Male Reproductive Toxicity And Mechanisms Of Chlordecone In Caenorhabditis Elegans

BackgroundChlordecone(CLD),also name Kepone,which is a synthetic organochlorine pesticide that has been banned to production and use by the international community due to its various toxic effects.As one of the common persistent organic pollutants(POPs)in nature,Chlordecone has a profound impact on the environment and human health.Investigations have found that the pollution of chlordecone in China has been increasing these years.However,studies on its toxic effects have not been systematically conducted in China.Therefore,research on the toxic effects of chlordecone cannot be delayed.PurposesIn this study,Caenorhabditis elegans was used as a model to investigate the toxic effects of chlordecone on the male reproductive system of nematodes and the mechanism of its induced reproductive toxicity from the individual,tissue,cell,and molecular levels.MethodsIn order to evaluate the effects of chlordecone on the fertility of nematodes on the basis of observing the number of offspring progeny of wild-type nematodes,generation time,and the number of offspring of the male crosses.The gonad area was observed through the stereomicroscope and observe the development of distal primordial(DTC)cells of JK2868 mutantsunder the fluorescence microscope.Determine the number of germ cells and sperm cells in the mitotic and meiotic regions by DAPI fluorescent staining.Determine the sperm abnormality rate and sperm activation rate under differential interference contrast microscopy(DIC).At the tissue and cell level to explore the target organs of male reproductive toxicity of chlordecone and its role in sperm cell development.Determination of CEP-8 pathway genes and regulation of DNA damage induced germ cell cycle arrest.Related gene expression during the critical step of spermatozoa activation after Chlordecone exposure at the molecular level by real-time fluorescent quantitative PCR.The activity of reactive oxygen species(ROS)in nematodes was detected by CM-H2 DCFDA probe,and the activity of catalase(CAT)and superoxide dismutase(SOD)was measured by antioxidase kit to determine the oxidation of chlordecone to nematodes.The expression levels of the related genes gas-1,mev-1,isp-1,sod-1,sod-2,and ctl-1 were measured by RT-qPCR,and exploredthe protective effect of mannitol to the reproductive toxicity ofChlordecone.ResultsThe number of offspring in each dose group of wild type nematode decreasedafter chlordecone exposure,and the high dose group was prolonged during the generation time.The results of the hybridization experiment showed that the number of offspring in each exposure group was reduced compared with the control group.At the tissue and cellular level,chlordecone exposure resulted in a decrease in nematode gonad area.The area of the lowdose group was reduced by approximately 20% compared to the control group,and the area of the medium and highdose group was reduced by approximately 40% compared with the control group.DTC cells were observed,compared with the control group,the fluorescence intensity of the cells also reduced.The number of germ cells in the mitotic area,meiotic area and the number of sperm cells decreased,and the descending trend was the same as the number of offsprings.The sperm deformity rate increased,and the activation rate decreased.At the molecular level,in studies on the mechanism of reproductive toxicity of chlordecone,it was found that the expression level of MSP-related genes spe-8,spe-12,spe-19,and spe-29 was decreased and spe-6 was increased,spe-27 was no change.In the DNA damage experiment,the number of fluorescent spot-polymerized cells in the exposed group increased,and the expression of the related genes cep-1,hus-1,and clk-2 was increased.In the further study of nematode oxidative stress,ROS levels were increased in the medium and highdose groups after exposure to chlordecone,SOD activity increased slightly in the lowdose group and decreased in the middle and high dose group,CAT content decreased.Oxidative stress-related Genes gas-1 and mev-1 gene expression were no changein the lowdose group,but moderate and high dose groups were decreased,isp-1 expression was increased,sod-1 and sod-2 genes were slightly increasedin the lowdose group,in high dose groups wasdecreased,ctl-1 gene expression was decreased.The mannitol treatment found that 5 mM and 20 mM concentrations of mannitol effectively reduced ROS levels in nematodeswhat exposed to chlordecone,and 20 mM mannitol restored ROS levels in nematodes to the same levels as in the control group.When the combination of chlordecone and mannitol was exposed,the number of germ cells in the mitotic area recovered,but there was still a difference compared to the control group.ConclusionChlordecone affects the development of gonadal structures by regulates oxidative stress-related genes in nematodes to causes oxidative damage in nematodes.At the same time,Chlordecone activates CEP-1 pathway causes DNA damage to the germ cells,leading to arrest of the germ cell cycle and reduction of the number of sperm cells.Radical scavenger mannitol can relieve the reproductive toxicity of Chlordecone.Chlordecone can inhibit the SPE-8 pathway,andcause MSP polymerization abnormalities,disturb the normal activation of sperm,generate malformed and incapacitated sperm,resulting in thecount of sperm reduced andshape deformity,finally lead in male reproductive toxicity.