The Male Reproductive Toxicity Of Perfluorooctane Sulfonates And Perfluorooctanoic Acid On C.Elegans

Research Background and Research PurposesPerfluorinated compounds(PFCs)are widely used in military and civilian production,due to its non-hydrophilic,non-lipophilic properties and favorable chemical stability.It’s believed that the general population can be exposed to PFCs through various exposure routes.Two representative PFCs,Perfluorooctane Sulfonates(PFOS)and Perfluorooctanoic Acid(PFOA)are widely used in industry and our daily life,the increasingly severe pollution of PFOS and PFOA have raised public awareness worldwide.More seriously,as organic pollutants,PFOS and PFOA are difficult to degrade.PFOS and PFOA have been measured in environmental media(water,soil,snowfall,etc.),organisms(mammals,fish,poultry,etc.)and human beings(blood,breast milk,urine,etc),which indicates that PFOS and PFOA exposure can happen anywhere,at any time.PFOS and PFOA exposure have caused a profound impact on ecosystems and human beings.In this study,we aimed to investigate the male reproductive toxicity of PFOS and PFOA,and explore the possible mechanism in Caenorhabditis elegans(C.elegans).Additionally,we also tried to identify the sensitivity indices to estimate the reproductive toxicity of PFOS and PFOA in C.elegans.Methods and Results1.Contamination of PFOS and PFOA in raw and finished water in JiangsuXAD-2 resins were used to collect the raw and finished water.The Contamination of PFOS and PFOA were mesured by LC/MS/MS.The concentration of PFOA in raw water and finished water were 12.62 ng/L and 10.18 ng/L respectively.PFOS was not detected in raw water and finished water.2.Toxicty of PFOS and PFOA on fertility of C.elegans2.1 Toxicty of PFOS and PFOA on fertility of L4 N2 C.elegansThree dose(0.1,0.01 and 0.001 mmol/L)and one control groups were setted to evaluate the fertility of L4 N2 C.elegans.The fertility of L4 N2 C.elegans were evaluated by calculating brood size and generation time.After synchronization,the L4 N2 C.elegans were exposed to PFOS or PFOA for 24 h in 24-well plates,then 10 C.elegans were picked to caculate the brood size and generation time from each well.Compared with the control group,0.1 and 0.01 mmol/L PFOS can statistically reduce the brood size of L4 N2 C.elegans(p<0.05).Similarly,three doses(0.1,0.01 and 0.001 mmol/L)of PFOA can statistically reduce the brood size of L4 N2 C.elegans.However,no statistical differrence of generation time were found between the treated and control groups of PFOS and PFOA.2.2 Toxicty of PFOS and PFOA on fertility of LI N2 C.elegansThree dose(0.1.0.01 and 0.001 mmol/L)and one control groups were setted to evaluate the fertility of L1 N2 C.elegans.After synchronization,L1 larvae were exposed to PFOS or PFOA for 48 h,then 10 C.elegans were picked to caculate the brood size and generation time from each well.The results showed that,the brood size of LI N2 C.elegans in middle and upper dose(0.1 and 0.01 mmol/L)groups treated with PFOS and PFOA were more than the control(p<0.05).The generation time were statistically different between three dose groups treated by PFOS and control(p<0.05).2.3 Toxicty of PFOS and PFOA on fertility of L1 him-5 C.elegansThree dose(0.1,0.01 and 0.001 mmol/L)and one control groups were setted to evaluate the fertility of LI him-5 C.elegans.After synchronization,L1 him-5 larve were exposed to PFOS or PFOA for 48 h,the males of him-5 and the young synchronized females of fog-2 were mixed in NGM Petri dish according to the proportion of 1:1 for 12h,after that,the females of fog-2 were picked into a new OP50 NGM Petri dish for every 36h,until the fog-2 females stop spawning,count the brood size of each female of fog-2.We found the brood size in all three dose groups teated by PFOS and PFOA were smaller than control(P<0.05).3.Toxcity of PFOS and PFOA on spermatogenesis of Caenorhabditis elegans3.1 Germ cell countDAPI Dyeing were used to evaluate the number of germ cell.After dyed,the germ cells of C.elegans were counted using fluorescence microscope.Compared with the control,the number of germ cells in all groups treated with PFOS were decreased(P<0.05).The results showed that,the number of germ cells in in middle and upper dose(0.1 and 0.01 mmol/L)groups