Iodine nutritional status and thyroid effects of exposure to ethylenebisdithiocarbamates

Environmental Research 154 (2017) 152–159

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Iodine nutritional status and thyroid effects of exposure to ethylenebisdithiocarbamates

MARK

Emanuela Meddaa,1, Ferruccio Santinib,1, Simona De Angelisc, Fabrizio Franzellind, Carla Fiumalbie, Andrea Pericof, Enzo Gilardic, Maria Teresa Mechie, Alessandro Marsilib, ⁎ Angela Citronie, Adaniele Leandrif, Alberto Mantovanig, Paolo Vittib, Antonella Olivieric, a

National Centre for Epidemiology Surveillance and Health Promotion, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy Endocrinology Unit, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy c Department of Cell Biology and Neuroscience, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy d Nuclear Medicine of Bolzano Hospital, Bolzano, Italy e Department of Prevention, ASL Firenze, Italy f Public Health Laboratory, ASL Firenze, Italy g Department of Food Safety and Veterinary Public Health, Italian National Institute of Health, Viale Regina Elena 299, 00161 Rome, Italy b

A R T I C L E I N F O

A BS T RAC T

keywords: Iodine Thyroid disruptors Mancozeb Ethylenthiourea Exposure

Introduction: Italy is still characterized by a mild iodine deficiency and is among the most intensive users of chemical products for agriculture in Europe. The aim of this study was i) to evaluate thyroid effects of exposure to mancozeb, a fungicide widely used in agriculture, in a sample of Italian grapevine workers, and ii) to verify whether the iodine intake may modulate the risk of thyroid disruption due to the mancozeb metabolite ethylenthiourea (ETU). Methods: One hundred seventy-seven occupationally exposed male workers (29 from Chianti, a mild iodine deficient area, and 148 from Bolzano an iodine sufficient province) and 74 non-occupationally exposed male controls (34 from Chianti and 40 from Bolzano) were enrolled in the study. Serum biomarkers of thyroid function, as well as urinary iodine and ETU concentrations were assessed. Moreover all the recruited subjects underwent clinical examination and thyroid ultrasound. Results: Multivariate comparisons showed lower mean serum levels of FT4 in Chianti-workers as compared to Bolzano-workers. Moreover, an increased urinary iodine excretion ( > 250 µg/L) was more frequently found among more exposed workers (ETU > 20 µg/L) than among less exposed ones and this effect was more pronounced in Chianti- than in Bolzano-workers. Chianti-workers also showed a significantly higher frequency of very low thyroid volume (≤6.0 ml) as compared to controls. Conclusions: These findings showed a mild thyroid disrupting effect due to occupational exposure to mancozeb, more pronounced in workers residing in an area characterized by a mild to moderate iodine deficiency as compared to workers residing in an area covered by a long-lasting iodine prophylaxis program.

1. Introduction Increasing evidence from in vivo and in vitro studies has demonstrated the vulnerability of the thyroid to endocrine disrupting effects due to environmental exposures. The most potent thyroid disruptor is iodine, a micronutrient essential for the thyroid hormone synthesis.

Both insufficient dietary intake and excessive exposure to iodine have thyroid disrupting effects (Laurberg et al., 2010). Furthermore in the last two decades an increasing body of data have demonstrated that a wide range of chemicals with endocrine disrupting activities (endocrine disruptors, EDs) are able to interfere with the thyroid function (Brucker-Davis, 1998; De Angelis et al., 2007; Boas et al., 2009).

Abbreviations: SD, Standard Deviation; CH, Chianti; BZ, Bolzano; ETU, Ethylenethiourea; CI, Confidence Interval; NR, Normal Range; OR, Odds Ratio; GM, geometric mean; EDs, Endocrine Disruptors; EBDC, Ethylenebisdithiocarbamates; T3, Triiodothyronine; T4, Thyroxine; Tg, Thyroglobulin; TSH, Thyroid Stimulating Hormone; FT3, free T3; FT4, free T4; TgAb, anti-thyroglobulin antibodies; TPOAb, anti-thyroid peroxidase antibodies; UIC, Urinary Iodine Concentration; ICP-MS, Inductively Coupled Plasma Mass Spectrometry; US, Ultrasonography; RIA, Radioimmunological Assay ⁎ Corresponding author. E-mail address: [email protected] (A. Olivieri). 1 Equally contributed http://dx.doi.org/10.1016/j.envres.2016.12.019 Received 25 August 2016; Received in revised form 29 November 2016; Accepted 21 December 2016 0013-9351/ © 2016 Published by Elsevier Inc.

