Impact Of Exposure To Phthalates And Bisphenol A On Growth And Development Of School Children

Phthalates (PAEs) and bisphenol A (BPA) are frequently used plasticizers with a history of more than50years of production and utilization. Environment and food are well documented to be contaminated with PAEs and BPA, and this results in human extensive exposure to them. Worryingly, PAEs and BPA are suspected endocrine disrupting chemicals (EDCs), and have been reported to interfere with homeostasis of sex hormones or thyroid function. In view of a critical role of sex and thyroid hormones in children’s growth and development, it is reasonably hypothesized that exposure to PAEs and BPA can have an adverse influence.However, studies on the associations of PAEs and BPA exposure with sex hormones, thyroid function, and children’s growth and development are scarce. In the current study, we first established a rapid and efficient analytical method for PAEs metabolites, BPA and endogenous sex hormones in urine, and then assessed the exposure levels of PAEs and BPA and investigated the associations of PAEs and BPA levels in urine with children’s sex hormones, and growth and development in nearly1000school children living in Haimen city in Jiangsu province, Minhang district in Shanghai city, and Yuhuan county in Zhejiang province.First section:Establishing a rapid and efficient analytical method for measurements of phthalate metabolites, BPA and endogenous sex hormones in urineA rapid and sensitive method for determination of14phthalate metabolites, BPA, and four endogenous steroid hormones in urine was developed and validated based on ultra-performance liquid chromatography coupled to electrospray ionization triple quadropole mass spectrometry (UPLC-ESI-MS/MS). After purified by the reversed-phase and anion-exchange mixed-mode solid phase-extraction (SPE), these target compounds were separated on C18chromatographic column and analyzed in multi-reaction monitoring MS mode. Phthalate metabolites were determined in negative mode with an acetonitrile/water mobile phase containing0.1%acetic acid, and dansylated BPA and steroid hormones were determined in positive ion mode with a methanol/water mobile phase containing10mM ammonium formate. Quantification was performed by isotope-labeled internal standard calibration curve, and most correlation coefficients were0.992or more. The limits of detection for phthalate metabolites were below0.84ng/mL, and those for endogenous steroid hormones and BPA were below0.04ng/mL. The inter-day recoveries at the lowest spiked concentration varied between79.4%and125.0%with relative standard deviations (RSDs) from9.1%to22.0%for phthalate metabolites, and between76.5%and113.6%with RSDs from3.7%to14.9%for endogenous steroid hormones and BPA. This proposed analytical method was rapid, sensitive and accurate in analyzing these target compounds in urine.Second section:Screening and assessing urine phthalate metabolites and BPA in school childrenFourteen urine phthalate metabolites and BPA were screened in968school children9-12years of age chosen from Haimen city in Jiangsu province, Minhang district in Shanghai city, and Yuhuan county in Zhejiang province. Except for MOP and MiNP, other12phthalate metabolites and BPA were detected in urine, and among them,10phthalate metabolites and BPA were detected in98.6%-100%of participants with the exception of MBzP (42.3%) and MCHP (85.2%). Primary urine phthalate metabolites were from DBP, DEHP and DiBP, which respectively accounted for36.5%,32.7%and17.9%of mass concentration sum of all metabolites in urine, followed by MEP (8.5%) and MMP (3.9%). There were significant area differences of urine concentrations of phthalate metabolites and BPA in school children. According to the daily intake reference doses for DBP, DEP and DEHP proposed by U.S. Environmental Protection Agency (EPA), DEHP estimated daily intake exceeded the reference dose in0.6%-2.6%of school children, but not for DBP and DEP. According to BPA tolerable daily intake of50μg/kg.day suggested by the European Food Safety Authority (EFSA), none of school children exceeded the values.Third section:The association of urine phthalate metabolites and BPA with sex hormones in childrenIn800school children9-12years of age chosen from Haimen city in Jiangsu province, Minhang district in Shanghai city, and Yuhuan county in Zhejiang province, we firstly investigated the association of urine phthalate metabolites and BPA with urine sex hormone levels. Logistic regression was used to analyze the relationship of the quartiles of urine phthalate metabolites and BPA with sex hormone level. Adjusted covariates included age, sex, urine specific gravity, urine iodine, BMI and sampling site. The results showed that urine BPA was negatively associated with PGT, and positively associated with E2, E1and TE (p=0.061). All phthalate metabolites were positively associated with E2except for MEHHP. Except for MEOHP, MCMHP, MCHP (p=0.053) and MECPP (p=0.093), other metabolites were positively associated with Ei. Only MEHP, MCMHP, MBP and MEP were positively associated with PGT. Only MEHP, MBP and MEP (p=0.055) were positively associated with TE. Phthalates and BPA might interfere with estrogen, androgen and progestogen, and the interference direction of BPA with progestogen was different from estrogen and androgen. There were significant differences in the associations of urine phthalate metabolites and BPA with sex hormones according to age, sex and area. Given that the cross-sectional nature of this study can not rule out reverse causation, more lab and epidemiological studies were needed to confirm these associations.Fourth section:The association of urine phthalate metabolites and BPA with children’s growth and developmentIn about940school children9-12years of age chosen from Haimen city in Jiangsu province, Minhang district in Shanghai city, and Yuhuan county in Zhejiang province, we investigated the association of urine phthalate metabolites and BPA with children’s thyroid gland volume, growth and development by using multiple linear regression. Adjusted covariates included age, sex, urine specific gravity, urine iodine, sampling site or body mass index (BMI). The results showed that urine BPA was positively associated with indicators reflecting children growth and development (height, weight, chest circumference, and height and chest circumference index (HCI)) and those reflecting fat accumulation (waist circumference, hip circumference, and BMI), but inversely associated with thyroid gland volume. DEHP metabolites were inversely associated with thyroid gland volume and indicators reflecting children’s growth and development (height, weight, and chest circumference), and also showed an inverse trend with those reflecting fat accumulation (hip circumference and BMI). MEP and MCHP were positively associated with indicators reflecting fat accumulation or growth and development, and their associations with indicators reflecting fat accumulation showed to be stronger than those with indicators reflecting growth and development. Both of MEP and MCHP were not significantly associated with thyroid gland volume. There were significant differences in the associations of urine phthalate metabolites and BPA with these indicators according to age, sex and area. Phthalates and BPA might interfere with children’s growth and development, and they might have different impacts. BPA exposure promoted children’s growth and development as well as body fat genesis. DEHP exposure mainly showed to inhibit children’s growth and development, while DEP or DCHP exposure mainly showed to promote body fat genesis. Given that the cross-sectional nature of this study can not rule out reverse causation, more lab and epidemiological studies were needed to confirm these associations.