Study On Oxidative Stress And The Injury Of The Cell Junctions Of Testis In Rare Minnow (Gobiocypris Rasus) Exposed To Bisphenol A

Bisphenol A（BPA）is a ubiquitous estrogen-like endocrine disrupting chemicals（EDCs）and widely used in plastic products for daily life and industrial production.Owing to the extensive use and environmental persistence of BPA,public concern about its environment pollution and health problems have been raised universally.Oxidative stress and immune response are two important parts of ecotoxicological research.Studies shown that BPA exposure can induce oxidative stress and immune response in fish while little is known about the relationship between oxidative stress and immune response.To explore the molecular mechanism of BPA on oxidative stress and inflammatory response in rare minnow（Gobiocypris rarus）larvae,the fish was exposed to 1,225 and 1000 μg/L BPA and solvent control（DMSO）for 7 days,respectively.The levels of H₂O₂ and NO were measured by spectrophotometry.Meanwhile,the transcripts of iNOS,TLRs-associated genes and cytokine-related genes were analyzed using real-time quantitative PCR（qRT-PCR）methods.Studies in recent years shown that cytokines can affect male reproductive function and may damage the spermatogenesis process which is mainly by regulating the integrity of blood-testis barrier（BTB）.Similar to mammals,fish also have the BTB structure,which called Sertoli cells（SCs）barrier.However,the molecular mechanisms of how cytokines affect the function of testes after BPA exposure in fish were still unknown.In the present study,adult male G.rarus were exposed to 15 μg/L BPA for 7 to 35 days.Gonadal histology and immunohistochemistry were used to detect the pathological changes of testes and the integrity of BTB,respectively.In addition,the levels of TNFα,a BTB-associated protein,was detected by Western Blot（WB）.Meanwhile,the gene expressions of the mitogen activated protein kinase（MAPK）pathway were detected by qRT-PCR.The main results of the present study were as follows:1.H₂O₂ content was significantly increased in a concentration-dependent manner by by all treatment groups.The content of NO was significantly increased by 225 μg/L BPA exposure while significantly decreased by 1000 μg/L BPA.Moreover,225 μg/L BPA significantly up-regulated iNOS expressions.The results suggested that BPA has induced oxidative stress in G.rarus larvae.In 225 and 1000 μg/L BPA groups,the transcripts of IFNγ,CXCR3 and CXCR5 were significantly decreased,indicating that BPA exposure could be produced immunosuppressive effects and participated in the regulation of T-lymphocyte function in G.rarus.After 7-day exposure,the expressions for TLR4,MyD88,TRAF6 and NF-κB were significantly decreased in 225 and 1000 μg/L BPA groups.The mRNA levels of TNFα,IL-1β and IL-8,which are downstream molecules of TLRs signaling pathway,were significantly inhibited by all concentrations of BPA.These results,suggested that TLRs signaling pathways might be involved in mediating the inflammatory response in G.rarus larvae by BPA exposure.2.Histological analysis demonstrated 15 μg/L BPA significantly increased the blood vessel congestion,inflammatory cells infiltration and the contents of TNFα in the testes after 7-days exposure.The results illustrated that G.rarus was sensitive to short-term acute BPA exposure for their quickly immune response and altered expression of cytokines.The Occludin and β-catenin proteins are important compositions of the SCs barrier in mammals.The significant decrease of Occludin and β-catenin expressions were observed in the testes of BPA group on days 7 and 21 compared to control group.Moreover,the distribution of Occludin was diffuse and scattered by 7-day BPA exposure.After 7-day exposure,the biotin tracer passed through the SCs junction barrier and entered and filled with specific staining in the spermatid of testes,suggesting that the complete SC junction barrier structure was destroyed by BPA exposure.p38 MAPK play an crucial roles in the regulation of BTB restructuring in the testes through the cytokines.The mRNA expression of p38 MAPK was significantly decreased by 7-day BPA while it was significantly increased by 35-day BPA,indicating that the MAPK signaling pathway might be involved in the TNFα-induced injury of the SCs barrier by BPA exposure.In summary,the study investigated the effect of BPA on the oxidative stress and the injury of the testis in G.rarus by detecting the mRNA expression and protein level of the key genes.These findings provide a foundation in the subsequent research work of BPA.