Carcinogenesis, Vol 18, 1623-1630, Copyright © 1997 by Oxford University Press
CJ Patten, TJ Smith, MJ Friesen, RE Tynes, CS Yang and SE Murphy
The tobacco specific carcinogen N'-nitrosonornicotine (NNN), is believed to
be a causative agent for esophageal cancer in smokers. NNN requires
metabolic activation to exert its carcinogenic potential. Metabolism occurs
through cytochrome P450 (P450) catalyzed 2'- and 5'- hydroxylation, which
generates unstable metabolites that decompose to 4-
hydroxy-1-(3-pyridyl)-1-butanone ('keto alcohol') and 4-hydroxy-4-(3-
pyridyl)butanal, respectively. The latter cyclyzes to 5-(3-pyridyl)-2-
hydroxytetrahydrofuran ('lactol'). 2'-Hydroxylation of NNN is believed to
be the pathway critical for esophogeal NNN carcinogenesis in the rat. The
ability of human liver microsomes and expressed human P450s to metabolize
[5-(3)H]NNN to keto alcohol and lactol was determined by reverse phase HPLC
with radioflow detection. At low NNN concentrations, 11 human liver
microsomes metabolized NNN primarily by 5'-hydroxylation to lactol. This
reaction was strongly correlated (r = 0.92) with coumarin 7-hydroxylation,
suggesting that NNN 5'-hydroxylation is catalyzed mainly by P450 2A6.
2'-Hydroxylation of NNN by human liver microsomes correlated with
6beta-hydroxylation of testosterone, a P450 3A4-specific activity (r =
0.94). The relative rates of 2'- and 5'- hydroxylation by human P450s 2A6,
2E1, 2D6 and 3A4 expressed in Sf9 cells by the baculovirus-insect cell
expression system, and human P450 3A4 produced by stable expression in
Chinese hamster ovary cells, were determined. Human P450 2A6 metabolized 1
microM NNN exclusively by 5'- hydroxylation. The rate of lactol formation
was 317 pmol/min per nmol P450. Human P450s 2E1 and 2D6 also metabolized
NNN only to lactol, but at much lower rates, 0.4 and 0.8 pmol/min per nmol
of P450 respectively. In contrast, the metabolism of NNN by expressed human
P450 3A4 was specific for keto alcohol formation. The Km for 5'-
hydroxylation by baculovirus-expressed P450 2A6 was 2.1 microM, and k(cat)
was 953 pmol/min per nmol of P450. The Km for lactol formation by human
liver microsomes containing high levels of P450 2A6, was 5 microM . Human
liver microsomes exhibited a Km of 312 microM for keto alcohol formation.
Coumarin, 8-methoxypsoralen (P450 2A6 inhibitors), and anti-2A6 monoclonal
antibody were strong inhibitors of NNN-derived lactol formation in human
liver microsomes. Troleandomycin, an inhibitor of P450 3A4, effectively
inhibited the metabolism of NNN to keto alcohol by human liver microsomes.
These results are consistent with P450 2A6 mediated 5'-hydroxylation and
P450 3A4 mediated 2'- hydroxylation of NNN in human liver microsomes.
ARTICLES
Evidence for cytochrome P450 2A6 and 3A4 as major catalysts for N'- nitrosonornicotine alpha-hydroxylation by human liver microsomes
Sandoz Pharmaceuticals Corporation, Sandoz Research Institute, Department of Drug Metabolism and Pharmacokinetics, E. Hanover, NJ 07936, USA.
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