/55-60 58-61/omitted 61-64/60-65 64-67/omitted 67-70/65-70 70-73/70-75 <55/reference 55-58/55-60 58-61/omitted 61-64/60-65 64-67/omitted 67-70/65-70 70-73/70-75 ≤50/reference 50-55/omitted 55-60/55-60 60-65/60-65 >65/65-70 <55/reference 55-60/55-60 >60/60-65

1.00 0.93 (0.77, 1.12) 0.85 (0.68, 1.05) 1.04 (0.84, 1.29) 1.10 (0.86, 1.41) 0.95 (0.70, 1.28) 0.94 (0.64, 1.39) 1.00 1.30 (1.07, 1.58) 1.12 (0.89, 1.41) 1.38 (1.12, 1.71) 1.53 (1.19, 1.95) 1.59 (1.19, 2.10) 1.66 (1.17, 2.36) 1.00 0.90 (0.78, 1.04) 0.89 (0.76, 1.05) 0.61 (0.44, 0.84) 0.95 (0.55, 1.66) 1.00 1.04 (1.02, 1.07) 1.05 (1.02, 1.09)

Odnos između izloženosti prometnoj buci i rizika od moždanog udara: sustavni pregled s metaanalizom Prometna je buka rizični čimbenik za nastanak bolesti krvožilja poput povišenoga krvnog tlaka i ishemijske bolesti srca, ali su saznanja vezana uz moždani udar još uvijek ograničena. Cilj je ovoga istraživanja bio napraviti sustavni pregled epidemioloških podataka i metaanalizu rizika od moždanog udara povezanoga s izloženošću buci cestovnog i zračnog prometa. Pretraživanje je provedeno 24. studenoga 2015., a obuhvatilo je članke na engleskom, španjolskom i ruskom jeziku koji su odgovarali kriterijima pretrage u bazama MEDLINE, EMBASE i Google Scholar. Kvalitativna sinteza obuhvatila je 13 istraživanja, od kojih je 11 obuhvaćeno metaanalizom kvalitativnih učinaka. U prosjeku su svi članci bili visokokvalitetni. Na temelju rezultata šest istraživanja (n ≈ 8.790.671 sudionik) vezanih uz buku cestovnog prometa, utvrdili smo da ukupni relativni rizik (RR) od moždanog udara prilikom porasta buke od 10 dB iznosi 1,01 (95 % CI: 0,96, 1,06). U rasponu buke od 70 do 75 dB (prema <55 dB) RR se povećao na 1,29 (95 % CI: 0,74; 2,24). Prema objedinjenim podacima o buci zračnoga prometa iz pet istraživanja (n ≈ 16.132.075 sudionika), RR za porast buke od 10 dB iznosio je 1,01 (95 % CI: 1,00; 1,02). Podaci iz istraživanja cestovne buke, za razliku od onih iz istraživanja zračne buke, bili su statistički izrazito heterogeni. Obje su skupine istraživanja iskazale odstupanje podataka zbog pretežitog objavljivanja određenog tipa istraživanja (tzv. publication bias). Ovim smo istraživanjem utvrdili donekle povišeni rizik od moždanog udara zbog izloženosti buci cestovnog i zračnog prometa, ali je ta povezanost bila statistički značajna samo kod potonjega. Učinci cestovne buke slijedili su nelinearni trend.

KLJUČNE RIJEČI: bolest krvožilja; cerebrovaskularna bolest; cestovni promet; prijevoz; zračni promet

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Knežević I, et al. Salience and conflict of work and family roles among employed men and women Arh Hig Rada Toksikol 2016;67:152-163

Original article

DOI: 10.1515/aiht-2016-67-2724

Salience and conflict of work and family roles among employed men and women Irena Knežević1, Ljiljana Gregov2, and Ana Šimunić2 Association of Hearing Impaired People1, Department of Psychology, University of Zadar2, Zadar, Croatia [Received in October 2015; CrossChecked in October 2015; Accepted in May 2016] The aim of this research was to determine the salience of work and family roles and to study the connection between role salience and the interference of different types of roles among working men and women. Self-assessment measurement scales were applied. The research involved 206 participants; 103 employed married couples from different regions of Croatia. The results show that roles closely connected to family are considered the most salient. However, men are mostly dedicated behaviourally to the role of a worker. Women dedicate more time and energy to the roles of a spouse, a parent, and a family member whereas men are more oriented towards the leisurite role. The highest level of conflict was perceived when it comes to work disturbing leisure. Gender differences appeared only for work-to-marriage conflict, with men reporting higher conflict than women. The research found proof of only some low correlations between the salience of different types of roles and work-family conflict.

KEY WORDS: role salience; work-family conflicts; gender; spouse; parent; colleague; leisure People perform many different roles throughout their lives, such as the role of a child, a student, a leisurite (engaging in free-time or leisure activities), a citizen, an employee, a partner, a houseworker, a parent, a pensioner (1). Roles can be defined as the sum of behaviours, rights, and duties that an individual should present in a given social situation (2). Although there may be several roles at once, they are not all equally important; there are usually two or three prominent roles (salient), while others are peripheral.

It has been shown that the importance of each life role depends on the perception of the possibilities of actualising salient values through these roles (3). Thus, the salience of a life role represents the motivational force for participating in the role. Salient roles form the core of a person; they are the basis of personal identity and are essential for life satisfaction (4).

Super (1) used a three-part model to explain role salience. According to the model, role salience reflects the knowledge, participation, and commitment we have for the role. Therefore, salience has three dimensions: cognitive, behavioural, and affective. It can be concluded that role salience is the highest when we have good knowledge of the role, when we participate in it, and when we feel that it is of personal importance to us.

When played simultaneously, roles interact and influence each other. Since work organisation and family are two central institutions in most men and women's lives (5-7), interactions of work and family life are commonly Correspondence to: Ana Šimunić, Department of Psychology, University of Zadar, e-mail: asimunic@unizd.hr

examined. They can support or complement each other but may also be in conflict and become a source of stress. When meeting the requirements of family roles becomes difficult or impossible due to the requirements of work roles, and vice versa, we speak of work-family conflict (8). This type of conflict has a negative impact on the quality of both family and work life (9, 10).

Work-family conflict in men and women A well-known and generally accepted model of workfamily conflict was proposed in 1992 by Frone et al. (11).

They assumed that there are two distinct forms of conflict between work and family roles: work-to-family conflict, i.e., the disruptive effect of work on the family role and family-to-work conflict, i.e., the disruptive effects of family on the work role. They have been identified as reciprocal constructs, which have independent antecedents and outcomes (11-13). Research results consistently show that work-to-family conflict is more common than family-towork conflict (14-16).

Most research on work-family conflict was conducted on groups of individuals, and only a smaller number on couples (6, 17). However, there are several reasons such research should be carried out on couples, and one of them is the assumption that work-family conflict will be higher in dual-earner couples, because of a larger number of interactions between the work and family domains. The experiences of one partner in his/her work and family domains will not only be influenced by their own work and family variables but also by the work and family variables of the other partner (18-21).

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Work-family conflict and role salience among men and women Work-family conflict is the highest when both work and family roles are highly salient to an individual (8, 22).

Although some researchers believe that women, compared to men, still consider their career less important (7, 22), other findings suggest that women with a salient work role often return to work after giving birth and prefer to work rather than stay at home (23). The importance of career also strongly differs between women with traditional gender ideology, and feminists (24, 25). In men, such a link between the listed variables was not manifested (26).

A greater relative importance of a domain does not necessarily mean a greater investment in it. The results of some previous studies (27, 22) have shown that people use some family time in order to deal with their job more often than they use the time devoted to work to spend it with their family. A very likely explanation for this could be the fact that the existence of an individual and his/her family often depends on the work role, regardless of its salience.

Therefore, the conclusion of many researchers that work affects family more than family affects work is not surprising, at least when it comes to the time invested in these two domains (11, 27). Rothbard and Edwards (27) found that women with a salient work role, invested more of their time in work than in their family life. Men with a salient work role invested as much time in their family life as men with a salient family role. It is possible that the work role provides men self-esteem and social status, which increases their energy to invest more time in other roles (28). The results obtained in another research (29) suggest that family role is vital for the mental and physical health of men, and is more important for their psychological state than the work role.

When reviewing domestic and foreign literature it can be noted that role salience is rarely examined in Croatia (30-32). Ĺ verko (30) examined the salience of five roles: the role of a student, a worker, a citizen, a houseworker, and a leisurite. However, the existing studies have a common drawback, and that is that everything that was not part of the work domain was considered the family domain.

For example, a hobby does not belong to the work domain,

yet it cannot be classified as the family domain. Moreover, it should be taken into account that not all aspects of the work domain have the same disruptive effects on all aspects of the family domain or vice versa. For this reason, we attempted to divide work-to-family and family-to-work conflict into several components to create a more detailed picture of the impact of the work domain on family, and vice versa. The objective of present study was to examine the salience of different roles within the work and family domains, and to examine the relationship between the salience of these roles and their mutual interferences among employed men and women.

