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Association of serum organochlorine pesticides concentrations with reproductive hormone levels and polycystic ovary syndrome in a Chinese population
Chemosphere 171 (2017) 595e600
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Association of serum organochlorine pesticides concentrations with reproductive hormone levels and polycystic ovary syndrome in a Chinese population Zhizhun Guo a, 1, Huiling Qiu b, 1, Lingling Wang b, Lijun Wang b, Chonggang Wang a, c, Meng Chen c, *, Zhenghong Zuo a, c, ** a b c
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361102, China Department of Gynaecology, The Affiliated Chenggong Hospital of Xiamen University, Xiamen 361002, China Key Laboratory of Ministry of Education for Subtropical Wetland Ecosystem Research, Xiamen University, Xiamen 361102, China
h i g h l i g h t s PCOS is caused by a combination of genetic susceptibility and environmental exposures. Patients with PCOS had higher p,p0 -DDT (P ¼ 0.016) and o,p0 -DDT levels than the controls. o,p0 -DDT may play a role in the pathogenesis of PCOS related with reproductive hormone levels.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2016 Received in revised form 28 November 2016 Accepted 24 December 2016 Available online 26 December 2016
To investigate the serum concentrations of organochlorine pesticides (OCPs) in patients with polycystic ovary syndrome (PCOS), a total of 178 women were studied. The concentrations of hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) in serum were determined using Gas Chromatography Mass-Mass Spectrometer. No differences with statistical significance in the mean HCH, p,p0 -DDD, p,p’-DDE concentrations were observed between the patients with PCOS and the control group. Serum p,p0 -DDT (P ¼ 0.016) and o,p0 -DDT (P ¼ 0.000) levels were significantly higher in patients with PCOS compared with the control group. The results of the association between OCPs levels and hormone levels indicated that o,p0 -DDT may play a role in the pathogenesis of PCOS by affecting hormones levels. Further trials should be investigated with the findings in this study to obtain new pathogenesis of PCOS. © 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Andreas Sjodin Keywords: Polycystic ovary syndrome Organochlorine pesticides Endocrine disrupting Serum
1. Introduction Polycystic ovary syndrome (PCOS) is a highly prevalent
heterogeneous syndrome of clinical and/or biochemical androgen excess, ovulatory dysfunction and polycystic ovaries (Goodarzi et al., 2011). PCOS is the most common cause of anovulatory
Abbreviations: BMI, body mass index; CVD, cardiovascular disease; DBP, diastolic blood pressure; DCM, dichloromethane; DDD, dichlorodiphenyldichloroethane; DDE, dichlorodiphenyldichloroethylene; DDT, dichlorodiphenyltrichloroethane; DHEAS, dehydroepiandrosterone-sulfate; E2, estradiol; EDCs, exogenous endocrine disrupting chemicals; FSH, follicle-stimulating hormone; GC-MS-MS, chromatography mass-mass spectrometer; GIR, fasting glucose/insulin ratio; HCH, hexachlorocyclohexane; HDL-C, high-density lipoprotein cholesterol; HOMA, homeostasis model assessment; IR, insulin resistance; LDL-C, low-density lipoprotein cholesterol; LH, luteinizing hormone; LOD, limit of detection; OCPs, organochlorine pesticides; PCOS, polycystic ovary syndrome; PLS-DA, partial least-squares-discriminant analysis; POPs, persistent organic pollutants; PRL, progesterone; QUICKI, quantitative insulin sensitivity check index; SBP, systolic blood pressure; SHBG, sex hormone binding globulin; T2DM, type 2 diabetes mellitus; TSH, thyroid-stimulating hormone; WHR, waist-to-hip ratio; YLDs, lived with disability. * Corresponding author. College of the Environment and Ecology, Xiamen University, 422 Siming South Road, Xiamen, Fujian Province 361005, China. ** Corresponding author. School of life sciences, Xiamen University, 422 Siming South Road, Xiamen, Fujian Province 361005, China. E-mail addresses: [email protected] (M. Chen), [email protected] (Z. Zuo). 1 These authors are co-first authors. http://dx.doi.org/10.1016/j.chemosphere.2016.12.127 0045-6535/© 2016 Elsevier Ltd. All rights reserved.
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infertility among all known causes of infertility (Teede et al., 2010). Two-thirds of women with PCOS have metabolic dysfunction and have an increased risk of developing type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). PCOS is also associated with obesity, insulin resistance (IR) and endometrial carcinoma (Lin et al., 2012). PCOS affects 5e10% of reproductive-aged women worldwide, with prevalence among the Han Chinese population of 5.6% (Li et al., 2013). The pathogenesis of PCOS has not been fully elucidated. However, the results of many studies demonstrated that PCOS is caused by a multifactorial etiology of genetic susceptibility and environmental exposures. A preliminary family studies have suggested the familial aggregation of hyperandrogenemia in PCOS kindreds (Legro et al., 1998). On the other hand, several preliminary studies show that environmental pollutants are involved in severe reproductive and endocrine disorders (Craig et al., 2011; Johansson et al., 2016; Yang et al., 2015). Persistent organic pollutants (POPs) are ubiquitous contaminants that are not readily degraded in the environment and can accumulate in biota, including humans. Most of these contaminants are endocrine-disrupting chemicals (EDCs) and may interfere with the synthesis, secretion, transport and metabolism of endogenous hormones (Yang et al., 2015). Prenatal exposure to mixtures of human relevant EDCs can have negative consequences on the female reproductive system later in life (Johansson et al., 2016). Organochlorine pesticides (OCPs) including dichlorodiphenyltrichloroethane (DDT) and hexachlorobenzene (HCH) are ubiquitous in the environment following many years of agriculture use. DDT is one of the most well known OCPs. It was applied worldwide as an insecticide for vector control until the 1970s, and is still used in some countries (van den Berg, 2009). A recent study shows that six OCPs including DDT and HCH are found in 8.2% nut samples of China, with the concentrations of 2.0 mg kg1 to 65.7 mg kg1 (Liu et al., 2016). In the environment and in living organisms, DDT is mainly degraded to p,p0 -dichlorodiphenyldichloroethylene (p,p0 -DDE), which is even more persistent than the parent compound. In China, DDT is still produced for export for malaria control and for domestic use in dicofol production. Unlike technical DDT, dicofol use in China is mainly in the southern and eastern provinces, especially in Fujian, mostly on litchi, longan, and citrus crops. DDT pollution in Fujian is very likely dominated by dicofol type DDT pollution, which means that the concentrations of o,p’-DDT are higher than normal area (Qiu et al., 2005; Zheng et al., 2016). Prenatal exposure to OCPs in Southern Spain has an impact on the weight of healthy full-term newborns (Monteagudo et al., 2016). DDT has been found to interact with androgen receptors and estrogen receptors (Li et al., 2008; Soto et al., 1995). DDT induces adverse effects on male rat fertility by acting directly on the testes and altering the neuroendocrine function (Patrick et al., 2016; Rhouma et al., 2001). There are several evidences suggesting that OCPs might be involved in reproductive hormone levels and thereby possibly also in the development of PCOS. A study used Partial least-squares-discriminant analysis (PLS-DA) confirm that serum concentrations of p,p0 -DDE are associated with PCOS (Yang et al., 2015). However, studies on DDT pollution in China have paid little attention to o,p0 -DDT, and few research show the serum o,p0 -DDT levels in relation to PCOS. Keeping in view the dicofol type DDT pollution in Fujian, the serum OCPs concentrations associated with PCOS and hormone concentrations was investigated in the present study. Our objective in this study was to identify the relationship between serum concentrations of OCPs and hormones and to assess the possible impact of exposure to OCPs on PCOS.
