The Role And Related Mechanisms Of Mitochondrial Damage In B[a]P-induced Male Reproductive Toxicity

Background:Benzo[a]pyrene(B[a]P),one of the representative chemicals of polycyclic aromatic hydrocarbons(PAHs),is mainly produced by incomplete combustion of organic substance.B[a]P is widely distributed in the environment and enters the body through the respiratory tract,digestive tract and skin contamination.B[a]P is converted into a biologically active form,Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide(BPDE),by metabolic enzymes in the body.Increasing evidences demonstrated that B[a]P and BPDE are also important environmental endocrine disruptors in addition to their mutagenicity and carcinogenicity.B[a]P can affect spermatogenesis and hormone synthesis,resulting in male reproductive damage,and the corresponding targets and precise mechanisms are still obscure.Mitochondria are special organelles of eukaryotes and sensitive targets for a variety of environmental toxic substances.Mitochondria functionality of sperm is not only closely related to sperm quality,but also plays a critical role in spermatogenesis and hormone synthesis.Therefore,alterations in mitochondrial structure and function may be the important mechanisms of B[a]P leading to male reproductive toxicity.Mitochondrial biogenesis is one of the important factors affecting mitochondrial function,and inhibition of mitochondrial biogenesis can cause mitochondrial dysfunction.Meanwhile,mitochondrial clearance also participates in mitochondrial quality control.Mitophagy is the major way to clear damaged mitochondria,while over-activated mitophagy can increase mitochondrial loss and aggravate mitochondrial damage.Objective:1.To elucidate the effects and related mechanism of B[a]P and BPDE on mitochondria in spermatogenic cells.2.To clarify the effect of BPDE on testosterone synthesis in Leydig cells as well as the involved target organelles and mechanisms.3.To further verify the relationship between mitochondrial functionality of human sperm and semen quality,and initially explore the potential mechanism(s)how mitochondrial functionality influences semen quality.Methods:1.Mouse spermatocyte derived GC-2 cells were treated with various concentrations of BPDE to construct a model of spermatogenic cells in vitro.GC-2 cells were treated with different concentrations of BPDE for 72 h.The mitochondrial DNA copy number was determined by quantitative PCR.The mitochondrial mass was measured by NAO staining.SIRT1 activity was detected using SIRT1 activity detection kit.Mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit,ATP content was measured using ATP detection kit,ROS production was determined by ROS detection kit,caspase-9/-3 activity were measured using caspase-9/-3 activity detection kit detects;immunoprecipitation combined with immunoblotting were performed to determine expression of mitochondrial biogenesis-related proteins,key enzyme of mitochondrial oxidative phosphorylation,and proteins in relation to mitochondria-dependent apoptosis,which could help to clarify the effects of BPDE treatment on mitochondrial function and biogenesis in GC-2 cells.After pre-treatment with the SIRT1 agonist CAY10602 and SIRT1 siRNA,the above index changes were examined to evaluate the role of SIRT1 in the mitochondrial damage induced by BPDE.In addition,cells were co-treated with resveratrol and BPDE to reveal the role of ROS overproduction in mitochondrial biogenesis and function of GC-2 cells.2.Based on the tests in vitro,an animal model of B[a]P-exposure was established to further validate the results in vivo.Male SD rats were administered with different doses of B[a]P for 4 weeks,with intraperitoneal injection of ZLN005 or resveratrol at the same time.After 4 weeks,the rats were sacrificed and the testicular index was detected by weighing.The pathological alterations of testis were reflected by HE staining.The apoptosis of testicular spermatogenic cells was observed by TUNEL staining.The expression of SIRT1 in spermatogenic cells was detected by immunohistochemistry.After isolating spermatogenic cells by gradient centrifugation,the mitochondrial DNA copy number,mitochondrial mass,ATP content,expression and activity of proteins related to mitochondria-dependent apoptosis,mitochondrial biogenesis-related proteins and protein expression level of key enzyme of oxidative phosphorylation were measured.3.Mouse Leydig cells(TM3 cells)were treated with BPDE to construct a model in vitro.The cell viability of TM3 cells was detected by trypan blue staining after BPDE treatment,and the subsequent experiments were performed at doses which did not cause obvious cell death.After treated with various concentrations of BPDE for 48 h,testosterone ELISA kit was used to measure testosterone levels in TM3 cells.Western blot was used to detect the expression levels of proteins related to testosterone synthesis to evaluate the effects of BPDE treatment on testosterone levels and synthesis in TM3 cells.Transmission electron microscopy was used to reflect mitochondrial ultrastructural changes,and mitochondrial membrane potential,ATP content,ROS production,mitochondrial biogenesis-related proteins,mitochondrial DNA copy number and mitochondrial mass were determined in TM3 cells.TM3 cells were pretreated with ZLN005,a PGC-1? agonist,and the indicators mentioned above were assessed to explore the role of mitochondrial biogenesis in BPDE-induced inhibition of cellular testosterone synthesis.Furthermore,the expression of proteins relevant to mitophagy were detected by immunofluorescence co-localization and Western blot.The autophagy inhibitor(3-MA)and mitophagy inducer(CCCP)were used to inhibit or promote mitophagy in TM3 cells,and the related indicators were detected to confirm the role of mitophagy in BPDE-induced inhibition of cellular testosterone synthesis4.Based on the epidemiological studies and previous data,we initially explored the relationship between human