Role Of DNA Methylation In The Regulation Of Differential Lung CYP2E1 Expression By Nrf2 In PM_2.5 Exposed Mice

Objective:Fine particulate matter(PM_2.5)is the main harmful factor affecting the health of the respiratory system,and short-term exposure to PM_2.5 can cause a decline in lung function.The more toxic substances damage the lung health.CYP2E1 is an important phase I metabolic enzyme in CYP450,which is mainly involved in the metabolism of environmental toxicants and precarcinogens.The expression level of CYP2E1 was strongly correlated with the degree of lung damage caused by PM_2.5 exposure.When the body is exposed to some harmful substances,the expression of CYP2E1 in lung tissue will increase significantly,which leads to lung damage through increased oxidative stress and oxygen free radicals.In our previous study,The low expression of CYP2E1 in the lung tissue of Nrf2 knockout mice after PM_2.5exposure is closely related to the lack of obvious pathological injury of lung tissue.This study was conducted to elucidate the regulatory mechanism of CYP2E1 expression by Nrf2 in the lungs of PM_2.5 exposed mice.The degree of DNA methylation in the promoter region is closely associated with gene expression.Numerous studies have shown that PM_2.5 exposure can regulate gene expression through altered DNA methylation of specific genes,and it is unclear whether the low expression of CYP2E1 in lung tissues of Nrf2 knockout mice exposed to PM_2.5 is affected by its promoter region methylation status.In this study,we investigated the relationship between Nrf2regulation of CYP450 enzyme expression and the degree of DNA methylation by establishing a real-ambient PM_2.5 exposure mouse model.Methods:In this study,Shijiazhuang city was selected as the exposure site and experiments were conducted using a real-ambient atmospheric exposure system between November 2017 and January 2018.Animals were selected from Nrf2-knockout（KO）mice and C57BL/6J wild-type（WT）mice were placed in the PM_2.5 exposure chamber（PM）or filtered air（FA）chamber of this system for 12 weeks.The temperature and humidity in the animal cages were maintained at a constant 12-hour night/12-hour day rhythm during the exposure period,and lung samples were stored at-80°C at the end of the exposure.The expression of relevant indicators of antioxidant enzymes,CYP450 enzymes,DNA methylation transferase and demethylation were measured in mouse lung tissues using RT-PCR and Western Blot,immunohistochemistry,and enzyme-linked immunosorbent assay.The degree of DNA methylation was detected by the Mass ARRAY technique.Graph Pad Prism（8.3）software was used to analyze the difference in data between the exposed group and the control group by two-way analysis of variance or Kruskal-Wallis.Results:1.PM_2.5 exposure affected the expression levels of CYP1A1,CYP2E1 and CYP2S1in the lung tissues of both WT and KO mice.the levels of Cyp2e1 m RNA and protein expression were elevated in the lungs of WT mice after PM_2.5 exposure,but decreased in the lungs of KO mice.Cyp1a1 m RNA and protein expression were elevated in the lungs of both WT and KO mice after PM_2.5 exposure,while Cyp2s1 m RNA and protein level expression were decreased in both lungs.The results suggest that Nrf2 regulates the expression of CYP2E1 in mouse lung tissue during PM_2.5 exposure.2.The level of Cp G2 unit methylation in the Cyp2e1 promoter region showed a decreasing trend in the lungs of WT mice after PM_2.5 exposure,while the level of Cp G2unit methylation in the lungs of KO mice showed an increasing trend.the level of Cp G2unit methylation in the Cyp2e1 promoter region showed an opposite trend to the m RNA expression of Cyp2e1.the level of Cp G3 unit methylation in the Cyp1a1 promoter region showed a decreasing trend in the lungs of both WT and KO mice after PM_2.5 exposure.The level of Cp G3 unit methylation in the Cyp1a1 promoter region showed a decreasing trend,while the Cyp2s1 promoter Cp G1 unit methylation showed an increasing trend in both WT and KO mice after PM_2.5 exposure.the same more obvious relationship existed between Cp G3 unit methylation in the Cyp1a1 promoter and its m RNA expression,and Cp G1 unit methylation in the Cyp2s1 promoter and its m RNA expression.The results suggested that methylation of Cp G2 in the Cyp2e1 promoter region,Cp G3 in the Cyp1a1 promoter and Cp G1 unit in the Cyp2s1 promoter regulated the expression of the corresponding genes.3.The expression of TET3,DNMT1 and 5hm C was decreased in the lungs of WT mice and increased in the lungs of KO mice after PM_2.5 exposure,and the SAM/SAH values were increased in the lungs of WT mice and decreased in the lungs of KO mice after PM_2.5exposure.The results suggest that PM_2.5 exposure can affect DNA methylation-related indicators.4.γ-H2AX expression was significantly increased in the lungs of WT mice after PM_2.5exposure,but not in the lungs of KO mice.homologous recombinant repair markers in the lung tissue of WT mice after PM_2.5 exposure included DNA repair protein RAD51 homolog（RAD51）,breast cancer（BRCA1）,ataxia-telangiectasia mutated（ATM）,and ataxia-telangiectasia and Rad3-associated kinase（ATM）.The expression of ataxia-telangiectasia mutated（ATM）and ataxia-telangiectasia and Rad3 related（ATR）kinases was significantly reduced,while the expression was increased in the lung tissues of KO mice.The results suggest that Nrf2 may influence DNA methylation by affecting DNA damage repair in mouse lung tissues after PM_2.5 exposure.Conclusion:Changes in CYP2E1,CYP1A1 and CYP2S1 expression in WT and Nrf2 knockout mice after PM_2.5 exposure may be related to the specific methylation pattern of their promoter Cp G units.nrf2 may regulate CYP2E1 expression by affecting the methylation level of Cp G2 units by PM_2.5 exposure.Our study reveals the mechanism of the role of Cp G methylation in the promoter region in the regulation of CYP2E1 differential expression in the lungs of PM_2.5-exposed mice by Nrf2.