2. Materials and methods 2.1. Study subjects and data collection Blood serum samples were recruited from women at the Reproductive Medicine Center of 174th Hospital of PLA (Xiamen, China) between 1 March 2013 and 31 August 2013, and stored at 80 C. All of women were from Xiamen and its surrounding cities and this area is not an industrial area. Among these samples, 84 women were PCOS patients. The patients of PCOS was defined when at least two of the following three features were present: oligo-/amenorrhea (<8 menstrual cycles in the presenting year); hyperandrogenism (and/or hirsutism); and polycystic ovaries (Rotterdam EA-SPcwg, 2004). In order to carry out control, we randomly selected 94 samples. None of the controls had symptoms of hyperandrogenism, a history of menstrual dysfunction, infertility, or sonographic signs of PCOS. All subjects were nonpregnant, non-smokers. All women were studied within the first 10 days after onset of menstruation in the case of mild oligomenorrhea or at random in those suffering from severe oligoor amenorrhea. The characteristics of PCOS women and controls were summarized in Table 1, and their concentrations were determined following the published methods (Zheng et al., 2015). 2.2. Ethical approval The present study was approved by the Reproductive Medicine Center of 174th Hospital of PLA (Xiamen, China), and informed consent was obtained from all participants prior to their recruitment into the study. 2.3. Analysis of OCPs The methods were based on previous study with some modifications (Hagmar et al., 2006). Before extraction, the surrogates 13Cb-HCH, 13C-p,p0 -DDT(20 ng) were spiked separately into the serum. Samples are kept at 4 C overnight for equilibrium before sample extraction. The HLB column (500 mg, Waters, USA) was washed with dichloromethane (DCM) and activated with methanol and Milli-Q water. After conditioning, 1 mL serum was added into column with moist column. The column was then dried for 30 min by aspiration of ambient air. Subsequently, 18 mL of DCM: n-hexane (1:1, v/v) was added to the column for elution. Dried sodium sulfate column was used for the cleaning of extract. After extraction, the solvents were evaporated down to 0.5 mL under a gentle stream of nitrogen gas, and then, 20 ng of internal standard PCB-103 was added before analysis by Gas Chromatography Mass-Mass Spectrometer (GC-MSMS). OCPs were determined with a Trace 1310 gas chromatograph (GC) coupled with TSQ 8000 mass selective detector (MSD) and a 50 m 0.25 mm DB-5 capillary column (Agilent, USA). The GC injector temperature was 290 C with splitless mode, and the GC oven temperature was programmed as follows: 80 C for 2 min, increased 20 C/min to 200 C, held for 2 min, increased to 250 C at a rate of 2 C/min, followed by a rate of 25 C/min to 300 C, and then held for 15 min. 2.4. Quality assurance/Quality control A solvent blank and a procedural blank were added for every sequence of 10 samples to ensure that the samples and the analysis process were free of contamination. The retention times matched with those of the authentic reference compounds and the signal to noise (S/N) ratio was greater than three for the selected ions. The
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Table 1 Clinical characteristics for controls and PCOS patients.
Age (years) Weight (kg) Height (cm) BMI (kg/m2) Waist (cm) Hip (cm) WHR Menarche (years) Hirsutism score Acne score SBP(mmHg) DBP(mmHg) FSH(mIU mL1) LH(mIU mL1) LH/FSH E2(pg mL1) PRL(ng mL1) TT(nmol L1) P(ng mL1) TSH(mIU mL1) DHEAS(mg dL1) SHBG(nmol L1) Fasting insulin (mU mL1) Fasting glucose (mg dL1) GIR HOMA-IR QUICKI Cholesterol (mmol L1) Triglycerides (mmol L1) LDL-C(mmol L1) HDL-C(mmol L1)
Control(n ¼ 94)
PCOS patients(n ¼ 84)
Pa
29(22e35) 54.16(38e93) 1.60(1.48e1.7) 21.28(15.82e37.17) 73.50(60e99) 87.21(74e108) 0.84(0.69e0.99) 14(12e17) 2(0e10) 1(0e15) 113(90e152) 71(50e98) 7.30(2.38e15.82) 4.86(1.36e26.15) 0.72(0.13e3.37) 39.22(12e222) 17.99(4.55e61.12) 1.22(0.32e2.26) 1.18(0.11e25.20) 2.22(0.031e7.69) 291.64(51.82e900.57) 67.23(17.63e391.51) 8.16(2.3e63) 82.71(28.8e98.64) 12.90(1.31e33.26) 1.70(0.4e12.8) 0.36(0.27e0.45) 4.80(3.22e6.53) 0.92(0.35e3.25) 2.89(1.46e4.57) 1.50(0.70e2.26)
28(20e35) 57.24(40e91) 1.60(1.47e1.73) 22.46(15.06e36.92) 76.36(56e102) 88.98(70e116) 0.86(0.69e1.14) 15(12e18) 6(0e18) 2(0e16) 117(93e142) 74(50e96) 6.60(0.98e12.44) 10.81(2.47e35.25) 1.67(0.40e4.66) 39.49(13e140) 16.75(1.18e177.00) 2.14(0.66e8.40) 0.92(0.18e4.21) 2.97(0.005e31.94) 309.77(124.28e463.91) 53.83(3.15e409.95) 11.18(2.40e38.40) 87.52(68.4e119.7) 10.23(2.87e29.70) 2.49(0.42e10.44) 0.34(0.28e0.45) 4.77(3.03e7.10) 1.79(0.30e40.5) 2.82(1.73e4.81) 1.42(0.72e3.14)
0.166 0.016 0.967 0.006 0.010 0.070 0.193 0.198 0.000 0.000 0.013 0.013 0.164 0.000 0.000 0.712 0.028 0.000 0.790 0.133 0.000 0.000 0.000 0.012 0.000 0.000 0.000 0.771 0.000 0.321 0.034
Data are presented as median (25e75% quartiles). a P-values were calculated by ManneWhitney U test and Chi-square test.
limit of detection (LOD) of DDTs and HCHs was 0.1 ng mL1. The recoveries of spiked serum samples for DDTs ranged from 78% to 98%, HCHs from 81% to 94%. Reported concentrations were not corrected with recovery rates. Six-point calibration curves with good to excellent linearity (r2 > 0.99) were constructed for the quantification. 2.5. Statistical analyses Data were presented as median, mean ± SD, and analyzed using SPSS 17.0 software. The reproductive hormones and metabolites data were log-transformed prior to conducting statistical tests. The differences in clinical characteristics and OCPs concentrations between the PCOS group and the control group were compared using ManneWhitney U. Appropriate correlation tests (Pearson or Spearman) were performed to analysis relationship among OCPs and anthropometric, hormonal and biochemical parameters. The association between OCPs concentrations and hormone using linear regression with hormone levels was examined. Statistical significance was set at P < 0.05. 3. Results Clinical characteristics of the case and control participants were shown in Table 1. The age, height, hip, waist-to-hip ratio (WHR), menarche, FSH, E2, P, TSH, cholesterol and LDL-C had no statistically significant differences between the PCOS group and the control group. The median PRL, SHBG, GIR, QUICKI and HDL-C were lower in the PCOS group than that in the control group. The other characteristics were higher in the PCOS group than that in the control group (Table 1). Concentrations of OCPs in serum samples were shown in Table 2
and Fig. 1. OCPs were not detected in all blanks. HCHs, o,p’-DDT, p,p’DDD and p,p’-DDE were detected in all serum groups, while p,p’DDT in 82% and 63% in different groups. OCPs which detected lower than 30% were not showed in this table. Previous studies also reveal that DDE is the most commonly detected congener of DDTs in the general population (Thomas et al., 2006). We observed the mean o,p’-DDT (P ¼ 0.000) and p,p’-DDT (P ¼ 0.016) concentrations in the PCOS group (0.53 ng mL1, 0.77 ng mL1) were higher than those of the control group (0.44 ng mL1, 0.58 ng mL1). Keeping in view the detection rates, we used appropriate correlation tests to analysis the relation between o,p’-DDT and all clinical and laboratory parameters investigated in the study (p,p’-DDT was not analyzed due to the detection rate). In all serum samples, o,p’-DDT levels had positive correlation with LSH/FH, testosterone, triglycerides), and negative correlation with FSH and SHBG. As shown in Table 3, a 1 ng mL1 increase in o,p’-DDT was associated with a 0.037% increase in FSH levels in unadjusted analyses. The association was strengthened with a 0.410% increase after adjusting for age, BMI and WHR. A 1 ng mL1 increase in o,p’-DDT was associated with a 0.997% increase in FSH/LH levels in unadjusted analyses. The association was strengthened with a 0.999% increase after adjusting for age, BMI and WHR. A 1 ng mL1 increase in o,p’-DDT was associated with a 0.823% increase in testosterone levels in unadjusted analyses. The association was attenuated with a 0.810% increase after adjusting for age, BMI and WHR. A 1 ng mL1 increase in o,p’-DDT was associated with a 1.241% increase in SHBG levels in unadjusted analyses. The association was strengthened with a 1.300% increase after adjusting for age, BMI and WHR.
