Enantioselectivity In Endocrine Disruption Of Two Typical Chiral Pesticides

Widespread application of chiral pesticides results in their occurrence in environmenat systems,wildlife and humans.In general,the enantiomers of chiral pesticides are different in biological processes,thus pose enantioselective ecotoxicity to nontarget biology.Nowadays,the application and environmental safety of chiral pesticides have been a new point of scientific researches worldwide.Several recent studies focused on the occurrence of enantioselectivity of chiral pesticides in environmental fate and acute aquatic toxicity.However,so far little attention has been given to enantioselectivity in chronic toxicities such as endocrine disruption activity.In this study,we chose two typical chiral pesticides,i.e., o,p′-DDT,an important member of the persistent organic pollutants(POPs),and bifenthrin,a newer and widely used pyrethroid insecticide,as model compounds to evaluate enantioselectivity in some endocrine disruption processes,such as mimicing the activity of endogenous estrogen 17β-estradiol(E₂),affecting the breast carcinoma tumour development and affecting the occurrence process of placenta.Based on the preparation and identification of enantiomers by the high performance liquid chromatography(HPLC)and gas chromatograph(GC),what we have investigated are as follows:(1)The estrogen-dependent MCF-7 human breast carcinoma cell line was chosen as the model to evaluate the enantioselectivity in estrogenic effects of o,p′-DDT by the MCF-7 cell proliferation assay(i.e.,the E-SCREEN assay)and the real-time quantitative polymerase chain reaction(qRT-PCR).The results show that R-(-)-o,p′-DDT can more significantly stimulate the MCF-7 cell proliferation and induce the estrogen-dependent report gene pS2 mRNA expression than S-(+)-o,p′-DDT.At the same time,the inhibition of mRNA expressions of ERαand ERβby R-(-)-o,p′-DDT was stronger than that by S-(+)-o,p′-DDT.Therefore,it was revealed that significant enantioselectivity existed in the estrogenic effect of o,p′-DDT,i.e.,the estrogen activity of R-(-)-o,p′-DDT was much higher than that of S-(+)-o,p′-DDT.Furthermore,the enantioselective estrogenicity of o,p′-DDT was likely through the ERαand ERβsignaling pathways.(2)Previous researches have showed that environmental estrogens are positively correlated with the occurrence and development of breast cancer,and that o,p′-DDT is a typical environmental estrogen.We evaluated the enantioselectivity in the effect on the development of breast cancer of o,p′-DDT for the first time based on our above study about the enantioselectivity in estrogenic activity.We used human breast cancer MCF-7 cells as an in vitro model to preliminarily study the enantioselectivity and mechanism of o,p′-DDT in breast cancer development,by means of intercellular adhesion analysis and qRT-PCR to determine mRNA expression levels of MMP-9,MMP-2,TIMP-1,TIMP-2,E-cadherin,β-catenin and hTERT which are related to invasion,adhesion and proliferation of breast cancer. The results show that o,p′-DDT can promote the invasion ability of MCF-7 breast cancer cells,which was associated with the up-regulation of MMP-9,MMP-2 gene expressions and the reduction of intercellular adhesion.The promotion to cell invasion by R-(-)-o,p′-DDT was higher than that by S-(+)-o,p′-DDT.In addition,the frequent cell proliferation is an obvious feature of the development of tumor,while our results show that the racemate and enantiomers of o,p′-DDT are all able to increase human telomerase reverse transcriptase(hTERT)gene expression,and the effects induced by R-(-)-o,p′-DDT is higher than that by S-(+)-o,p′-DDT.This is a possible mechanism of the proliferation of the breast cancer cell induced by the o,p′-DDT enantiomers.Combined with our studies shown in(1),we think that the enantioselective estrogenicity of o,p′-DDT may be one main reason of the enantioselective effects in breast tumor development.(3)Placentogenesis,fine regulated by various hormones and other factors,is the important physiological guarantee of normal pregnancy.Nowadays,few researches have referred to whether and how this progress be interfered by environmental estrogens.We used the human choriocarcinoma JEG-3 cell lines as the human trophoblastic cell model to investigate enantioselectivity of chiral contaminants(e.g.,o,p′-DDT)in human reproductive health as well as the mechanism, by means of enzyme-linked immunosorbent assay(ELISA)to determine the secretion level of progesterone(P4)and qRT-PCR to assess the expression levels of progesterone receptor(PR),HILA-G,GnRH-I and GnRHR-I which are related to placentogenesis.It was found that o,p′-DDT can increase the P4 secretion in JEG-3 cells and the expression levels of PR and immune molecule of pregnancy HILA-G,as well as the expression level of polypeptide hormone GnRH-I.All the results show a uniform enantioselectivity,namely,the endocrine disruption potential of R-(-)-o,p′-DDT was greater than that of S-(+)-o,p′-DDT in human trophoblastic cells.Integrated with the results shown in above(1),(2)and(3),we can get the conclusion that the estrogenic activity of o,p′-DDT is significantly enantioselective, while this endocrine disruption potentials in different tissues(for example,human breast and placenta)display consistent enantioselectivity,namely,the endocrine disruption potential of R-(-)-o,p′-DDT was always greater than that of S-(+)-o,p′-DDT. Consequently,when talked to enantioselectivity in estrogenic activity of chiral compounds,we should pay more attention to a series of problems concerning human health in health risk assessment.(4)The little attention about the enantioselectivity in endocrine disrupting effects are restricted to o,p′-DDT and its analogs so far.Although research increasingly suggests that synthetic pyrethroids(SPs),the widely used insecticides, possess hormone agonist or antagonist activities,no report has been found about their enantioselective endocrine disruption.Therefore,we chose bifenthrin(BF),a newer and widely used pyrethroid insecticide,to investigate the enantioselectivity in estrogenic potential and uptake.Significant differences in estrogenic potential were observed between the two enantiomers in the in vitro human breast carcinoma MCF-7 cell proliferation assay and the in vivo aquatic vertebrate vitellogenin ELISA. In the E-SCREEN assay,the relative proliferative effect ratios of 1S-cis-BF and 1R-cis-BF were 74.2%and 20.9%,respectively,and the relative proliferative potency ratios were 10%and 1%,respectively.The cell proliferation induced by the two BF enantiomers may be through the classical estrogen response pathway via the estrogen receptor(ER)as the proliferation induced by the enantiomers could be completely blocked when combined with 10^-6mol/L of the ER antagonist ICI 182,780.Measurement of vitellogenin induction in Japanese medaka(Oryzias latipes)showed that at an exposure level of 10 ng/mL,the response to 1S-cis-BF was about 123 times greater than that to the R enantiomer.Significant selectivity also occurred in the uptake of BF enantiomers in the liver and other tissues of J.medaka.These results together suggest that assessment of the environmental safety of chiral insecticides should consider enantioselectivity in acute and chronic ecotoxicities such as endocrine disruption.