Adverse Outcome Pathway Construction And Quantitative Analysis Based On Toxic Pathways

ObjectiveCurrently,the construction and application of Adverse Outcome Pathway(AOP)are mainly focused on ecological environmental toxicity,and the construction of AOP related to human health,especially the quantitative analysis method,is still being explored.This study utilizes high-throughput omics techniques and computational models,based toxicity pathway to construct an adverse outcome pathway linking AHR activation leading to lung damage,and evaluation and quantitative study of the model.This study provides a new clue and research means for the application of AOP framework in toxicity prediction and safety evaluation of exogenous chemicals.MethodsBased on the previously proposed 5 key toxicity pathways associated with Aryl Hydrocarbon Receptor(AHR)activation leading to lung injury,an AOP network was constructed,with AHR activation as the molecular initiating event,5 key toxicity pathways as the molecular key events,and multiple kinds of lung injury as the adverse outcomes.Evaluation of the AOP network was based on weight of evidence according to OECD guidelines.Multi-chemical validation of AOP networks was performed using highthroughput data from Toxcast,Pubchem and Comparative Toxicogenomics Database(CTD)combined with machine learning models.The binding ability of benzo(a)pyrene(Ba P)to AHR receptor protein was investigated by molecular docking and Surface Plasmon Resonance(SPR)experiments.An AOP network was quantitatively described using dose-dependent transcriptomics dataset of16HBE-CYP1A1 cells exposed to Ba P.Gene Set Enrichment Analysis(GSEA)and BMDexpress softs were used for pathway enrichment,benchmark dose and Point of Departure(PoD)analysis.The responsiveness of 5 toxic pathways was analyzed by selecting specific pattern genes from the toxic pathways.Cellular assays were used to further quantify key events such as the production of ROS(Reactive Oxygen Species),IL-6 release,8-OHDG release and collagen I production as presented in the AOP network.Finally,the relationship of between events was formulated using nonlinear fitting equations.ResultsWe constructed 7 linear AOP and integrated them into an AOP network,which started from AHR activation as the molecular initiation event,linking five toxicity pathway,nine cellular or tissue level key events to three adverse outcomes concerning lung injury.Evaluation of the AOP based on weight of evidence showed that 18 of the 22 key event relationships had high biological rationality,and 4 of them had medium biological rationality.7 machine learning models were developed using 32 in vitro high-throughput data,and the phenotypic and disease analysis data of CTD database were used to verify 6critical events and 3 adverse outcomes in AOP network.The results suggest in 2629 AHR reactive chemicals,a total of 6,8,2,28,30,20,and 72 chemicals can respectively activate each of the 7 linear AOPs.Molecular docking results showed the binding fraction of Ba P and AHR protein was– 4.38 kcal/mol,and SPR results showed that Ba P binding with AHR receptor in vitro was dose-dependent,suggesting AHR ligand property of Ba P.In addition,Nuclear and cytosolic fraction separation,PCR and Western-blotting results suggested that Ba P could activate AHR nuclear translocation followed by immediate intracellular degradation.GSEA analysis demonstrated that Ba P exposure-induced transcriptome data could be significantly enriched in the five key toxicity pathways.In order to explore the responsiveness of the five toxicity pathways,time-effect analysis of toxicity pathways was performed,suggesting AHR pathway as the first response pathway,followed by NRF2 pathway and IL-6 pathway,then HF pathway and p53 pathway.Further dose-response analysis showed that AHR gene had the lowest benchmark dose among transcriptomic data,and AHR pathway was the most sensitive pathway,with the PoD identified at 0.0044 μM.In addition,Ba P exposure resulted in a dose-dependent action in cellular critical events including ROS,8-OHDG,IL-6 and collagen.Finally,the relationships between key events were developed.ConclusionA human health-related AOP network linking AHR activation to lung damages were developed,evaluated and quantitatively analyzed.This study for the first time proposed a research strategy that integrated the concept of toxicity pathway into AOP framework,which is likely to be adopted for chemical safety assessment.