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et al., 2014). Our country is also among the most intensive users of chemical products for agriculture in Europe (EUROSTAT, 2012). Therefore the aim of this study was to evaluate thyroid effects of exposure to mancozeb in a sample of Italian grapevine workers, and to verify whether the iodine intake may modulate the risk of thyroid disruption due to the mancozeb metabolite ETU. To this end, workers and non-occupationally exposed controls were recruited in two areas with different iodine nutritional status: the autonomous province of Bolzano, which is iodine sufficient because of an efficient province-wide iodine prophylaxis program introduced in 1982 (Franzellin, 1998; Olivieri et al., 2015), and the Chianti area historically recognized as a mild iodine deficient area in the Tuscany, a region in the Central Italy (Aghini Lombardi et al., 1995; Maccherini et al., 1998).

Among EDs, pesticides are of particular concern due to their extensive worldwide utilization (EFSA, 2013), which leads to a potential widespread exposure involving occupational settings, residues in food (Mantovani 2015) and living environment in agricultural areas (Bradman et al., 2007; Fantke et al., 2011). Once these xenobiotics have entered the body, they usually undergo metabolic processes before excretion and only few compounds are excreted unmodified. Ethylenebisdithiocarbamates (EBDC), such as maneb and mancozeb, are a group of fungicides widely used in agriculture because of their low acute toxicity and short environmental persistence. In some countries EBDC are aerially sprayed by a light aircraft (van Wendel de Joode et al., 2014). Although EBDC have a low acute toxicity, this highly dispersive technique of application is of concern for people living near fields where EBDC are aerially sprayed as it can increase the EBDC environmental exposure. EBDC are broken down into ethylenthiourea (ETU), which is both the main degradation product of EBDC and the most important metabolite from a toxicological perspective (Houeto et al., 1995). Furthermore, ETU is present as an impurity in several EBDC formulations and it is formed when cooking food with residues of mancozeb and other EBDC (US EPA, 2005). EBDC are used on grapes and tobacco crops. For this reason ETU can be present in wine and cigarette smoke (Aprea et al., 1996). As ETU is a specific EBDC metabolite its urine concentration has been suggested as a sensitive indicator for biological monitoring of occupational and environmental exposure to ETU and/or EBDC (Colosio et al., 2002; Fustinoni et al., 2008). In humans, the elimination half-life of ETU in urine has been estimated to be in the range of 19–100 h depending on the exposure routes (Kurttio and Savolainen, 1990; Lindh et al., 2008). Although neither EBDC nor ETU accumulate within the body, ETU can cross the placenta and can be secreted into breast milk (NTP, 1992). In a previous evaluation it was classified as possible carcinogenic to humans (group 2B) by IARC (IARC, 1987), but subsequently it has been re-classified in the group 3 due to an inadequate evidence of carcinogenicity (IARC, 2001). ETU is also known for its teratogenic properties (US EPA, 1996; NTP, 1992; Iwase et al., 1997), slight and transient immunomodulating capabilities (Colosio et al., 2007), and anti-thyroid activity. The latter is due to its ability to interfere with thyroid hormone biosynthesis by inhibition of thyroid peroxidase, an enzyme involved in the iodination of thyroglobulin which is the precursor of thyroid hormones triiodothyronine (T3) and thyroxine (T4) (Marinovich et al., 1997; Freyberger and Ahr, 2006). The capability to regulate the expression of genes involved in the thyroid function has also been reported in non-mammalian vertebrates (Opitz et al., 2009). Moreover alterations in thyroid weight, cells, hormones, iodine uptake, and thyroid tumors have been reported in chronic mancozeb and ETU exposed rats, mice, and dogs (Axelstad et al., 2011; Belpoggi et al., 2002; Chhabra et al., 1992; IARC, 2001). Although in vivo studies have confirmed a thyreostatic effect of ETU at non teratogenic doses (Maranghi et al., 2013), only few studies have been conducted in humans. These have only revealed mild alterations in thyroid physiology and structure associated with mancozeb and ETU occupational exposure (Steenland et al., 1997; Panganiban et al., 2004). A cross-sectional study of EBDC-exposed Mexican backpack applicators has reported higher mean serum thyroid stimulating hormone (TSH) concentrations compared to non-exposed workers, as well as higher mean sister chromatic exchanges and chromosome translocations (Steenland et al., 1997). In another study conducted on mancozeb exposed Philippine banana plantation workers a positive correlation between ETU concentrations and size of solitary nodules measured with thyroid ultrasounds was observed (Panganiban et al., 2004). However, no information is available on whether an inadequate iodine intake, a condition still prevalent worldwide and still representing a major health concern in most countries (Zimmermann, 2013), may play a role in rendering the human thyroid more vulnerable to thyroid disrupting effects of EDs exposure. Italy is still characterized by a mild iodine deficiency (Pastorelli

