Effects Of Low-dose Octylphenol Exposure On Male Reproduction And Mechanism Study Based On Metabolomics

Part Ⅰ Metabolomics method establishmentIn order to study the metabolic changes of organism due to exogenous chemicals’ exposure or diseases,we established a high-throughput metabonomics analysis platform,including standard compound identification,biological sample pretreatment,relative quantification of metabolites in biological samples,bioinformatic analysis and quality control of the method.415 metabolites were qualitative accurately by confirming the accurate mass and retention time using HPLC-MS/MS method.These metabolites were mainly involved in carbohydrate metabolism,lipid metabolism,amino acid metabolism,nucleotide metabolism and phenol metabolism.Based on the metabolite standard compounds library,we established a rapid and stable sample pretreatment method,including the pretreatment of urine,serum,seminal plasma,tissue,cell and culture medium.The detectable metabolites in multisamples were analyzed under full-scan acquisition using HPLC-MS/MS.Among all the sample types,more than 200 kinds of metabolites were detected in serum,seminal plasma or tissue,and more than 180 kinds of metabolites were detected in urine,cell or culture medium.Metabolites detected in samples were identified by comparing with the metabolite standard compounds and the relative levels of metabolites in samples were quantitative using peak area normalization method.Bioinformatics analysis was conducted after enrichment of the changed metabolites in KEGG pathway.The stability of the process was evaluated by using the quality control(QC)samples.Results showed that the coefficient of variation(CV)of measured area and height in most detectable metabolites in QC samples were less than 20%,which indicated that the method was stable and reliable.The above results showed that a stable and high-throughput metabolomics method was established for our further study.Part Ⅱ Cytotoxicity screening and metabolic profiling analysis in low-dose OP treated GC-2 cellsGC-2 cell line was selected as an in vitro model to study the effects of low-dose OP on male reproduction and the underlying mechanism.We evaluated the low-dose effects of OP on spermatocytes by analyzing the effects of low-dose OP on cell proliferation,cell apoptosis,cell cycle,mitochondrial function,cellular ATP level and cell-based high-content screening(HCS)multi-parameter cytotoxicity.Meanwhile,non-target metabolomics approach was applied to study the low-dose effects of OP on metablism in spermatocytes and reveal the underlying mechanism.Results showed that cell proliferation,cell cycle and cellular ATP level were not affected in low-dose OP treated GC-2 cells.However,at the dose of 10-11 M and 10-8 M,spontaneous apoptosis of spermatocytes was inhibited with increased mitochondrial membrane potential in OP treated GC-2 cells.The results of HCS multi-parameter cytotoxicity analysis showed that nucleus size and permeability were not affected,while mitochondrial membrane potential was increased significantly in low-dose OP treated GC-2 cells.Results of metabolomics showed that the levels of 3’-Adenosine monophosphate(3’-AMP)and adenosine were decreased significantly and the adenosine metabolism was disturbed in GC-2 cells after low-dose OP treatment.Previous study has shown that suppression of cell apoptosis was promoted by increasing mitochondrial membrane potential.Another study reported that adenosine could induce cell apoptosis by disturbing mitochondrial function.Interestingly,low-dose OP-induced increased mitochondrial membrane potential and suppression of cell apoptosis were reversed after adding adenosine into the medium.Thus,our study firstly revealed low-dose OP could suppress spermatocyte cell apoptosis by promoting mitochondrial membrane potential,in which adenosine might play a key role.Part Ⅲ Effects of low-dose OP on spermatogenesis in mice and the verification of target metabolite in testisAdult male ICR mice were selected as an animal model to verify the low-dose OP-induced effects on spermatocytes and metabolism changes in vitro.In an acute toxicity test,ICR mice were treated with low-dose OP,sacrificed,and serum and a variety of organs were collected.Histological evaluations and TUNEL assay were conducted to observe morphological changes and apoptotic germ cells in testis of OP treated mice.Meanwhile,the level of adenosine in testis of OP treated mice were detected using HPLC-MS/MS.Results showed that body weight and testis organ coefficients were not affected in low-dose OP treated male mouse.Results of H-E staining and TUNEL assay showed that morphology of seminiferous tubules and germ cells were not changed in low-dose OP treated mouse,while low-dose OP could inhibit apoptosis of spermatocytes and decrease the number of spermatids.The level of adenosine in testis was decreased significantly in low-dose OP treated mice detected by HPLC-MS/MS.The above results showed that low-dose OP could change adenosine metabolism in testis and disturb spermatogenesis in male mice,which verified the results in vitro.Part Ⅳ The relationship of urinary OP exposure level and sperm concentration and the verification of target metabolite in seminal plasma of idiopathic infertile menSubjects were selected from Nanjing Medical University(NJMU)Infertility Study to study the relationship between urinary OP exposure level and sperm concentration and verify the changed adenosine metabolism found in vitro and in animal model by detecting the adenosine concentration in seminal plasma.The exposure level of OP in urine and concentration of adenosine in seminal plasma were detected using HPLC-MS/MS.Meanwhile,semen parameters were detected using computer-assisted sperm analysis(CASA).According to the concentration of OP in urine,idiopathic infertile men were divided into subjects with undetectable urinary OP and subjects with detectable urinary OP.Results showed that comparing with subjects with undetectable urinary OP,semen volume and sperm motility were not changed obviously,while sperm number per ejaculation and sperm concentration were decreased significantly in subjects with detectable urinary OP.Meanwhile,concentration of adenosine in seminal plasma was decreased significantly in subjects with detectable urinary OP.In summary,adenosine may be used as a biomarker of effect related to male reproduction impairment caused by low-dose OP exposure and verified the results in vitro and in animal model.