Many arylalkyl isothiocyanates are potent inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in rats and mice. In the mouse, 4-phenylbutyl isothiocyanate (PBITC) and 6-phenylhexyl isothiocyanate (PHITC) exhibited greater inhibition than benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC). The present study was conducted to investigate the structure-activity relationships of these four arylalkyl isothiocyanates for their inhibition of NNK oxidation and effects on xenobiotic-metabolizing enzymes in rats and mice. A single dose (0.25 or 1.00 mmol/kg) of each isothiocyanate was given to F344 rats 6 or 24 h before death. The rates of NNK oxidation were decreased in microsomes from the liver, lung and nasal mucosa of rats. Generally, PEITC was more potent than BITC but less potent than PBITC and PHITC. The rates in rat liver microsomes were decreased at 6 h but recovered or increased at 24 h; the rates in rat lung microsomes were markedly decreased at both 6 and 24 h; and the rates in rat nasal mucosa microsomes were also significantly decreased. The same treatment decreased the rat liver N-nitrosodimethylamine demethylase activity dramatically and ethoxyresorufin O-dealkylase and erythromycin N-demethylase activities moderately. However, the rat liver microsomal pentoxy-resorufin O-dealkylase activity was decreased at 6 h but increased at 24 h, with PEITC showing the most marked induction. The rat liver NAD(P)H:quinone oxidoreductase activity was increased 1.4- to 3.3-fold, with PEITC being most effective; and the glutathione S-transferase activity was increased slightly. Similarly, at a single dose of 0.25 mmol/kg (5 mumol/mouse) 24 h before death, PEITC, PBITC, PHITC but not BITC, decreased NNK oxidation in mouse lung microsomes by 40-85%, with PBITC and PHITC showing greater inhibition. Furthermore, all four isothiocyanates extensively inhibited NNK oxidation in rat lung and nasal mucosa microsomes as well as mouse lung microsomes in vitro, with PEITC (IC50 of 120-300 nM) being more potent than BITC (IC50 of 500-1400 nM) but less potent than PBITC and PHITC (IC50 of 15-180 nM). PHITC was a very potent competitive inhibitor of NNK oxidation in mouse lung microsomes with apparent K(i) values of 11-16 nM. These results indicate that PBITC and PHITC are more potent inhibitors of NNK bioactivation in rats and mice than PEITC. In addition, these arylalkyl isothiocyanates could be effective in protecting against the actions of a broad spectrum of carcinogenic or toxic compounds.

Following analysis by reversed-phase HPLC, a previously uncharacterized metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was found in the urine of A/J mice treated with NNK. Treatment with beta-glucuronidase converted the metabolite to a peak that co-eluted with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Treatment with sulfatase or beta-glucuronidase plus saccharic acid 1,4-lactone did not change the retention time of the metabolite. These data suggested that the unknown metabolite was a glucuronic acid conjugate of NNAL. Upon isolation and purification of larger quantities of the metabolite from the urine of A/J mice, CD-1 mice and F344 rats, 1H and 13C NMR and MS confirmed that the unknown metabolite was 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl beta-D-glucopyranosiduronic acid (NNAL Glu). To determine the quantitative relationship between NNK dose and NNAL Glu production and to compare the importance of glucuronidation relative to other metabolic pathways, [5-3H]NNK was administered to F344 rats and A/J mice at doses of 500-0.005 mumol/kg. At 500 mumol/kg, NNAL Glu accounted for 22% of the total urinary excretion of NNK in A/J mice, and for 8% in F344 rats 48 h after dosing. The proportions of excreted glucuronide and NNAL decreased with diminishing doses of NNK, yielding undetectable levels of each metabolite in both mice and rats at a dose of 0.005 mumol/kg NNK. Since substantial amounts of metabolites formed via alpha-hydroxylation and N-oxidation pathways were observed at the lower doses of NNK, these data demonstrate that NNAL glucuronidation is a quantitatively unimportant metabolic pathway at low doses of NNK.

The distribution and metabolism of phenethyl isothiocyanate (PEITC), a naturally occurring anticarcinogen, was investigated in A/J mice. Mice were administered 5 mumol of [14C]PEITC (2 microCi/mouse) by gavage and killed at 1, 2, 4, 8, 24, 48 or 72 h after dosing. Radioactivity present in the spleen, heart, liver, lung, kidney, brain, urine and feces was measured. Lung, the target tissue of PEITC inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) lung tumorigenesis, showed maximum radioactivity between 4 and 8 h after dosing, suggesting this time period would be optimal for maximal inhibition by PEITC in A/J mice. Approximately 50% of the total radioactivity was excreted within 24 h after dosing with nearly 80% of radioactivity found in urine and feces at 72 h. Two metabolites were isolated by reverse-phase HPLC from urine of mice treated with PEITC. The identities of these metabolites were determined by comparison with synthetic standards and by NMR and MS. The major metabolite was a cyclic mercaptopyruvic acid conjugate, whereas the minor metabolite was an N-acetylcysteine conjugate. Approximately 25% of the administered dose of PEITC was excreted as the cyclic mercaptopyruvic acid conjugate and 10% as the N-acetylcysteine conjugate. These results suggest that urinary metabolites of PEITC may provide potentially useful dosimeters for this natural anticarcinogen.