Associations of urinary phthalate metabolites with risk of papillary thyroid cancer

Associations of urinary phthalate metabolites with risk of papillary thyroid cancer

Chemosphere 241 (2020) 125093 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Associati...

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Chemosphere 241 (2020) 125093

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Associations of urinary phthalate metabolites with risk of papillary thyroid cancer Hongjian Miao a, 1, Xin Liu a, b, 1, Jingguang Li a, Lei Zhang a, *, Yunfeng Zhao a, Shaoyan Liu c, Song Ni c, **, Yongning Wu a a

NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing, 100021, China Institute of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023, Hubei, China Department of Head and Neck Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Science, Peking Union Medical College, Beijing, China b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Associations of urinary phthalate metabolites with PTC risk were examined.  DEHP metabolites were positively associated with increased risk of PTC.  The associations were influenced by urinary iodine levels.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 June 2019 Received in revised form 8 October 2019 Accepted 9 October 2019 Available online 11 October 2019

Some studies have revealed thyrotoxicity of phthalates; however, associations of phthalate exposure with papillary thyroid cancer (PTC) remain unclear. We conducted a pair-matching case-control study of 111 PTC cases and 111 age- and sex-matched non-PTC controls to examine associations between urinary concentrations of phthalate metabolites and PTC. Phthalate metabolites were determined in fasting urine specimens by ultra-performance liquid chromatography e tandem mass spectrometry (UPLC-MS/MS). After adjusting for potential confounders and other phthalate metabolites, the concentrations of the sum of di (2-ethylhexly) phthalate (DEHP) metabolites in urine were positively associated with PTC [odds ratio (OR) ¼ 5.35; 95% confidence interval (CI): 1.61e17.83], suggesting the effect of phthalates exposure on PTC development. The findings require confirmation. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: A. Gies Keywords: Thyroid cancer Case-control study Phthalate Urinary metabolites

1. Introduction * Corresponding author. No. 7 Panjiayuan Nanli, Chaoyang District, Beijing, 100021, China. ** Corresponding author. 17 Pan Jia Yuan Nan Road, Chaoyang District, Beijing, 100021, China. E-mail addresses: [email protected] (L. Zhang), [email protected] (S. Ni). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.chemosphere.2019.125093 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

Phthalates are a class of synthetic chemicals that have been manufactured (several million tons per year) largely and used widely as plasticizers and softeners in multiple commercial products. Diethyl phthalate (DEP) and di-n-butyl phthalate (DBP) with low molecular weights are mainly used to manufacture personal

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care products, while di (2-ethylhexyl) phthalate (DEHP) with high molecular weight phthalates being mainly used in the manufacture of polyvinyl chloride (PVC) plastics for food packaging, building materials, and medical devices (Kim and Park, 2014). Phthalates are non-chemical bound, and therefore, can be continuously emitted from end-products, resulting in ubiquitous contamination in the environment, including in food, water, air and dust. Moreover, due to the high amount of ingestion, microplatics as the carrier of phthalates should be taken in to consideration (Zuccarello et al., 2019). Although some phthalates can enter the human body through dermal absorption and inhalation, the main route of exposure of the non-occupational population is oral ingestion (Zuccarello et al., 2018). After exposure, phthalates are rapidly converted into their metabolites and urinary phthalate metabolites are the most useful biomarkers for detecting human exposures over several weeks or months (Teitelbaum et al., 2008; Wittassek and Angerer, 2010). Thyroid cancer (TC) is the commonest endocrine malignancy worldwide. Approximately 90,000 new cases of TC were diagnosed in China in 2015, accounting for ~2.1% new cancer diagnoses (Chen et al., 2016). In recent decades, a dramatic increase in the incidence of TC has been observed in many countries, which is largely due to the increased detection of papillary thyroid cancer (PTC) (Morris et al., 2013; Veiga et al., 2013; Wiltshire et al., 2016). (Chen et al., 2016) In general, increased detection was primarily due to more sensitive diagnostic procedures and access to care, resulting in some over-diagnosis. However, some reports support a real increase in incidence (Ron and Schneider, 2006). Many studies have been conducted to identify potential risk factors, including ionizing radiation (especially nuclear radiation) (Brenner et al., 2011), iodine ro et al., 2012), diet (Cho and Kim, 2015; Peterson et al., intake (Cle 2012), physical activity (Fiore et al., 2019a), obesity (Pappa and Alevizaki, 2014), and environmental endocrine disrupting chemicals (EDCs) (Fiore et al., 2019a). Phthalates and their metabolites are typical EDCs. Like some EDCs, phthalates and their metabolites could interfere the homeostasis of thyroid hormones at various levels (Boas et al., 2012; Dong et al., 2017; Fiore et al., 2019a, 2019b; Huang et al., 2017; Liu et al., 2015; Nassan et al., 2019). Moreover, only one study reported the association of di (2-ethylhexyl) phthalate (DEHP) with high risk of thyroid cancer (TC) in patients with thyroid nodules (Marotta et al., 2019). The associations between phthalates and their metabolites and TC remains unclear. Thus, we conducted a hospital based case-control study with age- and sex-matched controls to evaluate the associations of urinary concentrations of phthalates metabolites with the risk of PTC.

