The Mechanisms Of Telomere And Mitochondrial Damge In PAHs-induced Male Reproductive Toxicity

BackgroudPolycyclic aromatic hydrocarbons(PAHs)are a kind of important environmental pollutants,which inevitably enter the human body through inhalation,ingestion,and dermal contact.Because of the widespread availability of PAHs in the environment,the great potential for human exposure and their mutagenicity and carcinogenicity,many chemicals of PAHs are classified as probable human carcinogens by the International Agency for Research on Cancer(IARC).Moreover,exposure to PAHs or their reactive intermediates has been found to affect the integrity of reproductive functions in mammals,and it ultimately contributes to male infertility.In particular,both epidemiological studies and animal experiments have reported that increased PAHs levels were associated with poor sperm quality and sperm DNAdamage,and a correlation between exposure to PAHs and an increased risk of male idiopathic infertility was also observed.However,the potential impact of exposure to PAHs on human fertility remains controversial and inconclusive,especially among the general population exposed at low levels of pollutants,the weak correlations between PAHs exposure and reproductive damage might not be identified by conventional semen quality detection.Thus,it is necessary to explore more sensitive biomarkers of male reproductive damage at the low levels of PAHs exposure.In addition,the events and underlying molecular mechanisms in PAHs-induced male reproductive toxicity remains unclear,therefore,tracking the targets and molecular mechanisms of genetic damage in germ cells,may provide important evidence for exploring the biomarkers to reflect the PAHs reproductive damage.Telomeric DNA and mitochondrial DNA play an important role in maintaining genome integrity and physiological functions of cells.The previous studies found that PAHs induced telomeric DNA and mitochrondrial damage,suggesting that the changes of telomere and mitochrondria integrity may be the important targets of male reproductive damage induced by PAHs.Based on the Male Reproductive Health in Chongqing College Students(MARHCS)prospective cohort study,we investigated the relationships between urinary PAHs metabolites and sperm telomeres and mitochondria in the current study.Meanwhile,B[a]P,the typical representative of PAHs,and its active metabolites BPDE were selected to confirm their effetcs on telomeres and mitochondria,and further to explore their roles in the mechanisms of male reproductive damage in vitro and in vivo experiments.Contents1.Study on the association between PAHs exposure and sperm telomeres and mitochondria damage and semen parameters in college students.Based on the MARHCS cohort study carried out in 2013-2015.We assessed the potential associations between PAHs internal exposure and genetic damage by analyzing biological samples(urine and semen)collected in the first follow-up in 2014.PAHs exposure was characterized by measuring the corresponding urinary metabolites using GC-MS,and its effects on telomeres and mitochondria were quantified through analyses of sperm telomere length and sperm mt DNA copy number using quantitative real-time PCR,sperm mitochrondrial membrane potential(MMP)using JC-1,mt DNA integrity using Long-PCR,sperm apoptosis using Annexin V-FITC,and semen quality of each subject.Specially,we hope to explore the biomarkers of male reproductive damage at the environmental exposure levels of PAHs.2.Study on the mechanism of telomere dysfunction induced by BPDE and B[a]P.In order to evaluate the toxic effects in vitro model of BPDE exposed mouse spermatocytederived GC-2 cells,we detected the cell survival,senescence and apoptosis by Trypan blue exclusion assay,SA-?-gal staining and Annexin V-FITC,respectively.To further evaluate the effects of BPDE on telomere dysfunction in GC-2 cells,quantitative real-time PCR and TRF assay were used to measure telomere length,FISH combined with ?-H2 AX immunofluorescence was used to dectect telomeric DNA damage,and the detection of telomerase activity and the expression levels of TERT protein were used by TRAPPCR-ELISA and Western blotting,respectively.Moreover,in order to find whether BPDE activated DNA damage response in GC-2 cells,we examined DNA damage response related protein expression by Western blotting.In addition,by establishing cell models of TERT down-expression and re-expression by shRNA-TERT and pLV-EGFP-TERT vectors,respectively,we explored the roles of TERT in BPDE-induced senescence and apoptosis.We also established in vivo model of B[a]P(0,5,10,and 20 mg/kg)exposed Sprague Dawley(SD)rats by oral gavage once daily for 7 days to further verify the results of GC-2 cells.3.Study on the mechanism of mitochrondrial damage induced by BPDE.Based on in vitro model of BPDE exposed GC-2 cells,we examined the mitochondrial mass using NAO staning,mt DNA copy number using quantitative real-time PCR,mitochondrial membrane potential using JC-1,COX IV expression and caspase cascade by Western blotting in the BPDE-treated GC-2 cells to assess the effects of BPDE on mitochondrial function,biosynthesis and mitochondrial pathway apoptosis.ZLN005 pretreatment was employed to explore the roles of PGC-1? in mitochondrial