2. Patients and methods 2.1. Study population One hundred and seventy-seven occupationally exposed grapevine workers and 74 non-occupationally exposed controls were enrolled in the study. Specifically, 29 out of 177 grapevine workers resided in the Chianti area (CH-workers), whereas the remaining 148 were recruited in Bolzano province (BZ-workers). All the workers were male (age range: 21–71 years) and were randomly recruited from vineyard farms where mancozeb was systematically used. All of them had at least 1 year history of direct exposure to mancozeb. In all the recruited workers pesticide exposure occurred during preparation of pesticide mixture and its application in vineyards or during re-entry activities, i.e. activities involving workers’ entry in the crops after pesticide application. Given the short half-life of ETU and the seasonal use of mancozeb between June and September, urine samples for the detection of exposure biomarkers (urinary iodine and ETU) were collected during the period of treatments (July–August). Spot urine samples were collected the day after the treatment in workers engaged in application of mancozeb (n=170) and the day after the re-entry in culture in workers engaged only in plant maintenance (n=7). Blood samples for thyroid function tests were collected after six weeks from the last treatment, in October, at the time of grape harvest. At this time all the recruited workers underwent a complete clinical examination and thyroid ultrasound. The control group consisted of 74 non-occupationally exposed male subjects (age range: 29-59 years) recruited from the healthcare personnel of the Bolzano Hospital and the Local Health Unit of Florence. Specifically, 34 resided in the Chianti area (CH-controls) and 40 in the Bolzano province (BZ-controls). None of the recruited subjects were known to be affected by thyroid diseases or were taking drugs interfering with thyroid function at the recruitment. In this study the sample size was the largest we could obtain taking into consideration available funds, availability of farmers where the use of mancozeb was exclusive or prevalent, and availability of workers and controls without thyroid diseases. Moreover, since the goal of the study was to evaluate thyroid effects of occupational exposure to mancozeb in iodine sufficient and iodine deficient areas, the study was designed with a larger number of workers. A structured questionnaire was administered to all participants by medical personnel. Information on drinking and smoking habits, dietary pattern, additional pesticides besides mancozeb, pesticide use (occupational and non-occupational use), and individual protection devices use was collected. All the recruited subjects signed an informed consent form in acceptance of entering the study. This study was approved by the ethics committees of Bolzano Hospital and the Local Health Unit of Florence. 2.2. Evaluation of urinary biomarkers (iodine and ETU) Urinary iodine concentration (UIC) has been indicated by WHO 153

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confounders, it was also taken into account the effect of the years worked in agriculture, the effect of the use of personal protective equipment, and of the non-occupational exposure to mancozeb (Table 1). A backward stepwise regression model, with a significance level of 0.05 for entering and 0.10 for removing the variables, was used to select the best subset of variables for each model. Distributions of potential confounders were compared by use of Student t test or median test. Data were normalized when necessary by means of a ln transformation (UIC, ETU, thyroid volume, TSH, Tg). Chi-square test was used to compare frequency distributions and to test for linear trend across groups (Chi-square for trend). A backward stepwise logistic regression model was also performed to estimate the risk of high values of UIC in workers with a higher exposure. The dependent variable was dichotomized into two categories: urinary iodine values ≤250 μg/L (coded as 0) and urinary iodine values > 250 μg/L (coded as 1). ETU exposure and covariates were included in the model as independent variables. Three levels of ETU were considered: 1–10 μg/L, reference category; 10.1–20 μg/L, low exposure; > 20 μg/L, high exposure. In a second logistic model, the effect of CH- versus BZ-workers on UIC levels was tested adjusting by covariates. An odds ratio (OR) was considered significant when 1.0 was not included in the 95% confidence interval (CI). Statistical analyses were conducted using Intercooled STATA for Windows (version 11.0; StataCorp, College Station, TX, USA). A P value < 0.05 was considered significant.

and International Council for Control of Iodine Deficiency Disorders (ICCIDD) as a highly sensitive biomarker to evaluate iodine intake in representative samples of subjects (WHO, 2007). In our study UIC was measured by using inductively coupled plasma mass spectrometry (ICP-MS) with a preliminary dilution of the sample 1/10 (v/v) at the Water Analysis Laboratory of the Environment Agency, Bolzano, Italy. The equipment used was supplied by Perkin Elmer Sciex (model Elan DRC 6100). The laboratory is accredited according to the UNI CEI EN ISO/IEC 17025 from SINAL “National System for the Laboratory Accreditation”. The laboratory also participated at the EQUIP (Ensuring the Quality of Urinary Iodine Procedures) of the CDC (Center for Disease Control and Prevention), Atlanta (USA). Assessment of urinary ETU concentration was performed at the Department of Prevention of the ASL of Florence. ETU was isolated from urine by liquid/liquid extraction using dichloromethane and analyzed via gas chromatography-mass spectrometry (Aprea et al., 2004). Urinary ETU concentration was expressed as μg/L after verifying the absence of significant differences in urinary dilution between the groups. This evaluation was performed by analysing urinary ETU corrected per creatinine (data not shown). ETU concentration expressed as μg/L was preferred because this unit of measure was the same used to calculate the reference limits for ETU in the Italian population (SIVR, 2011). 2.3. Thyroid function evaluation