2. Material and methods 2.1. Study population We conducted a 1:1 pair-matched case-control study to determine the exposure of the non-occupational population to phthalates and the risk of PTC. The study was approved by the Ethics Committee of China National Center for Food Safety Risk Assessment (No. 2015006). A total of 116 patients with newly diagnosed TC were enrolled in the Cancer Hospital of Chinese Academy of Medical Sciences during the period of JuneeSeptember 2017. Thyroid cancer was classified with code C73.9, according to the International Classification of Disease, 10th edition (ICD-10) (Fritz et al., 2000). Health subjects (non-PTCs) were unrelated healthy individuals and enrolled in the hospital during the same study period, and medical history inquiry and the performance of thyroid ultrasonography and serum THs

determination for excluding the subject with history of endocrine diseases, severe organic diseases or aberrant thyroid function. To avoid possible interference, no participants had a family history of TC, a history of radiation exposure during childhood, therapeutic exposure to iodine, and potential occupation exposure. Moreover, due to the associations between age/sex and PTC prevalence (Ron and Schneider, 2006), cases and controls were matched by age (differences between birthdays within 1 year) and sex. With participants’ informed consent, a single, fasting, morning urine specimen (approximately 10 mL) was collected from each subject. Among the cases, urine specimens were sampled before the thyroidectomy to exclude the possibility that phthalate exposure may have been influenced by cancer treatment. All samples were stored at 40  C until analysis. In our previous study (Zhang et al., 2018a), we determined the urinary concentrations of iodine, perchlorate, thiocyanate, and creatinine in same samples, during which 5 pairs of samples were used entirely. Thus, 111 pairs of matched subjects were involved in the present study. 2.2. Covariates Epidemiological studies have indicated associations of age and sex with TC prevalence. In general, ~25% of all TC occurs in patients< 35 years of age, with incidence rising comparatively slowly with age (Ron and Schneider, 2006). Moreover, females have an approximately 3-times higher incidence of TC than males (American Cancer Society, 2018). To account for such confounders, we employed a pair-matching design for age and sex, with the stratified control subjects at risk at the same sex and age. We also considered multiple covariates and potential confounders for the association between phthalates exposure and the risk of PTC. Several human epidemiological studies identified obesity and smoking habits as factors influencing the development of PTC (Wiersinga, 2013; Xu et al., 2014). Thus, body mass index (BMI) and cigarette smoking status were selected as confounders. Moreover, in our previous study (Zhang et al., 2018a), we observed significant associations between the risk of PTC and urinary concentrations of iodine, perchlorate, and thiocyanate that were also selected as potential confounders in the present study. 2.3. Assessment of phthalate metabolite urinary concentrations The urinary concentrations of six phthalate metabolites [monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-2ethylhexyl phthalate (MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and mono-(2-ethylpentyl-5-carboxy) phthalate (MECPP)] were measured according to methodologies published elsewhere that included solid-phase extraction (Waters Oasis Prime HLB cartridge, 3 mL, 60 mg) coupled with Shimadzu ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) (Miao et al., 2019). 2.4. Statistical analysis Descriptive statistics on subject demographics and potential confounding factors were tabulated with the distributions of phthalate metabolite concentrations by TC status. The MannWhitney U test and Chi-square test were used to evaluate the differences between cases and controls. Due to severely skewed distribution, urinary concentrations of phthalates were transformed to a natural logarithmic scale to perform logistic regression analyses. First, we performed univariable conditional logistic regression analyses to evaluate the rough associations between phthalate metabolites and PTC. We then ran two multivariable conditional

