Effects And Mechanisms Of Microcystin-LR On Reproduction,Growth And Development In Zebrafish

Nowadays,eutrophication and the frequent occurrence of cyanobacterial blooms are receiving increasing global attentions as urgent environmental issues,with the production of microcystins(MCs)being one of their major hazards.Numerous studies have documented that MCs,as typical hepatotoxins,can also exert significant reproductive toxicity and adverse impacts on the growth and developmental process of fish.In order to systematically assess toxic effects of MCs on fish reproduction,growth and development,the present study used zebrafish(Danio rerio)as a model organism.First of all,ovarian pathological damages and antioxidant system responses in zebrafish were studied after intraperitoneally(i.p.)injected with MC-LR in an acute toxicity experiment.And then through a life cycle assessment experiment,endocrine disrupting effects of environmental relevant concentrations of MC-LR on zebrafish growth and reproduction were systematically analyzed from growth parameters,ultrapathological damages,sex hormone contents as well as gene expressions along the regulatory pathways.At last,testicular histology and sex hormone contents as well as related gene and protein expression levels were examined in a subchronic exposure experiment to provide a comprehensive assessment of the toxic and endocrine disrupting effects of MC-LR on the reproductive system in adult male zebrafish,and the estrogenic potency of MC-LR and possible mechanisms were also concluded.The present study makes contributions to experimental evidence for the comprehensive knowledge on reproductive toxicity,endocrine disruption and estrogenic risks of MC-LR under natural conditions.The main results were as follows,1.In an acute toxic experiment,female zebrafish were i.p.injected at doses of 50 and 200 ?g MC-LR/kg body weight(BW)respectively,and histopathological lesions and antioxidant enzymatic activities and gene expression in the ovary were studied at 1,3,12,24,48 and 168 h post injection.Pathological lesions of zebrafish ovary progressed in severity and extent with the increasing exposure time and dose within 12 hpi.Concurrently,the increases in malondialdehyde(MDA)contents as well as the enzymatic activities and transcriptional levels of antioxidant enzymes catalase(CAT),superoxide dismutase(SOD)and glutathione peroxidase(GPx)showed the occurrence of oxidative stress,indicating that MC-LR induced adverse effects on the structure and functional activity of zebrafish ovary.Oxidative stress plays a significant role in the reproductive toxicity of MC-LR.The significant decrease of glutathione(GSH)content in zebrafish ovary suggested the importance of MC-LR detoxification by glutathione S-transferases(GST)via GSH.The final recovery of histostructure and antioxidative indices indicated that ovarian efficient antioxidant defense system might be an important mechanism of zebrafish to counteract MC-LR.2.In a life-cycle assessment,zebrafish hatchlings(5 d post-fertilization)were exposed to 0,0.3,3 and 30 ?g/L MC-LR for 90 d until reaching sexual maturity.The results showed a life cycle exposure to MC-LR(above 3 ?g/L)caused significant growth inhibition,brain and liver ultrapathological lesions,and retardation of gonadal development.The sex-dependent growth inhibitory effect might be related to decreased up-stream ghrh,pacap1,gh expression on the GH/IGF axis as well as ghra,igf3,igf2r(females and males)and igf1(males only)expression in the gonads.In the 3 and 30 ?g/L groups,ovarian T contents decreased while E2 remained steady,significantly up-regulated transcription of brain fsh? and lh? suggested a nagative feedback regulation in the HPGL-axis was induced as a compensatory mechanism for promoting the transformation of T to E2.Moreover,it was also noted that ovarian VTG content and hepatic er?,vtg1 expression were all down-regulated,which might be responsible for reduced vitellus storage and could further impair the oocyte maturation and reproductive function of female zebrafish.3.In a subchronic exposure experiment,adult male zebrafish were exposed to 0,0.3,1,3,10 and 30 ?g/L MC-LR for 30 d.Our results showed MC-LR exposure resulted in reduced GSI,widened cellular gaps and decreased mature spearm ratio in the testis of zebrafish.MC-LR could also interference with gene expressions along the HPGL axis,such as the dose-dependent down-regulation of gnrh2,gnrh3,cyp19 b,er?,er?,ar,fsh? and lh? expression in the brain.The gene expressions of steroidogenesis including star,cyp11 a,cyp17,cyp19 a and fshr showed non-dose-dependent increase at 3 ?g/L,and then gradually returned back with the increase of MC-LR concentrations.Testicular E2 and T contents also exhibited non-dose-dependent increase and peaked at 10 ?g/L,implying that the endocrine disrupting effect of MC-LR is an embodiment of its estrogenic potency and cytotoxicity.Moreover,the estrogenic potency was also indicated by the significant up-regulation of vtg1 in the liver after a long-tern exposure to 3,10 and 30 ?g/L MC-LR.Correlation analysis between testicular sex hormone and gene expression along HPGL axis revealed that the changes of E2 and T exhibited little correlation to most gene expression in the brain and liver as well as hepatic ER protein levels,but showed considerable correlation to the gene expressions concerning steroidogenesis in the testis.Thus,we speculated that the estrogenic potency of MC-LR might be induced via steroidogenesis disruption rather than ER-mediated pathway.