Differences In Endocrine Disrupting Effects Caused By Different Exposure Sequences Of Nanoparticles And Bisphenol Analogs

Endocrine disrupting chemicals(EDCs)are a class of exogenous substances that can interfere with the physiological processes of natural hormones in the body.Humans are often subjected to combined exposure or sequential exposure to exogenous EDCs and other environmental pollutants.However,most studies now focus on the combined effects of coexposure to multiple environmental pollutants(including EDCs).There is a lack of data on differences in endocrine disrupting effects due to different exposure sequences.Therefore,in this study,we investigated the endocrine effects induced by different exposure sequences of five bisphenol analogs(BPA,BPAF,BPAP,BPF,and BPS)and nanoparticles(CuO NPs and ZnO NPs)in vitro and in vivo using human breast cancer cells(MCF-7)and zebrafish embryos/larvae as models,respectively.The different exposure sequences in in vitro experiments were: 1)24 h exposure to NPs followed by 24 h exposure to BPs,2)24 h exposure to BPs followed by 24 h exposure to NPs,and 3)48 h co-exposure to NPs and BPs.The results showed that the cytotoxicity induced by the co-exposure of BPs and NPs was not significantly different from those caused by the exposure of BPs or NPs alone.However,significant differences were found in the cytotoxicity caused by the exposure of BPs and CuO NPs in different sequences.Furthermore,as compared to BPs and ZnO NPs,the combination of BPs and CuO NPs significantly influenced the expression of estrogen receptors(ER)on MCF-7 cells.The in vivo experiments were then performed using a zebrafish model with different exposure sequences of CuO NPs & BPF combinations: 1)3 d of BPF exposure followed by 3d of CuO NPs exposure(BPF-CuO);2)3 d of CuO NPs exposure followed by 3 d of BPF exposure(CuO-BPF);3)6 d co-exposure of CuO NPs and BPF(BPF+CuO).By measuring three sensitive biological indicators of endocrine disruption in vivo(estradiol(E2),testosterone(TTE),and vitellogenin(VTG)),we found that the different exposure sequences of BPF and CuO NPs disrupted the hormonal levels of E2 in zebrafish larvae.Changes in the E2 with TTE ratio further confirmed that the different exposure sequences of the two contaminants caused different endocrine effects.Compared with the CuO-BPF treatment group,BPF-CuO still caused hypopigmentation in zebrafish larvae,but the inhibition of tyrosinase activity caused by BPF-CuO could be reversed.With the help of transcriptomics,we found that endocrine-related signalling pathways were active in both BPF+CuO and CuO-BPF treatment groups,mainly regulated by hypothalamic-pituitary-related neuroendocrine signalling pathways.Furthermore,BPF-CuO may have a potential carcinogenic effect on zebrafish larvae.The major window of action of BPF on pigment inhibition in larvae is earlier than 4 dpf,according to the upregulation of the tyr,tyr1 b,and dct genes in the CuO-BPF treated group.The different exposure sequences of BPF and CuO NPs interfere with the melanocortical system,resulting in differences in melanin expression on the body surface of zebrafish,which may also be involved in the regulation of sex hormones.In summary,the results of the present work suggest that the different exposure sequences of BPF and CuO NPs cause different endocrine effects,possibly through diverse molecular mechanisms.In addition,the exposure sequence is a time-dependent factor and therefore the coupling with the growth and developmental window of the organism needs to be further considered in in vivo experiments.This study provides fundamental theoretical data to support the study of the possible multiple toxicity targets between chemical contaminants due to the sequence of exposure and the underlying physicochemical and physiological mechanisms.