First of all, gender differences in the salience (behavioural, affective, and cognitive) of the roles of a worker, a colleague, a spouse, a parent, a family member, and a leisurite were examined. Next, gender differences in the assessments of different types of role conflict (work-tomarriage, work-to-parent, work-to-housework, work-toleisure, family-to-worker, family-to-colleague) were examined. Finally, the relationships between the salience of the roles of a worker, a colleague, a spouse, a parent, a family member, and a leisurite and the assessments of different types of work-family conflict (work-to-marriage, work-to-parent, work-to-housework, work-to-leisure, family-to-work, and family-to-colleague relations) separately for men and women were examined.

METHODS Participants The study was conducted on 103 couples from different regions in the Republic of Croatia. The average age of women was 39.05 (SD=9.41) and ranged from 22 to 62 years, while the average age of men was 41.6 (SD=10.05), ranging from 23 to 62 years. Table 1 presents a more detailed view of demographic characteristics (age and education level). In order to participate in the study, participants had to meet two criteria: both spouses had to be employed and they had to have at least one child.

Table 1 The characteristics of the tested sample of employed couples

Age

Education level

Women

Men

Total

22-30

N=24 (23.3%)

N=14 (13.59%)

N=38 (18.45%)

31-40

N=32 (31.07%)

N=36 (34.95%)

N=68 (33.01%)

41-50

N=32 (31.07%)

N=27 (36.21%)

N=59 (28.64%)

51-62

N=12 (11.65%)

N=24 (23.3%)

N=36 (17.47%)

not stated

N=3 (2.91%)

N=2 (1.94%)

N=5 (2.43%)

Secondary

N=52 (50.49%)

N=68 (66.02%)

N=120 (58.25%)

Higher

N=11 (10.68%)

N=10 (9.71%)

N=21 (10.19%)

High

N=39 (37.86%)

N=23 (22.33%)

N=62 (30.1%)

not stated

N=1 (0.97%)

N=2 (1.94%)

N=3 (1.46%)

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Data collection The sample was collected mainly through personal contacts, with individuals or couples being asked to assist in the distribution of questionnaires to other spouses that they know. Data from some respondents was collected after visiting the organisations they work in. Prior to this, their supervisors were asked permission for the researcher's arrival at the workplace. This method of data collection results in a convenience sample, which is one of the limitations of this study, but due to the economic crisis, finding married couples who are both employed is a real challenge in Croatia. Unfortunately, there are much more examples of only one working spouse. Respondents filled out the questionnaires in their spare time and had at least one week to complete them. Questionnaires were distributed in envelopes to ensure anonymity and respondents were also asked to pick and write down a common code for easier determination of the pairing individuals.

Measures A set of socio-demographic type questions aimed at collecting data on the age and education level of the respondents.

Role Salience Inventory (SI) (33) originally measures one's participation in, commitment to, and expectation of the ability to fulfil a value in each of five life roles – the role of a student, a worker, a citizen, a houseworker, and a leisurite. It was developed within the Work importance study (WIS) project. The questionnaire consists of three subscales giving three measures of importance of each of the five roles. First, the behavioural (participation) component scale consists of 10 items and is a measure of the respondents' estimates on how much time and effort is put in action or in thinking about each role. On the affective (commitment) component scale, which also consists of 10 items, respondents estimate how important it is to them to be successful in a variety of roles. Third, the respondents estimate the expected possibilities of achieving different values in each of the five roles on the cognitive component scale (2). This scale originally has 14 items, but the number of items differs from one country to another, depending on the values perceived as significant in each culture (2). For example, the Croatian version of SI is originally composed of 10 items in the behavioural scale, 10 in the affective scale, and as many as 20 items in the cognitive scale (34).

Each role is assessed on a four-level Likert scale, and the final result for each of the three scales is obtained by summing the respondents' estimates for each role separately.

Alpha coefficients of the questionnaire have so far proved to be sufficiently high. For example, for each of the five roles in each of the three scales, alpha coefficients ranged from .82 to .95 (33, 34).

An adapted version of the Role Salience Inventory based on the Croatian validation by Šverko and colleagues (34)

was used in this study. The behavioural, affective, and cognitive components of salience of the following roles were measured: a colleague, a worker (employee), a spouse, a parent, a family member, and a leisurite. The behavioural component scale consisted of seven items as three items from the original version which could not be applied to all of the roles assessed were excluded from the scale. The affective component scale consisted of all the original 10 items. On these two scales, the behavioural and the affective component scales, respondents evaluated each role by choosing one out of four possible answers (1-not at all, never; 2-small, sometimes; 3-quite often; 4-very many, very often). The third, cognitive component scale, consisted of all the 20 items from the original Croatian version of the scale, but items 14 (''to feel the lure of danger...'') and 19 (''to be with people of my origin...'') were excluded since the correlations of these items with the overall score were lower than 0.30, resulting in 18 items for further analysis.

Here, respondents evaluated each role by choosing one out of four possible answers (1-nothing or little; 2-partly; 3-rather; 4-very many).

The Work-family conflict scale (35) is used for measuring the degree of conflict between work and family life, i.e., the interaction of work and family life, taking into account the time, effort, and organisation of activities as the causes of conflict. The Croatian adaptation of this scale (36) consists of 12 items, or two sets of six items that differ only in the direction of conflict. The first concerns the effect of work on family life and the other the effect of family on work life. Respondents express their agreement/ disagreement with a particular item on a seven-degree scale (1-strongly disagree; 7-strongly agree).

A modified version of this scale was used in this study.

It did not measure the general work-to-family and familyto-work conflict, as in previous research. Instead, work-tofamily conflict was divided into four types (work-tomarriage, work-to-parenting, work-to-houseworking, work-to-leisure) and family-to-work conflict into two types (family-to-worker and family-to-co-worker) giving a total of six subscales. Unlike the Croatian adaptation of the scale which has six items for each conflict, the scales used in this study have five items for each conflict. Specifically, one item (“My family members do not respect nor accept the demands and obligations included in my job.”) could not be adjusted to be applicable to all types of conflict and was excluded from this study. Respondents expressed their agreement/disagreement with a particular item on a 4-degree scale (1-nothing or little; 2-partly, 3-rather; 4-very much).

Six exploratory factor analyses (principal components) with the Kaiser-Guttman criterion of factor extraction (latent root > 1) were conducted to check whether the items of each of the subscales indeed belonged to one factor.

KneĹževiÄ&#x2021; I, et al. Salience and conflict of work and family roles among employed men and women Arh Hig Rada Toksikol 2016;67:152-163

Ethical approval This research was approved by the Ethical Research Committee in the area of psychology at the University of Zadar. It had been planned and was carried out according to the ethical principles of the Croatian Psychological Society, which are in accordance with the ethical principles, concerning psychological research, of the American Psychological Association, the British Psychological Society, and also of the Declaration of Helsinki. The subjects gave their informed consent to participate in this research.

RESULTS AND DISCUSSION In this study, the generally recognised conflicts such as work-to-family and family-to-work were examined with respect to several individual roles within a specific domain, as were also their relations to the salience of these different roles. Although the research was conducted on married couples, differences between men and women, rather than between couples, were examined. This type of sample was chosen to assure a greater control of the variables that could influence the results.

Role salience among men and women According to Super (1), all three components of the salience of a role should be considered separately and we have held this view in this research. Figures 1, 2, and 3 represent the levels and differences in the reported behavioural, affective, and cognitive components of the salience of six different roles in men and women: the role of a co-worker, a worker, a spouse, a parent, a family

155

member, and a leisurite. According to the aim of this research, we first performed one-way analyses of variance to determine whether men and women differed in the severity of the three components for each role individually (Table 2)1.Given the fact that the men and women in this sample differ in their qualifications, the analyses of covariance with education as a covariate have also been performed along with the one-way analyses of variance (Table 2). We additionally examined the differences between the salience of different roles with the one-way analyses of variance and post-hoc Sheffe procedures (these results will be presented in the text ).

The results showed that, expectedly, the roles closely related to family are more salient to the respondents than those related to work (F(5/960)=116.95; p=.000). In fact, respondents provided the highest estimates on all three components of salience precisely for these roles. Therefore, most respondents invest and find that being successful in the roles they have in the family (particularly the role of a parent) is the most important, and believe that they, at least in comparison with the roles offered in this study, could provide the best opportunity for achieving salient values.

Although Croatia underwent significant transformations in the political, economic, and cultural aspects in the last 20 years, resulting in the transformation of the system of individual values, family is still ranked first in this very much Christian country (37, 32, 7). However, there are differences between women and men in the expression of Although it is more usual to use t-tests in such case, analyses of variance were conducted to make a comparison with the results of analyses of covariance.

