Yoo J S, Guengerich F P, Yang C S
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103.
Cancer Res. 1988 Mar 15;48(6):1499-504.
The metabolism of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine, N-nitrosobenzylmethylamine, and N-nitrosobutylmethylamine was investigated in incubations with human liver microsomes. All of the 16 microsomal samples studied were able to oxidize NDMA to both formaldehyde and nitrite at NDMA concentrations as low as 0.2 mM; the rates of product formation of the samples ranged from 0.18 to 2.99 nmol formaldehyde/min/mg microsomal protein (median, 0.53 nmol). At a concentration of 0.2 mM NDMA, the rates of denitrosation (nitrite formation) were 5 to 10% (median, 6.3%) those of demethylation (formaldehyde formation); the ratio of denitrosation to demethylation increased with increases in NDMA concentration, in a similar manner to rat liver microsomes. Immunoblot analysis with antibodies prepared against rat P-450ac (an acetone-inducible form of cytochrome P-450) indicated that the P-450ac [P-450j (isoniazid-inducible form)] orthologue in human liver microsomes had a slightly higher molecular weight than rat P-450ac and the amounts of P-450ac orthologue in human liver microsomes were highly correlated with NDMA demethylase activities (r = 0.971; P less than 0.001). Analysis of four selected microsomal samples showed that human liver microsomes exhibited at least three apparent Km and corresponding Vmax values for NDMA demethylase. This result, suggesting the metabolism of NDMA by different P-450 enzymes, is similar to that obtained with rat liver microsomes, even though most of the human samples had lower activities than did the rat liver microsomes. The high affinity Km values of the four human samples ranged from 27 to 48 microM (median, 35 microM), which were similar to or slightly lower than those observed in rat liver microsomes, indicating that human liver microsomes are as efficient as rat liver microsomes in the metabolism of NDMA. The human liver microsomes also catalyzed the dealkylation and denitrosation of other nitrosamines examined. The rates of product formation and the ratios of denitrosation to dealkylation varied with the structures and concentrations of the substrates as well as with the microsomal samples tested. The results indicate that human liver microsomes are capable of metabolizing N-nitrosodialkylamines via the pathways that have been established with rat liver microsomes.
在与人类肝微粒体的孵育实验中，研究了N-亚硝基二甲胺（NDMA）、N-亚硝基二乙胺、N-亚硝基苄基甲胺和N-亚硝基丁基甲胺的代谢情况。所研究的16个微粒体样本均能够在低至0.2 mM的NDMA浓度下将其氧化为甲醛和亚硝酸盐；样本中产物生成速率范围为0.18至2.99 nmol甲醛/分钟/毫克微粒体蛋白（中位数为0.53 nmol）。在0.2 mM的NDMA浓度下，脱亚硝化作用（亚硝酸盐生成）速率为去甲基化作用（甲醛生成）速率的5%至10%（中位数为6.3%）；脱亚硝化作用与去甲基化作用的比率随NDMA浓度的增加而增加，这与大鼠肝微粒体的情况类似。用针对大鼠P-450ac（细胞色素P-450的一种丙酮诱导形式）制备的抗体进行免疫印迹分析表明，人类肝微粒体中的P-450ac [P-450j（异烟肼诱导形式）] 同源物分子量略高于大鼠P-450ac，且人类肝微粒体中P-450ac同源物的量与NDMA去甲基酶活性高度相关（r = 0.971；P小于0.001）。对四个选定的微粒体样本进行分析表明，人类肝微粒体对NDMA去甲基酶表现出至少三个明显的Km值和相应的Vmax值。这一结果表明NDMA由不同的P-450酶代谢，与大鼠肝微粒体的情况相似，尽管大多数人类样本的活性低于大鼠肝微粒体。四个人类样本的高亲和力Km值范围为27至48 μM（中位数为35 μM），与大鼠肝微粒体中观察到的值相似或略低，表明人类肝微粒体在NDMA代谢方面与大鼠肝微粒体一样高效。人类肝微粒体还催化了所检测的其他亚硝胺的脱烷基化和脱亚硝化作用。产物生成速率以及脱亚硝化作用与脱烷基化作用的比率随底物的结构和浓度以及所测试的微粒体样本而变化。结果表明，人类肝微粒体能够通过已在大鼠肝微粒体中确立的途径代谢N-亚硝基二烷基胺。
