A New Method Of Generating Adverse Outcome Pathway(AOP) Via Phenotypic Analysis And Its Application In Male Reproductive Impairment

BackgroundWith the rapid development of economic society and industrial production,the species and quantities of chemicals are surging constantly.Facing the potential adverse effects of these chemicals on human health and environment,it is a major challenge for toxicology to conduct their risk assessment efficiently.Traditional toxicological tests based on animal experiments have limitations of low-throughput,time-wasting,high cost and low sensitivity,which are increasingly unable to meet the needs of current toxicity tests for explosive growing chemicals.In 2007,The US National Research Council blueprint for change,entitled Toxicity Testing in the 21st Century:A Vision and Strategy（TT21C）which described the necessity,feasibility and development prospect of the transformation from traditional toxicology system to a new toxic-pathway-based toxicity testing strategy conducting on human cells,cell lines and cell components.The OECD（Organization for Economic Co-operation and Development）officially put forward the concept of the adverse outcome pathway（AOP）in 2013,Which refers to a series of association between direct molecular initiation events caused by chemicals and the final adverse outcome,reflecting as key events that occur sequentially at different levels of biological structure,such as tissues and organs.In recent years,the rapid development of international public databases such as CTD（Comparative Toxicogenomics Database）,Tox Cast and AOPWiki in toxicology and the improvement of computer science have provided rich information resources and theoretical basis for the construction of AOP,which make it possible to construct AOP networks and solve the biological complexity problems of chemical toxicity through computational toxicology or bioinformatics analysis methods.In this research,noticing that phenotypic data refers a biological outcome spanning various levels from cellular to system,and have great potential to illustrate key events consisting of AOPs,we have innovatively established a new method for generation of AOPs based on chemicals-phenotype-disease framework.Contents1.A new approach to construct an adverse outcome pathway（AOP）based on chemical-phenotypic-disease frameworkAdverse outcome pathways（AOPs）reflecting a series of biological processes of chemical-phenotypic-disease was established,through integration of CTD and AOP Wiki database.The significance of chemical-phenotypic and phenotypic-disease associations were analyzed using topological local network algorithm.The filtered phenotypic information in CTD were mapped into key events in AOP Wiki through GO（Gene Ontology）ID identifier.Then predicted AOPs were enriched based on matching information and phenotypic significance.Then the molecular initiation events（MIEs）were inferred through enriched AOPs.In accordance with OECD guidelines on AOP construction,the predicted AOP:64 was comprehensively evaluated using the evidence weighting method.2.Exploring the effects and toxicity pathways of arsenic-induced decreasing testosterone in vitro under the guidance of AOPBased on the toxicity pathway suggested by AOP,the testosterone production model of mouse Leydig cells（TM3）in vitro was established for verification.After being treated with0-5μM gradient doses of Na As O₂,the effect and toxicity pathways of arsenic-induced testosterone reduction were analyzed at 24 hours and 48 hours.The testosterone level was quantitatively analyzed by ELISA.Transcriptome sequencing was used to dynamically predict the transcriptional regulation of TM3 cells treated at different time points/doses.Ingenuity Pathway Analysis（IPA）was used to analyze pathway enrichment and upstream factor regulation of different gene sets.With the test in Hep G2 cells in vitro from Tox Cast program,the activation of PPARαwas screened with the treatment of dimethyl arsenic acid（DMA）,a major arsenic metabolite in human body.The potential of direct binding between PPARαreceptor and arsenic metabolite DMA was analyzed by energy minimization molecular docking analysis.3.Effects of low dose exposure to heavy metals including arsenic on male reproductive health in a college-students cohortOur research group recruited 796 male college students in Chongqing University City in2013 to study the impact of environmental,social,psychological and behavioral factors on reproductive health.In the second year,666 of them（83.7%）were successfully followed up.The volunteers completed psychological behavior information collection,serum sex hormone levels,semen quality and other reproductive health outcomes