Shertzer H G, Puga A, Chang C, Smith P, Nebert D W, Setchell K D, Dalton T P
Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, OH 45267-0056, USA.
Chem Biol Interact. 1999 Nov 15;123(1):31-49. doi: 10.1016/s0009-2797(99)00121-0.
The mechanisms by which soybean- and soybean isoflavone-enriched diets inhibit carcinogenesis are not known. We found that the isoflavones genistin and daidzin, and their respective aglucone forms daidzein and genistein, block 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin)-induced CYP1A1 enzyme activity. This inhibition is correlated with the capacity of the isoflavones to prevent CYP1A1-mediated covalent binding of benzo[a]pyrene (BaP) metabolites to DNA. We further evaluated daidzein and genistein, believed to be the active forms of the isoflavones, for the mechanism of the inhibitory process. Although daidzein and genistein appear structurally similar to known aromatic hydrocarbon receptor (AHR) agonists and antagonists, gel mobility shift assays indicated that the isoflavones do not inhibit dioxin-induced activation of the AHR or the accumulation of CYP1A1 mRNA, suggesting that the isoflavones do not act at the transcriptional level. We therefore evaluated the isoflavones for direct effects on the CYP1A1 enzyme. Daidzein and genistein non-competitive with the CYP1A1 substrate BaP for microsomal BaP hydroxylation, with apparent Ki values of 325 microM and 140 microM, respectively. The extent of CYP1A1 inhibition increases with time of preincubation at 37 degrees C, but not at 4 degrees C, in the presence of isoflavone plus NADPH; after 60 min preincubation the inhibition remains non-competitive, with apparent Ki values of 55 microM and 50 microM, respectively. Inhibition is neither prevented nor reversed by the thiol antioxidant dithiothreitol, nor by the iron chelator deferoxamine. Repeated washing of the microsomes does not reverse the inhibition. The dependency on NADPH, temperature and time for inhibition of CYP1A1 suggests that metabolism of either isoflavone or molecular oxygen to reactive species is required. Isoflavone-mediated inhibition of CYP1A1 activity may contribute to the mechanism by which these soybean isoflavones protect against carcinogenesis.
富含大豆及大豆异黄酮的饮食抑制致癌作用的机制尚不清楚。我们发现，异黄酮染料木苷和黄豆苷，以及它们各自的苷元形式大豆苷元和染料木素，可阻断2,3,7,8 - 四氯二苯并 - p - 二恶英（TCDD；二恶英）诱导的CYP1A1酶活性。这种抑制作用与异黄酮预防CYP1A1介导的苯并[a]芘（BaP）代谢产物与DNA共价结合的能力相关。我们进一步评估了被认为是异黄酮活性形式的大豆苷元和染料木素的抑制过程机制。尽管大豆苷元和染料木素在结构上看似与已知的芳烃受体（AHR）激动剂和拮抗剂相似，但凝胶迁移率变动分析表明，异黄酮不会抑制二恶英诱导的AHR激活或CYP1A1 mRNA的积累，这表明异黄酮并非在转录水平起作用。因此，我们评估了异黄酮对CYP1A1酶的直接作用。大豆苷元和染料木素对微粒体BaP羟基化的CYP1A1底物BaP无竞争性，其表观Ki值分别为325 microM和140 microM。在存在异黄酮加NADPH的情况下，CYP1A1抑制程度随37℃预孵育时间增加，但在4℃时不增加；预孵育60分钟后，抑制作用仍无竞争性，表观Ki值分别为55 microM和50 microM。硫醇抗氧化剂二硫苏糖醇以及铁螯合剂去铁胺均不能阻止或逆转这种抑制作用。对微粒体进行反复洗涤也不能逆转抑制作用。对NADPH、温度和时间的依赖性表明，CYP1A1抑制需要异黄酮或分子氧代谢为活性物质。异黄酮介导的CYP1A1活性抑制可能有助于这些大豆异黄酮预防致癌作用的机制。