1

Figure 1 The degree of the behavioural component of salience of different roles (co-worker, worker, spouse, parent, family member, and leisurite) in men and women

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Table 2 Testing differences (ANOVA and ANCOVA - covariate: education level) between women and men in the behavioural, affective, and cognitive component of each role's salience (co-worker, spouse, parent, family member, and leisurite)

Cognitive

Affective

Behavioural

Women

Men

ANOVA

ANCOVA

Variables

M

SD

M

SD

df

F

p

df

F

p

Co-worker (N=97)

2.57

0.62

2.65

0.72

1/192

0.66

0.419

1/188

2.36

0.126

Worker (N=103)

3.18

0.57

3.26

0.53

1/204

0.99

0.322

1/200

0.41

0.525

Spouse (N=103)

3.19

0.54

2.98

0.58

1/204

7.01

0.009

1/200

3.36

0.068

3.63

0.36

3.17

0.60

1/204

44.9

0.000

1/200

0.44

0.506

3.26

0.54

2.93

0.57

1/204

17.5

0.000

1/200

0.54

0.463

2.19

0.72

2.51

0.67

1/204

10.8

0.001

1/200

0.19

0.663

Co-worker (N=97)

2.78

0.66

2.85

0.64

1/192

0.53

0.470

1/188

0.17

0.681

Worker (N=103)

3.28

0.58

3.31

0.54

1/204

0.12

0.734

1/200

1.88

0.172

Spouse (N=103)

3.57

0.45

3.49

0.47

1/204

1.28

0.259

1/200

4.51

0.035

3.83

0.25

3.64

0.41

1/204

15.9

0.000

1/200

1.39

0.239

3.51

0.49

3.39

0.50

1/204

3.33

0.069

1/200

0.65

0.421

2.87

0.67

2.99

0.65

1/204

1.57

0.211

1/200

0.00

0.986

Co-worker (N=97)

2.59

0.65

2.67

0.64

1/192

0.88

0.348

1/188

2.50

0.115

Worker (N=103)

2.88

0.62

2.92

0.62

1/204

0.22

0.642

1/200

0.82

0.365

Spouse (N=103)

3.26

0.51

3.21

0.55

1/204

0.59

0.442

1/200

0.91

0.342

3.44

0.41

3.32

0.50

1/204

4.05

0.046

1/200

0.99

0.320

3.19

0.52

3.15

0.51

1/204

0.40

0.526

1/200

0.28

0.601

2.43

0.59

2.54

0.58

1/204

1.76

0.187

1/200

5.12

0.025

Parent (N=103) Family member (N=103) Leisurite (N=103)

Parent (N=103) Family member (N=103) Leisurite (N=103)

Parent (N=103) Family member (N=103) Leisurite (N=103)

individual role salience components closely related to family.

We can start with the role of a parent, since it proved to be the most salient. Jankovic et al. (37) have already demonstrated that the value ''child'' stands out among other values. There is no difference between men and women in believing in the possibility of realising values (cognitive component) in the parental role, but women are much more committed to this role (affective component), and invest much more time and energy into it (behavioural component) (Table 2). This can be explained by different socialisation

of men and women. Men are socialised to be wage earners, while women are socialised to be full of love and dedication, caring mothers and wives who take care of the quality of family member interactions. The Croatian society is still a quite traditional one, but it is suggested that, even in the most egalitarian relationships, women would, due to their socialisation and psychological orientation on the caretaking role, feel a special connection with the role of being a mother (38).

As for the role of a spouse and a family member, men and women do not differ neither in the assessments of their

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commitment to these roles, nor in the belief that they will be able to achieve personal values through these. However, it can be noted that women, compared to men, invest more time and energy in participating in these roles (Table 2), which is again in line with the traditional socialisation of men and women. Both recognise the importance of family and are highly committed to it, but when it comes to taking care of other family members, women mainly do the job.

The results of previous research indeed show that men are significantly less involved in housework and childcare, especially when it comes to housework (39, 15, 40). Most couples cooperate in social contacts with relatives and institutions, and in the improvement of financial issues.

However, when it comes to the traditionally “female” tasks - cooking, washing dishes, and cleaning, there is a much lower extent of symmetrical division of labour. Men are more likely to perform traditional “male” tasks such as easy repairs of household appliances. Topolčić (40) explains that even when their levels of participation in housework increase, men usually remain only the helpers. They »help their wives'' and their wives are concerned that they perform these tasks in a timely and satisfactory manner.

Within the roles closely related to the job, at least as far as the investment of time and energy is concerned, the role of a worker proved to be the most salient (Figure 3).

Moreover, men and women invest an equal amount of time and energy into this role, are equally devoted to it, and equally believe in the possibility of achieving values in it (Table 2). This proves that women appreciate work as much as men. In particular, the existence of an individual and his family depends on the work role. Insufficient investment in this role can result in job loss, which may adversely affect the quality of life, not only of the individual but also of his family.

In comparing these highly salient four roles (the role of a worker, spouse, parent, and family member), women, compared to men, invest more time and energy in the roles

that are closely related to the family, while men, although they also invest much in these roles, invest most of their time and energy in the role of a worker (Table 2). This result can also be attributed to the aforementioned socialisation of women and men, which is more traditionally perceived in Croatia. Results of studies are consistent in indicating that women are more involved in family roles and have higher expectations of these roles than men (39, 38, 41, 42).

Interestingly, women invest as much time in the role of a worker as they invest in the roles closely related to family (except for the role of a parent in which they invest by far the most time and energy). Employment is an additional source of self-esteem for working mothers (43) so such a result may not be surprising. Moreover, success at the workplace is often more visible than success in the household, so maybe we can expect that success on the job has a stronger effect on the self-esteem of working mothers, since they can prove their capabilities at work, while no one has the habit of complimenting them for, let's say, a well cleaned living room.

The roles of a co-worker and a leisurite showed to be less salient than the roles closely related to family and the role of a worker. Respondents are more affectively attached to the role of a free-time consumer, but they invest more time and energy in the role of a co-worker (since this is imposed to them by their working hours and the type of work they perform) and believe that this role provides greater opportunities for the realisation of appreciated values (Figures 1, 2, and 3). As for the differences between men and women (Table 2) the results show that they invest the same amount of time and energy into, are equally devoted, and equally believe in the possibility of realising values in the role of a co-worker. As for leisure, there are also no differences in the affective and cognitive components of role salience, but men invest significantly more time and energy in this role than women. Men have more time for themselves and their desires and needs, which

Table 3 Testing differences (ANOVA and ANCOVA - covariate: education level) between men and women in the amount of certain types of work-family conflict (work-to-marriage, work-to-parenting, work-to-housework, work-to-leisure, family-to-work, and familyto-relations with co-workers) Women Men ANOVA ANCOVA Variables M SD M SD df F p df F p Work-to-marriage (N=103)

2.12

0.76

2.35

0.81

1/204

4.31

0.039

1/200

0.69

0.404

Work-to-parenting (N=103)

2.21

0.77

2.39

0.76

1/204

2.89

0.091

1/200

0.12

0.734

Work-to-housework (N=103)

2.42

0.83

2.57

0.80

1/204

1.59

0.208

1/200

0.32

0.572

Work-to-leisure (N=103)

2.62

0.91

2.61

0.79

1/204

0.01

0.915

1/200

1.33

0.250

1.57

0.58

1.43

0.58

1/204

2.79

0.096

1.48

0.55

1.38

0.52

1/192

1.59

0.208

Family-to-work (N=103) Family-to-relations with co-workers (N=97)

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is not surprising given the fact that caring for the family is usually left to women. They fulfil the task required based on their gender role at work, take care of children at home, most likely while the wife is at work or performing other house tasks, and then they are free to do whatever they want. Women work, but the task they are required to perform based on their gender role is at home, making them less able to dedicate time to themselves and their other needs and desires.

Work-family conflicts in men and women Figure 4 represents a display of the average assessments of the examined types of work-family conflict. Similar to the results of previous research (11, 12), it has been shown that work roles have a more harmful effect on family than family roles on the examined work roles (Figure 4). As for the differences between men and women in different types of work-to-family conflict, men reported a higher level of work-to-marriage conflict than women, while there were no differences obtained in other types of work-to-family conflict (Table 3; Figure 4). It is possible that men, when it comes to social support, are more dependent on their wives than their wives on them. Research has shown that wives are the main source of social support to men, while husbands are only one of such sources to women (44). Women generally have a wider network of social support within and outside the family, and when they fail to get support from their husband, they will look elsewhere. The results of this study also show that work mostly interferes with leisure activities (F(3/612)=32,78; p=.000; Figure 4), which may not be surprising given the fact that an individual is obliged to participate in other roles taken account of in this

study; we have to interact with our spouse, children need to be taken care of, and housework needs to be done from time to time. Of all of these roles, leisure activities are not obligatory and are often neglected or sacrificed for the sake of other obligations.

As for the perception of the two types of family-to-work conflict, it seems that there is no difference between men and women (Table 3; Figure 4). However, respondents estimate that their family has a greater influence on their role as a worker than as a co-worker [F(1/192)=5,46; p=.021]. Presenting ourselves as valuable workers can be difficult when a problem occurs in our family, which we constantly have in mind and which distracts us. As for the relations with co-workers, we do not have to work on these if we do not feel like it that day, of course, if it is not required by the job. The relations with co-workers are much more flexible and easier to ''avoid'' than our job activities. On the other hand, our co-workers can be a source of social support in which case our family issues may actually encourage entering into a relationship with them.