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Table 2 Concentrations of OCPs in serum samples from PCOS patients and controls. Control N(%)>LOD
Pa
PCOS patients mean
SD
25th
50th
75th
N(%)>LOD
mean
SD
1
b-HCH(ng mL ) g-HCH(ng mL1)
94(100) 0.69 0.31 0.46 0.63 0.85 84(100) 0.86 0.60 94(100) 0.81 0.32 0.58 0.73 0.93 84(100) 0.96 0.58 P 1 94(100) 1.50 0.63 1.03 1.39 1.80 84(100) 1.82 1.19 HCHs(ng mL ) 1 p,p’-DDT(ng mL ) 77(81.9) 0.58 0.27 0.42 0.51 0.62 53(63.1) 0.77 0.44 p,p’-DDD(ng mL1) 94(100) 0.42 0.05 0.38 0.42 0.45 84(100) 0.42 0.05 p,p’-DDE(ng mL1) 94(100) 2.50 1.82 1.19 2.03 2.97 84(100) 2.42 1.65 o,p’-DDT(ng mL1) 94(100) 0.44 0.08 0.39 0.42 0.46 84(100) 0.53 0.16 P DDTs(ng mL1) 94(100) 3.84 2.02 2.40 3.53 4.65 84(100) 3.86 2.02 P P HCHs refer to the sum of b-HCH and g-HCH, and DDTs refer to the sum of p,p0 -DDT, p,p’-DDD, p,p0 -DDE and o,p0 -DDT. a P-values were calculated by ManneWhitney U test and Chi-square test.
25th
50th
75th
0.45 0.58 1.02 0.44 0.38 1.09 0.41 2.20
0.64 0.74 1.39 0.65 0.41 2.16 0.46 3.62
1.08 1.22 2.32 1.00 0.45 3.37 0.61 5.46
Controls
0.271 0.353 0.316 0.016 0.780 0.981 0.000 0.991
Max
Concentrations (ng mL-1)
PCOS Patients
75% Mean Median 25% Min
Fig. 1. Box Plots of EDCs Concentrations in serum. Data presented as mean ± SD, (*P < 0.05).
A 1 ng mL1 increase in o,p’-DDT was associated with a 0.705% increase in triglycerides levels in unadjusted analyses. The association was attenuated with a 0.752% increase after adjusting for age, BMI and WHR.
4. Discussion PCOS is a common reproductive endocrine disease that is seen among adolescent women. Environmental factors based on genetic
Table 3 Unadjusted and adjusted results for the percentage difference in serum hormone levels or metabolic profile per unit change in serum o,p’-DDT levels. Adjusteda
Unadjusted
FSH LH LH/FSH E2 PRL TT P TSH DHEAS SHBG Fasting insulin Fasting glucose GIR HOMA-IR QUICKI Cholesterol Triglycerides LDL-C HDL-C a
% difference
95% CI
0.388 0.610 0.997 0.171 0.198 0.823 0.143 0.263 0.307 1.241 0.102 0.007 0.094 0.109 0.017 0.017 0.705 0.030 0.100
0.032 0.103 0.010 0.531 0.511 0.003 0.708 0.567 0.100 0.031 0.739 0.919 0.739 0.750 0.740 0.852 0.037 0.827 0.446
Adjusted for age, BMI and WHR.
0.742 0.124 0.240 0.708 0.792 0.279 0.609 1.169 0.059 2.365 0.501 0.138 0.652 0.567 0.121 0.167 0.042 0.242 0.357
P
% difference
95% CI
0.033 1.343 1.755 0.367 0.396 1.367 0.895 0.643 0.673 0.117 0.704 0.153 0.463 0.785 0.086 0.201 1.368 0.302 0.158
0.410 0.589 0.999 0.193 0.203 0.810 0.089 0.272 0.299 1.300 0.154 0.019 0.135 0.173 0.027 0.027 0.752 0.038 0.097
0.022 0.114 0.010 0.481 0.497 0.003 0.815 0.558 0.113 0.024 0.558 0.794 0.583 0.560 0.546 0.773 0.015 0.782 0.424
P 0.759 0.143 0.242 0.734 0.790 0.273 0.666 1.189 0.072 2.430 0.364 0.124 0.622 0.412 0.117 0.157 0.148 0.231 0.336
0.061 1.321 1.756 0.347 0.385 1.348 0.844 0.644 0.669 0.169 0.673 0.162 0.351 0.758 0.062 0.211 1.355 0.307 0.142
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predisposition have been thought to play a role in the aetiopathogenesis of PCOS in recent years (Pasquali et al., 2011). The main environmental factors include environmental toxins, diet and nutrition, socioeconomic status, and geography (Merkin et al., 2016). The ovaries seem to be a prime target for endocrine disndez et al., 2010; Newbold et al., 2007). In ruptor compounds (Ferna this study, we examined the relationship between OCPs and PCOS. Statistically significant higher serum o,p’-DDT levels in the PCOS group compared to the controls were observed. As a member of EDCs, o,p’-DDT may play a role in the pathogenesis of PCOS by affecting the reproductive hormones and metabolites. Before the ban of DDT use in agriculture, more than 60% of the technical DDT had been used on cotton fields, mainly in central and eastern China. In comparison to central and eastern China, less technical DDT was used in southern China, such as Guangdong, Guangxi, and Fujian provinces (Qiu et al., 2005; Zheng et al., 2016). Considering the difference between the spatial distribution of technical DDT and dicofol use DDT pollution in Fujian is very likely dominated by dicofol type DDT pollution. Our study also showed the high levels of o,p’-DDT in serums of females in Fujian province. However, until now, few study investigating the endocrine disrupting function of o,p’-DDT. In the present study, statistically significant higher serum o,p’-DDT levels in the PCOS group (0.53 ng mL1) compared to the control group (0.44 ng mL1) were found (P ¼ 0.000). DDT exhibit properties which are very similar to E2 (Diel et al., 2002), and some studies have proved that o,p’-DDT enantiomer is a more active estrogen-mimic than the (þ)-enantiomer in rats (Hoekstra et al., 2001). o,p’-DDT has a similar function with bisphenol A (BPA), which is known to interfere with androgen catabolism. A previous study has demonstrated that BPA stimulates ovarian -theca-interstitial cells to produce androgens by dysregulation of 17a-hydroxylase, a key enzyme in gonadal steroidogenesis (Zhou et al., 2008). The activation of this steroidogenic pathway has a role in PCOS ovarian hyperandrogenism (Nelson et al., 2001). There might be a similar mechanism for the association between o,p’-DDT and androgens in these patients. A mice experiment suggests that perinatally exposed to DDT reduced core body temperature, decreased energy expenditure, impaired cold tolerance, and produced a transient early-life increase in body fat in female offspring (La Merrill et al., 2014). When challenged with a high fat diet for 12 weeks in adulthood, perinatal DDT exposure developed glucose intolerance, hyperinsulinemia, dyslipidemia, and altered bile acid metabolism in female offspring (La Merrill et al., 2014). Exposure adult rodents to high doses of DDT have impaired insulin secretion, glucose intolerance, and elevated gluconeoegenesis (Yau and Mennear, 1977). The results of epidemiological studies have showed the associations between DDT and DDE exposures and diabetes in humans (Lee et al., 2011; Patel et al., 2010). However, our study show that there was no correlation between o,p’-DDT concentrations and insulin resistance and sensitivity indexes. Our study showed the positive correlation between o,p’-DDT concentrations and LSH/FH(P ¼ 0.009), testosterone (P ¼ 0.003), triglycerides (P ¼ 0.035), and the negative correlation between o,p’DDT concentrations and FSH (P ¼ 0.013), SHBG (P ¼ 0.037). A epidemiological study suggests a positive relationship of baseline E2 and baseline testosterone with DDT compounds (Dalvie et al., 2004). We speculate o,p’-DDT may specifically bind to SHBG, resulting in elevated levels of testosterone; o,p’-DDT may cause a decline in FSH levels through the involvement of steroid enzyme (Gregoraszczuk and Ptak, 2013), affecting the development of follicles, while causing high levels of LH. Elevated LH levels can also lead to excessive secretion of male hormones, thus disrupt or inhibit the development of the female reproductive system. All the conditions causing symptoms such as ovarian cysts, which play an