H. Miao et al. / Chemosphere 241 (2020) 125093

logistic regression models for each detected phthalate metabolite. One model was adjusted for BMI, smoking habits, urinary iodine, urinary thiocyanate, urinary perchlorate, and urinary creatinine. In the second model, all phthalate metabolites and previous variables were input into the model, but due to severe multicollinearity (VIF>4.0) among MEHP, MEOHP, MECPP and MEHHP, the summaP tion of DEHP metabolites, DEHP, was used to present the DEHP exposure. The results of collinearity statistics are shown in Supplementary Table S1. To evaluate potential dose-response relationship, tertiles of phthalate exposure were created based on the observed concentration distribution of phthalate metabolites concentrations in urine. Median values were assigned to phthalate metabolites concentration tertiles, and multivariable linear regression analyses were preformed to estimate trend p-values. Moreover, natural cubic spline regression analyses were used to visualize and examine the dose-response relationships between the urinary concentrations of phthalate metabolites and the risk of PTC. The degrees of freedom of urinary analytes were set at three. Moreover, dichotomous variables of iodine status were based on the observed urinary iodine concentration distributions in our previous study (Zhang et al., 2018a). Binary logistic regression models were stratified by iodine status. Data analyses were performed using SPSS software (version 22) and R (version 3.4.1).

3. Results This study included 111 PTC cases and 111 non-PTC controls comprised of 25 sets of men and 86 sets of women. By design, the mean age of the study populations was similar in both groups: 42.5 ± 11.4 years in cases and 42.5 ± 11.1 years in controls. Participants with PTC had higher BMI, higher urinary concentrations of iodine, and lower urinary concentrations of perchlorate and thiocyanate. No difference in active smoking habits was observed between cases and controls (Table 1). The distributions of phthalate metabolite concentrations are presented in Table 2. Among the 222 urine samples, phthalate metabolites were detected in >97% of samples. Significant differences were observed in urinary concentrations of DEHP metabolites, including MEHP, MEOHP, MECPP, and MEHHP, between groups (p < 0.001), but not for MBP and MEP, which are metabolites of DBP and DEP, respectively (p > 0.05). PTC cases tended to be with higher concentrations of DEHP metabolites. In addition, significant (p < 0.01) and strong correlation coefficients (>0.7) were observed among DEHP metabolites, and significant but moderate coefficients (<0.6) were observed between MBP and MEP, MBP/MEP and DEHP metabolites (Supplementary Table S2), indicating simultaneous exposure to these phthalates. Therefore, the PTC subjects might not

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have extra and strengthened exposure of DEHP, although DEHP has been largely used in various medical devices. No significant differences were observed in urinary levels of all phthalate metabolites between men and women (Supplementary Table S3). Conditional logistic regression models were used to evaluate the associations between urinary concentrations of phthalate metabolites and the risk of PTC (Table 3). There was no significant association between MBP and PTC using the univariable model, but after adjusting for covariates, the association approached suggestive significance (p ¼ 0.06). MEP did not show an association with PTC in univariable or multivariable models (p > 0.05). DEHP metabolites, including MEHP, MEOHP, MECPP, and MEHHP, were all significantly associated with PTC. After adjusting for covariates and P other phthalate metabolites, DEHP metabolites ( DEHP) remained positively associated with PTC [odds ratio (OR) ¼ 5.35; 95% confidence interval (CI): 1.61e17.83]. When examining the ORs of PTC according to tertiles (Supplementary Table S4), subjects in the higher tertiles of DEHP exposure had significantly elevated ORs compared to the reference group (the first tertile). Only MEHP showed a significant linear trend (p ¼ 0.02), while the other metabolites showed suggestive significance. These results suggested nonmonotonic relationships between phthalate exposure and PTC that were confirmed by performing natural cubic spline regression analyses (Fig. 1). With increased urinary concentrations of DEHP metabolites, PTC risk rose sharply and remained stable (or very slow elevation). However, an invert-U relationships observed for MEP. Iodine status may influence associations of phthalates exposure with PTC (Table 4). At lower urinary concentrations of iodine, MBP was positively associated with PTC (p ¼ 0.03) after adjusting for covariates, while MEP showed suggestive significance (p ¼ 0.06). However, at higher iodine level, MBP and MEP were not significantly associated with PTC. For DEHP metabolites, the significance of relationship was not changed by iodine status.

