Arlt Volker M, Hewer Alan, Sorg Bernd L, Schmeiser Heinz H, Phillips David H, Stiborova Marie
Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom.
Chem Res Toxicol. 2004 Aug;17(8):1092-101. doi: 10.1021/tx049912v.
3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with beta-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a genotoxic potential of 3-ABA for humans. Moreover, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA.
3-硝基苯并蒽酮（3-NBA）是一种在柴油尾气和环境空气污染中发现的疑似人类致癌物。3-NBA的主要代谢产物3-氨基苯并蒽酮（3-ABA）最近在职业性接触柴油排放物的盐矿工人尿液中被检测到。确定人类代谢3-ABA的能力以及了解哪些人类酶参与其活化过程对于评估个体易感性很重要。我们比较了八个人肝微粒体样品催化3-ABA形成DNA加合物的能力。使用（32）P后标记法，我们发现所有肝微粒体都能够活化3-ABA。观察到具有多个加合物的DNA加合物模式，其在质量上与用3-ABA处理的大鼠体内形成的模式相似。这些模式也与硝基芳烃对应物3-NBA形成的模式相似，并且源自与DNA中嘌呤碱基结合的3-NBA的还原代谢产物。通过将每个微粒体样品中与细胞色素P450（P450）相关的催化活性与相同微粒体形成的DNA加合物水平相关联，研究了人肝微粒体样品中特定细胞色素P450（P450）在3-ABA活化中的作用。基于该分析，3-ABA的大部分肝微粒体活化归因于P450 1A1和1A2酶活性。分别用P450 1A1和1A2的抑制剂α-萘黄酮和呋拉茶碱以及单独的P450 1A2抑制人肝微粒体中DNA加合物的形成，支持了这一发现。使用在中国仓鼠V79细胞中表达的重组人P450 1A1和1A2以及杆状病毒转染昆虫细胞的微粒体（超微粒体），我们证实了这些酶参与3-ABA衍生的DNA加合物的形成。此外，在表达P450 1A1和1A2的具有代谢活性的人淋巴母细胞MCL-5细胞中检测到与微粒体中发现的基本相同的DNA加合物模式。使用大鼠肝微粒体，我们表明人和大鼠微粒体都会导致相同的3-ABA衍生的DNA加合物。分别用P450 1A1和1A2的诱导剂β-萘黄酮或苏丹I以及单独的P450 1A1预处理大鼠，显著刺激大鼠肝微粒体形成的3-ABA衍生的DNA加合物水平。利用纯化的大鼠重组P450 1A1，证实了该酶参与3-ABA形成DNA加合物的过程。总之，我们的结果强烈表明3-ABA对人类具有遗传毒性潜力。此外，3-ABA不仅是接触3-NBA的合适生物标志物，而且可能直接导致3-NBA的高遗传毒性潜力。