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important role in the pathogenesis of PCOS. The elevated triglyceride levels affected by o,p’-DDT may also lead to obesity, cardiovascular disease and other illnesses. A rats animal model study shows that ancestral exposure to DDT can cause metabolic syndrome-related lesions in the testis, ovary, and promote obesity and associated disease in offspring generation (Skinner et al., 2013). 5. Conclusion In conclusion, our results indicate that o,p’-DDT concentrations in PCOS group were higher than the control group. Many studies support that androgen catabolism may play an important role in the pathogenesis of PCOS. o,p’-DDT may have positive effects on PCOS by increasing or decreasing reproductive hormone levels related with androgen catabolism. However, larger trials are required before reaching definitive conclusions. Further mechanistic studies are needed to confirm the findings in our study. Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported in part by the Program for Xiamen Southern Oceanographic Center (14GST69NF33), and the project of the Xiamen Science and Technology Program (2013Z20134027). References Craig, Z.R., Wang, W., Flaws, J.A., 2011. Endocrine-disrupting chemicals in ovarian function: effects on steroidogenesis, metabolism and nuclear receptor signaling. Reproduction 142 (5), 633e646. Dalvie, M.A., Myers, J.E., Thompson, M.L., Dyer, S., Robins, T.G., Omar, S., Riebow, J., Molekwa, J., Kruger, P., Millar, R., 2004. The hormonal effects of long-term DDT exposure on malaria vector-control workers in Limpopo Province, South Africa. Environ. Res. 96 (1), 9e19. Diel, P., Olff, S., Schmidt, S., Michna, H., 2002. Effects of the environmental estrogens bisphenol A, o, p0 -DDT, p-tert-octylphenol and coumestrol on apoptosis induction, cell proliferation and the expression of estrogen sensitive molecular parameters in the human breast cancer cell line MCF-7. J. Steroid. Biochem. Mol. Biol. 80 (1), 61e70. ndez, M., Bourguignon, N., Lux-Lantos, V., Libertun, C., 2010. Neonatal expoFerna sure to bisphenol A and reproductive and endocrine alterations resembling the polycystic ovarian syndrome in adult rats. Environ. Health Perspect. 118, 1217e1222. Gregoraszczuk, E.L., Ptak, A., 2013. Endocrine-disrupting chemicals: some actions of POPs on female reproduction. Int. J. Endocrinol. 2013, 1e9. Goodarzi, M.O., Dumesic, D.A., Chazenbalk, G., Azziz, R., 2011. Polycystic ovary syndrome: etiology, pathogenesis and diagnosis. Nat. Rev. Endocrinol. 7 (4), 219e231. € nsson, B.A., Bergman, Å., Rylander, L., 2006. Hagmar, L., Wallin, E., Vessby, B., Jo Intra-individual variations and time trends 1991e2001 in human serum levels of PCB, DDE and hexachlorobenzene. Chemosphere 64 (9), 1507e1513. Hoekstra, P.F., Burnison, B.K., Neheli, T., Muir, D.C., 2001. Enantiomer-specific activity of o, p0 -DDT with the human estrogen receptor. Toxicol. Lett. 125 (1), 75e81. Johansson, H.K.L., Jacobsen, P.R., Hass, U., Svingen, T., Vinggaard, A.M., Isling, L.K., Axelstad, M., Christiansen, S., Boberg, J., 2016. Perinatal exposure to mixtures of endocrine disrupting chemicals reduces female rat follicle reserves and accelerates reproductive aging. Reprod. Toxicol. 61, 186e194. La Merrill, M., Karey, E., Moshier, E., Lindtner, C., La Frano, M.R., Newman, J.W., Buettner, C., 2014. Perinatal exposure of mice to the pesticide DDT impairs energy expenditure and metabolism in adult female offspring. PLoS One 9 (7), e103337. €din, A., Jones, R.S., Needham, L.L., Jacobs Jr., D.R., 2011. Lee, D.H., Steffes, M.W., Sjo Low dose organochlorine pesticides and polychlorinated biphenyls predict obesity, dyslipidemia, and insulin resistance among people free of diabetes. PLoS One 6 (1), e15977. Legro, R.S., Driscoll, D., Strauss, J.F., Fox, J., Dunaif, A., 1998. Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc. Natl. Acad. Sci. U. S. A. 95 (25), 14956e14960. Li, J., Li, N., Ma, M., Giesy, J.P., Wang, Z., 2008. In vitro profiling of the endocrine disrupting potency of organochlorine pesticides. Toxicol. Lett. 183 (1), 65e71. Li, R., Zhang, Q., Yang, D., Li, S., Lu, S., Wu, X., Wei, Z., Song, X., Wang, X., Fu, S., Lin, J.,
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Zhu, Y., Jiang, Y., Feng, H., Qiao, J., 2013. Prevalence of polycystic ovary syndrome in women in China: a large community-based study. Hum. Reprod. 28 (9), 2562e2569. Lin, M.W., Tsai, S.J., Chou, P.Y., Huang, M.F., Sun, H.S., Wu, M.H., 2012. Vitamin D receptor 1a promotor 1521 G/C and 1012 A/G polymorphisms in polycystic ovary syndrome. Taiwan J. Obstet. Gynecol. 51 (4), 565e571. Liu, Y.H., Shen, D.Y., Li, S.L., Ni, Z.L., Ding, M., Ye, C.F., Tang, F.B., 2016. Residue levels and risk assessment of pesticides in nuts of China. Chemosphere 144, 645e651. Merkin, S.S., Phy, J.L., Sites, C.K., Yang, D.z., 2016. Environmental determinants of polycycstic ovary syndrome. Fertn. Stert. 106 (1), 16e24. Monteagudo, C., Mariscal-Arcas, M., Heras-Gonzalez, L., Ibanez-Peinado, D., Rivas, A., Olea-Serrano, F., 2016. Effects of maternal diet and environmental exposure t organochlorine pesticides on newborn weight in Southern Spain. Chemosphere 156, 135e142. Nelson, V.L., Qin, K.N., Rosenfield, R.L., Wood, J.R., Penning, T.M., Legro, R.S., Strauss, J.F., Mcallister, J.M., 2001. The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome. J. Clin. Endocrinol. Metab. 86 (12), 5925e5933. Newbold, R.R., Jefferson, W.N., Padilla-Banks, E., 2007. Long-term adverse effects of neonatal exposure to bisphenol A on the murine female reproductive tract. Reprod. Toxicol. 24 (2), 253e258. Pasquali, R., Stener-Victorin, E., Yildiz, B.O., Duleba, A.J., Hoeger, K., Mason, H., Homburg, R., Hickey, T., Franks, S., Tapanainen, J., Balen, A., Abbott, D., Legro, R., 2011. PCOS Forum: research in polycystic ovary syndrome today and tomorrow. Clin. Endocrinol. 74 (4), 424e433. Patrick, S.M., Bornman, M.S., Joubert, A.M., Pitts, N., Naidoo, V., de Jager, C., 2016. Effects of environmental endocrine disruptors, including insecticides used for malaria vector control on reproductive parameters of male rats. Reprod. Toxicol. 61, 19e27. Patel, C.J., Bhattacharya, J., Butte, A.J., 2010. An environment-wide association study (EWAS) on type 2 diabetes mellitus. PLoS One 5 (5), e10746. Qiu, X., Zhu, T., Yao, B., Hu, J., Hu, S., 2005. Contribution of dicofol to the current DDT pollution in China. Environ. Sci. Technol. 39 (12), 4385e4390. bourbi, O., Krichah, R., Sakly, M., 2001. Reproductive toxicity of DDT Rhouma, K.B., Te
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Association of serum organochlorine pesticides concentrations with reproductive hormone levels and polycystic ovary syndrome in a Chinese population Zhizhun Guo a, 1, Huiling Qiu b, 1, Lingling Wang b, Lijun Wang b, Chonggang Wang a, c, Meng Chen c, *, Zhenghong Zuo a, c, ** a b c
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361102, China Department of Gynaecology, The Affiliated Chenggong Hospital of Xiamen University, Xiamen 361002, China Key Laboratory of Ministry of Education for Subtropical Wetland Ecosystem Research, Xiamen University, Xiamen 361102, China