4. Discussion This study suggests that the urinary concentrations of phthalate metabolites may be related to PTC. MBP, the main metabolite of DBP, was significantly associated with PTC in subjects with lower urinary iodine concentrations, while metabolites of DEHP (MEHP, MEOHP, MECPP, and MEHHP) showed positive associations with PTC that was not influenced by stratification by iodine status. Phthalates are well-known endocrine-disrupting chemicals and showed anti-thyroid activity in experimental and epidemiological studies. Due to the vital role of thyroid hormones in many physiological processes, most studies focused on the aberrant change in thyroid hormones homeostasis arising from phthalates exposure.

Table 1 Characteristics of papillary thyroid cancer (PTC) subjects and corresponding health controls. Characteristic

PTC subjects (n ¼ 111)

Non-PTC controls (n ¼ 111)

p-value

Gender ratio (Male: Female)a Age (years)b Height (cm)b Bodyweight (kg)b Body Mass Index (BMI)b Smoking habits (%) Urinary iodine (mg/L)c Urinary thiocyanate (mg/L)c Urinary perchlorate (mg/L)c Urinary creatinine (mmol/L)c

25:86 42.5 ± 11.4 163.7 ± 7.0 66.2 ± 11.5 24.7 ± 4.1 10.0% 570.03 (141.7, 2292.8) 467.5 (205.7, 1095.7) 4.7 (1.6, 13.6) 6.2 (3.0, 12.9)

25:86 42.5 ± 11.1 164.2 ± 7.1 63.6 ± 11.5 23.5 ± 3.0 9.0% 199.5 (111.5, 357.0) 809.4 (384.2, 1705.0) 9.6 (5.0, 18.6) 9.1 (4.7, 17.5)

e e 0.56 0.10 0.01 0.82 <0.001 <0.001 <0.001 <0.001

a b c

25 males and 86 females were recruited. Arithmetic mean ± standard deviation. Geometric mean with ±1 GSD (geometric standard deviation) in parentheses.

4 H. Miao et al. / Chemosphere 241 (2020) 125093 Table 2 Distribution of phthalate metabolites in urine (ng/mL, n ¼ 222). Phthalate metabolites

Subgroups

MBP

Case Control Case Control Case Control Case Control Case Control Case Control Case Control

MEP MEHP MEOHP MECPP MEHHP P DEHPb a b

Detection rate

99.1% 100% 97.3% 96.4% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% e e

Geometric Mean

Percentiles

88.9 (17.8, 444.5) 78.9 (21.3, 291.9) 9.3 (2.3, 37.2) 8.7 (1.7, 43.5) 5.0 (2.8, 9.0) 2.6 (1.2, 5.5) 9.4 (2.5, 35.7) 4.5 (1.3, 16.2) 15.7 (5.6, 44.0) 6.1 (1.8, 20.1) 11 (4.1, 29.7) 3.2 (0.9, 10.9) 44.5 (17.1, 115.7) 17.5 (6.0, 50.8)

