| Arsenic(As)is a ubiquitous environmental contaminant.Arsenic can enter human body and cause multiple organ damage through drinking water or inhalation.Chronic exposure to arsenic has been linked with increased risks of various cancers and diseases.In recent years,the potential harm of arsenic to male reproductive health has attracted increasing attention.Animal studies have proved that arsenic can interfere with normal spermatogenesis by inhibiting testosterone synthesis,reducing sperm quality,inducing oxidative stress after short-term arsenic exposure.However,the toxicity and the related mechanisms after long-term arsenic exposure are still unclear,and effective prevention and treatment measures are still lacking.Currently,it is agreed that oxidative stress is closely related to arsenic-induced organ damage,suggesting that antioxidants can alleviate arsenic-induced oxidative damage.Therefore,the search for safe and effective antioxidants becomes the focus of attention.Grape skin contains a large amount of anthocyanidin,which has a strong antioxidant capacity.However,whether Grape Skin Extract(GSE)can alleviate arsenic-induced oxidative damage and other damages and protect normal spermatogenesis has not been unexplored.Epidemiologic studies found increased prevalences of diabetes in populations exposed to high levels arsenic in drinking water.Oxidative stress is closely related to the occurrence of diabetes.Increasing evidence shows that arsenic disrupts glucose homeostasis.The liver is an important organ for glucose metabolism.Up to now,there are few studies on the effects of long-term arsenic exposure on glucose homeostasis,oxidative stress,liver injury,inflammatory response,insulin sensitivity and glucose transport in liver.The target organs of arsenic accumulation in mice are consistent with those of arsenic poisoning in humans.In this study,we used mice as experimental materials,to explore the male reproductive toxicity induced by long-term exposure to sodium arsenite(NaAsO2)and its underlying mechanisms,to analyze alleviation effect of GSE on arsenic-induced male reproductive toxicity,to study effect of arsenic exposure on glucose homeostasis.Results demonstrated that:Firstly,in order to investigate the effects of long-term arsenic exposure on spermatogenesis,testosterone synthesis,the expression of androgen binding protein(ABP)and Ddx3y,and the underlying mechanisms,male C57BL/6 mice were treated with 5 and 50 ppm arsenic for 6 months via drinking water.The results showed that arsenic reduced sperm count and sperm motility and enhanced the abnormal sperm percentage.The decrease in the number of spermatogenic cells and sperm in seminiferous tubules and the decline in the Johnsen score were observed in both arsenic-treated groups,suggesting spermatogenesis disorders.Moreover,arsenic diminished serum testosterone,along with the reduced expression of luteinizing hormone receptor(LHR),steroidogenic acute regulatory protein(StAR)and 17?-hydroxysteroid dehydrogenase(17?-HSD)genes.Arsenic also down-regulated mRNA levels of ABP and Ddx3y in a dose-dependent manner.Meanwhile,the protein levels of StAR,17B-HSD and Ddx3y were significantly reduced in arsenic-treated groups.Taken together,these results suggest that the reduced testosterone by the inhibition of multiple genes involved in steroidogenesis,the damaged androgen homeostasis partially via lessening the expression of the ABP gene and the down-regulated expression of Ddx3y,might contribute to spermatogenesis disorders in mice exposed to arsenic.Secondly,in order to elucidate the possible mechanisms of arsenic-induced male reproductive toxicity,the effects of long-term arsenic exposure on oxidative stress,cell cycle arrest and apoptosis were evaluated in testes.Our results showed that arsenic significantly decreased the activity of total superoxide dismutase(T-SOD)and glutathione(GSH)content but increased the levels of reactive oxygen species(ROS)and malondialdehyde(MDA)content,suggesting that arsenic induced oxidative stress in testes Pearson correlation analysis showed that there was a significantly negative correlation between the antioxidants(GSH and T-SOD)and ROS.It was noteworthy that the Pearson correlation coefficient(r=-0.822)between GSH and ROS was higher than that between T-SOD and ROS(r=-0.718).In addition,arsenic could induce G2/M phase arrest in testes,concurrent with a significant increase in mRNA levels of p53 and p21,decrease in mRNA and protein levels of cyclin-dependent