3. Results

2.3.1. Clinical evaluation All recruited subjects underwent a complete medical examination.

3.1. Comparison between workers and controls 2.3.2. Thyroid ultrasonography The thyroid gland ultrasonography (US) was performed by two trained endocrinologists, one for each geographic area. To avoid any bias due to inter-observer variability (Whiting et al., 2004; Santini et al., 2008), US findings on thyroid volume and hypoechoic pattern were compared only between groups residing in the same geographic area. As the presence of measurable nodules ( > 5.0 mm maximum diameter) is a parameter less biased by inter-observer variability (Park et al., 2012), the frequency of subjects with nodules was compared between groups residing in the different areas.

Clinical examination findings showed hypertension in 5.6% (n=10), psoriasis in 4% (n=7), and respiratory allergic disease in 4% (n=7) of workers. Among controls only one subject had hypertension at the recruitment. Sociodemographic characteristics and family history of thyroid diseases in workers and controls are shown in Table 1. The mean age at enrolment was comparable between the groups, as well as the prevalence of wine consumers and the family history of thyroid diseases in first degree relatives. However the frequency of tobacco smokers was higher in workers than in controls (39.8% vs 23.6%, P=0.02). Also the prevalence of subjects with non-occupational exposure to mancozeb was significantly higher in workers than in controls (84.9% vs 22.4%, P < 0.01). Multivariate comparisons of urinary iodine and ETU concentrations, thyroid US findings, and thyroid function tests between workers and controls are shown in Table 2. Specifically, urinary ETU concentrations were found to be significantly higher in workers (geometric mean, GM: 12.2 μg/L) than in controls (GM: 8.2 μg/L, β=0.35, P=0.03). Whereas median urinary iodine values did not show any significant difference between the groups, as well as the frequency of measurable nodules on thyroid US. Similarly no significant differences were observed in the frequency of positivity for serum thyroid antibodies (TgAb and/or TPOAb), and in the mean levels of serum TSH, Tg and T3, whereas lower T4 (88.8 ± 17.9 and 94.2 ± 16.4 ng/ml, β=-5.41, P=0.03) and higher FT4 (12.8 ± 2.0 and 12.0 ± 1.6 pg/ml, β=0.92, P=0.001) and FT3 (3.8 ± 0.6 and 3.6 ± 0.4 pg/ml, β=0.20, P=0.008) serum mean values were found in workers as compared to controls.

2.3.3. Thyroid function tests Serum samples were stored at -80 °C until examination. All thyroid function tests were performed at Metabolism and Endocrinology Unit of the Istituto Superiore di Sanità. Radioimmunological Assay (RIA) kits for detection of serum Triiodothyronine (T3), Thyroxine (T4), Thyroglobulin (Tg), and TSH were purchased from Radim (Pomezia, Italy). BRAHMS RIA kits (Berlin, Germany) were used for detection of free T3 (FT3), free T4 (FT4), anti-Thyroglobulin antibodies (TgAb), and anti-thyroid peroxidase antibodies (TPOAb). The intraassay variability for RIA assays was less than 7%. 2.4. Statistical analysis Arithmetic and geometric means and percentage distribution of variables by residential area and professional exposure were used to describe the data. Multivariate comparisons of urinary iodine and ETU concentrations, thyroid US findings, and thyroid function tests between workers and controls were carried out by using separate linear (for continuous dependent variables) or logistic (for binary dependent variables) regression models as appropriate, after checking for potential confounders (age at enrolment measured on an ordinal scale; wine consumption and tobacco smoking, history of thyroid disorders in first degree relatives measured as “yes” or “no”) (Table 1). Multivariate comparisons between the two groups of workers with different iodine nutritional status were also performed by using separate linear or logistic regression models. Beside the above mentioned potential

3.2. Comparison between the groups of workers To verify whether the iodine intake may modulate the risk of thyroid disruption due to occupational exposure to ETU, a comparison between the two groups of workers was performed. Analysis of potential confounders (Table 1) showed a significantly higher number of years worked in agriculture (27.9 ± 10.3 vs 11.0 ± 10.6, P < 0.01) 154

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Table 1 Sociodemographic characteristics and family history of thyroid diseases in workers and controls, and in workers grouped by geographical area.