p-value

Min

10th

25th

50th

75th

90th

Max

NDa 4.9 NDa NDa 1.8 0.5 0.3 0.1 1.3 0.2 1.0 0.1 5.9 1.5

11.8 14.5 2.3 1.5 2.5 1.2 1.7 1.2 5.0 1.6 3.1 0.8 13.9 5.6

39.6 32.1 4.4 3.7 3.2 1.5 3.8 2.4 7.9 3.0 6.2 1.4 22.5 8.9

108.7 72.4 10.3 8.1 4.8 2.2 9.8 4.0 13.5 5.4 11.3 3.0 39.4 15.3

315.7 221.7 19.1 25.6 7.4 4.4 30.7 10.6 34.7 14.0 21.5 7.8 85.9 37.4

508.7 488.4 41.6 62.1 10.1 7.9 48.7 23.3 57.4 27.7 34.7 17.5 145.0 70.1

882.5 1446.3 222.2 341.5 46.0 14.3 376.7 76.5 434.4 92.0 131.3 41.6 988.4 216.3

0.20 0.62 <0.001 <0.001 <0.001 <0.001 <0.001

Not detection. Summation of metabolites of DEHP, including MEHP, MEOHP, MECPP, and MEHHP.

Table 3 Odds ratios (ORs) of papillary thyroid cancer risk for phthalate metabolites (PMs) with 95% confidence intervals (CI) (n ¼ 222). PMsa

Univariable Model Rough ORs

p-value

Adjusted ORs

p-value

Adjusted ORs

p-value

MBP MEP MEHP MEOHP MECPP MEHHP P DEHPc

1.07 1.03 6.22 1.50 2.06 2.57 2.30

0.49 0.74 <0.001 <0.001 <0.001 <0.001 <0.001

1.48 1.40 7.30 2.07 3.11 3.63 3.51

0.06 0.14 0.001 0.008 0.001 0.001 0.001

0.60 (0.25e1.41) 1.04 (0.54e2.02) e e e e 5.35 (1.61e17.83)

0.26 0.90 e e e e 0.006

a b c

(0.89e1.28) (0.86e1.23) (3.21e12.05) (1.20e1.89) (1.53e2.77) (1.83e3.62) (1.66e3.20)

Multivariable Model 1b

(0.98e2.24) (0.90e2.19) (2.17e24.56) (1.21e3.53) (1.56e6.19) (1.69e7.74) (1.64e7.49)

Multivariable Model 2c

Values were transformed to a natural logarithmic scale. For each PMs, these variables were applied for adjustment, including BMI, urinary iodine, urinary thiocyanate, urinary perchlorate, urinary creatinine and smoking habits. P Variables enter the Model 3: MBP, MEP, DEHP, BMI, urinary iodine, urinary thiocyanate, urinary perchlorate, urinary creatinine and smoking habits.

Fig. 1. The concentration-response curves for the associations of papillary thyroid cancer risk with urinary phthalate metabolite concentrations (adjusted for BMI, smoking habits, urinary iodine, urinary perchlorate, urinary thiocyanate, and urinary creatinine. Dashed lines ¼ 95% CI, gray circles ¼ partial residues).

Phthalates could inhibit the activity of thyroid peroxidase (TPO) and the expression of sodium iodine symporter (NIS) to reduce the production and secretion of thyroxine or triiodothyronine, and increase levels of some hepatic enzymes in the liver to reduce the half-life of thyroid hormones (Dong et al., 2017; Liu et al., 2015). Experimental studies showed that the levels of thyroid hormones were suppressed after phthalates exposure (Dong et al., 2017;

Zhang et al., 2018b). Several human studies have examine the relationships between urinary concentrations of phthalates/phthalate metabolites and serum thyroid hormone profiles, but inconsistent results shown (Kim et al., 2019; Wu et al., 2013). The types of metabolites associated and the direction of associations are different among studies, with null associations also being reported. Nevertheless, after a meta-analysis of several human

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Table 4 ORs of papillary thyroid cancer risk for phthalate metabolites with 95% CI in subjects with different urinary iodine levelsa. PMs

MBP MEP MEHP MEOHP MECPP MEHHP P DEHP

Iodine Level 1 (<247 mg/L, n ¼ 111)

Iodine level 2 (>247 mg/L, n ¼ 111)

