N'-Nitrosonornicotine (NNN) was the first tobacco-specific
nitrosamine (TSNA) identified as
carcinogen in tobacco
smoke, but no data exist on in vivo interactions between NNN and other tobacco
alkaloids, TSNA or
phenethyl isothiocyanate (
PEITC) which have been demonstrated in various studies on
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Acute effects on NNN metabolism were tested in male Fischer F344 rats injected s.c. with 30nmol/kg
body weight (bw) [5-(3)H]NNN either alone or simultaneously with 15mumol/kg bw
nicotine,
nornicotine,
anatabine, or
anabasine, 150mumol/kg bw
cotinine, 3mumol/kg bw
myosmine, or 300nmol/kg bw of either
N'-nitrosoanatabine or
N'-nitrosoanabasine. Another group of rats was fed a diet supplemented with
PEITC at 1mumol/g diet starting 24h before NNN treatment. Within 24h more than 80% and about 10% of the radioactivity was excreted with urine and feces, respectively. Urinary metabolites were separated by reversed-phase radio-HPLC and identified by co-chromatography with UV standards. In two sets of experiments with control rats treated with NNN only,
4-hydroxy-4-(3-pyridyl)butanoic acid (
hydroxy acid, 44.4/44.8%),
4-oxo-4-(3-pyridyl)butanoic acid (keto
acid, 32.4/31.5%), NNN-N-
oxide (5.0/3.8%), 4-(3-pyridyl)butane-1,4-diol (diol, 1.1/1.0%) and
norcotinine (2.3/1.0%) were consistently detected besides unmetabolised NNN (4.7/3.3%). Co-treatment with
nicotine,
cotinine,
nornicotine and
PEITC shifted the contribution of the two major metabolites significantly in favor of
hydroxy acid (108-113% of control) as compared to keto
acid (86-90% of control). The same treatments also increased
norcotinine (135-170% of control). These changes are consistent with a decreased metabolic activation of NNN. In subacute studies rats received NNN in
drinking water for 4 weeks at a daily dose of 30 nmol/kg bw with or without
nornicotine at 15 micromol/kg bw or
myosmine at 3 micromol/kg bw. On the last day of the experiment all rats received [5-(3)H]NNN at 30 nmol/kg bw with a contaminated apple
bite followed by collection of urine and feces for 18h. Most of the radioactivity, 87-96% of the dose, was recovered in urine and only minor amounts have been excreted in feces or persisted in blood. In urine of the NNN-control group keto
acid (32.2%) and unmetabolised NNN (3.9%) were present in identical amounts as in the acute experiment whereas
hydroxy acid (41.4% of total radioactivity in urine, 93% of acute NNN control) was reduced in expense of the minor NNN metabolites. Co-administration of
nornicotine resulted in a small but significant rise of keto
acid (107% of control) and a significant decrease in NNN-N-
oxide (76% of control). After co-treatment with
myosmine the increase of keto
acid (104% of control) was even less but still significant whereas NNN-N-
oxide and diol were significantly reduced to 72% and 79% of control, respectively. Our experiments with rats indicate significant mutual effects of some of the major tobacco
alkaloids and most relevant TSNA. Further studies on the impact on smokers and the inhibitory effects of
isothiocyanates are needed for a final risk assessment.