N. subjects Age at enrolment (years) Mean ± SD Wine consumers % Tobacco smokers % Family history of thyroid diseases % Years of agricultural activity Mean ± SD Use of protective devices % Non-occupational exposure* %

Workers

Controls

p-Value

CH-workers

BZ-workers

177 45.3 ± 10.5

74 44.4 ± 8.0

89.3

p-Value

0.55

29 43.0 ± 15.7

148 45.7 ± 9.1

0.20

90.5

0.76

82.8

90.5

0.22

39.8

23.6

0.02

53.9

37.1

0.11

21.3

28.8

0.20

10.3

23.5

0.14

26.5 ± 11.4

-

-

11.0 ± 10.6

27.9 ± 10.3

95.9

-

-

85.2

97.9

0.01

84.9

22.4

< 0.01

58.3

89.4

< 0.01

< 0.01

Abbreviations: SD: Standard Deviation; CH: Chianti; BZ: Bolzano * Activity in the own vegetable garden

Results of multivariate comparisons between CH- and BZ-workers are shown in Table 3. Urinary ETU concentration resulted to be significantly higher in BZ- (GM: 13.8 μg/L) than in CH-workers (GM: 6.4 μg/L, β=-0.81, P=0.03). With regard to thyroid function tests, all mean hormone values were in the normal range in both groups (Table 3). Nevertheless CH-workers showed significantly lower mean serum levels of FT4 compared to BZ-workers (11.1 ± 1.4 and 13.2 ± 1.9 pg/ml, β=-2.42, P < 0.001). Since thyroid US is highly dependent on the observer, in this study no statistical test was performed to compare the thyroid volume and the frequency of subjects with thyroid hypoechoic pattern in the two groups of workers. Among BZ-workers only 2 subjects (1.4%) had a thyroid volume ≤6.0 ml (5.9 ml in both). None of them were positive for TgAb and/or TPOAb or had a hypoechoic pattern on US. When the CH-workers were analysed the frequency of subjects with thyroid volume ≤6.0 ml (range: 4.2–5.9 ml) was 24.1% (7/29). None of these CH-workers were positive for TgAb and/or TPOAb. However, this subset of CH-workers with a reduced thyroid volume showed a significantly higher frequency of thyroid hypoechoic pattern on US (86% vs 14%, P < 0.01) and a significantly higher mean serum TSH level (1.9– 0.7 vs 1.2–0.5 μUI/ml, P=0.03) as compared to the remaining CHworkers. Although none of the CH-workers declared to use iodized salt and iodine-containing medicine at the time of the recruitment, the median value of UIC observed in this group was similar to that observed in BZworkers (108 and 122 μg/L, respectively). Some experimental studies have demonstrated that both mancozeb and ETU are able to reduce iodide uptake into thyroid cells (Kackar et al., 1997; Hurley et al., 1998). Therefore it was hypothesized that ETU exposure may cause an increased urinary iodine excretion because of the reduced iodide uptake into thyrocytes. To verify this hypothesis, the distribution of UIC values was analyzed in all the workers grouped by increasing values of ETU. Three classes of ETU exposure were identified: low, ETU < 10 μg/L; medium, ETU 10.1–20 μg/L; high, ETU > 20 μg/L (Fig. 1). The frequency of high values of UIC ( > 250 μg/L) was significantly associated with the increasing exposure to ETU (χ2trend = 2.20, P=0.027). The adjusted risk of high UIC values ( > 250 μg/L) was also calculated (Table 4). It was more than 4-fold higher among workers with high ETU exposure than in those with low exposure. To verify whether the iodine nutritional status may modulate the ETUmediated urinary iodine excretion, the distribution of UIC was analysed separately in BZ- and CH-workers with high exposure (ETU > 20 μg/L). Median values of urinary ETU were 45 μg/L (range 23–134 μg/L) in CH-workers and 46 μg/L (range 21-441 μg/L) in BZ-workers. Although not significant, the frequency of high UIC values ( > 250 μg/ L) was higher in CH- than in BZ-workers (43% and 15%, respectively).

Table 2 Multivariate comparisons (separate linear or logistic regression models) of urinary iodine and ETU concentrations, thyroid ultrasound findings, and thyroid function tests between workers and controls.

N. subjects a Urinary iodine concentration (µg/L) Geometric mean (95% CI) Median Urinary ETU concentration (µg/L) Geometric mean (95% CI) b Thyroid volume (ml) Geometric mean (95% CI) Median Frequency of subjects with thyroid nodules Frequency of subjects with a solitary thyroid nodule b Frequency of subjects with thyroid hypoechoic pattern T3 (NR: 0.6–2.1 ng/ml) Mean ± SD T4 (NR: 60–120 ng/ml) Mean ± SD FT3 (NR: 2.3–5.3 pg/ml) Mean ± SD FT4 (NR: 7.8–19.4 pg/ml) Mean ± SD TSH (NR: 0.2–3.2 µUI/ml) Mean ± SD Tg (NR: 0–40 ng/ml) Mean ± SD Frequency of subjects positive for TgAb and/or TPOAbc

β Coeff., p-Value

Workers

Controls

177

74

114.8 (101.6–129.6) 119

97.3 (82.3–115.1) 104

0.18, 0.11

12.2 (10.2–14.6)

8.2 (6.7–10.0)

