大鼠食管和肝脏微粒体以及大鼠和人类细胞色素P450同工型对[3H-戊基]甲基-n-戊基亚硝胺的去戊基化作用。

Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms.

作者信息

Chen S C, Wang X, Xu G, Zhou L, Vennerstrom J L, Gonzalez F, Gelboin H V, Mirvish S S

机构信息

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha 68198-6805, USA.

出版信息

Cancer Res. 1999 Jan 1;59(1):91-8.

PMID:9892192

Abstract

Methyl-n-amylnitrosamine (MNAN) induces esophageal cancer in rats, probably involving activation by cytochromes P450. We studied the metabolic depentylation of MNAN. [3H-4,5-pentyl]MNAN and [3H-2,3-pentyl]-MNAN were synthesized, purified, and incubated with rat esophageal microsomes (REM) or rat liver microsomes (RLM) to give [3H]pentaldehyde (depentylation), an indicator of MNAN activation. [3H]Pentaldehyde was determined by high-performance liquid chromatography of its 2,4-dinitrophenylhydrazone. Adding 5 mM semicarbazide to incubations increased the observed depentylation (except that due to CYP2E1) by >60%. MNAN depentylation by REM and uninduced and induced RLM showed Km values of 64, 610, and 170-330 microM, respectively (Vmax: 20, 220, and 160-1270 pmol/mg protein/min, respectively). The depentylation of 100 microM MNAN by REM was inhibited 98% by CO and 65% by coumarin preincubated for 15 min with REM (Ki, 120 microM) but was unaffected by antibodies inhibitory to various P450s. MNAN inhibited coumarin 7-hydroxylation by RLM and CYP2A6 (Ki, 3000 and 320 microM, respectively). REM showed slight coumarin 7-hydroxylase activity. MNAN depentylation by RLM was 41% inhibited by an antibody to CYP2C11. Km for rat CYP2E1, human CYP2E1, and human CYP2A6 was 210, 115, and 17 microM, respectively (Vmax: 900, 570, and 120 pmol/nmol P450/min, respectively). We conclude that MNAN activation by REM is probably due to a P450 related to CYP2A3, a rodent nasal P450.

摘要

甲基正戊基亚硝胺（MNAN）可诱导大鼠患食管癌，这可能涉及细胞色素P450的激活作用。我们研究了MNAN的代谢脱戊基化反应。合成、纯化了[3H - 4,5 - 戊基]MNAN和[3H - 2,3 - 戊基] - MNAN，并将它们与大鼠食管微粒体（REM）或大鼠肝微粒体（RLM）一起孵育，以生成[3H]戊醛（脱戊基化反应），这是MNAN激活的一个指标。通过高效液相色谱法测定其2,4 - 二硝基苯腙来确定[3H]戊醛。在孵育体系中加入5 mM氨基脲后，观察到的脱戊基化反应（CYP2E1介导的除外）增加了60%以上。REM以及未诱导和诱导的RLM对MNAN的脱戊基化反应的Km值分别为64、610和170 - 330 μM（Vmax分别为20、220和160 - 1270 pmol/mg蛋白质/分钟）。REM对100 μM MNAN的脱戊基化反应可被CO抑制98%，被与REM预孵育15分钟的香豆素抑制65%（Ki为120 μM），但不受针对各种P450的抑制性抗体的影响。MNAN可抑制RLM和CYP2A6对香豆素的7 - 羟化反应（Ki分别为3000和320 μM）。REM显示出轻微的香豆素7 - 羟化酶活性。RLM对MNAN的脱戊基化反应可被抗CYP2C11抗体抑制41%。大鼠CYP2E1、人CYP2E1和人CYP2A6的Km值分别为210、115和17 μM（Vmax分别为900、570和120 pmol/nmol P450/分钟）。我们得出结论，REM对MNAN的激活作用可能归因于一种与CYP2A3相关的P450，CYP2A3是一种啮齿动物的鼻腔P450。