Mode Of Action Of Benzo[a]pyrene In Cigarette Induced Lung Cancer Based On The Strategy Of Toxicity Testing 21st Century

Objective:Benzo[a]pyrene is a kind of pentacyclic polycyclic aromatic hydrocarbon and group 1 carcinogens.It is a recognized harmful component of cigarettes,and smoking is the main way in which benzo[a]pyrene is exposed through the respiratory tract.Quantitative assessment of the health risks posed by benzo[a]pyrene in cigarettes is an important public health requirement.Traditional toxicological risk assessment methods rely on animal experiments,which have disadvantages such as long cycles,high costs,low throughput,inability to extrapolate fully to humans,and being restricted by animal welfare and ethics.In recent years,the "21st-century toxicity testing strategy" has emerged,which has the characteristic of predicting the early events of chemical toxicity on humans being rapidly.This strategy has increasingly important application value in chemical toxicity testing and risk assessment,but the rationality of the prediction process and the reliability of the prediction results still need to be tested and improved in practical applications.Therefore,based on transcriptome sequencing and bioinformatics analysis of benzo[a]pyrene-exposed bronchial epithelial cells,this study established a framework for the carcinogenic effects of benzo[a]pyrene in cigarettes,and investigated the mechanism of benzo[a]pyrene-induced lung cancer,evaluating the health risks posed by benzo[a]pyrene in cigarettes.On one hand,this study provides information for a comprehensive evaluation of the health hazards of smoking,and on the other hand,it accumulates data for evaluating the effectiveness,reliability,and practicality of the "21stcentury toxicity testing strategy.”Methods:(1)Proposing the MOA hypothesis for B[a]P-induced lung cancer via respiratory tract exposure:The genes and phenotypes related to respiratory tract exposure of B[a]P were screened by the systematic literature reviewing and Comparative Toxicogenomics Database analysing.KEGG pathway enrichment analysis was performed,and the top ten pathways were selected for subsequent analysis.Transcriptome sequencing and differential analysis were performed on B[a]P-exposed epithelial bronchial cells and normal epithelial bronchial cells.The main pertured genes and major toxic pathways of B[a]P exposed through respiratory tract were identified by combining the differential analysis results and the enrichment analysis results.The Mode of Action(MOA)hypothesis composed of molecular initiation events and critical events was established on the basis of toxic pathway,and the quantitative evidence weight of MOA hypothesis was evaluated according to Bradford Hill principle.(2)Verifying key events of MOA through in vitro experiments:Based on the molecular initiation and critical events identified in the MOA hypothesis established in the previous section,the effect of benzo[a]pyrene on the activity of BEAS-2B cells and HBE135-E6E7 cells were detected by CCK-8 assay,the mRNA expression of CYP1A1 gene was detected by qPCR,the production of reactive oxygen species(ROS)was detected by DCFH-DA fluorescence probe,and the 8-OHdG release was detected by ELISA method.The occurrence of early key events in the MOA hypothesis would be verified through these experiments.(3)Quantitative assessment of the health effects of benzo[a]pyrene in cigarettes onthe population:Meta analysis of the relative risk caused by active smoking among Chinese population was conducted.Based on the average B[a]P content in cigarette smoke and the B[a]P daily exposure limit stipulated in the National Ambient Air Quality Standard,the proportion of the additional B[a]P intake from smoking to the daily exposure limit was calculated,and the impact of B[a]P in cigarette on population health was evaluated on this basis.By compared the population data with the predicted results of MOA model,the reliability,accuracy and practicability of "21st Century Toxicity testing strategy" were further validated.Results:(1)Establishing the MOA hypothesis of lung injury caused by respiratory tract exposure of B[a]P:According to the results of systematic literature review,transcriptome sequencing,and bioinformatics analysis,the main target organ of benzo[a]pyrene via respiratory exposure was determined to be the lungs,and the adverse outcome was cell mutation.The following MOA hypothesis was established:The molecular initiating event is the activation of lung AhR by B[a]P,the first key event is the up-regulation of CYP1A1 to initiate B[a]P metabolism,the second key event is oxidative stress caused by benzo[a]pyrene diol-epoxide(BPDE),a metabolic product of benzo[a]pyrene,and the third key event is DNA oxidative damage.The MOA score is 75,indicating good reliability.(2)Verifying key events of MOA through in vitro experiments:In vitro experimental results showed that exposure to benzo[a]pyrene led to increased CYP1A1 expression,ROS production,and DNA oxidative damage in BEAS-2B and HBE135E6E7 cells.The in vitro experimental results validated the key events in the MOA hypothesis.(3)Quantitative assessment of the health effects of benzo[a]pyrene in cigarettes on the population:The average benzo[a]pyrene content of different brands of cigarettes in the past twenty years is about 9 ng.According to the national environmental air quality standards,the average daily limit of benzo[a]pyrene concentration is 0.01 μg/m3.According to the daily ventilation volume of adult,it can be calculated that the daily limit of exposure to benzo[a]pyrene for adults is 10-20 ng,which is approximately equal to the amount of benzo[a]pyrene in one or two cigarettes.Results of meta-analysis showed that benzo[a]pyrene in cigarettes significantly increases the risk of lung cancer in smokers,which was consistent with the prediction of MOAConclusion:(1)The molecular initiating event of MOA for benzo[a]pyrene-induced lung cancer via respiratory exposure is the activation of lung AhR by benzo[a]pyrene.The key event is the upregulation of CYP1A1,which initiates benzo[a]pyrene metabolism.The benzo[a]pyrene metabolite BPDE leads to oxidative stress and DNA oxidative damage.(2)In vitro experiments have confirmed the established MOA for benzo[a]pyreneinduced lung cancer,and predict that the presence of benzo[a]pyrene in cigarettes significantly increases the risk of lung cancer in smokers.(3)The results of population data analysis are consistent with the predicted results of MOA established in this study,indicating that the "21st Century toxicity testing strategy" could predict early events of chemical hazards to human health,and the MOA model prediction is relatively fast and reliable.