黃酮類對人體細胞色素P450代謝黃麴毒素之調節作用

The Modulatory Effects of Flavones on Human P450-Catalyzed Aflatoxin B1 Oxidation

黃闢類是許多醫用植物之主要成份，可抑制或促進肝微粒體活化黃麵毒素AFB1之能力。以重組酵素系統研究人體細胞色素P450 1A2, 3A4 及3A5 氧化代謝AFB1作用。分析AFB1氧化代謝之代謝產物，結果顯示經P450 1A2 作用產生AFM1，少量之AFQl及AFBl-exo-epoxide 與endo-epoxide；P450 3A4 產生AFQl及exoepoxide0 P450 3A5與3A4之胺基酸序列有85%的相似性，但P450 3A5 產生之主要代謝產物為AFBl-exo-epoxide 。產生exo-epoxide 之速率，P450 1A2 與P4503A5 分別約為3A4之14%與43%。這結果與基因毒性測試結果具一致性，均支持P4503 A4為主要活化AFB1毒性之人體P450。α-Naphthoflavone (αNF)為合成之黃酬類化合物，抑制所有P450 1A2 催化之AFB1氧化反應；但相反的，將αNF 加入P450 3A4 反應溶液中，可增加生成epoxide 之速率達2倍，但降低AFQl之生成。然而，AFBl對3A4氧化αNF 產生αNF-5，6 -oxide之活性無影響。分析AFQl及exoepoxide之生成速率與AFBl 濃度之相關性，結果顯示其Hil1係數分別為2 .4及4.1。αNF 可降低此合作係數並使速率與濃度之相關性圖趨近hyperbolic 性質。這種雙向調節作用不因增加P450還原酵素／P450之莫耳比例而改變，在P450 3A4 與P450還原酵素之融合蛋白系統中，αNF 亦具相同之作用。改變還原酵素系統，產物中AFQl與epoxide 之組成比率不同，使用flavodoxin 及ferrodoxin 為還原系統，只進行過氧化作用。同時，以iodosylbenzene 及cumene hydroperoxide 為oxygen surrogate 時，只出現過氧化作用，但αNF 抑制此過氧化作用。這些結果顯示αNF可能以異位方式調節P450 3A4氧化AFBl之作用。

Flavonoids are major components in a large number of medicinal herbs and showed inhibitory and stimulatory effects on the activation of aflatoxin (AF) B1 by liver microsomes. AFB1 oxidation was studied with purified recombinant human P450 1A2, 3A4, and 3A5 in reconstituted systems. Analysis of the AFB1 oxidation products showed that P450 1A2 formed AFM1, a small amount of AFQ1, and both exo- and endo-8,9-epoxides; P450 3A4 formed AFQ1 and exo-8,9-epoxide. P450 3A5, with 85% amino acid sequence identity to P450 3A4, formed mainly the exooxide and only a small amount of the detoxication product AFQ1. The exo epoxide is the most potent genotoxic and mutagenic metabolite of AFB1. The exo-oxide formation rates of 1A2 and 3A5 were - 14% and 43% of 3A4. These results coincide with the view that P450 3A4 is a major human liver P450 involved in AFB1 activation. A synthetic flavone, o-naphthoflavone ( oNF) caused potent inhibition of all oxidation reactions catalyzed by P450 1A2. In contrast to P450 1A2, addition of oNF to P450 3A4 resulted in a 2-fold increase of oxide formation but decreased AFQ1 formation. However, there was no obvious effect of AFB1 on the oxidation of oNF by P450 3A4 to its 5,6-oxide. Plots of AFQ1 and oxide formation rates vs AFB1 concentration were sigmoidal with Hill coefficients (n) of 2.4 and 4.1. Velocity vs AFB1 concentration plots were shifted to a more hyperbolic nature in the presence of oNF (n=l.4 for AFQ1, 1.6 for AFB1-oxide). This bidirectional modulatory effect was not changed by increasing P450 reductase/ P450 molar ratio. oNF showed the same modulatory effects in a P450 3A4/ NADPH-P450 reductase fusion protein system. The balance between 3o-hydroxylation (AFQ1) and epoxidation was influenced by changing the reduction system; flavodoxin-and ferredoxindependent systems yield only epoxidation. Also, only epoxidation was seen with P450 3A4 supported by the oxygen surrogates iodosylbenzene and cumene hydroperoxide. These results support a model that o NF affects AFB1 oxidation of P450 3A4 allosterically.