Rough ORs

p-value

Adjusted ORs

p-value

Rough ORs

p-value

Adjusted ORs

p-value

1.29 1.05 3.27 1.78 2.07 2.32 2.56

0.09 0.71 <0.01 <0.01 <0.01 <0.01 <0.01

1.68 1.44 4.31 2.33 2.94 3.14 3.91

0.03 0.06 0.02 <0.01 <0.01 <0.01 <0.01

0.71 0.82 4.74 1.13 1.82 2.58 2.17

0.04 0.25 <0.01 0.44 <0.01 <0.01 <0.01

1.03 (0.50e2.10) 0.99 (0.92e1.07) 15.53 (2.20e109.4) 2.50 (1.11e5.67) 5.00 (1.52e16.5) 15.0 (2.12e106.6) 7.74 (1.80e33.24)

0.94 0.87 <0.01 0.03 <0.01 <0.01 <0.01

(0.96e1.75) (0.82e1.33) (1.72e6.21) (1.25e2.53) (1.38e3.09) (1.54e3.51) (1.57e4.18)

(1.05e2.69) (0.98e2.14) (1.74e10.69) (1.39e3.92) (1.63e5.32) (1.70e5.77) (1.87e8.18)

(0.51e0.99) (0.58e1.15) (2.15e10.46) (0.82e1.56) (1.18e2.79) (1.62e4.11) (1.30e3.63)

a Binary logistic regression analyses were preformed. Adjusted ORs were adjusted for sex, age, BMI, smoking habits, urinary iodine, urinary thiocyanate, urinary perchlorate, and urinary creatinine.

epidemiological studies, Kim et al. (2019) observed negative associations of several DEHP metabolites with total T4 and positive associations with TSH. Those data suggested a significant association between human phthalate exposure and thyroid function. The relationships between phthalates exposure and cancer have not been clarified and human epidemiologic evidence is limited. A recent study reported a possible association between TC and breast cancer, while breast cancer has been linked with higher urinary concentrations of DEHP metabolites in several human epidemiopezcarrillo et al., 2010; Zuccarello logic studies (An et al., 2015; Lo et al., 2018). The association between TC and breast cancer as well as higher incidence of TC in female might suggest a role for higher expression of sex hormone receptors, such as estrogen receptors, in the development of those cancers. DEHP and their metabolites are estrogen-like compounds could act as estrogen receptor agonists. However, abundant expression and activation of sex hormone receptors could not directly cause cancer. It is known that the action of phthalates, including DEHP, are carried out on different cellular pathways and receptors. Phthalates, such as DBP and DEHP, could induce oxidative stress in rat thyroid (Erkekoglu et al., 2012; Wu et al., 2017), and oxidative stress is involved in the pathophysiology of TC (Wang et al., 2011). The reactive oxygen species (ROS) may enhance the mitogenactivated protein (MAP) kinase and phosphatidylinositol-3-kinase (PI3K) pathways, propelling thyroid tumorigenesis (Xing, 2012). The activation of MAP kinase signaling pathway is central to PTC tumorigenesis (Knauf and Fagin, 2009; Sturgis and Li, 2009). Moreover, DEHP metabolites, such as MEHP, could activate human peroxisome proliferator-activated receptor (PPAR)-a and PPARg. The overexpression of PPARg could activate the MAPK(Mírian et al., 2013). In papillary thyroid cancer cells, PPARg overexpression can also promote the growth and invasion of cancer cells (Wood et al., 2011). However, a reciprocal interaction between PPAR and ER has been observed in human PTC cells. PPARg protein and activity were reduced by the over-expression of either ERa or ERb, whereas repression of ERa or ERb increased PPARg expression (Ryan et al., 2013). Additionally, MBP is an PPARg antagonist but an ER agonist (Venkata et al., 2006). Thus, there is a complex series of competitive underlying mechanisms resulting from phthalate exposure that complicates investigations aimed at determining the effects of phthalates exposure on PTC. Collectively, the net effects likely depend on exposure doses and profiles. Differences in the concentrations and profiles of phthalate metabolites among individuals from various countries or regions have been observed (listed in Supplementary Table S5). For the present study, the urinary concentrations of MBP were higher than those reported in non-occupational populations from North American studies, while that of DEHP metabolites were lower or comparable (US CDC, 2013). Additionally, the concentrations-response curves (Fig. 1) revealed that the risk of PTC rose sharp with elevating exposure dose when subjects were exposed to low levels of