at each phase.Eighteen metals/metal-like elements including arsenic in urine samples were quantified by ICP-MS and used as the basis of metal internal exposure.Through cluster analysis,the population was divided according to the overall exposure level,and the reproductive outcomes of different subgroups were compared.The effects of various metals/metal-like elements were analyzed by using multiple statistical strategies such as single-element mixing effect model and multi-elements mixing model.The Jonckheere-Terpstra test was used to verify the associations between arsenic exposure and reproductive outcomes including the predicted key events of testosterone reduction in college students,and an internal exposure threshold dose analysis was performed using EPA BMDs 3.20 reference dose model.4.Application of chemical-phenotype-disease AOP framework in the study of reproductive toxicity of B[a]PAOP Wiki and CTD database were integrated to construct screening adverse outcome pathways（AOPs）network for key phenotypes related to the reproductive toxicity of B[a]P.The upstream and downstream correlations of testosterone synthesis,mitochondrial function and energy metabolism clued by AOP network were studied by in vitro and in vivo experiments.After 35 days of intragastric treatment,the levels of testosterone in serum and interstitial testis of mice were detected.Western blot was used to detect the expression level of testosterone synthesis transporter in the testis tissues after treatment.The ultrastructural changes of mitochondria in TM3 cells were observed by electron microscopy.The key events and toxicity pathway suggested by AOP were analyzed and verified by downloading the testis omics data of mouse treated with B[a]P from GEO database.The mouse Leydig cells（TM3cell）were cultured in vitro.After being treated with different doses of BPDE（Benzo[a]Pyrene-7,8-di-hydrodiol-9,10-Epoxide,one of the most active metabolites in vivo for B[a]P exposure）,the testosterone synthesis level was detected by ELISA.Western blot was used to detect the expression levels of key proteins in the pathway regulating mitochondrial synthetic proteins and testosterone synthesis.Further,other mitochondrial function-related molecular indicators were detected including mitochondrial mass,mitochondrial ATP synthesis rate.Then NAD⁺protective agent KL1333 was pretreated to observe the alleviating effect of BPDE-induced mitochondrial function and testosterone synthesis damage in the protected state of energy metabolism.Results:1.Construction of chemical-phenotypic-disease prediction pathway（AOP）approachA total of 655 phenotypes related to arsenic-induced male reproductive damage were filtered from the CTD database as key event sets for constructing AOP.After computing the significance of phenotype based on scale-free random network algorithm,39 key events with most significance were filtered.Cluster analysis of these events was applied and be mainly focused on the function/process related oxidative stress,steroid metabolism and cell cycle.Furthermore,AOPs network was constructed by combining prior knowledge in AOP Wiki.A total of 16 predicted AOPs were enriched and a set of possible initiation molecular events（MIE）can be inferred,including PPARαactivation,NADPH oxidase activation,glucocorticoid receptor agonists,and histone deacetylase inhibition.The network of 6 AOPs with coverage greater than 0.4 was presented as two relatively independent subgraphs,with ROS mediated apoptosis and decreased testosterone synthesis as the core key events,respectively.Then AOP:64 was evaluated via the principle of overall weight of evidence.2.Exploring the effects and toxicity pathways of arsenic-induced decreasing testosterone in vitro under the guidance of AOPTM3 cells were treated by Na As O₂ in vitro for 24h and 48h,and the testosterone synthesis level of TM3 cells decreased significantly,and the testosterone-biosynthetic protein of CYP11A1 and St AR decreased.Transcriptome analysis found that the pathways activated at different time points were largely overlapped with the molecular pathway events predicted by AOP.IPA analysis showed that PPARαactivated pathway has the most significant upstream regulatory effect,which is a possible molecular initiation event.Then dimethylarsenic acid（DMA）,the main arsenide metabolite in mammals was also verified the capacity to activate PPARαpathway in anthropogenic Hep G2 in vitro.Molecular docking simulation also suggests the potential of direct binding between DMA and PPARαreceptor.3.Effects of low dose exposure to heavy metals including arsenic on male reproductive health in a college-students cohortThe metal exposure level of college students is