h i g h l i g h t s PCOS is caused by a combination of genetic susceptibility and environmental exposures. Patients with PCOS had higher p,p0 -DDT (P ¼ 0.016) and o,p0 -DDT levels than the controls. o,p0 -DDT may play a role in the pathogenesis of PCOS related with reproductive hormone levels.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2016 Received in revised form 28 November 2016 Accepted 24 December 2016 Available online 26 December 2016
To investigate the serum concentrations of organochlorine pesticides (OCPs) in patients with polycystic ovary syndrome (PCOS), a total of 178 women were studied. The concentrations of hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) in serum were determined using Gas Chromatography Mass-Mass Spectrometer. No differences with statistical significance in the mean HCH, p,p0 -DDD, p,p’-DDE concentrations were observed between the patients with PCOS and the control group. Serum p,p0 -DDT (P ¼ 0.016) and o,p0 -DDT (P ¼ 0.000) levels were significantly higher in patients with PCOS compared with the control group. The results of the association between OCPs levels and hormone levels indicated that o,p0 -DDT may play a role in the pathogenesis of PCOS by affecting hormones levels. Further trials should be investigated with the findings in this study to obtain new pathogenesis of PCOS. © 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Andreas Sjodin Keywords: Polycystic ovary syndrome Organochlorine pesticides Endocrine disrupting Serum
1. Introduction Polycystic ovary syndrome (PCOS) is a highly prevalent
heterogeneous syndrome of clinical and/or biochemical androgen excess, ovulatory dysfunction and polycystic ovaries (Goodarzi et al., 2011). PCOS is the most common cause of anovulatory
Abbreviations: BMI, body mass index; CVD, cardiovascular disease; DBP, diastolic blood pressure; DCM, dichloromethane; DDD, dichlorodiphenyldichloroethane; DDE, dichlorodiphenyldichloroethylene; DDT, dichlorodiphenyltrichloroethane; DHEAS, dehydroepiandrosterone-sulfate; E2, estradiol; EDCs, exogenous endocrine disrupting chemicals; FSH, follicle-stimulating hormone; GC-MS-MS, chromatography mass-mass spectrometer; GIR, fasting glucose/insulin ratio; HCH, hexachlorocyclohexane; HDL-C, high-density lipoprotein cholesterol; HOMA, homeostasis model assessment; IR, insulin resistance; LDL-C, low-density lipoprotein cholesterol; LH, luteinizing hormone; LOD, limit of detection; OCPs, organochlorine pesticides; PCOS, polycystic ovary syndrome; PLS-DA, partial least-squares-discriminant analysis; POPs, persistent organic pollutants; PRL, progesterone; QUICKI, quantitative insulin sensitivity check index; SBP, systolic blood pressure; SHBG, sex hormone binding globulin; T2DM, type 2 diabetes mellitus; TSH, thyroid-stimulating hormone; WHR, waist-to-hip ratio; YLDs, lived with disability. * Corresponding author. College of the Environment and Ecology, Xiamen University, 422 Siming South Road, Xiamen, Fujian Province 361005, China. ** Corresponding author. School of life sciences, Xiamen University, 422 Siming South Road, Xiamen, Fujian Province 361005, China. E-mail addresses: [email protected] (M. Chen), [email protected] (Z. Zuo). 1 These authors are co-first authors. http://dx.doi.org/10.1016/j.chemosphere.2016.12.127 0045-6535/© 2016 Elsevier Ltd. All rights reserved.
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infertility among all known causes of infertility (Teede et al., 2010). Two-thirds of women with PCOS have metabolic dysfunction and have an increased risk of developing type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). PCOS is also associated with obesity, insulin resistance (IR) and endometrial carcinoma (Lin et al., 2012). PCOS affects 5e10% of reproductive-aged women worldwide, with prevalence among the Han Chinese population of 5.6% (Li et al., 2013). The pathogenesis of PCOS has not been fully elucidated. However, the results of many studies demonstrated that PCOS is caused by a multifactorial etiology of genetic susceptibility and environmental exposures. A preliminary family studies have suggested the familial aggregation of hyperandrogenemia in PCOS kindreds (Legro et al., 1998). On the other hand, several preliminary studies show that environmental pollutants are involved in severe reproductive and endocrine disorders (Craig et al., 2011; Johansson et al., 2016; Yang et al., 2015). Persistent organic pollutants (POPs) are ubiquitous contaminants that are not readily degraded in the environment and can accumulate in biota, including humans. Most of these contaminants are endocrine-disrupting chemicals (EDCs) and may interfere with the synthesis, secretion, transport and metabolism of endogenous hormones (Yang et al., 2015). Prenatal exposure to mixtures of human relevant EDCs can have negative consequences on the female reproductive system later in life (Johansson et al., 2016). Organochlorine pesticides (OCPs) including dichlorodiphenyltrichloroethane (DDT) and hexachlorobenzene (HCH) are ubiquitous in the environment following many years of agriculture use. DDT is one of the most well known OCPs. It was applied worldwide as an insecticide for vector control until the 1970s, and is still used in some countries (van den Berg, 2009). A recent study shows that six OCPs including DDT and HCH are found in 8.2% nut samples of China, with the concentrations of 2.0 mg kg1 to 65.7 mg kg1 (Liu et al., 2016). In the environment and in living organisms, DDT is mainly degraded to p,p0 -dichlorodiphenyldichloroethylene (p,p0 -DDE), which is even more persistent than the parent compound. In China, DDT is still produced for export for malaria control and for domestic use in dicofol production. Unlike technical DDT, dicofol use in China is mainly in the southern and eastern provinces, especially in Fujian, mostly on litchi, longan, and citrus crops. DDT pollution in Fujian is very likely dominated by dicofol type DDT pollution, which means that the concentrations of o,p’-DDT are higher than normal area (Qiu et al., 2005; Zheng et al., 2016). Prenatal exposure to OCPs in Southern Spain has an impact on the weight of healthy full-term newborns (Monteagudo et al., 2016). DDT has been found to interact with androgen receptors and estrogen receptors (Li et al., 2008; Soto et al., 1995). DDT induces adverse effects on male rat fertility by acting directly on the testes and altering the neuroendocrine function (Patrick et al., 2016; Rhouma et al., 2001). There are several evidences suggesting that OCPs might be involved in reproductive hormone levels and thereby possibly also in the development of PCOS. A study used Partial least-squares-discriminant analysis (PLS-DA) confirm that serum concentrations of p,p0 -DDE are associated with PCOS (Yang et al., 2015). However, studies on DDT pollution in China have paid little attention to o,p0 -DDT, and few research show the serum o,p0 -DDT levels in relation to PCOS. Keeping in view the dicofol type DDT pollution in Fujian, the serum OCPs concentrations associated with PCOS and hormone concentrations was investigated in the present study. Our objective in this study was to identify the relationship between serum concentrations of OCPs and hormones and to assess the possible impact of exposure to OCPs on PCOS.
