Yamazaki H, Oda Y, Funae Y, Imaoka S, Inui Y, Guengerich F P, Shimada T
Osaka Prefectural Institute of Public Health, Japan.
Carcinogenesis. 1992 Jun;13(6):979-85. doi: 10.1093/carcin/13.6.979.
The possible roles of cytochrome P450 (P450) enzymes in the metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by rat liver microsomes have been examined in a system containing the bacterial tester strain Salmonella typhimurium NM2009, a newly developed strain showing high O-acetyltransfer activities. The DNA-damaging activity could be determined by measuring expression of the umu gene in a plasmid containing the fused umuC-lacZ gene construct in the bacteria. The following lines of evidence support the view that both NDMA and NDEA are principally oxidized to reactive products by P450 2E1 in rat liver microsomes. First, NDMA and NDEA were activated by rat liver microsomes in a protein- and substrate-dependent manner and the former chemical was more active than the latter; both activities were induced in rats treated with P450 2E1 inducers such as ethanol, acetone and isoniazid and by starvation. Second, activation of NDMA and NDEA were both inhibited significantly by antibodies raised against rat P450 2E1 and by P450 2E1 inhibitors such as diethyldithiocarbamate and 4-methylpyrazole in rat liver microsomes. Finally, in reconstituted monooxygenase systems containing purified rat P450 enzymes, P450 2E1 gave the highest rates of the activation of both NDMA and NDEA; the addition of rabbit cytochrome b5 to the system caused about a 1.5-fold increase in both reactions. In separate experiments we also found that N-nitrosomethylacethoxymethylamine, a compound that reacts with DNA after ester cleavage, is more genotoxic in S.typhimurium NM2009 than in S.typhimurium NM2000, a strain that is defective in O-acetyltransferase activity. Part of the pathway involved in the activation of nitrosamines is suggested to be acetylation of alkyldiazohydroxides formed by P450 or acetylesterase, because the genotoxic activity of N-nitrosomethylacethoxymethylamine in S.typhimurium NM2009 could be inhibited by the O-acetyltransferase inhibitor pentachlorophenol. These results indicate that NDMA and NDEA are oxidized to gentoxoic products by rat liver microsomes and that a P450 2E1 enzyme plays a major role in the activation of these two potent carcinogens. The activation pathway of N-nitrosodialkylamines through acetylation by O-acetyltransferase has been proposed. This simple bacterial system for measuring genotoxicity should facilitate studies on the activation of N-nitroso alkylamines.
在一个含有细菌测试菌株鼠伤寒沙门氏菌NM2009（一种新开发的具有高O - 乙酰转移酶活性的菌株）的系统中，研究了细胞色素P450（P450）酶在大鼠肝微粒体对N - 亚硝基二甲胺（NDMA）和N - 亚硝基二乙胺（NDEA）的代谢活化中可能发挥的作用。DNA损伤活性可通过测量细菌中含有融合umuC - lacZ基因构建体的质粒中umu基因的表达来确定。以下一系列证据支持这样的观点，即NDMA和NDEA在大鼠肝微粒体中主要被P450 2E1氧化为反应性产物。首先，NDMA和NDEA被大鼠肝微粒体以蛋白质和底物依赖性方式活化，且前者比后者更具活性；在用乙醇、丙酮和异烟肼等P450 2E1诱导剂处理的大鼠以及饥饿大鼠中，这两种活性均被诱导。其次，NDMA和NDEA的活化在大鼠肝微粒体中均被针对大鼠P450 2E1产生的抗体以及P450 2E1抑制剂如二乙基二硫代氨基甲酸盐和4 - 甲基吡唑显著抑制。最后，在含有纯化大鼠P450酶的重组单加氧酶系统中，P450 2E1对NDMA和NDEA的活化速率最高；向系统中添加兔细胞色素b5导致两个反应的速率均增加约1.5倍。在单独的实验中，我们还发现N - 亚硝基甲基乙酰氧基甲基胺（一种在酯裂解后与DNA反应的化合物）在鼠伤寒沙门氏菌NM2009中比在O - 乙酰转移酶活性有缺陷的鼠伤寒沙门氏菌NM2000中具有更高的遗传毒性。亚硝胺活化所涉及的部分途径被认为是由P450或乙酰酯酶形成的烷基重氮氢氧化物的乙酰化，因为N - 亚硝基甲基乙酰氧基甲基胺在鼠伤寒沙门氏菌NM2009中的遗传毒性活性可被O - 乙酰转移酶抑制剂五氯苯酚抑制。这些结果表明，NDMA和NDEA被大鼠肝微粒体氧化为遗传毒性产物，且P450 2E1酶在这两种强效致癌物的活化中起主要作用。已经提出了通过O - 乙酰转移酶乙酰化的N - 亚硝基二烷基胺的活化途径。这种用于测量遗传毒性的简单细菌系统应有助于对N - 亚硝基烷基胺活化的研究。
