TNF-α Upregulates CYP2A13 To Contribute To Aflatoxin G₁-Induced DNA Damage In Alveolar Epithelial Cells

Aflatoxins G₁（AFG₁）is a highly toxic,mutagenic,and carcinogenic mycotoxin produced by specific species of fungi.A large number of studies have confirmed that AFG₁ is mainly metabolized by cytochrome P450（CYP450）,and then the AFG₁ can induce DNA damage,which is a key factor in the carcinogenicity of AFG₁.Our previous studies showed increased expression of cytochrome P450（CYP450）subtype CYP2A13 was associated with oxidative stress in AFG₁-induced inflamed lung tissues.Recently,we found TNF-αenhanced AFG₁-induced DNA damage associated with up-regulation of CYP2A13 in AFG₁-exposed human alveolar type II like cells（A549）.Several lines of studies have shown that lung inflammation could up-regulate CYP450 enzyme activity to promote the activation of benzopyrene in AT-II cells.Recently,we also found that AFG₁ induced TNF-α-dependent lung inflammation in vivo.However,whether AFG₁-induced TNF-α-dependent lung inflammation enhanced the activation of AFG₁ in AT-II cells by up-regulating CYP450 remains unknown.In this study,we found increased expression of CYP2A13 was inhibited by sTNFR:Fc in AFG₁-induced inflamed lung tissues by using IHC and western blot,which provides a link between TNF-α-dependent lung inflammation and CYP2A13 in promoting metabolic activation of AFG₁ in alveolar epithelial cells.Then,we treated A549 cells with AFG₁ and TNF-αto mimic an AFG₁-induced inflammatory response in vitro,and explored whether TNF-α/NF-κB-dependent lung inflammation up-regulate CYP450 to contribute to the activation of AFG₁ and enhanced AFG₁-induced oxidative DNA damage in AT-II cells.Flow cytometry（FCM）was used to detect ROS and apoptosis in A549 cells.Western blot was used to detect the expression of CYP2A13 and DNA damage associated proteins.Immunocytochemistry was used to detect the expression ofγ-H2AX and 8-OHdG,markers of oxidative DNA damage.Blocking of CYP2A13 or NF-κB by siRNAs inhibited TNF-α-enhanced DNA damage and apoptosis in AFG₁-exposed A549 cells.NF-κB blocking also inhibited CYP2A13 up-regulation by TNF-αtreatment,which suggests that up-regulation of 2A13 by TNF-αthrough NF-κB pathway plays an essential role in enhancement of DNA damage under AFG₁-induced inflammatory conditions.Taken together,our findings suggest that AFG₁ induced TNF-α-dependent lung inflammation up-regulates CYP2A13 to promote metabolic activation of AFG₁ and enhances oxidative DNA damage in alveolar epithelial cells.