The relationship between the salience and conflicts of work and family roles Considering the aim of the research, the relationships between the salience and conflicts of different types of roles, separately for men and women, were examined. Correlation analysis obtained several significant but low Pearson correlation coefficients (presented in Table 4). In women, the experience of disruption of marital relations due to the work role was correlated with lower levels of affective involvement in leisure activities, and disruption of parenting due to work was correlated with a lower level of affective

Figure 2 The degree of the affective component of salience of different roles (co-worker, worker, spouse, parent, family member, and free-time consumer) in men and women

KneĹževiÄ&#x2021; I, et al. Salience and conflict of work and family roles among employed men and women Arh Hig Rada Toksikol 2016;67:152-163

159

Figure 3 The degree of the cognitive component of salience of different roles (co-worker, worker, spouse, parent, family member, and leisurite) in men and women

and behavioural involvement in the same activities. It is not possible to make any exact conclusion of causal relations between the variables on the basis of correlations, but we can offer some assumptions about these. Perhaps the overload with work roles brings difficulties in fulfilling marital and parental roles, which women give high salience to. This leads to a lower possibility of engaging in leisure activities, which later on become less important to women.

Another assumption is that women who find leisure activities less important take in more responsibilities at their

job and at home. Additional analyses provide some support for the statements above. The affective and behavioural components of the salience of leisure are in a negative, although insignificant, correlation with the behavioural component of work role salience for women in this study (r=-.12; p>.05), and all the components of leisure role salience are in a negative and significant correlation with their age (correlation coefficients around -.25; p<.05). In men, the experiences of disruption of marital and family relationships because of work were associated with lower

Figure 4 Average levels of work-family conflict (work-marriage, work-parenting, work-housework, and work-leisure) and family-work (family-work and family-relationships with co-workers) in men and women

-0.02

-0.27*

-0.09

-0.07

-0.08

-0.18

-0.05

-0.03

-0.15

-0.15

-0.06

-0.23*

0.02

0.15

-0.01

-0.03

-0.01

-0.08

Spouse

Parent

Family member

Leisurite

Coworker

Worker

Spouse

Parent

Family member

Leisurite

Coworker

Worker

Spouse

Parent

Family member

Leisurite

-0.18

-0.03

-0.07

-0.08

0.09

-0.11

-0.11

-0.17

0.00

-0.08

0.02

0.02

-0.05

0.13

0.00

-0.12

-0.07

-0.06

-0.08

0.07

-0.11

-0.16

-0.22*

-0.16

-0.13

-0.07

-0.11

-0.09

-0.07

-0.15

0.02

-0.10

Workhousew.

-0.13

0.03

0.09

-0.01

0.02

-0.10

-0.05

-0.00

0.05

0.00

0.15

-0.04

-0.14

-0.11

0.03

0.01

0.08

-0.10

Workleisure

0.10

-0.01

-0.06

-0.09

-0.06

-0.02

-0.04

0.16

0.26* -0.04

-0.06

-0.18

0.

-0.02

-0.12

0.02

-0.07

0.08

0.04

-0.02

-0.06

0.05

0.05

0.10

-0.08

0.06

-0.06

-0.12

-0.02

-0.14

-0.00

0.10

0.04

0.08

-0.03

-0.08

Familywork

Familycoworker

0.07

0.00

-0.07

-0.05

-0.06

0.01

0.15

0.08

0.01

-0.01

0.02

-0.03

-0.15

0.06

-0.11

-0.12

-0.04

0.02

0.00

0.16

-0.04

-0.11

-0.05

0.08

-0.02

-0.08

-0.09

-0.22*

-0.28* -0.12

-0.13

-0.06

-0.05

Workparenting

-0.15

-0.07

-0.06

Workmarriage

-0.01

-0.12

-0.14

-0.13

-0.03

-0.07

0.18

-0.00

-0.01

0.07

0.10

-0.01

-0.08

-0.08

-0.17

-0.09

-0.05

-0.06

Workhousew.

Men

0.06

-0.06

-0.11

-0.06

-0.05

-0.07

0.20*

0.03

0.06

0.13

0.04

0.04

-0.15

0.04

-0.02

0.01

-0.06

-0.09

Workleisure

0.17

0.11

0.06

0.06

0.20*

0.23*

0.09

-0.06

-0.01

-0.16

0.07

-0.10

0.03

-0.03

-0.06

-0.10

0.04

-0.13

Familywork

0.12

0.09

0.05

0.06

0.10

0.05

0.05

-0.07

-0.01

-0.01

0.07

-0.17

0.06

0.12

0.02

0.04

0.03

-0.08

Familycoworker

Note: Correlations of the three components of salience of the co-worker role and family-co-working conflict with other variables have df= 95. since some respondents have no co-workers. Other degrees of freedom are 101. *p<0.05

Cognitive component

Affective component

Behavioural component

0.04

0.06

Worker

-0.08

-0.01

Workparenting

Coworker

Workmarriage

Women

Table 4 Correlations between 3 components (behavioural, affective, and cognitive) of salience of examined roles (co-worker, spouse, parent, family member, and leisurite) and different types of workfamily conflict (work-marriage, work-parenting, work-housework, work-leisure, family-work, and family-co-worker relationships) in men and women

160 KneĹževiÄ&#x2021; I, et al. Salience and conflict of work and family roles among employed men and women Arh Hig Rada Toksikol 2016;67:152-163

Knežević I, et al. Salience and conflict of work and family roles among employed men and women Arh Hig Rada Toksikol 2016;67:152-163

levels of behavioural involvement in the parental role. It could be that a man's lower engagement in the parental role causes problems in marital and family relationships additionally when he is preoccupied with his job.

Further on, the disruption of work because of family was associated with a greater cognitive involvement in the role of a worker for both men and women, and in the role of a co-worker for men. This is in accordance with the final assumption of this study, although the assumption was only made for men. The assumption was not confirmed when it comes to the other roles examined. It could be said that the more we expect to achieve different values in the work role, the more we find the disruption of our work role due to family issues difficult. The affective component of role salience was expected to be correlated with the perception of disruption of the role among women, but the only correlation confirming such an assumption was obtained among men. Greater experience of work disrupting leisure activities was associated with greater affective involvement in the role of a leisurite. It is probably more rational to assume that greater emotional commitment of men to enjoying time for themselves makes the disability to enjoy that time because of work more difficult, but then again, maybe their high workload makes them appreciate quality free-time more. It seems crucial to note here that affective leisure role salience in men is only correlated to the salience of the role of a co-worker, while in women it is also correlated to the roles connected to family life. This could mean that women include their family members in the activities they find as leisure, while men view leisure activities as those they spend with their colleagues and people besides their family members.

Other correlations between role conflict and role salience were not significant.

CONCLUSION The results of this study indicate that taking into account different roles included in the work and family life, the different dimensions of salience (importance) of those roles, and the connection of their importance with the perception of interrole disruption (conflict) separately for men and women is meaningful, not only in research, but also in everyday life.

Unfortunately, such as most research, this one has its certain shortcomings. One of them is also one of the most common problems associated with the use of questionnaires.

The subjects were given questionnaires, or self-assessment scales, which they were able to fill out wherever and whenever they wanted, resulting in unequal conditions of measurement and socially desirable responding. In addition, perhaps even more important, it was not possible to equalise the respondents with respect to the features of their work tasks and their working hours. Since these factors, as shown by previous studies, significantly contribute to the

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explanation of conflicts from one domain/role to the other, the limitations of the generalisation of the results obtained in this study should be noted.

As already suggested, future research should explore some of the variables that could determine the relationship between role salience and the conflict between different roles. Moreover, future research measuring role salience and different role conflicts should take into account the age of respondents because the salience of a role changes as people age (45), making the change in experiences of various role conflicts also possible. Although we had the information on respondents' age, we did not find the number of respondents sufficient, unfortunately, to divide them in age categories and conduct valid comparisons while taking gender into account simultaneously. In addition, it would be interesting to explore the conflicts between the roles that belong to the same domain, for example, parent-marriage or parent-leisure. It would also be interesting to seek potential causal relations between role salience and conflict by performing longitudinal research.

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Salijentnost i konflikt radnih i obiteljskih uloga među zaposlenim muškarcima i ženama Psiholozi su rano shvatili da radna i obiteljska uloga zauzimaju važno mjesto u životu pojedinca, no većina istraživanja nije uzimala u obzir činjenicu da se svaka od njih sastoji od niza specifičnijih uloga koje ne moraju biti jednake važnosti. Štoviše, nije se uzimalo u obzir da ne moraju svi aspekti radne domene jednako interferirati sa svim aspektima obiteljske domene ili obratno. U skladu s tim, i dosad provedena istraživanja pod obiteljskom su domenom podrazumijevala sve što nije spadalo u radnu, na primjer bavljenje hobijem ne spada ni u radnu ni u obiteljsku domenu. Stoga je cilj ovog istraživanja, u kojem su korištene skale samoprocjene, bio utvrditi salijentnost različitih uloga te ispitati povezanost salijentnosti tih uloga i interferencije različitih uloga kod zaposlenih žena i muškaraca. U istraživanju je sudjelovalo 206 ispitanika, odnosno 103 zaposlena bračna para iz različitih područja Republike Hrvatske. Rezultati su pokazali da ispitanici najsalijentnijima smatraju uloge usko vezane uz obitelj. Ipak, muškarci najviše vremena i energije ulažu u ulogu radnika. Žene više vremena i energije poklanjaju ulozi bračnog partnera, roditelja i člana obitelji, a muškarci ulozi korisnika slobodnog vremena. Ispitanici najvećim procjenjuju konflikt posao - slobodno vrijeme, a spolne su se razlike očitovale samo kod doživljaja ometanja bračnih odnosa zbog posla: muškarci su se izjasnili o većem ometanju nego žene. Ovo istraživanje ponudilo je dokaze o samo niskoj povezanosti salijentnosti nekih uloga s različitim vrstama interferencije između radne i obiteljske domene.