2. Materials and methods 2.1. Study subjects and data collection Blood serum samples were recruited from women at the Reproductive Medicine Center of 174th Hospital of PLA (Xiamen, China) between 1 March 2013 and 31 August 2013, and stored at 80 C. All of women were from Xiamen and its surrounding cities and this area is not an industrial area. Among these samples, 84 women were PCOS patients. The patients of PCOS was defined when at least two of the following three features were present: oligo-/amenorrhea (<8 menstrual cycles in the presenting year); hyperandrogenism (and/or hirsutism); and polycystic ovaries (Rotterdam EA-SPcwg, 2004). In order to carry out control, we randomly selected 94 samples. None of the controls had symptoms of hyperandrogenism, a history of menstrual dysfunction, infertility, or sonographic signs of PCOS. All subjects were nonpregnant, non-smokers. All women were studied within the first 10 days after onset of menstruation in the case of mild oligomenorrhea or at random in those suffering from severe oligoor amenorrhea. The characteristics of PCOS women and controls were summarized in Table 1, and their concentrations were determined following the published methods (Zheng et al., 2015). 2.2. Ethical approval The present study was approved by the Reproductive Medicine Center of 174th Hospital of PLA (Xiamen, China), and informed consent was obtained from all participants prior to their recruitment into the study. 2.3. Analysis of OCPs The methods were based on previous study with some modifications (Hagmar et al., 2006). Before extraction, the surrogates 13Cb-HCH, 13C-p,p0 -DDT(20 ng) were spiked separately into the serum. Samples are kept at 4 C overnight for equilibrium before sample extraction. The HLB column (500 mg, Waters, USA) was washed with dichloromethane (DCM) and activated with methanol and Milli-Q water. After conditioning, 1 mL serum was added into column with moist column. The column was then dried for 30 min by aspiration of ambient air. Subsequently, 18 mL of DCM: n-hexane (1:1, v/v) was added to the column for elution. Dried sodium sulfate column was used for the cleaning of extract. After extraction, the solvents were evaporated down to 0.5 mL under a gentle stream of nitrogen gas, and then, 20 ng of internal standard PCB-103 was added before analysis by Gas Chromatography Mass-Mass Spectrometer (GC-MSMS). OCPs were determined with a Trace 1310 gas chromatograph (GC) coupled with TSQ 8000 mass selective detector (MSD) and a 50 m 0.25 mm DB-5 capillary column (Agilent, USA). The GC injector temperature was 290 C with splitless mode, and the GC oven temperature was programmed as follows: 80 C for 2 min, increased 20 C/min to 200 C, held for 2 min, increased to 250 C at a rate of 2 C/min, followed by a rate of 25 C/min to 300 C, and then held for 15 min. 2.4. Quality assurance/Quality control A solvent blank and a procedural blank were added for every sequence of 10 samples to ensure that the samples and the analysis process were free of contamination. The retention times matched with those of the authentic reference compounds and the signal to noise (S/N) ratio was greater than three for the selected ions. The
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Table 1 Clinical characteristics for controls and PCOS patients.
Age (years) Weight (kg) Height (cm) BMI (kg/m2) Waist (cm) Hip (cm) WHR Menarche (years) Hirsutism score Acne score SBP(mmHg) DBP(mmHg) FSH(mIU mL1) LH(mIU mL1) LH/FSH E2(pg mL1) PRL(ng mL1) TT(nmol L1) P(ng mL1) TSH(mIU mL1) DHEAS(mg dL1) SHBG(nmol L1) Fasting insulin (mU mL1) Fasting glucose (mg dL1) GIR HOMA-IR QUICKI Cholesterol (mmol L1) Triglycerides (mmol L1) LDL-C(mmol L1) HDL-C(mmol L1)
Control(n ¼ 94)
PCOS patients(n ¼ 84)
Pa
29(22e35) 54.16(38e93) 1.60(1.48e1.7) 21.28(15.82e37.17) 73.50(60e99) 87.21(74e108) 0.84(0.69e0.99) 14(12e17) 2(0e10) 1(0e15) 113(90e152) 71(50e98) 7.30(2.38e15.82) 4.86(1.36e26.15) 0.72(0.13e3.37) 39.22(12e222) 17.99(4.55e61.12) 1.22(0.32e2.26) 1.18(0.11e25.20) 2.22(0.031e7.69) 291.64(51.82e900.57) 67.23(17.63e391.51) 8.16(2.3e63) 82.71(28.8e98.64) 12.90(1.31e33.26) 1.70(0.4e12.8) 0.36(0.27e0.45) 4.80(3.22e6.53) 0.92(0.35e3.25) 2.89(1.46e4.57) 1.50(0.70e2.26)
28(20e35) 57.24(40e91) 1.60(1.47e1.73) 22.46(15.06e36.92) 76.36(56e102) 88.98(70e116) 0.86(0.69e1.14) 15(12e18) 6(0e18) 2(0e16) 117(93e142) 74(50e96) 6.60(0.98e12.44) 10.81(2.47e35.25) 1.67(0.40e4.66) 39.49(13e140) 16.75(1.18e177.00) 2.14(0.66e8.40) 0.92(0.18e4.21) 2.97(0.005e31.94) 309.77(124.28e463.91) 53.83(3.15e409.95) 11.18(2.40e38.40) 87.52(68.4e119.7) 10.23(2.87e29.70) 2.49(0.42e10.44) 0.34(0.28e0.45) 4.77(3.03e7.10) 1.79(0.30e40.5) 2.82(1.73e4.81) 1.42(0.72e3.14)
0.166 0.016 0.967 0.006 0.010 0.070 0.193 0.198 0.000 0.000 0.013 0.013 0.164 0.000 0.000 0.712 0.028 0.000 0.790 0.133 0.000 0.000 0.000 0.012 0.000 0.000 0.000 0.771 0.000 0.321 0.034
Data are presented as median (25e75% quartiles). a P-values were calculated by ManneWhitney U test and Chi-square test.
