Reproductive Toxicity Caused By Embryonic Exposure To Phenanthrene In Zebrafish And Its Mechanism

PAHs(Polycyclic Aromatic Hydrocarbon,PAH)is a kind of organic pollutants which exist widely in the environment.Phenanthrene(Phe)is a representative PAH containing three benzene rings and has been proved to possess a variety of toxicity including reproductive toxicity,metabolic toxicity and cardiac toxicity.However,up to now,most studies on reproductive toxicity of Phe are based on the relatively high does exposure or the long-term exposure.There has been no research report on whether low-does Phe exposure at early life causes reproductive toxicity.In this study,zebrafish embryos with 0.5-1.5 hpf(hours post-fertilization)were exposed to Phe at environmental relevant concentrations(0.05,0.5,5,50 nmol/L)for 96 hours,and then the larvae were transferred to clean solution and maintained for 120 days.The reproductive function of adult zebrafish and the development of its offspring were analyzed using the histological,pathological,chemical and molecular biological methods.The major results are as follows:(1)The results indicated that both the number and the fertilization rate of eggs produced by the exposed female fish mated with the normal male fish were decreased.Histological examination showed that the relative numbers of mature oocytes in the ovary of exposed female fish were reduced.The levels of testosterone(T)and 17β-estradiol(E2)were decreased in the exposed female fish.The results of Real time PCR(RT-PCR)indicated that the transcriptional levels of GnRH3,Fshβ,LHβ,LHγ,cyp19a1a and cyp19b were decreased in the brains of exposed female fish.The transcriptional levels of cyp19a1a,cyp19b,esr1 and esr2β were decreased in female ovary.In addition,the transcriptional levels of vtgl,vtg2,esrl and esr2β in female liver were decreased.The Bisulfite sequencing PCR(BSP)results showed that promoter regions of GnRH3 in the exposed female zebrafish kept a high methylation status,which was matched with its decreased transcriptional level.The mechanism underlying the reproductive toxicity in adult female zebrafish might involve the down-regulated transcriptional level of GnRH3 cused by its hypermethylated.(2)Wild-type female zebrafish mated with the exposed adult males showed a decrease in the number and the fertilization rate of embryos.Histological examination showed that the relative numbers of mature spermatozoa in the testis of exposed male fish were decreased.The levels of T and E2 in the muscle tissue of exposed male fish were decreased.The transcriptional levels of GnRH3,Fshβ,LHβ,LHγ,cyp19a1a and cyp19b were decreased in the brains of exposed male fish.At the same time,the transcriptional levels of ar,esrl and esr2β in the testis of exposed male fish were decreased.The BSP results showed that promoter regions of GnRH3 in the exposed male zebrafish kept a high methylation status,which was matched with its decreased transcriptional level.The mechanism underlying the reproductive toxicity in adult male zebrafish might involve the hypermethylation of GnRH3 cused by Phe.(3)The exposed adult zebrafish were mated with different sex wild zebrafish respectively,and the embryos were collected and raised in clean water.The results showed that the mortality rate,yolk-sac edema rate,spinal curvature rate,cardiac hemorrhage rate,pericardial edema rate and heart rates of F1-generation embryos produced by the exposed adult females were increased,while the frequency of spontaneous movement was decreased.Alcian-blue staining showed that the development of craniofacial cartilage was inhibited.Similarly,the ratios of mortality and malformation in F1-generation embryos produced by the exposed males with the normal female fish were increased.These results suggested that embryonic Phe exposure with low concentrations could reduce the reproductive ability of adult zebrafish.In addition,it is related to the influence of development in F1-generation offspring.The results would provide a novel idea and theoretical foundation for Phe risk assessment.