KLJUČNE RIJEČI: bračni partner; kolega; roditelj; slobodno vrijeme; spol

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Purg D, et al. Low-dose intravenous lipid emulsion for the treatment of severe quetiapine and citalopram poisoning Arh Hig Rada Toksikol 2016;67:164-166

Case report

DOI: 10.1515/aiht-2016-67-2802

Low-dose intravenous lipid emulsion for the treatment of severe quetiapine and citalopram poisoning Darinka Purg1, Andrej Markota1, Damjan Grenc2, and Andreja Sinkovič1 Medical Intensive Care Unit, University Medical Centre Maribor, Maribor1, Poison Control Centre, University Medical Centre Ljubljana, Ljubljana2, Slovenia [Received in March 2016; CrossChecked in March 2016; Accepted in May 2016] The treatment of quetiapine and/or citalopram poisoning is mainly supportive and involves gastric lavage, activated charcoal, intubation, and mechanical ventilation. Recently, however, there were reports of successful treatment with intravenous lipid emulsion. Here we report a case of a 19-year-old Caucasian girl who ingested approximately 6000 mg of quetiapine, 400 mg of citalopram, and 45 mg of bromazepam in a suicide attempt. The patient developed ventricular tachycardia and epileptic seizures 12 h after admission to the hospital. As the patient’s condition deteriorated, we combined standard therapy (intubation, mechanical ventilation, and vasopressors) with low-dose intravenous lipid emulsion (ILE) (a total of 300 mL of 20 % lipid emulsion) and normalised her heart rhythm and stopped the seizures. She was discharged to the psychiatric ward after 48 h and home after a prolonged (2-month) psychiatric rehabilitation. Intravenous lipid emulsion turned out to be effective even in the lower dose range than previously reported for quetiapine poisoning in patients presenting with seizure and ventricular arrhythmia. To our knowledge, there are no case reports describing the use of ILE in treating citalopram poisoning.

KEY WORDS: cardiac arrhythmias; complementary therapies; emergency treatment; epilepsy; poisoning Severe poisoning with quetiapine or citalopram can lead to life-threatening dysrhythmias and epileptic seizures.

Therapy is mainly supportive (1-2). In recent years, there have been reports of successful use of intravenous lipid emulsions (ILE) to counter poisoning with lipophilic drugs, mainly local anaesthetics, beta blockers, and calcium channel blockers (3), including quetiapine (4-6). Here we present a patient with severe poisoning with quetiapine and citalopram (yet another lipophilic substance), who was successfully treated with ILE in the lower dose range than reported in most cases.

CASE REPORT A 19-year-old girl was admitted to the hospital after having ingested about 6000 mg of quetiapine (20 300-mg tablets), 400 mg of citalopram, and 45 mg of bromazepam in a suicide attempt. The doses of ingested drugs were determined from medical history and empty packaging. On admission the patient was aroused; her Glasgow coma scale was 11, blood pressure 128/60 mmHg (17.1/8 kPa), and peripheral oxygen saturation without supplemental oxygen was 95 %. The pupils were dilated, symmetrical, and poorly reactive to light. Electrocardiogram revealed sinus Correspondence to: Darinka Purg, MD, Medical Intensive Care Unit, University Medical Centre Maribor, Ljubljanska 5, 2000 Maribor, Slovenia, E-mail: darinka504@gmail.com

tachycardia with the heart rate of 150 min-1, incomplete right bundle branch block, and prolonged corrected QT interval of 510 ms. We performed gastric lavage, applied activated charcoal, and drew urine for toxicology testing, which turned positive for quetiapine and citalopram. The concentrations of quetiapine and citalopram were not determined.

Twelve hours after the admission, we registered monomorphic premature ventricular contractions (PVCs) and generalised epileptic seizures. During the hour that followed, PVCs became more frequent and progressed to ventricular tachycardia with pulse, whereas epileptic seizures progressed to generalised convulsive status epilepticus, which was unresponsive to multiple bolus doses of diazepam. Potassium was just below the normal range, and magnesium was normal (3.71 mmol L -1 , and 0.78 mmol L-1, respectively). She was intubated and ventilated mechanically. The maximum prolongation of corrected QT interval was 570 ms. Because of the combination of life-threatening arrhythmia and epileptic seizures we decided to infuse 100 mL of 20 % lipids (1.5 mL kg-1 of Lipofundin MCT/LCT 20 %, B. Braun Melsungen AG, Melsungen, Germany, composed of soya oil 100 g per 1000 mL of emulsion, medium-chain triglycerides 100 g per 1000 mL of emulsion, glycerol, egg-lecithin, α-tocopherol, and sodium oleate) over 10 min, followed by additional 200 mL over the following 2 h. As soon as the first 100 mL were infused, the corrected QT interval dropped to normal (370 ms), and ventricular

Purg D, et al. Low-dose intravenous lipid emulsion for the treatment of severe quetiapine and citalopram poisoning Arh Hig Rada Toksikol 2016;67:164-166

tachycardia and epileptic seizures stopped entirely.

Subsequent treatment in the ICU was uneventful. The patient was extubated 24 h later, and her level of consciousness returned to normal (Glasgow coma scale 15).

She was transferred to the psychiatric ward on the following day and was discharged home after two months of psychiatric rehabilitation.

DISCUSSION Quetiapine is an atypical antipsychotic used for the treatment of schizophrenia, bipolar disorders, depression, and sleeping disturbances. Its antipsychotic effects are mediated via serotonin 5HT2 and dopamine D1 and D2 receptors. It also has affinity for serotonin 5HT1A, muscarinic M1, adrenergic α-1 and α-2, and histamine H1 receptors. An overdose results in hypotension, sinus tachycardia, cardiac dysrhythmias, prolongation of the corrected QT interval, delirium, seizures, central nervous system depression, respiratory depression, coma, and death (1, 5, 7).

Citalopram is a selective serotonin reuptake inhibitor (SSRI). In overdosed patients it has shown higher toxicity than other SSRIs, manifesting in the prolongation of corrected QT interval, widening of the QRS complexes, tachycardia, generalised convulsions, acute respiratory distress syndrome, rhabdomyolysis with acute renal failure, and CNS depression (2, 8). To our knowledge, there are no case reports describing the use of ILE in treating citalopram poisoning.

ILE is a treatment strategy for lipophilic drug poisonings. At least two explanations exist regarding the mechanism of action: firstly, the lipid sink theory proposes a separate pharmacologic compartment into which the lipophilic drug may diffuse from the tissues, and secondly, the delivery of energy-rich substrate to energy depleted cells (e.g. myocardium) might improve their function. The lipid sink theory is the most widely accepted mechanism of action. A number of commercial products with different lipid formulations have been successfully used to treat lipophilic drug poisonings. However, not all effects of ILE can be explained solely by the lipid sink theory, especially rapid effects immediately after the start of the infusion (3, 4).

The decision to use ILE in our patient was based on the occurrence of sustained ventricular tachycardia and generalised epileptic seizures. Standard treatment (gastric lavage, activated charcoal, intubation, mechanical ventilation) did not succeed in preventing life-threatening complications. Immediately after the infusion of ILE our patient’s condition improved, and we observed no complications attributable to ILE. Successful use of ILE in quetiapine poisoning has been described before. Finn et al.

(6) described a patient who attempted suicide by ingestion of 4300 mg of quetiapine and 3100 mg of sertraline. The

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patient presented with hypotension and hypothermia but had no epileptic seizures or arrhythmias. The patient received symptomatic treatment and infusion of 20 % ILE. Like in our case, the initial bolus dose was 1.5 mL kg-1 (100 mL), followed by an infusion of 6 mL kg-1 h-1 for 1 h (in total 400 mL). His level of consciousness improved soon after the administration of ILE, which removed the need for intubation and mechanical ventilation. Eren Cevik et al.

(9) reported on a patient who ingested 2400 mg of quetiapine. That patient presented with hypotension and sinus tachycardia. The patient received ILE consisting of 100 mL bolus, followed by 30 mL kg-1 h-1infusion over 2 h (total dose of 3580 mL). Bartos and Knudsen (5) presented a female patient who ingested 24000 mg of quetiapine. Her cardiovascular collapse was refractory to all standard symptomatic treatment (intubation, mechanical ventilation, vasopressors). She was treated with 20 % ILE 170 mL bolus, followed by an infusion of 500 mL over 1 h. In all these cases treatment with ILE was reported efficient.

We used a similar initial bolus compared to other authors, but due to a clear clinical improvement, we continued the treatment at lower doses (cumulatively 300 mL). The treatment showed no adverse effects such as allergic reactions, fat overload syndrome with hepatosplenomegaly, jaundice, acute pancreatitis, seizures, fat embolism, coagulopathies or alteration of laboratory tests (10-11).

