Cherng Shur-Hueih, Hsu Shih-Lan, Yang Jia-Ling, Yu Chang-Tze Ricky, Lee Huei
Department of Biotechnology, Hung Kuang University, Taichung, Taiwan, ROC.
Toxicol Lett. 2006 Mar 1;161(3):236-43. doi: 10.1016/j.toxlet.2005.10.002. Epub 2005 Nov 8.
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 (BaP)-DNA adduct levels were decreased in a dose-dependent manner when the human hepatoma cell line HepG2 simultaneously treated with BaP and 1-nitropyrene (1-NP). To further investigate the molecular mechanism by which 1-NP interferes with the covalent binding of BaP 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 BaP, leading to the generation of BaP-DNA adducts. Northern blot analysis presented that 1-NP attenuated BaP-induced CYP1A1 mRNA expression by 30.4-39.6% (p < 0.05). Western blot analysis revealed that the co-treatment with BaP and 1-NP resulted in a significant inhibition of BaP-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 BaP 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 BaP-DNA adduct levels in the presence of binary mixtures of 1-NP and BaP.
多环芳烃（PAHs）和硝化多环芳烃的遗传毒性可能会受到这些化合物之间相互作用的影响。在本研究中，我们的数据表明，当人肝癌细胞系HepG2同时用苯并[a]芘（BaP）和1-硝基芘（1-NP）处理时，BaP-DNA加合物水平呈剂量依赖性降低。为了进一步研究1-NP干扰BaP与DNA共价结合的分子机制，我们进行了实验以分析细胞色素P450 1A1（CYP1A1）的mRNA水平和蛋白质稳定性，CYP1A1参与BaP的活化，导致BaP-DNA加合物的产生。Northern印迹分析表明，1-NP使BaP诱导的CYP1A1 mRNA表达降低了30.4-39.6%（p<0.05）。Western印迹分析显示，BaP与1-NP共同处理导致BaP诱导的CYP1A1蛋白表达受到显著抑制（70.7-88.2%，p<0.05）。然而，CYP1A1蛋白水平的降低明显大于CYP1A1 mRNA水平的降低。为了证实1-NP对CYP1A1蛋白表达的影响，对CYP1A1蛋白进行了体外蛋白水解评估。结果表明，添加1-NP增强了CYP1A1蛋白降解，而抗氧化剂二硫苏糖醇的添加抑制了CYP1A1蛋白的蛋白水解。此外，在BaP与1-NP共同处理的HepG2细胞中，活性氧（ROS）的相对水平升高，表明CYP1A1蛋白水平的降低可能归因于二元混合物产生的ROS。综上所述，这些发现表明转录抑制和翻译后机制可能参与了CYP1A1蛋白的缺失，导致在1-NP和BaP二元混合物存在的情况下BaP-DNA加合物水平降低。
