| Bisphenol AF(BPAF)has been widely used in the rubber fluorine chemical industry and tropical agricultural production as a kind of important flame retardant,which has been confirmed that it has the similar estrogen-like effect as bisphenol A.Thyroid hormone plays an important role in the growth and development of fish,however,it has not been reported in the literatures that whether the BPAF has effects on the thyroid axis or not.By using zebrafish as subjects of aquatic organisms,the effects of BPAF exposure on zebrafish embryos,larvae and adults were examined in different developmental stages through individual,tissue,cell and molecular levels,respectively.In order to elucidate the mechanism of BPAF exposure on the thyroid axis of zebrafish,we detected the effects of thyroid hormone on the secretion level and the expression of HPT axis gene.In this study,the methods of ecological toxicology,molecular biology,cytology and electrophysiology were used.Besides,through the research on the interaction of endocrine system and the cholinergic system,the potential impact of BPAF stress on the neural memory function was investigated to provide important theoretical basis for the ecological risk assessment of BPAF pollution on the environment.The results are shown as followed.(1)After 48-hour exposure to zebrafish embryos in 5,50 and 500?g/L BPAF,respectively,it was found that the exposures of 50 and 500 ?g/L resulted in a slow development of zebrafish embryos,and significantly reduced their 72-hour hatching rate.The results showed that the expression of crh mRNA and tsh-? mRNA were significantly up-regulated by 50 and 500 ?g/L BPAF,in which the crh mRNA showed more sensitive.Therefore,low concentration and short time exposure of BPAF to zebrafish embryos has a potential thyroid interference effect and non lethal but delayed development interference effect.(2)After 7-day exposure to zebrafish embryos in 5,50 and 500 ?g/L BPAF,no significant changes of body length,body weight and survival rate on zebrafish larvae were observed,while the contents of THs(including total thyroxine TT4,total triiodothyronine three TT3 free thyroxine FT4 and free triiodothyronine three FT3)in early development stage were decreased by 50 and 500 ?g/L,especially a significant decline of FT3 content was observed with the exposure to 5 ?g/L BPAF.The relative genes on synthesis and transport in HPT axis are relatively sensitive,the expression of tsh?,diol and dio2 were significantly increased by 50 ?g/L BPAF.In addition to the increased levels of dio1,dio2 and tsh?,the down-regulated levels of TT3,TT4,FT3,FT4 may be related to the expression of ttr?slc5a5?tg?crh?traand tr-? mRNA,while it is not sensitive to the expression of UGT1ab mRNA.It is believed that the zebrafish larvae exposed to BPAF prone to thyroid function decline,and which impact the expression genes related to synthesis,transformation and transport.(3)Adult zebrafish were exposed to 5,50 and 500?g/L BPAF for 14 days.The results showed that in the thyroid,some symptoms of pathologic changes were observed including increased number of epithelial cells,cell colloid deficiency and obvious follicle tissue deformation.After exposure of BPAF,the contents of THs in male zebrafish plasma were significantly increased with the increase of exposure concentration and time,showing the interference effect of thyroid function excitement.In females,the results are generally consistent with males,but there is a non monotonic dose-response relationship.For example,the expression of TT3 was down-regulated after the low dose BPAF exposure,while a significant upgrade was observed with the exposure to higher level.As far as the THs interferences are concerned,adult zebrafish are more sensitive than larvae at the same exposure concentration.(4)Many genes related to HPT axis in adult zebrafish were sensitive to BPAF exposure,especially which are related to the synthesis,transformation and metabolism functions.After exposure to BPAF for 7 days and 14 days,the contents of TT4 and TT3 in adult zebrafish were up-regulated,the expressions of diol and dio2 mRNAs were reduced in liver by BPAF,and the negative feedback regulation of CRH and TSH on THs also indirectly affected the contents of THs.Even the significant down-regulation of UGT1ab mRNA in liver decreased the metabolic rate of THs,which is also the reason for the increase of THs content in the body.It is also found that the responsible of genes related to the HPT axis to BPAF exposure are slightly different due to sex differences in zebrafish.The expression of dio2 and ttr mRNAs in female zebrafish was more sensitive to BPAF exposure,while the sclc5a5 and tg mRNAs expression in male was more sensitive.(5)In this paper,a T type maze model was developed to study the cognitive memory ability of zebrafish.When exposed to 50 and 500 ?g/L BPAF,the results showed that the zebrafishes entered the nutrient-rich region in the right arm of maze no longer faster than before with the increase of training times,on the contrary,the zebrafish appeared to run back,could not find the right arm or stagnant nutrient-rich region.It is also evaluated that the activity of nAChR decreased significantly in each exposed group.In this paper,the sensitivity of two different kinds of central nAChRs subtype ion channels expressed in vitro to BPAF exposure was detected by electrophysiological methods.And it was shown that BPAF had a significant blocking effect on the ?3?2 nAChR,while no significant blockade of ?3?4 nAChR.Therefore,it is speculated that BPAF has the interference effect of cognitive ability,and could be affected by nAChR activity and selective blocking subtype receptor.In conclusion,BPAF exposure has potential interference effects on zebrafish embryos,larvae,adult males and adult females in thyroid axis,which causes the levels of THs in disorder.The expression of related genes in HPT axis was found to be more sensitive to BPAF exposure.Because of the different developmental stages of zebrafish and the different genders,the interference effects presented by the present study may not be the same.Furthermore,it was found that BPAF had a certain disturbance in the neural memory of zebrafish,especially the activity of nAChR in the cholinergic system.In addition,it is proposed that BPAF interferes with the ability of zebrafish memory by inhibiting the activity of nAChR in the brain,which is likely to have a direct blocking effect on the specific subtype of nAChR. |
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