at a very low level,which is far below the national safety limit and comparable with the survey results of normal people in other developed countries.There was a significant correlation between urine metal concentrations,and cluster analysis was used to divided the population into high and low subgroups according to the overall exposure level of 18 metals.Serum testosterone levels in the high exposure subgroup were significantly lower than those in the low exposure subgroup（4.29vs.4.49 nmol/L,P=0.026）.After adjusting for confounding factors such as age,smoking and alcohol consumption,a stable positive correlation was observed between urinary silver concentration and several semen parameters（including semen volume,sperm concentration and total sperm count）.There was a significant negative correlation between urine vanadium/nickel concentration and normal sperm morphology rate.There was a significant negative correlation between serum testosterone level and lithium concentration.Jonckheere-Terpstra test showed that urinary arsenic concentration was monotonically correlated with serum testosterone level and normal sperm shape rate（P<0.001;P=0.004）.BMD model was used to further fit the dose-response relationship between urinary arsenic and serum testosterone,and the corresponding internal exposure dose BMDL was calculated to be 4.886mg/g creatinine when testosterone began to be disturbed in college students.4.Application of chemical-phenotype-disease AOP framework in the study of reproductive toxicity of B[a]PA total of 71 key phenotypes of B[a P induced male reproductive damage were filtered using chemical-phenotypic-disease AOP framework.Cluster analysis based on phenotypic similarity suggested that key events such as testosterone reduction,mitochondrial function impairment and ATP synthesis disorder played a key role in the process of male reproductive damage induced by B[A]P.In addition,multiple AOPs pointed to decreased testosterone synthesis in Leydig cells.According to the guidance of AOP,the mechanism of B[a]P-induced decreased testosterone synthesis through mitochondrial injury was further explored.Animal model experiments with the treatment of 50n M and 100nm B[a]P showed a decrease in circulating testosterone,and histological sections showed the morphological changes of mitochondrial crest,the formation of mitochondrial vacuoles and other mitochondrial structure destruction.B[a]P treated testicular tissue transcriptome results showed that transcription levels of several NADH dehydrogenase subunits comprising mitochondrial complexⅠwere significantly down-regulated,including Ndufa5,Ndufa6,Ndufa9 and Ndufa10.In vitro,BPDE induced decreased testosterone synthesis and down-regulated expression of key proteins of testosterone synthesis were also significantly improved by NAD⁺targeted small molecule modulator pretreatment.BPDE induced decreased testosterone synthesis and down-regulated expression of key proteins of testosterone synthesis were also significantly alleviated by NAD+targeted small molecule modulator pretreatment.Our results verified the AOPs’prediction that BPDE-mediated the imbalance of ATP metabolism and mitochondrial damage and ultimately impaired testosterone synthesis in TM3 cells.ConclusionIn this study,a new method for predicting AOP based on chemical-phenotypic-disease biological process was established.In the application of AOP to construct arsenic-induced male reproductive damage,testosterone reduction was accessed as key event in AOP networks,while activation of PPARαtoxicity pathway may be the relevant upstream molecular initial event.In the population study of college students,the dose-response relationship between arsenic exposure and serum testosterone reduction was verified,and the corresponding internal exposure dose BMDL at the beginning of testosterone disturbance was predicted to be4.886mg/g creatinine,which was lower than the urine arsenic exposure level associated with harmful outcomes of other systems.It is suggested that sex hormone level disturbance may be the key events that are more sensitive for low dose arsenic exposure compared to other reported arsenic toxic effects.The safety limits established based on sex hormone disturbance would be more protective.This AOP method has also been successfully applied in the reproductive toxicity study of B[a]P.In conclusion,In conclusion,this study proposed a new method for predicting AOP based on chemical-phenotypic-disease biological process using local network topology algorithm,which provided important references for subsequent study for key molecules and pathways of arsenic toxicity,as well as a brand new strategy for AOP construction of chemicals.