To conclude, treatment with ILE for severe poisoning with quetiapine and citalopram could be effective even in the lower dose range than previously reported.

REFERENCES 1. Ngo A, Ciranni M, Olson KR. Acute quetiapine overdose in adults: a 5-year retrospective case series. Ann Emerg Med 2008;52:541-7. doi: 10.1016/j.annemergmed.2008.03.016 2. Liotier J, Coudoré F. Drug monitoring of a case of citalopram overdosage. Drug ChemToxicol 2011;34:420-3. doi: 10.3109/01480545.2011.566571 3. Buys M, Scheepers PA, Levin AI. Lipid emulsion therapy: non-nutritive uses of lipid emulsions in anaesthesia and intensive care. South Afr J Anaesth Analg 2015;21:124-30. doi: 10.1080/22201181.2015.1095470 4. Cao D, Heard K, Foran M, Koyfman A. Intravenous lipid emulsion in the emergency department: a systematic review of recent literature. J Emerg Med 2015;48:387-97. doi: 10.1016/j.jemermed.2014.10.009 5. Bartos M, Knudsen K. Use of intravenous lipid emulsion in the resuscitation of a patient with cardiovascular collapse after a severe overdose of quetiapine. Clin Toxicol (Phila) 2013;51:501-4. doi: 10.3109/15563650.2013.803229 6. Finn SD, Uncles DR, Willers J, Sable N. Early treatment of a quetiapine and sertraline overdose with Intralipid.

Anaesthesia 2009;64:191-4. doi: 10.1111/j.1365-2044.2008.

05744.x 7. Balit CR, Isbister GK, Hackett LP, Whyte IM. Quetiapine poisoning: a case series. Ann Emerg Med 2003;42:751-8. doi: 10.1016/S0196-0644(03)00600-0

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8. Personne M, Sjöberg G, Persson H. Citalopram overdose review of cases treated in Swedish hospitals. J Toxicol Clin Toxicol 1997;35:237-40. doi: 10.3109/15563659709001206 9. Eren Cevik S, Tasyurek T, Guneysel O. Intralipid emulsion treatment as an antidote in lipophilic drug intoxications. Am J Emerg Med 2014;32:1103-8. doi: 10.1016/j. ajem.2014.05.019

10. Levine M, Skolnik AB, Ruha AM, Bosak A, Menke N, Pizon AF. Complications following antidotal use of intravenous lipid emulsion therapy. J Med Toxicol 2014;10:10-4. doi: 10.1007/s13181-013-0356-1 11. Punja M, Neill SG, Wong S. Caution with interpreting laboratory results after lipid rescue therapy. Am J Emerg Med 2013;31:1536.e1-2. doi: 10.1016/j.ajem.2013.05.009

Zdravljenje hude zastrupitve s kvetiapinom in citalopramom z nizkim odmerkom intravenske lipidne emulzije Zastrupitve s kvetiapinom in/ali citalopramom večinoma zdravimo podporno. Ob zdravljenju s spiranjem želodca, aktivnim ogljem, intubacijo in mehansko ventilacijo pa so opisani primeri uspešnega zdravljenja z intravensko lipidno emulzijo.

Predstavljamo primer 19-letne ženske, ki je v samomorilne namene zaužila približno 6000 mg kvetiapina, 400 mg citaloprama in 45 mg bromazepama. Bolnica je imela 12 ur po sprejemu prekatno tahikardijo in epileptične napade. Ob kliničnem poslabšanju stanja smo jo zdravili s standardno terapijo (intubacija, mehanska ventilacija, vazopresorna podpora) in nizkim odmerkom intravenske lipidne emulzije (celokupno 300 mL 20 % lipidne emulzije). Srčni ritem se je po terapiji normaliziral, epileptični krči so prenehali. Po 48 urah zdravljenja v enoti intenzivne terapije je bila premeščena na oddelek za psihiatrijo in po 2-mesečnem zdravljenju domov. Zastrupitev s kvetiapinom in citalopramom, ki se pri bolniku kaže z epileptičnimi krči in prekatnimi motnjami ritma, lahko uspešno zdravimo z nizkimi odmerki intravenske lipidne emulzije.

KLJUČNE BESEDE: dopolnilna terapija; epilepsija; srčne aritmije; urgentno zdravljenje; zastrupitev

Marashi SM and Nasri-Nasrabadi Z. Is there a role for sildenafil in the management of paraquat-induced lung fibrosis?

Arh Hig Rada Toksikol 2016;67:167-168

Letter to the Editor

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DOI: 10.1515/aiht-2016-67-2804

Is there a role for sildenafil in the management of paraquat-induced lung fibrosis?

Sayed Mahdi Marashi1 and Zeynab Nasri-Nasrabadi2 Shiraz University of Medical Sciences, Shiraz1, Tehran University of Medical Sciences, Tehran2, Iran Paraquat is a herbicide. It is a highly toxic compound for humans often used for suicide, especially by farmers, and is associated with high mortality rates (1). In fact, if one consumes more than 20 millilitres of its 20Â % solution, a rapid onset of multi-organ dysfunction will cause death within a few days. In contrast, consumption of smaller amounts of paraquat may respond to supportive care and the patient could survive the acute phase (2).

Unfortunately, the polyamine reuptake system, which is mainly expressed in the membrane of alveolar cells, is responsible for toxin accumulation in the lungs during the acute phase. Consequently, lung fibrosis may occur as a delayed complication, which may potentially be fatal (3, 4). However, scientists propose to use anti-inflammatory drugs as the main part of treatment for preventing pulmonary fibrosis, progression to end-stage respiratory failure, and death within 2-3 weeks after paraquat intoxication (2).

Fortunately, if the patient overcomes this phase, pulmonary fibrotic tissue remodelling can lead to functional regeneration within about 6 months (5).

To date, no clearly efficacious therapies have definitively been shown to alter fibrosis progression. Moreover, no treatment for respiratory failure due to paraquat-induced lung fibrosis till date has proven effective (6).

However, respiratory failure is the main reason for invasive mechanical ventilation, and there is no published study on the outcome of ventilator support. Nevertheless, according to recent studies evaluating the prognosis, respiratory failure and the need for respiratory support were the determinants of the fatal outcome (1). This finding is similar to those reported previously in patients with idiopathic pulmonary fibrosis (7). The probable mechanism is overdistension of relatively intact parts of pulmonary tissue and a consequent ventilator-induced lung injury or an increase in the right-to-left shunt flow in response to the prostanoid (8, 9).

In addition to fibrotic damage of the vasculature and elevation of vascular resistance, patients with lung fibrosis Correspondence to: Dr Sayed Mahdi Marashi, Trauma Research Center, Medical School, Emergency Room/Division of Medical Toxicology, Hazrat Ali-Asghar (p) Hospital, Shiraz University of Medical Sciences, Shiraz, Iran, E-mail: marashi@sums.ac.ir

show decreased levels of nitric oxide (NO) production (NO being a significant pulmonary vasodilator), which contributes to pulmonary vasoconstriction and consequently impaired gas exchange (10, 11). This can explain a relatively swift progression of respiratory dysfunction in patients with moderate to severe paraquat toxicity.

It has previously been demonstrated that inhaled NO leads to preferential vasodilation in well-ventilated lung tissue, as well as bronchodilation, anti-inflammatory and antiproliferative properties (12).

Interestingly, reviewing the literature on the internet, one case report of successful treatment of massive paraquat ingestion by inhaled NO exists (13). Moreover, Cho and colleagues demonstrated that inhaled NO was associated with survival improvement in paraquat-injured rats (14).

Unfortunately, considering that fibrosis requires a long time to develop, continuous inhalation NO therapy presents technical difficulties. Sildenafil as a phosphodiesterase-5 inhibitor stabilises cyclic guanosine monophosphate, helps increase NO levels in the lungs, and has selective pulmonary vasodilatation properties for well-ventilated lung areas (15).

Ghofrani et al. (15) demonstrated a significant improvement in gas exchange in patients with severe lung fibrosis treated with sildenafil; moreover, a controlled trial conducted by the idiopathic pulmonary fibrosis clinical research network showed a significant improvement of dyspnoea and quality of life, as well as pulmonary function stabilisation, improvement of arterial blood gas, and carbon monoxide diffusion capacity at 12 weeks. However, the latter study was unable to show a significant improvement of survival rates because it enrolled too few patients (11, 15).

Moreover, it should be kept in mind that these two trials involved patients with severe idiopathic pulmonary fibrosis, which is a chronic and progressive lung disease, whereas, as mentioned earlier, the remodelling of fibrotic tissue can lead to functional regeneration in paraquat poisoning survivors.

According to the available safety and efficacy data, it seems that administration of sildenafil can theoretically improve ventilationâ&#x20AC;&#x201C;perfusion matching and thus gas exchange in paraquat poisoning patients (16). Therefore, a

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randomised controlled trial should be conducted in such patients.

Conflicts of interests None declared.

