Effect Of Triclosan Exposure On Embryonic Development And Its Mechanism

Triclosan(2,4,4′-trichloro-2′-hydroxydiphenyl ether, TCS), was firstly introduced in the 1960 s for use in personal care products as an antimicrobial and preservative, and has been increasingly used over the past 20 years. TCS, a halogenated phenol, is a nonionic, broad spectrum antibacterial used in personal care, household and industrial products such as soap, toothpaste, deodorants, cosmetics, shampoo, dishwashing liquids, disinfectants, and an additive in many plastics and textiles. Widespread use of TCS has led to ubiquitous exposure of the general population. TCS, as a contaminant, was detected in human tissues and body fluid including fat tissue, umbilical cord blood, serum, urine, and breast milk. The effects of TCS on the reproductive endocrine system have been widely reported in the literatures, and there is sufficiently experimental evidence showing that TCS is one kind of Endocrine Disrupting Chemicals(EDCs). EDCs refer to a large category of chemicals which, naturally existing in or released into the environment, simulate natural hormones in their physiological and biological actions upon living organisms, and interfere with or suppress the function of the nervous, endocrine, immune andreproductive systems, producing reversible or irreversible biological effects. In certain cases, EDCs may lead to dysfunction of a tissue or organ tumorigenesis. EDCs can lead to pregnancy failure by causing chromosomal aberrations of oocytes and developmental arrest of embryos. EDCs can affect the development of fertilized eggs and embryonic implantation, and then result in early pregnancy loss. They can also cause embryonic dysplasia and lead to abortion. The interference of EDCs on key gene expression results in abnormalities of embryonic development and differentiation. In present study, we detected urinary TCS exposure levels and investigated the correlation between TCS exposure and spontaneous abortion. Next, we also adopted in vivo model(pregnant mice) and in vitro models(mouse embryonic stem cells and zebrafish embryo) to further clarify the effects and related mechanisms of different windows of TCS exposure on embryonic development. This will help us fully understand the effects and related mechanisms of TCS on embryonic development.Part I The relationship between triclosan levels and spontaneous abortion among non-occupational exposure femaleWidespread use of triclosan(TCS) has led to ubiquitous exposure of the general population. In vitro and animal studies have demonstrated that TCS has adverse effects on reproduction and development. However, there is no epidemiological evidence of the relationship between exposure to TCS and spontaneous abortion. We evaluated the association between maternal urinary levels of TCS and spontaneous abortion in a non-occupational exposure population. A case-control study was conducted that included 113 cases with medically unexplained spontaneous abortion and 339 controls who didn’t have a history of spontaneous abortion and had at leastone living child. Maternal urinary TCS levels were measured by ultra-high performance liquid chromatography-tandem mass spectrometry. Multivariate logistic regression and tests for trend were performed to analyze the relationship between TCS exposure and spontaneous abortion. Compared with females in the lowest exposure group, females in both groups of median and high TCS exposure were more likely to suffer spontaneous abortion {ORs for increasing exposure levels= 1.00, 2.00 [95% confidence interval(CI), 1.08-3.70], 2.36(95% CI, 1.29-4.34), p value for trend=0.003}. We provide the first human study that maternal urinary TCS exposure is associated with spontaneous abortion. These findings provide the evidence suggesting adverse effects of TCS on female reproductive and developmental health in humans.Part II Second trimester exposure to triclosan induced spontaneous fetal loss in mouseIn vivo and vitro studies have demonstrated that TCS caused adverse effect on the reproduction and development. The first part of our study shows that high exposure of TCS increases the risk of spontaneous abortion in the study population. The window period in which environmental chemicals have toxic effects on embryonic development mainly includes developmental stage of oocytes, embryonic preimplantation, implantation, and post-implantation stages. They may play a role in any period of the above stages or in multiple periods. In order to confirm the results of the first part and investigate the exact window period and toxicity dose of TCS on embryonic development, we selected GD5-15 as window period and doses of 1, 10, 100 mg/kg/day as oral exposure doses. Meanwhile, in order to determine whether thedoses which mice were exposed to could simulate exposure levels of the population, we detected urinary TCS exposure levels in mice. The results showed that mean weights of fetus in the exposure group of 10 and 100 mg/kg/day were significantly lower than that of the control group. Fetal loss rate of 10 and 100 mg/kg/day exposure group was significantly higher than that in the control group, and fetal loss rate significantly increased along with the increase of dose and exposure time, which demonstrated that the post-implantation stage was the toxic window period of TCS on embryonic development. Basis for species differences, by conversion of an uncertainty factor(100 times) we inferred that urinary TCS level of 10mg/kg/d dose group in mice was extrapolated to the high exposure level of our study population, and urinary TCS level of 100mg/kg/d group in mice was extrapolated to the high exposure level of Mexican Americans. In this study, the model of mice exposure to TCS could simulate the state of population exposure to TCS. TCS could increase fetal loss rate and reduce fetal weight. This provided the certain evidence for the causal association between TCS exposure and spontaneous abortion. Next, further investigation is required to elucidate the possible mechanisms.PartⅢ Effect of triclosan on pluripotency of mouse embryonic stem cells and embryonic development of zebrafishEmbryonic stem cells(ESC) and zebrafish embryo provide valuable in vitro models for testing the toxic effects of environmental chemicals on early embryogenesis. TCS poses potential risks to reproduction and development due to its endocrine disruption. However, the mechanism of TCS effects on early embryonicdevelopment is still unknown. In this study, mouse embryonic stem cells(m ESC) were acutely exposed to TCS for 24 h, and general cytotoxicity and the effect of TCS on pluripotency were then evaluated. Meanwhile, zebrafish embryos were exposed to TCS from 2h to 24 h and their morphology was evaluated. In m ESC, the significantly decreased AP staining and activity were observed in TCS treatment of the highest concentration(50μM) compared with the control. The expression levels of Sox2 m RNA were not obviously changed. However, the m RNA levels of Oct4 and Nanog in TCS-treated groups were significantly decreased, compared to the control. The protein levels of Oct4, Sox2 and Nanog were significantly reduced in response to TCS treatment. Furthermore, when compared with that in the control, micro RNA(mi R)-134, an expression inhibitor of pluripotency markers including Oct4, Sox2 and Nanog, was significantly increased in TCS-treated m ESC. In zebrafish experiments, after 24 h of TCS exposure, there were no changes of morphology between dose groups and control group. TCS exposure led to a decrease in the expression level of Oct4 and Nanog but no change was found in the expression of Sox2. After 24 h of TCS exposure, embryos exposed to 300μg/L of TCS all stayed at the early stage of somitogenesis when compared with the controls where they had developed to the late stage of somitogenesis. Three genes(Oct4, Sox2 and Nanog) were upregulated when compared with the controls. In summary, these results firstly indicate that TCS may disturb pluripotency in m ESC while mi R-134 may play a key role by inhibiting the expression of downstream transcription factor(Oct4, Sox2 and Nanog). In addition, TCS at the highest dose group(300μg/L) causes developmental arrest of zebrafish embryos at 24 h.