treated with PFOA were statistically smaller than control(p<0.05),the number of germ cells decreased in a dose dependent manner(R2=0.963).3.2 Sperm morphology observationAfter exposure,the washed Caenorhabditis elegans were picked to make slide specimens,then add 10μL SM buffer Solution into the slide specimens,select 10 male C.elegans,syringe pinhead were used to make a laceration at no more than one-third of the tail of C.elegans,cover the C.elegan with a coverslip.Sperm morphology were observed under the mode of differential interference contrast(DIC)with fluorescence microscopy.Count 200 sperm cells for each C.elegans to calculate the sperm size and malformation rate.After 48h exposure,non-circular sperm cells were found with him-5 male from each dose group of PFOS and PFOA.Compared with the control,the sperm cell size(diameter)of male him-5 were decreased(P<0.05)in the dose group of PFOS(0.1,0.01 mmol/L)and PFOA(0.1mmol/L).Non-circular sperm cell rate of C.elegans in all dose groups of PFOS and PFOAwere higher than control(P<0.05),and the rate increased in a dose dependent manner(R2=0.995;R2’=0.991).3.3 Sperm activation experimentAfter exposure,the washed C.elegans were picked to make slide specimens,then add 10μL M9 buffer Solution and 10μl 200 μg/ml of Pronase into the slide specimens,cover the C.elegan with a coverslip after 5 minutes.Sperm activation were observed under the mode of differential interference contrast(DIC)with fluorescence microscopy.Results showed Sperm activation rates of C.elegans trated with 0.1 and 0.01 mmol/L of PFOS and PFOA were lower than control(P<0.05),and the rate decreased in a dose dependent manner(R2=0.890,R2=0.938).4.Mechanisms of Toxic Damage of PFOS and PFOA to Spermatogenesis of C.elegans4.1 Effect of PFOS and PFOA on germ cell meiosis related gene expression in C.elegansThe expression of wee-1.3 and puf-8 were measured by quantitative RT-qPCR.Results showed that,the expression of wee-1.3 were were up-regulated in all dose groups of PFOS and PFOA(P<0.05).The expression of puf-8 were down-regulated in the groups treated with high dose of PFOS(0.1 and O.Olmmol/L)and PFOA(0.1mmol/L).4.2 Effect of PFOS and PFOA on FB-MOs related gene expression in C.elegansNo statistical difference were found among PFOS treated and the control groups of the expression of spe-4,in high PFOA dose(0.1mmol/L)treated group the expression of spe-4 was up-regulated(P<0.05).The expression of spe-5,spe-6 and spe-17 were all upregulated in all dose groups treated with PFOS and PFOA(P<0.05).The expression of spe-10 were down-regulated in all PFOS and high PFOA dose(0.1 and 0.01 mmol/L)treated group(P<0.05).The expression of fer-1 were down-regulated in all PFOS and PFOA treated groups than control(P<0.05).4.3 Effect of PFOS and PFOA on sperm activation related gene expression in C.elegansThe expression of swm-1 and try-5 were down-regulated in all PFOS and PFOA treated groups compared with control(P<0.05).4.4 Effect of PFOS and PFOA on sperm material transportation related gene expression in C.elegansCompared with control group,the expression of spe-15 were down-regulated in group treated with middle and upper-dose of PFOS and PFOA(P<0.05).The expression of spe-26 were up-regulated in all PFOS and PFOA treated groups compared with control(P<0.05).ConclusionPFOA was the major important kind of PFCs in raw and finished water of the Water Supply Plant in Jiangsu.The number of germ cells change,sperm cell abnormality and sperm activation rate are more sensitive than brood size and generation time to evaluate the reproductive toxicity of PFOS and PFOA in C.elegans.L1 larvae C.elegans are more sensitive to PFOS and PFOA than L4 larvae,PFOS and PFOA have reproductive toxicity on the development of C.elegans in infancy stage.PFOS and PFOA may caused smaller,deformed sperm cells and lower activation rate by disrupting the expression of Meiosis,FB-MO complex and Sperm activation related genes,the the the number of germ cells decreased,and finally the brood size reduced.In addition,we found the reproductive toxicity of PFOS was stronger than PFOA’s,the inhibition of PFOS on puf-8 and spe-10 are stronger than PFOA,PFOA can significantly increase spe-4 expression,however no effect on spe-4 was found with PFOS.