0.35, 0.03

14.3 (13.3–15.3) 15.1 15.3%

12.0 (10.9–13.2) 12.0 20.6%

-

11.3%

10.8%

10.2%

9.6%

1.9 ± 0.3

1.9 ± 0.2

88.8 ± 17.9

94.2 ± 16.4

3.8 ± 0.6

3.6 ± 0.4

12.8 ± 2.0

12.0 ± 1.6

1.6 ± 0.8

1.6 ± 0.8

-0.01, 0.77 -5.41, 0.03 0.20, 0.008 0.92, 0.001 0.01, 0.84

14.6 ± 44.9

11.7 ± 22.9

0.05, 0.73

10.2%

13.5%

-0.25, 0.57

-0.73, 0.06 -0.32, 0.50 -

Abbreviations: ETU: Ethylenethiourea; CI: Confidence Interval; SD: Standard Deviation; NR: Normal Range. Covariates: age at enrolment, wine consumption, tobacco smoking, history of thyroid disorders in first degree relatives. a Urinary iodine concentration was detected in a lower number of subjects: 159 workers and 73 controls. b Comparison between groups was not performed to avoid any possible bias due to the inter-observer variability. c Positive values of TgAb and TPOAb: > 60 U/ml.

among BZ-workers compared to CH-workers, as well as a more frequent daily use of personal protective equipment (97.9% vs 85.2%, P=0.01), and a higher frequency of subjects non-occupationally exposed to mancozeb (89.4% vs 58.3%, P < 0.01). 155

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Table 3 Multivariate comparisons (separate linear or logistic regression models) of urinary iodine and ETU concentrations, thyroid ultrasound findings and thyroid function tests between Chianti (CH) and Bolzano (BZ) populations. Chianti area

N. subjects a Urinary iodine concentration (µg/L) Geometric mean (95% CI) Median Urinary ETU concentration (µg/L) Geometric mean (95% CI) Selected percentiles of ETU (µg/L) 10th percentile 25th percentile 50th percentile 75th percentile 90th percentile 95th percentile Thyroid volume (ml) Geometric mean (95% CI) Median Thyroid volume < 6 ml Frequency of subjects with thyroid nodules Frequency of subjects with a solitary thyroid nodule Frequency of subjects with thyroid hypoechoic pattern T3 (NR: 0.6–2.1 ng/ml) Mean ± SD T4 (NR: 60–120 ng/ml) Mean ± SD FT3 (NR: 2.3–5.3 pg/ml) Mean ± SD FT4 (NR: 7.8–19.4 pg/ml) Mean ± SD TSH (NR: 0.2–3.2 µUI/ml) Mean ± SD Tg (NR: 0–40 ng/ml) Mean ± SD TgAb (positive > 60 U/ml) TPOAb (positive > 60 U/ml)