limit of detection (LOD) of DDTs and HCHs was 0.1 ng mL1. The recoveries of spiked serum samples for DDTs ranged from 78% to 98%, HCHs from 81% to 94%. Reported concentrations were not corrected with recovery rates. Six-point calibration curves with good to excellent linearity (r2 > 0.99) were constructed for the quantification. 2.5. Statistical analyses Data were presented as median, mean ± SD, and analyzed using SPSS 17.0 software. The reproductive hormones and metabolites data were log-transformed prior to conducting statistical tests. The differences in clinical characteristics and OCPs concentrations between the PCOS group and the control group were compared using ManneWhitney U. Appropriate correlation tests (Pearson or Spearman) were performed to analysis relationship among OCPs and anthropometric, hormonal and biochemical parameters. The association between OCPs concentrations and hormone using linear regression with hormone levels was examined. Statistical significance was set at P < 0.05. 3. Results Clinical characteristics of the case and control participants were shown in Table 1. The age, height, hip, waist-to-hip ratio (WHR), menarche, FSH, E2, P, TSH, cholesterol and LDL-C had no statistically significant differences between the PCOS group and the control group. The median PRL, SHBG, GIR, QUICKI and HDL-C were lower in the PCOS group than that in the control group. The other characteristics were higher in the PCOS group than that in the control group (Table 1). Concentrations of OCPs in serum samples were shown in Table 2
and Fig. 1. OCPs were not detected in all blanks. HCHs, o,p’-DDT, p,p’DDD and p,p’-DDE were detected in all serum groups, while p,p’DDT in 82% and 63% in different groups. OCPs which detected lower than 30% were not showed in this table. Previous studies also reveal that DDE is the most commonly detected congener of DDTs in the general population (Thomas et al., 2006). We observed the mean o,p’-DDT (P ¼ 0.000) and p,p’-DDT (P ¼ 0.016) concentrations in the PCOS group (0.53 ng mL1, 0.77 ng mL1) were higher than those of the control group (0.44 ng mL1, 0.58 ng mL1). Keeping in view the detection rates, we used appropriate correlation tests to analysis the relation between o,p’-DDT and all clinical and laboratory parameters investigated in the study (p,p’-DDT was not analyzed due to the detection rate). In all serum samples, o,p’-DDT levels had positive correlation with LSH/FH, testosterone, triglycerides), and negative correlation with FSH and SHBG. As shown in Table 3, a 1 ng mL1 increase in o,p’-DDT was associated with a 0.037% increase in FSH levels in unadjusted analyses. The association was strengthened with a 0.410% increase after adjusting for age, BMI and WHR. A 1 ng mL1 increase in o,p’-DDT was associated with a 0.997% increase in FSH/LH levels in unadjusted analyses. The association was strengthened with a 0.999% increase after adjusting for age, BMI and WHR. A 1 ng mL1 increase in o,p’-DDT was associated with a 0.823% increase in testosterone levels in unadjusted analyses. The association was attenuated with a 0.810% increase after adjusting for age, BMI and WHR. A 1 ng mL1 increase in o,p’-DDT was associated with a 1.241% increase in SHBG levels in unadjusted analyses. The association was strengthened with a 1.300% increase after adjusting for age, BMI and WHR.
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Table 2 Concentrations of OCPs in serum samples from PCOS patients and controls. Control N(%)>LOD
Pa
PCOS patients mean
SD
25th
50th
75th
N(%)>LOD
mean
SD
1
b-HCH(ng mL ) g-HCH(ng mL1)
94(100) 0.69 0.31 0.46 0.63 0.85 84(100) 0.86 0.60 94(100) 0.81 0.32 0.58 0.73 0.93 84(100) 0.96 0.58 P 1 94(100) 1.50 0.63 1.03 1.39 1.80 84(100) 1.82 1.19 HCHs(ng mL ) 1 p,p’-DDT(ng mL ) 77(81.9) 0.58 0.27 0.42 0.51 0.62 53(63.1) 0.77 0.44 p,p’-DDD(ng mL1) 94(100) 0.42 0.05 0.38 0.42 0.45 84(100) 0.42 0.05 p,p’-DDE(ng mL1) 94(100) 2.50 1.82 1.19 2.03 2.97 84(100) 2.42 1.65 o,p’-DDT(ng mL1) 94(100) 0.44 0.08 0.39 0.42 0.46 84(100) 0.53 0.16 P DDTs(ng mL1) 94(100) 3.84 2.02 2.40 3.53 4.65 84(100) 3.86 2.02 P P HCHs refer to the sum of b-HCH and g-HCH, and DDTs refer to the sum of p,p0 -DDT, p,p’-DDD, p,p0 -DDE and o,p0 -DDT. a P-values were calculated by ManneWhitney U test and Chi-square test.
25th
50th
75th
0.45 0.58 1.02 0.44 0.38 1.09 0.41 2.20
0.64 0.74 1.39 0.65 0.41 2.16 0.46 3.62
1.08 1.22 2.32 1.00 0.45 3.37 0.61 5.46
Controls
0.271 0.353 0.316 0.016 0.780 0.981 0.000 0.991
Max
Concentrations (ng mL-1)
PCOS Patients
75% Mean Median 25% Min
Fig. 1. Box Plots of EDCs Concentrations in serum. Data presented as mean ± SD, (*P < 0.05).
A 1 ng mL1 increase in o,p’-DDT was associated with a 0.705% increase in triglycerides levels in unadjusted analyses. The association was attenuated with a 0.752% increase after adjusting for age, BMI and WHR.
4. Discussion PCOS is a common reproductive endocrine disease that is seen among adolescent women. Environmental factors based on genetic
Table 3 Unadjusted and adjusted results for the percentage difference in serum hormone levels or metabolic profile per unit change in serum o,p’-DDT levels. Adjusteda
Unadjusted
FSH LH LH/FSH E2 PRL TT P TSH DHEAS SHBG Fasting insulin Fasting glucose GIR HOMA-IR QUICKI Cholesterol Triglycerides LDL-C HDL-C a
% difference
95% CI
0.388 0.610 0.997 0.171 0.198 0.823 0.143 0.263 0.307 1.241 0.102 0.007 0.094 0.109 0.017 0.017 0.705 0.030 0.100
0.032 0.103 0.010 0.531 0.511 0.003 0.708 0.567 0.100 0.031 0.739 0.919 0.739 0.750 0.740 0.852 0.037 0.827 0.446
Adjusted for age, BMI and WHR.
