Inhibition of benzo[a]pyrene-activating enzymes and DNA binding in human bronchial epithelial BEAS-2B cells by methoxylated flavonoids.
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AbstractCigarette smoking is a major risk factor in lung carcinogenesis via carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and nitrosamines. In this study, we used benzo[a]pyrene (BaP) as the classic PAH compound and BEAS-2B cells, a model of normal human bronchial epithelial cells, to investigate whether 5,7-dimethoxyflavone (5,7-DMF) and 3',4'-DMF compared with resveratrol (RV) have chemopreventive properties in this cancer. Exposure of BEAS-2B cells to [(3)H]BaP (1 microM) showed increasing binding to DNA up to 72 h of exposure, about 20-fold higher than that at 0.5 h exposure. BaP exposure also increased both CYP1A1/1B1 and microsomal epoxide hydrolase (mEH) enzyme activities with a maximum 10-fold increase at 48 h. BaP induced CYP1A1 protein and mRNA levels maximally after 48 h. In contrast, although CYP1B1 mRNA was rapidly induced, its protein expression showed a very poor response. Simultaneous treatment with BaP and 5,7-DMF, 3',4'-DMF or RV for 48 h inhibited BaP-DNA binding by > or =75%, with 3',4'-DMF being the most effective. 5,7-DMF affected CYP1A1 mRNA levels only modestly, whereas 3',4'-DMF was a potent inhibitor. The catalytic activity of CYP1A1/1B1 was reduced over 95% after exposure to 5,7-DMF, 3',4'-DMF or RV, most effectively by 3',4'-DMF. BaP-induced mEH activity was not affected by treatment with 5,7-DMF, but was significantly inhibited by 3',4'-DMF. In contrast, mEH activity was notably increased by RV. Most importantly, western blotting showed all three polyphenols dramatically reducing BaP-induced CYP1A1 protein expression. Both 5,7-DMF and 3',4'-DMF demonstrated very high, about 40-fold, accumulation in BEAS-2B cells. In summary, BaP exposure results in a high level of DNA binding in BEAS-2B cells, which is mainly mediated by induction of CYP1A1 protein, just as in the human lung. Two methoxylated dietary flavonoids with highly specific effects on BaP bioactivation block this DNA binding and CYP1A1 protein expression as effectively as RV, thus making them potential chemopreventive agents for BaP-induced lung carcinogenesis.
CitationCarcinogenesis 2006, 27 (8):1579-1585
DescriptionDietary modulation of carcinogenesis-related pathwaysDietary item or component studied: 5,7-dimethoxyflavone (5,7 DMF), 3,4-dimethoxyflavone (3,4DMF)Pathways studied: DNA binding and expression of carcinogen-metabolizing enzymesStudy type (in vitro, animals, humans): in vitroImpact on pathway (including dose-response): All polyphenols decreased the BaP-DNA binding, with _75% in the presence of 5,7-DMF or RV and >90%inhibition in the presence of 30,40-DMF (P<0.01)Both methoxylated flavonoids as well as RV effectively inhibited the BaP-induced EROD activity at 97% (5,7-DMF), 96% (RV) and >99%(30,40-DMF) (P<0.001)25 mM 5,7-DMF and 1 mM BaP for 48 h resulted in a modest, non-significant reduction of CYP1A1 at the mRNA level (P<0.001). On the other hand, 30,40-DMF was an extremely potent inhibitor of CYP1A1 mRNA. KEYWORDS CLASSIFICATION: antagonists & inhibitors;Anticarcinogenic Agents;Benzo(a)pyrene;Bronchi;Cells,Cultured;Cytochrome P-450 CYP1A1;Cytochrome P-450 Enzyme System;drug effects;dietary modulation of carcinogenesis-related pathways;Dna;Enzyme Induction;Epithelial Cells;Epoxide Hydrolases;Flavones;Flavonoids;genetics;Humans;Hydrolases;metabolism;pharmacology;Research.