Shu L, Hollenberg P F
Department of Pharmacology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.
Carcinogenesis. 1996 Apr;17(4):839-48. doi: 10.1093/carcin/17.4.839.
The metabolism of N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitroso-n-butyl-n-propylamine (NBPA) was investigated in vitro using liver microsomes and purified isoforms of cytochrome P450 in a reconstituted system. Liver microsomes were prepared from rats pretreated with phenobarbital (PB), pyridine (PYR), beta-naphthoflavone (BNF), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), clofibrate (CLO) or from untreated rats. The purified cytochrome P450s used in the reconstituted system were rat 1A1 and 2B1 and rabbit 2E1. The rates of metabolism and the product profiles for NDPA, NDBA and NBPA changed significantly depending on the pretreatment of the rats or the identity of the purified cytochrome P450 isoforms. Induction by PB dramatically increased cleavage of NDPA, NDBA and NBPA at C-N bonds, leading to substantial increases in formation of the respective aldehydes and the overall metabolic rates. Microsomes from PYR-pretreated rats exhibited increased activities for formation of formaldehyde and propionaldehyde from NDPA and NBPA. Microsomes from BHT-pretreated rats showed a slight increase in activity for N-dealkylation of NDBA and BNPA. Treatment with BHA decreased the overall metabolism of NDBA, but slightly increased N-dealkylation of NBPA. Microsomal metabolism of NDPA, NDBA and NBPA was decreased by pretreatment with BNF and CLO. Results from studies using the reconstituted system with purified cytochrome P450 isoforms demonstrated that cytochrome P450 2B1 specifically catalyzed alpha-hydroxylation of these three long chain nitrosamines with high activity. Cytochrome P450 2E1 catalyzed formation of formaldehyde and propionaldehyde from NDPA and NBPA, but did not catalyze formation of acetaldehyde or butyraldehyde. Cytochrome P450 1A1 exhibited no activity for metabolism of NDPA, NDBA and NBPA. The contributions of cytochrome P450 2B1 and 2E1 to N-dealkylation reactions were determined using inhibitory monoclonal antibodies (mAb). With microsomes from PB-pretreated rats, inhibition by mAb-2B1 indicated a 62% contribution by cytochrome P450 2B1 to debutylation of NDBA and 65% to depropylation of NDPA. In microsomes from PYR-pretreated rats inhibition by mAbs also showed a role for cytochrome P450 2E1 in depropylation of NDPA. These studies provide a better understanding of the role of various forms of cytochrome P450 in metabolic activation of these long chain N-nitrosodialkylamines to potentially toxic, mutagenic and carcinogenic intermediates.
利用肝微粒体和重组系统中纯化的细胞色素P450同工型，在体外研究了N-亚硝基二丙胺（NDPA）、N-亚硝基二丁胺（NDBA）和N-亚硝基-n-丁基-n-丙胺（NBPA）的代谢。肝微粒体取自经苯巴比妥（PB）、吡啶（PYR）、β-萘黄酮（BNF）、丁基羟基甲苯（BHT）、丁基羟基茴香醚（BHA）、氯贝丁酯（CLO）预处理的大鼠或未经处理的大鼠。重组系统中使用的纯化细胞色素P450为大鼠1A1和2B1以及兔2E1。NDPA、NDBA和NBPA的代谢速率及产物谱因大鼠的预处理情况或纯化的细胞色素P450同工型的种类而有显著变化。PB诱导显著增加了NDPA、NDBA和NBPA在C-N键处的裂解，导致相应醛类的形成及总体代谢速率大幅增加。来自PYR预处理大鼠的微粒体对NDPA和NBPA形成甲醛和丙醛的活性增加。来自BHT预处理大鼠的微粒体对NDBA和BNPA的N-脱烷基化活性略有增加。BHA处理降低了NDBA的总体代谢，但略微增加了NBPA的N-脱烷基化。BNF和CLO预处理降低了NDPA、NDBA和NBPA的微粒体代谢。使用含有纯化细胞色素P450同工型的重组系统进行的研究结果表明，细胞色素P450 2B1以高活性特异性催化这三种长链亚硝胺的α-羟基化。细胞色素P450 2E1催化NDPA和NBPA形成甲醛和丙醛，但不催化乙醛或丁醛的形成。细胞色素P450 1A1对NDPA、NDBA和NBPA的代谢无活性。使用抑制性单克隆抗体（mAb）确定了细胞色素P450 2B1和2E1对N-脱烷基化反应的贡献。对于来自PB预处理大鼠的微粒体，mAb-2B1的抑制作用表明细胞色素P450 2B1对NDBA的脱丁基化贡献为62%，对NDPA的脱丙基化贡献为65%。对于来自PYR预处理大鼠的微粒体，mAb的抑制作用也表明细胞色素P450 2E1在NDPA的脱丙基化中起作用。这些研究有助于更好地理解各种形式的细胞色素P450在这些长链N-亚硝基二烷基胺代谢活化成潜在有毒、致突变和致癌中间体过程中的作用。
