Perfluorooctanoic Acid-induced Testicular Damage And Its Mechanism Of Toxicity In Mice

Objective:Perfluorooctanic acid?PFOA?is a synthetic organofluorine compound which has widely attracted attention due to the adverse effect on ecological environment and human health.PFOA is enriched and amplified in the food chain,resulting in damage to many tissues and organs of organism.Previous studies have showed that exposure to PFOA is associated with cardiovascular disease,chronic kidney disease,thyroid disease and liver damage.In recent years,studies have found that PFOA exerts a negative effect on male reproductive function.However,the mechanism of reproductive toxicology of PFOA has not been cleared.This study was designed to observe the toxicological effect of PFOA on male reproductive system at the organic and molecular levels,and to investigate the influence of PFOA on testicular oxidative stress and apoptosis in mice.Meanwhile,we focused on the relationship between the mechanism of PFOA toxicity and transcription factor NRF2.The aim of this study was to reveal the molecular mechanism of PFOA-induced male reproductive damage and to provide a new target for the prevention and treatment of sperm abnormalities and male infertility caused by perfluorinated compounds?PFCs?.Methods:Male Kunming mice?8 weeks old?were orally administrated different concentrations of PFOA?0,2.5,5,10 mg/kg/d?for 14 consecutive days after acclimatization for 1 week.At the end of treatment period,the testes and epididymides were quickly excised and collected.The right testes and epididymides were fixed in Bouin's fluid.The left epididymides were separated for sperm count and the left testes were frozen in liquid nitrogen for subsequent biochemical experiments.HE staining was used to observe the pathological changes in testis and epididymis tissues.Colorimetry was applied to detect the activity of testicular superoxide dismutase?SOD?and catalase?CAT?and the generation of hydrogen peroxide?H₂O₂?and malondialdehyde?MDA?.Real-time PCR and Western blot were performed to measure the expression of NRF2,p-p53,BAX and BCL-2 in the testis.TUNEL staining was carried out to determine the apoptosis of testicular cells.Results:?1?Effect of PFOA on testicular weight:Compared with the control group,PFOA treatment for 14 consecutive days significantly diminished absolute testis weight at the highest concentration?10 mg/kg/day??p<0.05?.The lowest and median doses of PFOA had no obvious effect on absolute testicular weight?p>0.05?.However,PFOA treatment did not alter the testis weight relative to body weight?p>0.05?.?2?Effect of PFOA on testicular histology:After exposure to PFOA for 14consecutive days,HE staining showed obvious atrophy of seminiferous tubules,disrupted arrangement of spermatogenic cells and detachment of germ cells from seminiferous epithelium.The maximal effect was observed at the highest concentration?10 mg/kg/day?.?3?Effect of PFOA on epididymal sperm count:Compared with the control group,exposure to PFOA for 14 consecutive days caused a dose-dependent decrease in epididymal sperm count?p<0.05?.Furthermore,epididymal histopathology also revealed a significant decrease in sperm density in PFOA-treated mice after 14 days of exposure.?4?Effect of PFOA on testicular oxidative stress:Compared with the control group,MDA formation and H₂O₂ generation in the testis were significantly increased after exposure to 5 and 10 mg/kg/d PFOA for 14 days?p<0.05?.In addition,the activity of SOD and CAT in the testis was significantly decreased after exposure to PFOA for 14 days?p<0.05?.?5?Effect of PFOA on testicular NRF2 expression:Compared with the control group,the mRNA and protein expression of NRF2 in the testis was significantly down-regulated in a dose-dependent manner after exposure to PFOA for 14consecutive days?p<0.05?.?6?Effect of PFOA on testicular cell apoptosis:Compared with the control group,PFOA administration for 14 days significantly up-regulated the expression of BAX and p-p53 and down-regulated the expression of BCL-2 in the testis of mice?p<0.05?.Moreover,TUNEL staining showed that PFOA treatment significantly increased the number of positive apoptotic cells?p<0.05?Conclusion:Exposure to PFOA for 14 consecutive days can result in testicular pathological damage,reduce the expression of NRF2 and its target protein activity of SOD and CAT,increase the generation of H₂O₂ and MDA,up-regulate the expression of p-p53and BAX,and down-regulate the expression of BCL-2.These results suggest that PFOA can cause testicular injury and disrupt spermatogenesis through inducing testicular oxidative stress and cell apoptosis,and the mechanism of PFOA-induced male reproductive toxicity be involved in the inhibition NRF2 expression.