Effects Of Bisphenol A On Social Recognition In Female Mice

Bisphenol A（BPA）is a typical environmental endocrine disruptor that binds to Estrogen receptors（ERs）and plays an estrogen-like and anti-estrogen-like role through ERs-dependent signaling pathways.Humans are exposed to BPA through multiple pathways,including oral and nasal inhalation and skin contact.Therefore,BPA is closely associated with some endocrine diseases and developmental disorders of the nervous system,leading to cognitive dysfunction,behavioral disorders,and impaired social abilities.Social recognition is the foundation of social behavior.The neuropeptide Oxytocin（OT）has been shown to be a regulator of many social behaviors,and the OT system in the brain is regulated by estrogen.The m RNA levels of OT and its receptor（OTR）varied with the fluctuation of estrogen levels during the estrus cycle.Estrogens can enhance the excitability of OT neurons in the paraventricular nucleus（PVN）,and regulate the density of OTR and the expression of OTR genes in the amygdala.OT system,thus,may be an important target of BPA on influencing social recognition.In this study,female mice were used to establish Ovariectomy（OVX）and exogenous Estradiol benzoate（EB）models（30 ug/kg/d）to investigate the effects of different doses of BPA（4 and 0.4 mg/kg/day）on estrogen-regulated brain OT system,and to explore whether BPA affects social recognition through brain OT system.Methods:Three-week-old ICR female mice in clean grade were randomly divided into three groups:adolescent BPA exposure group（4 and 0.4 mg/kg/day）and control group（subcutaneous administration for 3 weeks）.From the 13th Week（adult）,experiment were performed by three chamber paradigm for social novel preference,individual habituation/dishabituation paradigm for recognition capability in female mouse,habituation/dishabituation paradigm in urine and bedding tendency model for testing the ability to distinguish social stimulus,and food buried test for the average olfactory function.All of assays above were to assess the influence of social recognition by the adolescence BPA exposure;The expression level of c-fos in the brain regions related to social recognition after social stimulation was detected by immunofluorescence in each group to determine which of the brain regions sensitive to BPA.In the second experiment,3-week-old female mice were treated with OVX,and some OVX mice were supplemented with EB（OVX+EB）until the end of the experiment.Female OVX and OVX+EB mice were exposed to BPA for 3 weeks.At 13weeks,the level of 17β-estradiol（17β-E₂）in serum was determined by enzyme-linked immunosorbent assay（ELISA）to verify the success of the modeling.Social recognition ability,the level of 17β-E₂ and OT in the whole brain were then determined.Immunohistochemistry and Western blotting（WB）were used to detect the expression levels of Estrogen receptorα（ERα）,Estrogen receptorβ（ERβ）and OTR in BPA-sensitive brain regions after social stimulation.Results:1.BPA（4 mg/kg/d）exposure significantly decreased the time ratio of female mice to sniff unfamiliar/familiar mice in three-chambered model（P<0.05）.The females exposed to 0.4 and 4 mg/kg/d BPA did not show dishabituation towards novel female mice,and did not affect any adaptation to the opposite sex urine in urine habituation/dishabituation,and reduced the time spent in bedding of female mice（P<0.05;P<0.001）,but BPA did not affect the general olfactory function of adult mice.These results suggest that adolescent exposure to BPA impairs the social olfactory and social recognition abilities of adult female mice.2.Further analysis of the behavior in OVX mice showed that BPA could reverse the recognition ability of OVX mice in the individual habituation/dishabituation test（P<0.05）,but attenuated the recovery effect of EB on the social recognition impairment of OVX mice（P<0.05）.BPA could reverse the ability of OVX to discriminate between male and female urines（P<0.05）,but significantly inhibited the recovery of EB to discriminate between male and female urines in OVX mice.OVX eliminated female mice preference for bedding in opposite sex（P<0.001,compared with Sham）,and BPA promoted OVX mice preference for bedding in opposite sex（P<0.05;P<0.05）,but eliminated the recovery of EB supplementation to the opposite sex cushion preference in OVX mice（P<0.05）.These results suggest that BPA can improve the reduction of social odor discrimination and social recognition ability induced by estrogen deprivation,whereas inhibiting the recovery of social odor discrimination and social recognition function in OVX mice by estrogen supplementation.3.Immunofluorescence analysis showed that the number of c-Fos-immunoreactive neurons（c-Fos-ir）in brain regions of Accessory olfactory bulb（AOB）,PVN and Medial amygdala（Me A）was significantly increased after social stimulation,indicating that these three brain regions were sensitive to social stimulation,and BPA significantly downregulated the number of c-Fos-ir cells in brain regions of PVN and Me A（P<0.001;P<0.001;P<0.001）,indicating that PVN and Me A brain regions were sensitive brain regions disturbed by BPA.4.BPA did not affect the level of 17β-E₂,the expression of ERαand ERβin PVN region,and the expression of ERβin Me A region,but significantly down-regulated the expression of ERαin Me A region（P<0.001;P<0.001）.OVX treatment decreased the expression levels of ERαand ERβin this two brain regions,and this effect could be reversed by EB supplementation.BPA upregulated the expression of ERαand ERβin PVN and Me A brain regions of OVX mice,but significantly inhibited the recovery of ERαexpression level in Me A brain region by EB（P<0.001;P<0.05）,and had no effect on PVN and Me A.These results suggest that BPA can dose-regulated and region-specific inhibitory effect of estrogen on the level of ERαin PVN and Me A regions related to social recognition.5.BPA did not affect the whole brain OT level,but significantly decreased the expression of OTR in Me A region（P<0.05;P<0.05）;Low dose of BPA significantly inhibited the recovery of OTR expression in PVN brain region of OVX mice supplemented with EB,but significantly promoted the OTR expression in Me A brain region of OVX mice（P<0.05;P<0.05）,suggesting that BPA could antagonize the regulation effect of estrogen on OTR in brain regions related to social recognition.ConclusionAdolescent BPA exposure can impair social odor discrimination and affect social recognition in adult female mice.BPA can play an estrogen-like role and promote the impairment of social recognition in OVX mice due to different estrogen environments in vivo.It can also play an anti-estrogenic role and inhibit the promotion of social recognition by estrogen.The down-regulation of ERαexpression in related brain regions interferes with the regulation of OT system by estrogen,which may be related to the impairment of social recognition by BPA exposure in adolescence.