Suppressive effect of 1-nitropyrene on benzo[a]pyrene-induced CYP1A1 protein expression in HepG2 cells.
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.
Cherng
Shur-Hueih
SH
Department of Biotechnology, Hung Kuang University, Taichung, Taiwan, ROC.
Hsu
Shih-Lan
SL
Yang
Jia-Ling
JL
Yu
Chang-Tze Ricky
CT
Lee
Huei
H
D001564:Benzo(a)pyrene / Q000633:toxicity*; D002273:Carcinogens / Q000633:toxicity*; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D018736:DNA Adducts / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D004338:Drug Combinations; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D009153:Mutagens / Q000494:pharmacology*; D011721:Pyrenes / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism
D002273:Carcinogens; D018736:DNA Adducts; D004338:Drug Combinations; D009153:Mutagens; D011721:Pyrenes; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D001564:Benzo(a)pyrene; D019363:Cytochrome P-450 CYP1A1; C032668:1-nitropyrene