Suppressive effect of 1-nitropyrene on benzo[ a]pyrene-induced CYP1A1 protein expression in HepG2 cells

Abstract

The genotoxicity of polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs may be influenced by the interaction of the compounds. In this study, our data showed that benzo[ a]pyrene (B aP)-DNA adduct levels were decreased in a dose-dependent manner when the human hepatoma cell line HepG2 simultaneously treated with B aP and 1-nitropyrene (1-NP). To further investigate the molecular mechanism by which 1-NP interferes with the covalent binding of B aP to DNA, we conducted experiments to analyze the mRNA level and protein stability of cytochrome P450 1A1 (CYP1A1), which is engaged in the activation of B aP, leading to the generation of B aP-DNA adducts. Northern blot analysis presented that 1-NP attenuated B aP-induced CYP1A1 mRNA expression by 30.4–39.6% ( p < 0.05). Western blot analysis revealed that the co-treatment with B aP and 1-NP resulted in a significant inhibition of B aP-induced CYP1A1 protein expression (70.7–88.2%, p < 0.05). However, the decrease in CYP1A1 protein levels was significantly larger than that in CYP1A1 mRNA levels. To confirm the effect of 1-NP on the CYP1A1 protein expression, in vitro proteolysis of CYP1A1 protein was evaluated. The results demonstrated that the addition of 1-NP enhanced CYP1A1 protein degradation and the proteolysis of CYP1A1 protein was inhibited by the addition of an antioxidant, dithiothreitol. In addition, the relative levels of reactive oxygen species (ROS) were elevated in HepG2 cells co-treated with B aP and 1-NP, indicating that the decrease of CYP1A1 protein level was probably attributed to the production of ROS generated by binary mixture. Taken together, these findings suggested that the transcriptional suppression and posttranslational mechanism may be involved in loss of CYP1A1 protein, causing the decrease of B aP-DNA adduct levels in the presence of binary mixtures of 1-NP and B aP.