Comparative Study On Toxicity Of Bisphenol A And Nitro-bisphenol A To Male Reproductive System Of Zebrafish

Objective: Bisphenol A(BPA)can be detected in a variety of environmental media such as surface water,drinking water,dust,sediment and sludge.As an environmental endocrine disruptor,BPA can interfere with the endocrine system and induce toxic effects in reproductive system.BPA can lead to lower sperm quality,lower serum testosterone level,damage the proliferation of male germ cells,and seriously affect the health of male reproductive system.In the process of BPA degradation,BPA is nitrated to produce nitro-BPA.nitro-BPA is also found in surface water and air at concentrations comparable to BPA.However,the epidemiological evidence and animal experiments on the toxic effects of nitro-BPA on male reproduction are lacking.In this study,zebrafish models exposed to BPA and nitro-BPA were compared for male reproductive toxicity,and the differences and similarities of subacute exposure to environmentally related concentrations of BPA and nitro-BPA on male reproductive toxicity and molecular mechanisms of zebrafish were analyzed from the aspects of cumulative spawning capacity,hormone metabolism and transcriptomic changes.In order to provide reference for the risk identification and management of BPA and its degradation products.Methods: 1.Exposure to BPA and nitro-BPA: Healthy male zebrafish at the spawning baseline were measured.After 17 days of exposure to BPA and nitro-BPA(2,20,200 n M),mating was conducted three times every other day to calculate the cumulative spawning capacity.2.Determination of physical constants: After exposure,body length and wet weight of parental male zebrafish were measured,fullness(K)was calculated,gonads were taken out and weighed,and gonad somatic index(GSI)was calculated.All zebrafish samples were frozen in liquid nitrogen and stored at-80 ℃.3.Steroid hormone determination: The expression levels of BPA and nitro-BPA at 2,20 and 200 n M were detected by ultra-high performance liquid chromatography and tandem mass spectrometer.Partial least squares analysis was performed to investigate the changes of steroid hormone metabolism in zebrafish caused by BPA and nitro-BPA.4.Untargeted metabolome analysis: Liquid chromatography and tandem mass spectrometer were used to detect the expression levels of BPA and nitro-BPA endogenous metabolites at 2,20 and 200 n M.Partial least squares analysis was used to investigate the changes of endogenous metabolites of zebrafish caused by BPA and nitro-BPA.5.Transcriptome sequencing analysis: Total RNA was extracted,and the quality and total amount of RNA were tested to be qualified.Then,illumina Nova Seq 6000 high-throughput sequencing platform was used for pairing and sequencing.For subsequent analysis using the clean reads to | log2(multiple)| > 0 and P < 0.05 for the standard,screening the differentially expressed genes(DEGs).Pan-partial least square analysis was used to analyze whether there were differences in gene expression levels of BPA and nitro-BPA in different dosage groups.cluster Profiler(v3.8.1)was used for enrichment analysis of KEGG pathway in DEGs sets.Results: 1.K in the BPA 200 n M exposure group was slightly lower than that in the control group,GSI in the nitro-BPA 200 n M exposure group was about 0.58 times lower than that in the control group,and the difference was statistically significant(P < 0.05).2.There was no significant difference in the cumulative spawning amount of zebrafish before exposure.Compared with before exposure,the cumulative spawning amount of zebrafish in BPA 2 n M exposure group was higher,while that in BPA 20 and 200 n M exposure groups were lower.The cumulative spawning amount of zebrafish in nitro-BPA2 and 20 n M exposure groups were decreased,while that in 200 n M exposure group was increased,with statistical significance(P < 0.05).3.A total of 43 kinds of cholesterol hormones were detected in this study,and a total of 20 kinds of hormones were detected,12 of which were effectively detected.Androstenedione was up-regulated in BPA 20 n M exposure group,androstenedione was up-regulated in BPA 200 n M exposure group,and7 a,27-dihydroxycholesterol and cholesterol were down-regulated.5β-androsterone was down-regulated in nitro-BPA 200 n M exposure group,and the difference was statistically significant compared with control group(P < 0.05).Compared with the control group,the numbers of differentially expressed substances in BPA 2,20 and 200 n M groups were5,7 and 6,respectively,and the numbers of differentially expressed substances in nitro-BPA 2,20 and 200 n M groups were 5,2 and 4,respectively.4.The results of metabolism showed that 149,91 and 127 different endogenous metabolites were found in the BPA low-medium-high dose groups compared with the control group in the positive ion mode,and 147,215 and 209 different endogenous metabolites were found in the nitro-BPA exposure groups,respectively.In the negative ion mode,84,68 and 80 were found in the BPA exposure groups,and 95,136 and 138 in the nitro-BPA exposure groups,respectively.Pathway enrichment analysis showed that BPA could regulate the expressions of deoxycorticosterone and 16α-hydroxydehydroepiandrosterone,while nitro-BPA could regulate the expressions of testosterone,deoxycorticosterone,16α-hydroxydehydroepiandrosterone and 18-hydroxycorticosterone,which interfered with the steroid biosynthesis pathway.5.Transcriptome results showed that there were916,1260 and 1193 DEGs in the low,medium and high dose BPA exposure groups and1065,3258 and 1171 DEGs in nitro-BPA exposure groups compared with the control groups,respectively.There were 27 DEGs of 20 nm BPA exposure groups,more than 12 DEGs of 20 n M nitro-BPA exposure groups.Pathway enrichment analysis showed that among the top 100 pathways enriched by BPA and nitro-BPA exposure groups,there were 16 common toxic pathways.There was one differential toxic pathway related to reproductive toxicity,which was steroid hormone biosynthesis,and it is mainly composed of seven genes(cyp11a2,cyp7a1,cyp17a1,cyp11c1,ugt1a1,ugt1b2,ugt2a2).Conclusions: Both BPA and nitro-BPA have toxic effects on the male reproductive system of zebrafish,and the phenotype shows that nitro-BPA is more toxic than BPA.In terms of hormone levels,nitro-BPA is less toxic than BPA.From the metabolome level,the endogenous metabolites affected by nitro-BPA are more diverse.From the transcriptomic level,both BPA and nitro-BPA can affect the male reproductive system through steroid hormone generation pathway,and BPA can mediate biological metabolism by regulating cytochrome P450,thus producing toxic effects on the male reproductive system.nitro-BPA may have toxic effects on the male reproductive system by affecting the metabolism of other enzymes.