0.742 0.124 0.240 0.708 0.792 0.279 0.609 1.169 0.059 2.365 0.501 0.138 0.652 0.567 0.121 0.167 0.042 0.242 0.357
P
% difference
95% CI
0.033 1.343 1.755 0.367 0.396 1.367 0.895 0.643 0.673 0.117 0.704 0.153 0.463 0.785 0.086 0.201 1.368 0.302 0.158
0.410 0.589 0.999 0.193 0.203 0.810 0.089 0.272 0.299 1.300 0.154 0.019 0.135 0.173 0.027 0.027 0.752 0.038 0.097
0.022 0.114 0.010 0.481 0.497 0.003 0.815 0.558 0.113 0.024 0.558 0.794 0.583 0.560 0.546 0.773 0.015 0.782 0.424
P 0.759 0.143 0.242 0.734 0.790 0.273 0.666 1.189 0.072 2.430 0.364 0.124 0.622 0.412 0.117 0.157 0.148 0.231 0.336
0.061 1.321 1.756 0.347 0.385 1.348 0.844 0.644 0.669 0.169 0.673 0.162 0.351 0.758 0.062 0.211 1.355 0.307 0.142
Z. Guo et al. / Chemosphere 171 (2017) 595e600
predisposition have been thought to play a role in the aetiopathogenesis of PCOS in recent years (Pasquali et al., 2011). The main environmental factors include environmental toxins, diet and nutrition, socioeconomic status, and geography (Merkin et al., 2016). The ovaries seem to be a prime target for endocrine disndez et al., 2010; Newbold et al., 2007). In ruptor compounds (Ferna this study, we examined the relationship between OCPs and PCOS. Statistically significant higher serum o,p’-DDT levels in the PCOS group compared to the controls were observed. As a member of EDCs, o,p’-DDT may play a role in the pathogenesis of PCOS by affecting the reproductive hormones and metabolites. Before the ban of DDT use in agriculture, more than 60% of the technical DDT had been used on cotton fields, mainly in central and eastern China. In comparison to central and eastern China, less technical DDT was used in southern China, such as Guangdong, Guangxi, and Fujian provinces (Qiu et al., 2005; Zheng et al., 2016). Considering the difference between the spatial distribution of technical DDT and dicofol use DDT pollution in Fujian is very likely dominated by dicofol type DDT pollution. Our study also showed the high levels of o,p’-DDT in serums of females in Fujian province. However, until now, few study investigating the endocrine disrupting function of o,p’-DDT. In the present study, statistically significant higher serum o,p’-DDT levels in the PCOS group (0.53 ng mL1) compared to the control group (0.44 ng mL1) were found (P ¼ 0.000). DDT exhibit properties which are very similar to E2 (Diel et al., 2002), and some studies have proved that o,p’-DDT enantiomer is a more active estrogen-mimic than the (þ)-enantiomer in rats (Hoekstra et al., 2001). o,p’-DDT has a similar function with bisphenol A (BPA), which is known to interfere with androgen catabolism. A previous study has demonstrated that BPA stimulates ovarian -theca-interstitial cells to produce androgens by dysregulation of 17a-hydroxylase, a key enzyme in gonadal steroidogenesis (Zhou et al., 2008). The activation of this steroidogenic pathway has a role in PCOS ovarian hyperandrogenism (Nelson et al., 2001). There might be a similar mechanism for the association between o,p’-DDT and androgens in these patients. A mice experiment suggests that perinatally exposed to DDT reduced core body temperature, decreased energy expenditure, impaired cold tolerance, and produced a transient early-life increase in body fat in female offspring (La Merrill et al., 2014). When challenged with a high fat diet for 12 weeks in adulthood, perinatal DDT exposure developed glucose intolerance, hyperinsulinemia, dyslipidemia, and altered bile acid metabolism in female offspring (La Merrill et al., 2014). Exposure adult rodents to high doses of DDT have impaired insulin secretion, glucose intolerance, and elevated gluconeoegenesis (Yau and Mennear, 1977). The results of epidemiological studies have showed the associations between DDT and DDE exposures and diabetes in humans (Lee et al., 2011; Patel et al., 2010). However, our study show that there was no correlation between o,p’-DDT concentrations and insulin resistance and sensitivity indexes. Our study showed the positive correlation between o,p’-DDT concentrations and LSH/FH(P ¼ 0.009), testosterone (P ¼ 0.003), triglycerides (P ¼ 0.035), and the negative correlation between o,p’DDT concentrations and FSH (P ¼ 0.013), SHBG (P ¼ 0.037). A epidemiological study suggests a positive relationship of baseline E2 and baseline testosterone with DDT compounds (Dalvie et al., 2004). We speculate o,p’-DDT may specifically bind to SHBG, resulting in elevated levels of testosterone; o,p’-DDT may cause a decline in FSH levels through the involvement of steroid enzyme (Gregoraszczuk and Ptak, 2013), affecting the development of follicles, while causing high levels of LH. Elevated LH levels can also lead to excessive secretion of male hormones, thus disrupt or inhibit the development of the female reproductive system. All the conditions causing symptoms such as ovarian cysts, which play an
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important role in the pathogenesis of PCOS. The elevated triglyceride levels affected by o,p’-DDT may also lead to obesity, cardiovascular disease and other illnesses. A rats animal model study shows that ancestral exposure to DDT can cause metabolic syndrome-related lesions in the testis, ovary, and promote obesity and associated disease in offspring generation (Skinner et al., 2013). 5. Conclusion In conclusion, our results indicate that o,p’-DDT concentrations in PCOS group were higher than the control group. Many studies support that androgen catabolism may play an important role in the pathogenesis of PCOS. o,p’-DDT may have positive effects on PCOS by increasing or decreasing reproductive hormone levels related with androgen catabolism. However, larger trials are required before reaching definitive conclusions. Further mechanistic studies are needed to confirm the findings in our study. Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported in part by the Program for Xiamen Southern Oceanographic Center (14GST69NF33), and the project of the Xiamen Science and Technology Program (2013Z20134027). References Craig, Z.R., Wang, W., Flaws, J.A., 2011. Endocrine-disrupting chemicals in ovarian function: effects on steroidogenesis, metabolism and nuclear receptor signaling. Reproduction 142 (5), 633e646. Dalvie, M.A., Myers, J.E., Thompson, M.L., Dyer, S., Robins, T.G., Omar, S., Riebow, J., Molekwa, J., Kruger, P., Millar, R., 2004. The hormonal effects of long-term DDT exposure on malaria vector-control workers in Limpopo Province, South Africa. Environ. Res. 96 (1), 9e19. Diel, P., Olff, S., Schmidt, S., Michna, H., 2002. Effects of the environmental estrogens bisphenol A, o, p0 -DDT, p-tert-octylphenol and coumestrol on apoptosis induction, cell proliferation and the expression of estrogen sensitive molecular parameters in the human breast cancer cell line MCF-7. J. Steroid. Biochem. Mol. Biol. 80 (1), 61e70. ndez, M., Bourguignon, N., Lux-Lantos, V., Libertun, C., 2010. Neonatal expoFerna sure to bisphenol A and reproductive and endocrine alterations resembling the polycystic ovarian syndrome in adult rats. Environ. Health Perspect. 118, 1217e1222. Gregoraszczuk, E.L., Ptak, A., 2013. Endocrine-disrupting chemicals: some actions of POPs on female reproduction. Int. J. Endocrinol. 2013, 1e9. Goodarzi, M.O., Dumesic, D.A., Chazenbalk, G., Azziz, R., 2011. Polycystic ovary syndrome: etiology, pathogenesis and diagnosis. Nat. Rev. Endocrinol. 7 (4), 219e231. € nsson, B.A., Bergman, Å., Rylander, L., 2006. Hagmar, L., Wallin, E., Vessby, B., Jo Intra-individual variations and time trends 1991e2001 in human serum levels of PCB, DDE and hexachlorobenzene. Chemosphere 64 (9), 1507e1513. Hoekstra, P.F., Burnison, B.K., Neheli, T., Muir, D.C., 2001. Enantiomer-specific activity of o, p0 -DDT with the human estrogen receptor. Toxicol. Lett. 125 (1), 75e81. Johansson, H.K.L., Jacobsen, P.R., Hass, U., Svingen, T., Vinggaard, A.M., Isling, L.K., Axelstad, M., Christiansen, S., Boberg, J., 2016. Perinatal exposure to mixtures of endocrine disrupting chemicals reduces female rat follicle reserves and accelerates reproductive aging. Reprod. Toxicol. 61, 186e194. La Merrill, M., Karey, E., Moshier, E., Lindtner, C., La Frano, M.R., Newman, J.W., Buettner, C., 2014. Perinatal exposure of mice to the pesticide DDT impairs energy expenditure and metabolism in adult female offspring. PLoS One 9 (7), e103337. €din, A., Jones, R.S., Needham, L.L., Jacobs Jr., D.R., 2011. Lee, D.H., Steffes, M.W., Sjo Low dose organochlorine pesticides and polychlorinated biphenyls predict obesity, dyslipidemia, and insulin resistance among people free of diabetes. PLoS One 6 (1), e15977. Legro, R.S., Driscoll, D., Strauss, J.F., Fox, J., Dunaif, A., 1998. Evidence for a genetic basis for hyperandrogenemia in polycystic ovary syndrome. Proc. Natl. Acad. Sci. U. S. A. 95 (25), 14956e14960. Li, J., Li, N., Ma, M., Giesy, J.P., Wang, Z., 2008. In vitro profiling of the endocrine disrupting potency of organochlorine pesticides. Toxicol. Lett. 183 (1), 65e71. Li, R., Zhang, Q., Yang, D., Li, S., Lu, S., Wu, X., Wei, Z., Song, X., Wang, X., Fu, S., Lin, J.,
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