phthalates. Those data suggested that considerable portion of general population may be with higher risk of PTC with current exposure levels. Additionally, inadequate nutritional iodine intake could render individuals more vulnerable to the adverse effects of thyroid disrupting chemicals, although underlying mechanisms by which this could occur remain unclear (Leung et al., 2016). MBP and MEP was positively associated with PTC prevalence at lower iodine level (lower than the median, median: 247 mg/L), while at higher iodine level (higher than the median), the associations not significant. Those data suggested that adequate iodine intake may counter the thyrotoxicity of these phthalates. However, for DEHP metabolites, significances were not changed with iodine status, but stronger associations were observed at higher iodine level. Unfortunately, the relatively small sample size prevented an accurate evaluation of the interaction between iodine and those phthalate metabolites. This study was the first to evaluate the associations between phthalate metabolites and PTC risk. We matched cases and controls on potentially important aspects such as age and sex, as well as adjusted for influencing factors and potential confounders identified in our previous study. Human exposure to phthalate was measured using UPLC-MS/MS, while the body weights and heights of subjects were measured by physical examination. Furthermore, urine specimens for phthalate metabolites determination were obtained in all cases before treatment began; thus, making it unlikely that phthalate exposure was influenced by treatment. This study had several limitations. First, the cross-sectional design is a major limitation. The temporality of the association could not be assessed because of simultaneous exposure and outcome measurements. Second, hospital-based study could have collection bias, although some measurments including serious recruiting criteria were done. Third, other thyroid disrupting chemicals like bisphenols and polychlorinated biphenyls as well as some nutrients like selenium and Vitamin D may have acted as confounders, affecting the observed associations. Additionally, phthalate metabolism might be influenced by the variation of sex hormones. In a recent study, Zhao et al. (2018) systematically evaluated DEHP metabolism during the period of pregnancy and variations in urinary concentrations of DEHP metabolites were observed. Therefore, further studies with a larger sample size and wider range of exposure doses are necessary to replicate the present results with considering other thyroid disrupting chemicals exposure, including nutrient intake, and sex hormones. 5. Conclusion This hospital-based case-control study reported that exposure to DEHP and DBP, as assessed by urinary metabolites, was associated with increased risk of PTC. The data suggested that specific phthalates were involved in the PTC tumorigenesis. The findings require that the possibility that phthalate metabolites are

