Rich K J, Murray B P, Lewis I, Rendell N B, Davies D S, Gooderham N J, Boobis A R
Clinical Pharmacology, Royal Postgraduate Medical School, London, UK.
Carcinogenesis. 1992 Dec;13(12):2221-6. doi: 10.1093/carcin/13.12.2221.
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (N-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [13C,15N2]MeIQx and [14C]MeIQx was used as substrate and the products analysed by HPLC-thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M+H]+) from the parent compound, MeIQx and at 230/233 ([M+H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M+H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 microM) was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 microM) inhibited the N-hydroxylation of MeIQx by > 90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.
2-氨基-3,8-二甲基咪唑并[4,5-f]喹喔啉（MeIQx）是肉类烹饪过程中形成的最丰富的杂环芳香胺之一，在啮齿动物中具有遗传毒性和致癌性。MeIQx在发挥这些作用之前需要通过P450进行代谢活化。虽然有间接证据表明诱变产物是N-羟基-MeIQx（N-OHMeIQx），但我们现在在将该胺与人肝微粒体部分孵育后已明确鉴定出这一点。未标记的MeIQx、[13C,15N2]MeIQx和[14C]MeIQx的混合物用作底物，并通过HPLC-热喷雾质谱分析产物。在母化合物MeIQx的m/z 214/217（[M+H]+）处发现了相隔3个质量单位的特征性双峰离子，在N-OHMeIQx的m/z 230/233（[M+H]+）处也发现了该特征双峰离子。在m/z 214/217处存在双峰离子以及在230/233 [M+H+]处存在双峰，这提供了额外证据，证明这就是N-OHMeIQx，因为“O”的容易丢失是N-羟基胺的特征。通过比较使用对N-羟基胺特别敏感的鼠伤寒沙门氏菌YG1024和对大多数N-羟基胺有抗性的TA98/1,8-DNP6对HPLC洗脱液的诱变性，获得了关于主要代谢产物身份的进一步证据，该主要代谢产物约占所有微粒体代谢的90%。如质谱所示，反应混合物中存在的95%的直接作用诱变性与一个单一峰相关，该峰与N-OHMeIQx共洗脱。在存在代谢活化系统的情况下，仅能检测到一个额外的诱变峰，对应于未变化的MeIQx。人肝微粒体以77±11 pmol/mg/分钟（平均值±标准误，n = 4）的速率将MeIQx（5 microM）N-羟基化。人CYP1A2的特异性抑制剂呋拉茶碱（5 microM）抑制MeIQx的N-羟基化超过90%。这些数据表明，N-OHMeIQx既是人肝微粒体部分产生的MeIQx的主要氧化产物，也是主要遗传毒性产物，并且该反应几乎完全由CYP1A2催化。
