Toxicity Of Bisphenol A And Norfloxacin Exposed Female Zebrafish Parents To Progeny Bone Development

Bisphenol A（BPA）and fluoroquinolone（FQs）antibiotics are typical important pollutants in the environment,often exist in water.Studies have shown that a certain concentration of FQs antibiotics or BPA can interfere with bone metabolism and normal bone development in higher vertebrates.Fish live in water all their lives,and the widespread FQs and BPA in aquatic environment may into the fish body throμgh gill respiration or skin penetration,interfere with bone metabolism and normal bone development of fish,and threaten their survival and population maintenance.However,the toxic effects of BPA on bone development of aquatic animals at environmental concentrations are still unclear.In this study,zebrafish were used as research subjects.Female zebrafish were exposed to 1μg/L BPA and NOR（environmentally relevant concentration）alone for 90 days before mating with normal male zebrafish to produce offspring.The skeletal development and histopathological changes of early offspring were observed by alizarin red,Alsinlan,HE and TUNNEL staining.The changes of gene expression pattern and its toxic mechanism were analyzed by ELISA,transcriptional sequencing（RNA-Seq）and bioinformatics.The main research results are as follows:（1）After exposure to BPA or NOR at 1μg/L for 90 days,female zebrafish parents were exposed to BPA,which resulted in decreased frequency of the ability of autonomic movement in the tail of offspring embryos,delayed incubation,and increased malformation rate of juvenile zebrafish.NOR exposed female parent fish had no significant effects on the developmental indexes of their offspring embryos.These results sμggest that maternal exposure to low concentrations of BPA may affect the reproductive ability of zebrafish and inhibit embryo development.Alxin blue cartilage staining showed that after exposure to drugs,the length of the ceratohyal cartilage and the palatal square cartilage in the offspring of the two treatment groups shortened,and the angle between the two ceratohyal cartilages,the angle between the two michael cartilage and the palatal square cartilage increased,and the overall structure changed from long and narrow to short and wide.Moreover,the angle difference of the cartilage in the offspring exposed to BPA was more significant.The toxic effects of both decreased gradually with the passage of time.Alizarin red bone staining showed that the ossification area and optical density（IOD）of the offspring decreased in both maternal treatment groups,and maternal BPA exposure had a more significant effect on the offspring.These results sμggest that exposure to BPA or NOR can lead to chondrodysplasia of the head,delayed ossification time and decreased bone density in the offspring of juvenile fish,and BPA has greater toxic effects than NOR.（2）Transcriptome sequencing analysis showed that 448 and 376 genes were differentially expressed in the 4 dpf progeny and 1202 and 959 genes were differentially expressed in the 7 dpf progeny after the mother was exposed to 1μg/L BPA or NOR.The differentially expressed genes are mainly involved in cell replication,steroid biosynthesis and apoptosis.Cytochrome P450 family genes were significantly enriched in both treatment groups.Fluorescence quantification showed that maternal BPA exposure significantly down-regulated Cyp27b1,a gene associated with vitamin D synthesis in the progeny,while Cyp24a1,a gene that disposes its active form,was unchanged.The protein level of parathyroid hormone（PTH）,a strong inducer of Cyp27b1 that inhibits the expression of Cyp24a1,and the active form of vitamin D,1,25（OH）₂D₃,and the ratio of calciμm to phosphorus were significantly decreased.NOR maternal exposure did not change the expression of vitamin D-related genes in the offspring.The content of the active form1,25（OH）₂D₃ showed a decreasing trend but no significant difference,but decreased PTH and the ratio of calciμm to phosphorus.In addition,ELISA showed that the antitartrate acid phosphatase（TRAP）activity of osteoblast marker alkaline phosphatase（ALP）and osteoclast marker did not change after drug exposure.Therefore,it is inferred that drug exposure to female parents reduces the circulating level of 1,25（OH）₂D₃ in blood regulated by hormone-mediated enhancer PTH of Cyp27b1 in the offspring,changes calciμm and phosphorus homeostasis,causes loss of bone calciμm,and thus inhibits the bone mineralization process in the offspring.The delay effect of BPA exposure on ossification of zebrafish offspring was greater than that of NOR.（3）Fluoroquinolones acted as ion-chelating agents,and the magnesiμm content of offspring（7 dpf）was significantly decreased after exposure to NOR of 1μg/L,while the magnesiμm content of offspring was not significantly changed after exposure to BPA of1μg/L.The production of reactive oxygen species is the mediμm of cartilage injury,and the stress-related pathways（response to external biological stimuli,response to other organisms,and response to biological stimuli）were enriched in GO analysis.ELISA detection showed that MDA increased significantly in the offspring after exposure,while H₂O₂ content did not change significantly,SOD and CAT enzyme activities increased significantly.After exposure to BPA,the activity of related antioxidant enzymes in offspring larvae increased but had no significant change,and the content of oxide had no significant change.NOR maternal exposure significantly increased the progeneration apoptosis rate,and the expression of apoptosis-related gene caspase9 m RNA was significantly up-regulated,but there were no abnormalities in pathological sections of the two treatment groups.We hypothesized that long-term exposure of NOR to environmental dependent concentrations chelated Mg²⁺in the offspring cartilage and stimulated the production of reactive oxygen species in the fish,thus inducing chondrocyte apoptosis.In conclusion,long-term exposure of BPA or NOR to female zebrafish can significantly inhibit bone development in offspring,lead to cartilage distortion and delayed ossification,stimulate oxidative stress response in the fish body,and induce cell apoptosis.