sperm mitochondrial functionality and sperm acrosin activity as well as DNA fragmentation index.On this basis,human sperms were treated with CCCP in vitro,and sperm mitochondrial membrane potential,acrosin activity,acrosome reaction capability,DNA fragmentation index,ROS production and ATP content were determined subsequently to further confirm the relationships between sperm mitochondrial functionality and semen quality.Results:1.BPDE treatment resulted in a decrease of ATP content,increased release of mitochondrial Cyt C and activation of caspase-9 and caspase-3 in GC-2 cells.BPDE inhibited SIRT1 activity in GC-2 cells,hindered the deacetylation of PGC-1?,and caused reduction of the protein levels of SIRT1,PGC-1?,and NRF1.Up-regulation of SIRT1 expression significantly attenuated the increase of acetylated PGC-1? induced and the decrease of SIRT1,TERT,PGC-1? and NRF1 induced by BPDE.Activation of SIRT1 also notably alleviated the reduction of mitochondrial membrane potential,cellular ATP content and oxidative phosphorylation key protein Cox IV induced by BPDE.Up-regulation of SIRT1 expression reduced cytoplasmic Cyt C levels and inhibited activation of caspase-9 and caspase-3.On the contrary,down-regulation of SIRT1 expression aggravated BPDE-induced inhibition of mitochondrial biogenesis and mitochondrial dysfunction in GC-2 cells.2.BPDE treatment remarkably increased ROS levels in GC-2 cells.Co-treatment with resveratrol significantly increased mitochondrial membrane potential,cellular ATP content,Cox IV protein levels,mitochondrial DNA copy number,mitochondrial mass and SIRT1 activity in GC-2 cells.Resveratrol treatment also reduced acetylated PGC-1? levels and increased protein levels of SIRT1,TERT,PGC-1?,NRF1.In addition,resveratrol treatment decreased the level of Cyt C protein in cytoplasm of GC-2 cells and significantly inhibited the activation of caspase-9 and caspase-3.3.Tests in vivo showed that B[a]P exposure led to decreased testicular index,pathological changes of seminiferous tubules,increased number of apoptotic spermatogenic cells and caspase-9/-3 activation in male SD rats,while administration of PGC-1? activator(ZLN005)or resveratrol significantly ameliorated the alterations mentioned above.Further analysis in animal experiments revealed that B[a]P exposure resulted in a reduction of mitochondrial DNA copy number and mitochondrial mass in spermatocytes of rats,accompanied by inhibition of SIRT1 activity and decreased levels of SIRT1,TERT,PGC-1? and NRF1 proteins.Additionally,B[a]P exposure caused a dose-dependent decrease in Cox IV protein levels and ATP content in spermatogenic cells of rats,and ZLN005 or resveratrol administration significantly alleviated part or all of these changes.4.At the doses which did not affect cell viability,BPDE treatment led to decreased testosterone levels and reduced expression levels of testosterone synthesis related proteins(TSPO,StAR and CYP11A1).BPDE treatment resulted in ultrastructural injury of mitochondria,reduction of mitochondrial membrane potential and ATP content,and elevated ROS production in TM3 cells.In addition,BPDE treatment also resulted in decreased mitochondrial DNA copy number and mitochondrial mass,as well as decreased protein levels of PGC-1?,NRF1,and TFAM in TM3 cells.Pre-treatment with ZLN005 remarkably promoted mitochondrial biosynthesis,restored mitochondrial function,and increased testosterone synthesis-related protein expression as well as testosterone synthesis levels.5.BPDE treatment caused activation of the PINK1/Parkin/P62/LC-3 pathway,leading to increased levels of mitophagy in TM3 cells.Inhibition of mitophagy remarkably increased mitochondrial biogenesis,improved mitochondrial function,elevated expression of proteins involved in testosterone synthesis,and increased testosterone levels in cells.Conversely,enhancement of mitophagy aggravated BPDE-induced decreased mitochondrial biogenesis,mitochondrial dysfunction,reduced testosterone synthesis-related protein expression,and reduction of testosterone synthesis.6.The results of the population study showed that the acrosome activity of groups with medium and high mitochondrial membrane potential was significantly increased than that of groups with low mitochondrial membrane potential.The DNA fragmentation index of groups with medium and high mitochondrial membrane potential was notably lower than that of groups with low mitochondrial membrane potential.Further results in vitro showed that CCCP-induced reduction in human sperm mitochondrial membrane potential decreased sperm ATP content,increased ROS production,reduced capability of sperm acrosome reaction,and enhanced sperm DNA fragmentation.Conclusion:1.B[a]P and BPDE induces mitochondrial dysfunction by inhibiting mitochondrial biogenesis of spermatogenic cells,which causes mitochondria-dependent apoptosis and spermatogenic cell damage.Promotion of mitochondrial promotion can alleviate mitochondrial dysfunction and male reproductive toxicity induced by B[a]P and BPDE in spermatogenic cells.2.B[a]P and BPDE treatment can inhibit the expression and activity of SIRT1 which reduces PGC-1?-dependent mitochondrial biogenesis in spermatogenic cells by overproduction of ROS.3.BPDE damages the mitochondria of Leydig cells,leading to a decrease in the content of testosterone synthesis-related proteins in mitochondria,which ultimately resulted in a reduction of testosterone levels in mouse Leydig cells.Enhancement of mitochondrial biogenesis can notably increase testosterone levels in Leydig cells.4.BPDE induces mitochondrial damages in Leydig cells,which leads to excessive activation of the mitophagy pathway.Over-activated mitophagy aggravates BPDE-induced mitochondrial damage and inhibition of testosterone synthesis in Leydig cells.5.Mitochondrial functionality of human sperm is remarkably related to sperm quality.Decreased sperm mitochondrial function can reduce sperm fertilization potential and aggravate sperm DNA damage.