Bolzano area β Coeff., p-Value

BZ-workers

BZ-controls

β Coeff., p-Value

148 114.2 (100.4–129.9) 122 13.8# (11.6–16.5) 3.9 6.7 12.9 24.3 58.0 107.6

40 94.3 (70.8–125.6) 103

0.13, 0.37

10.8 (8.3–14.1) 3.3 6.0 13.3 20.5 27.0 30.8

0.21, 0.27

12.6 (10.8–14.8) 11.8 2.5% 20.0%

0.16, 0.05

-1.89, 0.05

15.5 (14.5–16.7) 15.9 1.4% 14.9%

11.8%

-1.27, 0.27

11.5%

10.0%

0.15, 0.80

34.5% 1.9 ± 0.4

15.2% 2.0 ± 0.2

1.99, 0.02 -0.04, 0.61

5.4% 1.9 ± 0.3

5.0% 1.8 ± 0.2

-0.09, 0.92 0.06, 0.31

81.2 ± 12.1

102.4 ± 12.4

-22.14, < 0.01

90.3 ± 18.5

87.2 ± 16.3

3.41, 0.27

3.7 ± 0.5

3.6 ± 0.4

0.02, 0.84

3.9 ± 0.6

3.6 ± 0.4

0.24, 0.02

11.1 ± 1.4##

11.0 ± 1.5

0.12, 0.77

13.2 ± 1.9##

12.8 ± 1.2

0.48, 0.15

1.4 ± 0.6

1.6 ± 0.9

-0.17, 0.18

1.7 ± 0.8

1.5 ± 0.8

0.09, 0.35

5.8 ± 4.5

12.1 ± 23.2

-0.70, < 0.01

16.3 ± 48.9

11.4 ± 22.9

0.25, 0.17

6.9% 0%

5.9% 23.5%

0.29, 0.78 -

6.1% 8.1%

2.5% 2.5%

1.15, 0.30 1.20, 0.27

CH-workers

CH-controls

29 120.4 (80.3–180.7) 108 6.4# (3.6-11.6) 0.9 2.4 4.7 22.6 88.7 133.2

34 101.2 (86.9–117.7) 106 5.8 (4.4–7.5) 2.4 4.6 6.3 9.3 12.3 13.4

9.4 (7.8–11.2) 9.3 24.1% 17.2%

11.2 (10.3–12.3) 12.0 0% 21.2%

-0.24, 0.02

10.3%

0.29, 0.10

0.02, 0.95

0.84, 0.51 -0.53, 0.27

Abbreviations: CI: Confidence Interval; SD: Standard Deviation; ETU: Ethylenethiourea; CH: Chianti; BZ: Bolzano; NR: Normal Range Covariates: age at enrolment, wine consumption, tobacco smoking, history of thyroid disorders in first degree relatives, years worked in agriculture, use of personal protective equipment, non-occupational exposure. a Urinary iodine concentration was detected in 15 CH-workers, 33-CH controls, 144 BZ-workers, 40 BZ-controls # CH-workers vs BZ-workers: β Coefficient -0.81, P=0.03; ## CH-workers vs BZ-workers: β Coefficient -2.42, P < 0.001

3.3. Comparison between workers and controls in each geographical area

Moreover the risk of a higher urinary iodine excretion was more than 10-fold higher in CH- than in BZ-workers with high ETU exposure (ORadj 11.6; 95% CI: 0.9-153.3).

To complete the study a comparison between workers and controls stratified by geographical provenance was also performed. No significant differences were found in mean urinary ETU concentrations between workers and controls both in Chianti and Bolzano areas (Table 3). However, the frequency of ETU values > 20 μg/L was 27.6% in CH-workers, whereas none of the CH-controls had so high Table 4 Adjusted Odds Ratios (OR) of high urinary iodine values ( > 250 μg/L) in workers with increasing exposure to ETU.

ETU (μg/L) 1–10 10.1–20 > 20

ORadj

(95% CI)

p-Value

Reference 2.7 4.1

0.6–12.0 1.0–17.1

0.20 0.05

Abbreviations: OR: Odds Ratio; CI: Confidence Interval; ETU: Ethylenethiourea Covariates: age at enrolment, wine consumption, tobacco smoking, history of thyroid disorders in first degree relatives, years worked in agriculture, use of personal protective equipment, non-occupational exposure.

Fig. 1. Frequency of low, normal and high urinary iodine concentration (UIC, µg/L) in the workers grouped by increasing exposure to Ethylenethiourea (ETU).

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(Jones et al., 2004; Krassas et al., 2010). Taken together these data strongly suggest that further studies are needed to evaluate the consequences on reproductive health and thyroid function of a longlasting exposure to EBDC in both male and female workers. Another result found in this study was the association of a higher exposure to ETU with an increased urinary iodine excretion among workers. This finding is in accordance with previously reported high values of urinary iodine levels in workers occupationally exposed to ETU (Panganiban et al., 2004). More specifically, in our study values of UIC > 250 μg/L were more frequently found among more exposed workers (ETU > 20 μg/L) than in less exposed ones and this effect was more pronounced in CH- than in BZ-workers. These results suggest that a high ETU exposure might increase the urinary iodine excretion leading to a reduced availability of iodine for thyroid hormone biosynthesis. This hypothesis is supported by experimental studies conducted in rats which have demonstrated that both mancozeb and ETU are able to reduce iodide uptake into the thyroid cell (Kackar et al., 1997; Hurley et al., 1998). However, it is still unclear whether the reduced iodide uptake is due to a specific block in the active transport of iodide into the cell (Na+/I- symporter) and/or it is a consequence of the inhibition of thyroid peroxidase, as the iodide is not trapped within the cell in an organic form (Doerge and Takazaka, 1990; Kackar et al., 1997; Freyberger and Ahr, 2006). Further studies are needed to differentiate these possibilities. Among the CH-workers a high frequency of very low thyroid volume (≤6.0 ml), which was associated to both high serum TSH levels and a high frequency of thyroid hypoechoic pattern, was also observed. The higher mean serum TSH levels found in this sub-group of CHworkers may be a response to a possible atrophic process of the gland due to ETU exposure in the presence of iodine deficiency. In fact these findings together with a more pronounced ETU-mediated effect on the urinary iodine excretion in the CH-workers led us to hypothesize that, in absence of an adequate iodine intake, a steady increase in urinary iodine excretion may mimic a condition of severe iodine deficiency. A variety of former studies from areas of very severe iodine deficiency report about the occurrence of a variable rate of thyroid atrophy associated with a severe iodine deficiency (Stanbury et al., 1974; Delange, 1994; Shankar et al., 1994). The mechanism of thyroid tissue loss due to sustained severe iodine deficiency has not been resolved so far. However, there are speculations about the role of elevated TSH levels mediating an increased intracellular oxidative stress via increased levels of O2- and H2O2 (Resch et al., 2002; Senou et al., 2010) and about the role of free radicals, which might possibly lead to an atrophic thyroid gland. In our study, because of the long-lasting occupational exposure to mancozeb, a direct toxic effect of ETU on thyroid cells cannot be excluded. In fact an oxidative and proapoptotic effect of mancozeb causing post-apoptotic necrotic alterations in cell membrane integrity has been reported (Calviello et al., 2006; Domico et al., 2007). In our study the prevalence of nodules was similar in workers and controls, as well as the prevalence of solitary nodules. This finding does not confirm what was found in other studies reporting a high frequency of solitary nodules among banana plantation workers exposed to ETU in the Philippines (Panganiban et al., 2004). The Authors hypothesized an increased risk of thyroid cancer for these workers because thyroid cancer generally presents as solitary nodule or lump in the thyroid gland (Studer and Gerber, 1991). Since the study conducted in the Philippines reported higher urinary ETU levels than those found in our workers, we cannot exclude that the lack of a high frequency of solitary nodules in our study may be due to a lower exposure to ETU.