REFERENCES 1. Delirrad M, Majidi M, Boushehri B. Clinical features and prognosis of paraquat poisoning: a review of 41 cases. Int J ClinExp Med 2015;8:8122-8.PMCID: PMC4509324 2. Gil HW, Hong JR, Jang SH, Hong SY. Diagnostic and therapeutic approach for acute paraquat intoxication. J Korean Med Sci 2014;29:1441-9.doi: 10.3346/ jkms.2014.29.11.1441 3. Dinis-Oliveira R, Duarte JA, Sánchez-Navarro A, RemiãoF, Bastos ML, Carvalho F. Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol 2008;38:13-71.doi: 10.1080/10408440701669959 4. Marashi SM, Raji H, Nasri-Nasrabadi Z, Majidi M, Vasheghani-Farahani M, Abbaspour A, Ghorbani A, Vasigh S. One lung circumvention, an interventional strategy for pulmonary salvage in acute paraquat poisoning: an evidence based review. Tzu Chi Med J 2015;27:99-101.doi:10.1016/j. tcmj.2015.06.002 5. Lee KH, Gil HW, Kim YT, Yang JO, Lee EY, Hong SY. Marked recovery from paraquat-induced lung injury during long-term follow-up. Korean J Intern Med 2009;24:95-100. doi: 10.3904/kjim.2009.24.2.95 6. Sanaei-Zadeh H. Can pirfenidone prevent paraquat-induced pulmonary fibrosis? A hypothesis. Tzu Chi Med J 2012;4:223. doi: 10.1016/j.tcmj.2012.07.001 7. Papiris SA, Manali ED, Kolilekas L, Kagouridis K, Triantafillidou C, Tsangaris I, Roussos C. Clinical review: idiopathic pulmonary fibrosis acute exacerbationsunravelling Ariadne’s thread. Crit Care 2010;14:246.doi: 10.1186/cc9241

8. Fernández-Pérez ER, Yilmaz M, Jenad H, Daniels CE, Ryu JH, Hubmayr RD, Gajic O. Ventilator settings and outcome of respiratory failure in chronic interstitial lung disease. Chest 2008;133:1113-9.doi: 10.1378/chest.07-1481 9. Olschewski H, Ghofrani HA, Walmrath D, Schermuly R, Temmesfeld-Wollbruck B, Grimminger F, Seeger W. Inhaled prostacyclin and iloprost in severe pulmonary hypertension secondary to lung fibrosis. Am J RespirCrit Care Med 1999;160:600-7.Doi: 10.1164/ajrccm.160.2.9810008 10. Patel NM, Lederer DJ, Borczuk AC, Kawut SM. Pulmonary hypertension in idiopathic pulmonary fibrosis. Chest 2007;132:998-1006.PMID: 17873194 11. Idiopathic Pulmonary Fibrosis Clinical Research Network, Zisman DA, Schwarz M, Anstrom KJ, Collard HR, Flaherty KR, Hunninghake GW. A controlled trial of sildenafil in advanced idiopathic pulmonary fibrosis. N Engl J Med 2010;363:620-8.doi: 10.1056/NEJMoa1002110 12. Ichinose F, Roberts JD Jr, Zapol WM. Inhaled nitric oxide: a selective pulmonary vasodilator: current uses and therapeutic potential. Circulation 2004;109:3106-11.PMID: 15226227 13. Eisenman A, Armali Z, Raikhlin-Eisenkraft B, Bentur L, Bentur Y, Guralnik L, Enat R. Nitric oxide inhalation for paraquat-induced lung injury. J ToxicolClinToxicol 1998;36:575-84.doi: 10.3109/15563659809028051 14. Cho JH, Yang DK, Kim L, Ryu JS, Lee HL, Lim CM, Koh YS. Inhaled nitric oxide improves the survival of the paraquat-injured rats. VasculPharmacol2005;42:171-8.doi: 10.1016/j.vph.2005.01.001 15. Ghofrani HA, Wiedemann R, Rose F, Schermuly RT, Olschewski H, Weissmann N, Gunther A, Walmrath D, Seeger W, Grimminger F. Sildenafil for treatment of lung fibrosis and pulmonary hypertension: a randomised controlled trial. Lancet 2002;360:895-900. doi: 10.1016/ S0140-6736(02)11024-5 16. Collard HR, Anstrom KJ, Schwarz MI, Zisman DA. Sildenafil improves walk distance in idiopathic pulmonary fibrosis.

Chest 2007;131:897-9. doi: 10.1378/chest.06-2101

OBITUARY Arh Hig Rada Toksikol 2016;67:A12

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OBITUARY Professor Alfred Michael Thiess, MD, PhD h. c.

(1921-2016) On February 3, 2016 Professor Alfred Michael Thiess passed away in Ludwigshafen, Germany, the legendary founder and Honorary President of the International Scientific Association for Occupational and Environmental Health in the Production and Use of Chemicals MEDICHEM. Professor Thiess was born near Hermannstadt, Romania. He came to Germany to study medicine in Frankfurt and stayed there his entire life. In 1954 he joined BASF where he was head of the Occupational Medical Department from 1963 until his retirement in 1986. During this time he was one of the major forces behind the development of occupational medicine in Germany. He intensified the application of occupational epidemiology in an industrial setting and fostered health promotion not only in the sense of “one-time” campaigns but with a focus on subsequent evaluation of its success. The results of his scientific work were published in more than two hundred articles in national and international occupational medical journals. Besides, he was a teacher at the Mannheim Medical Faculty of the University of Heidelberg and still found the time to actively participate in numerous national and international expert groups. In 1972 he inaugurated MEDICHEM, which also acts as the Scientific Committee on Occupational Health in the Chemical Industry of the International Commission for Occupational Health (ICOH).

He served as MEDICHEM Chairman from 1972 to 1986, and afterwards as Honorary President he always took a keen and active interest in supporting MEDICHEM’s goals and development.

In recognition of his achievements he received numerous awards and honours, among them the Grand Order of Merit of the Federal Republic of Germany. But one award, by far not the most prestigious one, just through its title characterized Professor Thiess so perfectly that it shall be mentioned here: It was the Service above Self

Award issued by the Rotary Club. Why that? Immediately after the fall of the iron curtain, Professor Thiess had started activities directed at helping schools, hospitals and other social institutions in Romania, to overcome the many shortcomings accumulated under the dictatorial regime of the past. It was for this reason that the German Society of Occupational and Environmental Medicine (DGAUM) had to wait for one more year until he found the time to accept the most prestigious award for his lifetime achievements to be issued by this society, the Franz-Kölsch Medal.

Coinciding with the intended date for the award ceremony Professor Thiess had already scheduled a truck convoy to Siebenbürgen, Romania. These activities were always more important to him than honours and recognitions directed to himself. In 2002 the city of his ancestors, Urwegen, Romania, bestowed on him honorary citizenship in recognition of his untiring and generous support over many years.

Professor Thiess has lived a long and fruitful life, and he was always aware of and grateful for this. While he received public honours with pride and grace, the prospect of public recognition was never the driver for his desire to influence and help. He was intrinsically motivated, and he was a formidable motivator of others. He was able to combine professional leadership with honest and warm personal interest in his co-workers, thus making it easy for them to adopt and actively pursue his sometimes indeed challenging goals. He has left deep marks not only in the history of German and international Occupational Medicine but also in the hearts of many people who had the privilege to work with him. Our thoughts and best wishes go to his wife, his life and professional companion, Dr Gisela Thiess and their family. We are sad to have lost him, but grateful that we had had him The connection with Professor Thiess and Archives of Industrial Hygiene and Toxicology is timeless because he was more than a colleague to a number of former and current members of the journal’s editorial boards. He will be remembered as an excellent professional and scientist, a passionate and motivating person with an exceptional personality and a true friend to everybody who had the privilege to meet him.

Dr Michael Nasterlack

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REPORTS Arh Hig Rada Toksikol 2016;67:A12-A13

REPORT The basic course “Principles of Toxicology” Zagreb, April 4-8, 2016 The basic course “Principles of Toxicology” was organized by the Croatian Society of Toxicology under the auspices and financial support of EUROTOX. The course was held in Zagreb April 4-8, 2016. Upon the announcement of the course at the websites of EUROTOX and the Croatian Society of Toxicology, an unusually high number of candidates applied for participation (88). Therefore, we faced a difficult choice because most of the candidates had an excellent CV and had worked in the field of toxicology.

The course was planned for 25-30 participants, but due to very high interest, we accepted 50 participants as suggested

by EUROTOX. The high number of applicants to “Principles of Toxicology” indicated to us that regular education in toxicology in Europe is necessary perhaps more than ever.

This is because, according to our knowledge, many Western countries have closed postdoctoral studies in toxicology, while such studies never even existed in Eastern Europe because of the small number of scientists interested in toxicology.

The participants at the course were from Austria, Bosnia and Herzegovina, Croatia, Denmark, Estonia, Germany, Italy, Poland, Serbia, Slovenia, Spain, Turkey, Ukraine and the South African Republic.

