Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms.
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.
Chen
S C
SC
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Wang
X
X
Xu
G
G
Zhou
L
L
Vennerstrom
J L
JL
Gonzalez
F
F
Gelboin
H V
HV
Mirvish
S S
SS
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004947:Esophagus / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D009602:Nitrosamines / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats
D007527:Isoenzymes; D009602:Nitrosamines; C025033:N-amyl-N-methylnitrosamine; D003577:Cytochrome P-450 Enzyme System