urinary ETU values. In Bolzano area the frequency of high ETU values was similar both in workers and controls (28.6% and 25%, respectively). This finding may be explained by the fact that 50% of the BZcontrols resided close to cultivated fields and vineyards as revealed by the questionnaires. History of known thyroid gland disorders in first degree relatives did not show any significant difference between workers and controls in both areas. Also urinary iodine median values were similar in workers and controls both in Chianti and Bolzano areas (Table 3). Thirty-eight percent of CH-controls and none of CH-workers declared to use iodized salt. The US findings showed a lower mean thyroid volume in CHworkers (GM: 9.4 ml) as compared to CH-controls (GM: 11.2 ml; β=0.24, P=0.02) with a higher frequency of very low thyroid volume (≤6.0 ml) among workers than in controls (24.1% vs 0%). Also the frequency of hypoechoic thyroid was higher in CH-workers than in controls (34.5% and 15.2%, β=1.99, P=0.02). No significant differences were found between workers and controls residing in Bolzano area. Both in Chianti and Bolzano areas the prevalence of subjects with nodules was similar in workers and in controls, as well as the prevalence of subjects with solitary nodules (Table 3). The analysis of thyroid function tests revealed a significantly lower mean value of serum T4 (81.2 ± 12.1 and 102.4 ± 12.4 ng/ml, β=22.14, P < 0.01) and Tg (5.8 ± 4.5 and 12.1 ± 23.2 ng/ml, β=-0.70, P < 0.01) in CH-workers compared with CH-controls. Moreover none of CH-workers was positive for TPOAb, whereas the frequency of subjects with TPOAb among CH-controls was 23.5%. No significant difference was observed between workers and controls in Bolzano area with the exception of the mean value of FT3 being higher in BZ-workers than in BZ-controls (3.9 ± 0.6 and 3.6 ± 0.4 pg/ml, β=0.24, P=0.02). 4. Discussion The results obtained in this study have confirmed a mild thyroid disrupting effect due to occupational exposure to mancozeb and its metabolite ETU. This effect was more evident in grapevine workers residing in an area characterized by a mild to moderate iodine deficiency (Chianti) than in workers residing in an area covered by a long-lasting iodine prophylaxis program (Bolzano autonomous province). Specifically, lower T4 and higher FT4 and FT3 serum levels were found in workers exposed to ETU in comparison to controls, even though all the workers were biochemically euthyroid at the recruitment. Differently from what has been previously reported by other authors (Panganiban et al., 2004; Steenland et al., 1997), no increased mean values of serum TSH, suggesting a possible subclinical hypothyroidism, were observed in our cohort of workers when compared to controls. This result may be explained by the fact that our workers were less exposed than workers recruited in previous studies as shown by urinary ETU levels (GM: 94.73 μg/L in Panganiban et al., 2004; GM:19.0 μg/L in Steenland et al., 1997). Moreover the increasing use of personal protective equipment over the years may have helped to reduce the exposure (Bohme, 2015). When the two groups of workers with different iodine nutritional status were compared, it was observed that the mild ETU-induced effects were more evident in workers coming from a historically mild iodine deficient area as compared to workers with an adequate iodine intake. Although in both groups the mean hormone values were in the normal range, CH-workers showed significantly lower mean serum levels of FT4 than BZ-workers. Disruption of thyroid homeostasis together with an altered reproductive hormone profile were observed in rats exposed in utero to a low dose of ETU (Maranghi et al., 2013). Low thyroid hormones may have implications for reproductive health as thyroid hormones regulate androgen biosynthesis and signalling through direct and indirect regulation of steroidogenic enzyme expression and activity (Flood et al., 2013). Normal levels of thyroid hormones are also needed to preserve a normal menstrual pattern

5. Conclusions Our results may have implications for communities living in intensive farming areas where mancozeb is aerially sprayed. In fact, contamination of the living environment by EBDC can cause the 157

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