The Effects And Action Mechanisms Of Bisphenol A And Bisphenol S On The Immune System And Reproductive Neuroendocrine System In Fish

Bisphenol A(BPA) is one of well-known endocrine disrupting chemicals(EDCs) which are ubiquitous in water, sediment and aquatic organisms in major rivers, lakes and other aquatic environments. Bisphenol A has been widely employed as a plastic monomer and plasticizer in production of epoxy resins and polycarbonate plastic. As a consequence of its ubiquitous existence in the environment, wide detection in human biological samples, and extensive application in our daily lives, BPA has raised considerable concerns for public health. As a result, some regulatory agencies, such as the European Commission, the US Food and Drug Administration, and Health Canada, during recent years have banned use of BPA in baby bottles. Given these restrictions and societal pressure, manufacturers seeking BPA alternatives have turned to primarily bisphenol S(BPS) to produce “BPA-free” products. However, the knowledge about toxicity of BPS is still limited all around the word. Thus, it is very necessary to study whether or not BPS, a common “safer” replacement for BPA, caused alterations in normal development in a similar manner as BPA. In the present study, we assessed the effects and action mechanisms of acute exposure to environmentally relevant concentrations of BPA and BPS on the immune system and the reproductive neuroendocrine system using the carp primary macrophages and zebrafish embryos as models. The results are shown as follows:1. Acute exposure(6 hours) to environmentally relevant concentrations of BPA(0.1,1,10,100 and 1000 ?g/L) had an immune modulatory effect on primary macrophages from red common carp(Cyprinus carpio). The results are shown as follows:(1) The viability of primary macrophages was not impaired after 6 h exposure to BPA based on the cytotoxicity assay;(2) A dynamic response process was observed in macrophages upon various concentrations of BPA exposure, which significantly enhanced the antibacterial activity of macrophages at 0.1, 1, or 10 μg/L, but instead induced the apoptosis at 100, 1,000, and 10 000 μg/L;(3) A potential pro-inflammatory effect of BPA exposure was suggested judging from the increased production of nitrite oxide and reactive oxygen species(ROS), the induction of interleukin-1β mRNA and protein, as well as NF-κB and other NF-κB-associated immune gene expression;(4)The alterations of most tested parameters were significantly correlated, indicating a coordinated modulation process in macrophages responded to increased BPA exposure. Following BPA coexposure with the ER or NF-κB antagonist, the induction of ROS, ERα, and NF-κB-associated immune gene expression was significantly inhibited, implying interaction between those two pathways. This study thus indicated that low doses of BPA exposure alone could significantly disturb the immune response of fish primary macrophages in vitro, and for the first time revealed the synergistic action of ERα and NF-κB transcription factors in the BPA effect.2. Acute exposure(6 hours) to environmentally relevant concentrations of BPA had a similar immune modulatory effect on primary macrophages from red common carp. The results are shown as follows:(1) Time-dependent lethal concentrations(LC), 50% lethal concentrations(LC50) and 5% lethal concentrations(LC5) of BPS were determined and at mg/L levels;(2) Exposure to the no-observed effect concentration of BPS(0.1,1,10,100,1,000 μg/L) was found to weaken the antibacterial activity of macrophages at 1,000 μg/L, but it enhanced the production of hydroxyl radicals and lipid peroxidation in a concentration-dependent manner;(3)The activity of total antioxidant capacity(T-AOC), as well as the content of nitric oxide(NO) and nitric oxide synthase(NOS) were all significantly induced after exposure to BPS, indicating the occurrence of oxidative stress;(4)Various concentrations of BPS exposure during development led to increased expression of immune related genes(Interloukin-1β, Tumour necrosis factor-α1, Interloukin-10, CXCL8, Interferonγ-1, Interloukin-12 p35, Interloukin-6, Interloukin-11). The present study also aimed to compare the impact of BPS and BPA on immune system in red carp primary macrophages. Notably, our results show that low levels of BPS exposure led to similar effects: increased the content of ROS, hydroxyl radicals, and increased expression of immune related genes. This study demonstrates that relatively low and environmentally relevant doses of BPS have a significant impact on multiple components of the immune system of macrophages, and Bisphenol S has similar effects as BPA on the effects of fish immune system. Antagonist of ER blocked many of the effects of BPS on immune-related gene expression, providing evidence that ER pathways play important role in mediating the actions of BPS on the immune system.3. The actions of BPA and BPS were similar on the reproductive neuroendocrine system during the early development of zebrafish embryos. Two-hour post-fertilization(hpf) zebrafish embryos were exposed to various concentrations of BPA, BPS until 120 h. The results are shown as follows:(1) Environmentally relevant, low levels of BPA exposure during development led to advanced hatching time, increased numbers of Gonadotropin-Releasing Hormone 3(GnRH3) neurons in both terminal nerve(TN) and hypothalamus(HYPO), as well as increased expression of a marker for synaptic transmission(sv2) and reproduction-related genes(kiss1, kiss1 r, gnrh3, lhβ, fshβ, and erα).(2) Low levels of BPS exposure led to similar effects: increased numbers of HYPO-GnRH3 neurons, and increased expression of kiss1, gnrh3, and erα.(3) Antagonists of ER, THRs and AroB, blocked many of the effects of BPA and BPS on reproduction-related gene expression, providing evidence that those three pathways play important roles in mediating the actions of BPA and BPS on the reproductive neuroendocrine system. This study demonstrates that relatively low and environmentally relevant doses of BPA have a significant impact on multiple components of the reproductive neuroendocrine system of developing zebrafish, and alternatives to BPA used in the manufacture of BPA-free products are not necessarily safer. The present study is the first report of interactions of multiple cellular pathways(ERα, THRs and AroB) mediating the effects of BPA and BPS during embryonic development in any species. It also provides foundational information using a unique model system for investigating mechanisms by which endocrine disrupting chemicals interfere with normal early-life development.In conclusion, our results indicated that acute exposure to BPA and BPS could interfere with the antioxidant defense system, immune function and reproduction-related genes in fish. Also, our results revealed a susceptibility of the innate immune system and reproductive neuroendocrine system in fish to estrogenic EDCs, and thus, the chronic impact of these compounds on aquatic organisms at even lower concentrations found in the aquatic environment had probably been greatly underestimated. In addition, the tested immune genes and reproduction-related genes should be useful as biomarkers for the risk assessment of BPA and NP in the aquatic environment. The present study also provides important supporting evidence that BPS is not necessarily a safer alternative to BPA, as suggested by earlier studies focusing solely on its estrogenic activities.