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surrogates of unrecognized PTC risk factors also be excluded. Declaration of competing interest The authors have no conflict of interest to declare. Acknowledgments We would like to express our gratitude to all volunteers who participated in this study. This project was founded by the National Key Research and Development Program of China [grant number 2017YFC1600500] and by the National Natural Science Foundation of China [grant numbers 21507018 and 21537001]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125093. References American Cancer Society 2018, 2018. Cancer facts & figures 2018. https://www. cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/ annual-cancer-facts-and-figures/2018/cancer-facts-and-figures-2018.pdf. An, J.H., Hwangbo, Y., Ahn, H.Y., Keam, B., Lee, K.E., Han, W., Park, D.J., Park, I.A., Noh, D.-Y., Youn, Y.-K., Cho, B.Y., Im, S.-A., Park, Y.J., 2015. A possible association between thyroid cancer and breast cancer. Thyroid 25, 1330e1338. Boas, M., Feldtrasmussen, U., Main, K.M., 2012. Thyroid effects of endocrine disrupting chemicals. Mol. Cell. Endocrinol. 355, 240e248. Brenner, A.V., Tronko, M.D., Hatch, M., Bogdanova, T.I., Oliynik, V.A., Lubin, J.H., Zablotska, L.B., Tereschenko, V.P., Mcconnell, R.J., Zamotaeva, G.A., 2011. I-131 dose response for incident thyroid cancers in Ukraine related to the chornobyl accident. Environ. Health Perspect. 119, 933. Chen, W., Zheng, R., Baade, P.D., Zhang, S., Zeng, H., Bray, F., Jemal, A., Yu, X.Q., He, J., 2016. Cancer statistics in China, 2015. Ca. Cancer J. Clin. 66, 115e132. Cho, Y., Kim, J., 2015. Dietary factors affecting thyroid cancer risk: a meta-analysis. Nutr. Cancer 67, 811e817.  Doyon, F., Chungue, V., Rache ro, E., di, F., Boissin, J., Sebbag, J., Shan, L., BostCle Bezeaud, F., Petitdidier, P., Dewailly, E., Rubino, C., de Vathaire, F., 2012. Dietary iodine and thyroid cancer risk in French Polynesia: a case-control study. Thyroid 22, 422e429. Dong, X., Dong, J., Zhao, Y., Guo, J., Wang, Z., Liu, M., Zhang, Y., Na, X., 2017. Effects of long-term in vivo exposure to di-2-ethylhexylphthalate on thyroid hormones and the TSH/TSHR signaling pathways in wistar rats. Int. J. Environ. Res. Public Health 14, 44. Erkekoglu, P., Giray, B.K., Kizilgun, M., Hiningerfavier, I., Rachidi, W., Roussel, A.M., Favier, A., Hincal, F., 2012. Thyroidal effects of di-(2-ethylhexyl) phthalate in rats of different selenium status. J. Environ. Pathol. Toxicol. 31, 143e153. Fiore, M., Cristaldi, A., Okatyeva, V., Lo Bianco, S., Oliveri Conti, G., Zuccarello, P., Copat, C., Caltabiano, R., Cannizzaro, M., Ferrante, M., 2019a. Physical activity and thyroid cancer risk: a case-control study in catania (south Italy). Int. J. Environ. Res. Public Health 16, E1428. Fiore, M., Oliveri Conti, G., Caltabiano, R., Buffone, A., Zuccarello, P., Cormaci, L., Cannizzaro, A.M., Ferrante, M., 2019b. Role of emerging environmental risk factors in thyroid cancer: a brief review. Int. J. Environ. Res. Public Health 16, E1185. Fritz, A., Percy, C., Jack, A., Shanmugaratnam, K., Sobin, L., Parkin, D.M., Whelan, S., 2000. International Classification of Diseases for Oncology. World Health Organization, Geneva, Switzerland. Huang, H.B., Pan, W.H., Chang, J.W., Chiang, H.C., Guo, Y.L., Jaakkola, J.J.K., Huang, P.C., 2017. Does exposure to phthalates influence thyroid function and growth hormone homeostasis? The Taiwan Environmental Survey for Toxicants (TEST) 2013. Environ. Res. 153, 63e72. Kim, M.J., Moon, S., Oh, B.-C., Jung, D., Choi, K., Park, Y.J., 2019. Association between diethylhexyl phthalate exposure and thyroid function: a meta-analysis. Thyroid. ahead of print. . Kim, S.H., Park, M.J., 2014. Phthalate exposure and childhood obesity. Ann. Pediatr. Endocrinol. Metab. 19, 69e75. Knauf, J.A., Fagin, J.A., 2009. Role of MAPK pathway oncoproteins in thyroid cancer pathogenesis and as drug targets. Curr. Opin. Cell Biol. 21, 296e303. pezcarrillo, L., Calafat, A.M., Torressa nchez, L., Galva nportillo, M., Needham, L.L., Lo n, M.E., 2010. Exposure to phthalates and breast cancer Ruizramos, R., Cebria risk in northern Mexico. Environ. Health Perspect. 118, 539e544. € hrle, J., Duntas, L.H., Leung, A.M., Korevaar, T.I.M., Peeters, R.P., Zoeller, R.T., Ko Brent, G.A., Demeneix, B.A., 2016. Exposure to thyroid-disrupting chemicals: a transatlantic call for action. Thyroid 26, 479e480. Liu, C., Zhao, L., Wei, L., Li, L., 2015. DEHP reduces thyroid hormones via interacting with hormone synthesis-related proteins, deiodinases, transthyretin, receptors,

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