kinase 2(cdc2)and cyclin Bl,and upregulation of p-cdc2(Tyr15).Arsenic markedly elevated Bax mRNA and protein levels,enhanced the ratio of Bax/Bcl-2,activated caspase-3,and induced testicular apoptosis.In conclusion,arsenic-induced testicular toxicity after long-term exposure might relate to oxidative damage,G2/M arrest and apoptosis in testes of mice,which contributed to the increased risk of spermatogenesis disorders and male infertility.Thirdly,previous studies have confirmed that oxidative stress is considered to be a major cause of male reproductive failure,suggesting that the arsenic-induced male reproductive injury can be alleviated by antioxidants.In this study,male kunming mice were given drinking water containing 10 ppm arsenic with or without Grape Skin Extract(GSE)for 60 days.The effects of GSE on the arsenic-induced male reproductive toxicity were evaluated.The results showed that arsenic exposure decreased the number of spermatogenic cells and sperm,lessened sperm count,and elevated the abnormal sperm percentage.These effects could be alleviated by co-treatment with arsenic and GSE.In addition,arsenic induced oxidative damage by increased H2O2 and MDA content,and decreased T-SOD activity and GSH content in testes,which could be alleviated by co-treatment with arsenic and GSE.Meanwhile,arsenic increased Bax mRNA and Bax/Bcl-2 ratio,but co-treatment with arsenic and GSE decreased Bax mRNA and Bax/Bcl-2 ratio.Arsenic downregulated key genes involved in testosterone synthesis(LHR and 17B-HSD)mRNA,but co-treatment with arsenic and GSE increased LHR and 176-HSD mRNA.Arsenic induced inflammatory response by the upregulated pro-inflammatory cytokines(TNF-? and IL-6)mRNA,which could be alleviated by co-treatment with arsenic and GSE.These results suggested that GSE might alleviate arsenic-induced male reproductive toxicity by reducing oxidative damage and inflammatory response,increasing key genes involved in testosterone synthesis(LHR and 17B-HSD)mRNA,and decreasing Bax mRNA and Bax/Bcl-2 ratio in testes.GSE has certain protective effects on arsenic-induced spermatogenesis disorders.Finally,in the present study,we aimed to study the effects of arsenic on glucose homeostasis and the underlying mechanisms.C57BL/6 mice were treated with 5 and 50 ppm arsenic for 6 months via drinking water.The results showed that no significant difference was observed in fasting blood glucose monthly and the glucose tolerance during 1-4 months of arsenic exposure.However,arsenic markedly reduced the glucose tolerance in a concentration-and time-dependent manner after 5 and 6 months,respectively Meanwhile,arsenic induced oxidative stress in the liver,which was positively correlated with the impaired glucose tolerance during the same period.In addition,activities of serum ALT and AST significantly increased in mice exposed to 50 ppm arsenic after 5 and 6 months.Hematoxylin-eosin(HE)staining showed the liver damages,inflammatory cells infiltration at both arsenic-treated groups.Meanwhile,tumor necrosis factor-?(TNF-?)mRNA significantly increased,while insulin receptor substrate 2(IRS2)and glucose transporter 2(GLUT2) mRNA decreased in liver.The results suggested that long-term arsenic exposure might lead to impaired glucose homeostasis by oxidative stress,structure and function injury,inflammatory response,the impaired insulin post-receptor signaling transduction and abnormal glucose transport in the liver.In summary,long-term exposure to arsenic caused testicular structural damage in mice,decreased sperm count,increased sperm malformation rate,leading to spermatogenesis disorders,and subsequent male reproductive toxicity.Further studies showed that arsenic exposure inhibited testosterone synthesis,caused oxidative damage,and induced G2/M cell arrest and apoptosis in testes.After GSE intervention,arsenic-induced male reproductive injury can be alleviated by reducing oxidative damage,inflammatory response,regulating key genes of testosterone synthesis and apoptosis-related genes,which will provide experimental evidences for the prevention and treatment of arsenic-induced male reproductive toxicity.In addition,arsenic exposure could induce the impaired glucose tolerance and disturbed glucose homeostasis in mice.The impaired glucose tolerance might be associated with oxidative stress,inflammatory response,the impaired insulin post-receptor signaling transduction and abnormal glucose transport in liver. |
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