The invited lecturers covered the most important issues in toxicology: Marina Goumenou (Introduction to toxicology and Scope and ethical principles of toxicology and 3 R), Biljana Antonijević (Role of metabolism), Benay Can Eke (Toxicokinetics), Maja Peraica (Factors affecting toxicity), Corrado Lodovico Galli (Target organ toxicity – liver and kidney, Reach and Risk assessment), Emanuela Corsini (Immunotoxicology), Eva Cecilie BonefeldJorgensen (Endocrine disruptors and Reproductive and developmental toxicity), Ksenija Durgo and Mirta Milić (Mutagenesis), Davor Želježić (Carcinogenesis), Alberto

Ritieni (Food toxicology), Bojan Hamer (Environmental exposure and biomonitoring) and Bas Blaauboer (Alternatives to animal testing in toxicological risk assessment). Their lectures were interesting and comprehensive. At the end of the course, i.e. at the final exam, the participants showed good knowledge about basic toxicology.

The participants were divided into seven working groups and each group had to prepare a 10 minute presentation on one toxic substance. Following the given instructions, they prepared excellent presentations to all of the other participants and lecturers.

Apart from education, the aim of such courses is to make personal acquaintances among the participants and lecturers (“networking”), which could be important for their careers – for instance, when looking for partners for projects. For this purpose, at the end of each presentation, the participants were asked to say a little about themselves as well as something about their research. Sightseeing and dinner were also organized and highly appreciated by the participants and lecturers. We would like to take this opportunity to once more thank all of the participants for their interest and helping to create a great atmosphere altogether.

The participation of eminent lecturers, their excellent lectures and all of their support during the course are also greatly acknowledged. This course could not be organized without the support of the EUROTOX Educational Committee, particularly its president Martin Wilks. Much due to the pleasant communication we had with the President of EUROTOX Prof Aristidis Tsatsakis and the members of the Educational Committee, the organization of the course Principles of Toxicology was a wonderful experience for us. Thank you all and see you at the next EUROTOX Congress.

President of the Croatian Society of Toxicology Maja Peraica, MD, PhD

REPORTS Arh Hig Rada Toksikol 2016;67:A13-A14

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REPORT 251 st American Chemical Society (ACS) National Meeting & Exposition: Computers in Chemistry San Diego, California, SAD, 13.-17. ožujka 2016.

Američko kemijsko društvo (ACS) godišnje održava dva najveća nacionalna znanstvena sastanka, koji zajedno privuku više od 25000 kemijskih i srodnih stručnjaka i studenata diljem američkih država. U okviru sastanka ACS ujedno se održavaju i znanstveni, stručni, društveni, obrazovni, gospodarski i brojni drugi susreti s međunarodnim sudjelovanjem u određenoj temi iz svijeta kemije. 251st Spring 2016 ACS National Meeting bio je posvećen utjecaju kompjuterske znanosti u kemijskim istraživanjima i okupio je više od 16000 sudionika. Kompjuterska tehnologija ima neprocjenjivu i rastuću ulogu u prirodnim znanostima, od prikupljanja i pohranjivanja podataka do dizajna novih materijala, kao i u brzini razmjene informacija i umrežavanju znanstvene zajednice. Opsežan tehnički program u organizaciji 29 tehničkih odjela ACS, sastavljenih iz svih grana i polja kemije, uključio je više od 12000 predavanja i gotovo 5000 posterskih priopćenja. Dva predavanja pod pokroviteljstvom The Kavli Foundation, organizacije koja podupire temeljna inovacijska istraživanja, nastavila su

tradiciju prezentiranja inovacija u kemiji i poticanja razumijevanja javnosti za najveće svjetske izazove i rješenja koja kemija može omogućiti. Organiziran je niz panela za raspravu aktualnih tema iz područja primjene znanja i novih metoda učenja te područja tržišta rada u kemijskoj znanosti i industriji. Brojni predstavnici izdavača predstavili su nove naslove iz cjelokupnog područja kemije. Konferencija je omogućila održavanje nekoliko desetaka kratkih stručnih tečajeva i radionica za pomoć u karijeri uz mogućnost povezivanja s potencijalnim poslodavcima. Više od 250 tvrtki instrumenata, laboratorijske opreme i servisa promoviralo je svoje nove proizvode i usluge na više od 400 reklamnih štandova. Kao delegat konferencije Pittcon 2016 na 251st ACS National Meeting S. Stipičević je sudjelovala u raznim sekcijama ACS posvećenim znanstvenom i obrazovnom radu u regijama Europa i Srednji Istok.

252nd Fall 2016 ACS National Meeting održat će se 21.-25. kolovoza 2016. u gradu Philadelphia, Pennsylvania, SAD, a najavljeni su i svi sljedeći skupovi do 270th ACS National Meeting. dr. sc. Sanja Stipičević

REPORT Pittsburgh Conference on Analytical Chemistry & Applied Spectroscopy 2016. (Pittcon 2016) Atlanta, Georgia, SAD, 6.-10. ožujka 2016.

S više od 12800 sudionika iz 88 država diljem svijeta, Pittcon 2016 predstavlja najveću konferenciju i izložbu laboratorijske znanosti u svijetu. Misija Pittcon-a kao neprofitne organizacije Društva spektroskopije Pittsburgh i Društva analitičkih kemičara Pittsburgh, Pensilvanija, SAD, koja djeluje već više od 60 godina, temelji se na unaprijeđenju obrazovanja u znanosti. Uz analitičke kemičare, sudionici konferencije Pittcon su svi istraživači koji analiziraju i testiraju kemijska i biološka svojstva molekula, kao i oni koji upravljaju znanošću, obrazovanjem i gospodarstvom na svim razinama. Većina sudionika (70 %) pripada sektoru industrije, obrazovanja i distribucije opreme.

Ovogodišnji program konferencije otvorio je prof. W. E. Moerner iz Sveučilišta Stanford, Kalifornija, SAD, dobitnik Nobelove nagrade za kemiju 2014. godine, s predavanjem iz područja fizikalne kemije, na temu razvoja i primjene nanoskopije visokog razlučivanja. Bogati tehnički program ponudio je preko 2000 prezentacija najnovijih analitičkih metodologija i njihovih primjena.

Prezentacije su uključile više od 350 plenarnih predavanja

(76 sekcija), 756 usmenih priopćenja (110 sekcija), 868 posterskih priopćenja (52 tematske sekcije), 147 poludnevnih do dvodnevnih stručnih tečajeva, 46 predavanja sponzora i 31 tematski sastanak sudionika kongresa. Za vrhunska postignuća u području analitičke kemije i spektroskopije dodijeljeno je 14 nagrada. Više od 800 tvrtki s 5000 sudionika predstavilo je najnoviju laboratorijsku i industrijsku tehnologiju i opremu te nakladničku djelatnost.

Program ACS Pittcon Travel Grant 2016, financiran iz fonda Wallace H. Coulter, Društva analitičkih kemičara Pittsburgh i Pittcon konferencije u organizaciji Američkog kemijskog društva, omogućio je sudjelovanje osam kandidata iz država Baltika i Balkana na ovogodišnjoj konferenciji. Među kandidatima s dva posterska priopćenja sudjelovale su S. Stipičević (Microwave-Assisted Extraction of Triketone and Pyrazole Corn Herbicides from Agricultural Soil) i D. Klinčić (PCBs and DDTs in Bluefin tuna from the Adriatic Sea). Oba priopćenja rezultat su istraživanja provedenih u okviru projekta OPENTOX Hrvatske zaklade za znanost (broj 8366, voditelj: D. Želježić).

Pittcon 2017 održat će se 5.-9. ožujka 2017. u gradu Chicago, Illinois, SAD, a registracija za konferenciju počinje 1. listopada 2016. dr. sc. Sanja Stipičević

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ANNOUNCEMENT Arh Hig Rada Toksikol 2016;67:A15

ANNOUNCEMENT PETI HRVATSKI TOKSIKOLOŠKI KONGRES S MEĐUNARODNIM SUDJELOVANJEM - CROTOX 2016 Poreč, Hrvatska, 9.-12. listopada 2016.

Hrvatsko toksikološko društvo organizira V. hrvatski toksikološki kongres pod pokroviteljstvom Instituta za medicinska istraživanja i medicinu rada (Zagreb) i Zavoda za toksikologiju i antidoping (Zagreb). Kongres će se održati u hotelu Valamar Diamant u Poreču od 9.-12. listopada 2016.

Teme kongresa podijeljene su u devet sekcija: Ekotoksikologija, Forenzička toksikologija, Genotoksičnost, Nanotoksikologija, Radiotoksikologija, Toksikologija hrane, Toksikologija metala, Zlouporaba nekih supstancija

i antidoti te Regulatorna toksikologija. U svakoj će sekciji predavanja održati istaknuti znanstvenici tog područja toksikologije, a omogućit će se i mladim znanstvenicima da predstave rezultate svojih istraživanja. Predviđeno je i održavanje posterske sekcije.

Službeni jezik Kongresa je engleski.

Kako bi omogućio mladim znanstvenicima sudjelovanje na Kongresu Organizacijski odbor dodijelit će pet stipendija doktorandima i post-doktorandima. Te se stipendije sastoje od kotizacije i smještaja u dvokrevetnoj sobi tijekom održavanja Kongresa.

Više informacija o Kongresu može se naći na web stranici Kongresa http://crotox2016.imi.hr/. dr. sc. Maja Peraica, dr. med.