damage and apoptosis in germ cells by analysis the effects of BPDE on mitochondrial-related indicators mentioned above.In additon,in order to clarify the roles of TERT in BPDE induced mitochondrial damage,cell models of TERT down-expression and re-expression were also used to further examnine the effects of BPDE on mitochondrial damage and investigate the associations between telomeres and mitochondria.Results1.Decreased sperm telomere length and mtDNA copy number in associations with exposure to polycyclic aromatic hydrocarbons.We measured eight urinary PAH metabolites(1-OHNap,2-OHNap,1-OHPhe,2-OHPhe,3-OHPhe,4-OHPhe,2-OHFlu and 1-OHPyr)to estimate PAHs exposure.The linear regression analysis revealed that increased levels of urinary 1-OHPyr and 1-OHNap were associated with decreased sperm telomere length(-0.385;95% CI,-0.749,-0.021 for 1-OHPyr;and-0.079;95% CI,-0.146,-0.011 for 1-OHNap).Increased levels of 2-OHPhe,3-OHPhe,?Phe metabolites and 2-OHFlu were found to be associated with decreased sperm mtDNAcn(-9.427;95% CI,-17.586,-0.459 for 2-OHPhe;-11.488;95% CI,-19.462,-2.725 for 3-OHPhe;-9.635;95% CI,-17.965,-0.688 for ?Phe metabolites;and-11.692;95% CI,-19.647,-2.949 for 2-OHFlu).The significant negative associations remained after adjusting for potential confounders.There were no significant associations between urinary PAH metabolites and sperm MMP or mt DNA integrity.Moreover,no significant associations were observed between urinary PAH metabolites and semen quality or sperm apoptosis.These results indicated that the low exposure levels of PAHs may cause abnormities in sperm telomere and mitochondria,and sperm telomere length and mtDNAcn may be the sensitive biomarkers of male reproductive damage in the condition of low-levels PAHs exposure.2.TERT regulates telomere-related senescence and apoptosis through the DNA damage response in BPDE-and B[a]P-exposed male germ cells.BPDE had toxic effects on the GC-2 cells,the results showed that BPDE inhibition cell proliferation,and induced senescence and apoptosis.Further study found that BPDE exposure induced shortened telomeres,telomere-associated DNA damage,triggered the DNA damage response(ATM/Chk1/p53/p21)in GC-2 cells.Telomerase played vital roles in regulating telomere length of germ cells,and the reduction of telomerase activity and TERT expression were also observed in BPDE-treated cells,suggesting that telomere dysfunction is the target of BPDE induced male reproductive cells damage.Moreover,by establishing cell models of TERT down-expression and re-expression,we found that BPDE aggravated telomere damage,cell senescence,cell apoptosis and activated DNA damage response pathway by down-expression of TERT,and re-expression of TERT partly rescued these damage,indicating that TERT-mediated telomere dysfunction contributes to BPDE-induced senescence and apoptosis in male reproductive cells.Furthermore,these results were confirmed in the testicular cells of rats that had been exposed to B[a]P.B[a]P treatment induced significant testicular injury,accompanied by shortened telomeres,reduced telomerase expression,senescence and apoptosis in testicular cells,which resulted in the male reproductive damage.3.TERT regulates PGC-1? mediated mitochrondrial damage and apoptosis in BPDE-exposed male germ cells.BPDE exposure decreased the mitochrondrial mass,mt DNA copy number,mitochondrial membrane potential,inhibited COX IV and PGC-1? expression,and caspase cascade,suggesting that BPDE damaged the mitochondrial function and biosynthesis.After pretreatment with ZLN005,the activator of PGC-1?,the mitochondrial damage and mitochondrial pathway apoptosis were significantly rescued,indicating that PGC-1? involved in BPDE induced mitochondria-mediated apoptosis in male reproductive cells.Moreover,to further analyzed the relationship between telomeres and mitochrondria,we used the cell models of TERT down-expression and re-expression metioned above,we found that BPDE aggravated mitochondrial damage by down-expression of TERT,including and mitochrondrial mass,mt DNA copy number,mitochondrial membrane potential and mitochondrial pathway apoptosis,however,reexpression of TERT partly rescued mitochondrial damage.The results showed that TERT participated in BPDE-induced mitochondria damage by regulating the expression of PGC-1?.ConclusionOur investigation of college students in Chongqing with PAHs enviromental exposure demonstrated that decreased sperm telomere length and sperm mt DNA copy number were in associations with urinary PAH metabolites,suggesting that sperm telomere legnth and sperm mt DNA copy number may be the sensitive biomarkers of PAHs chemicals induced male reproductive damage.In vitro and in vivo experiments found that exposure to B[a]P and its active metabolite,BPDE,induces telomere and mitochondrial dysfuntion in germ cells,eventually resulting in germ cell senescence and apoptosis.In these process,BPDE and B[a]P inhibited the expression of TERT,impaired telomere function and activated the DNA damage response,and then caused male reproductive damage.For another,BPDE inhibited the expression of PGC-1?,induced mitochondrial damge and apoptosis,resulting in the male reproductive toxicity.Wherein,TERT gene plays a key role in regulating the two pathways.