In vitro metabolism of bladder carcinogenic nitrosamines by rat liver and urothelial cells

Abstract

In order to establish the importance of the target organ in the activation of bladder carcinogens, we compared rat liver and urothelial cell α-hydroxylation activities using as substrates N-nitrosobutyl(4-hydroxybuty)amine and its metabolite N-nitrosobutyl(3-carboxypropyl)amine, two potent urinary bladder carcinogens in animals. Previous studies have shown that the production of molecular nitrogen can serve as an indicator of nitrosamine α-hydroxylation. The use of doubly 15N-labelled nitrosamines and the gas chromatography-mass spectrometric detection of 15N 2 formed gives a measurement of the extent of this metabolic step. Various amounts of 15N-labelled substrates were incubated for 60 min at 37°C with rat liver S9 preparations or urothelial cell homogenates in the presence of a NADPH generating system. Both enzyme sources metabolized 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine through the α-hydroxylation pathway. Using hepatic S9 fractions, 15N 2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine increased from 1.69 ± 0.02 nmol/h per mg protein (mean ± S.E.) to 5.78 ± 0.5 with substrate concentrations ranging between 0.55 and 5.55 mM. 15N 2 produced by urothelial cell homogenates was about 40–50% that of the liver S9. 15N-labelled N-nitrosobutyl(3-carboxypropyl)amine was also metabolized through the α-hydroxylation pathway both by hepatic S9 and urothelial cell homogenates, though to a lesser extent. 15N 2 production was about 10-times less than from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine, but again urothelial cell 15N 2 production was about 40–50% that of the liver. Treatment with phenobarbital resulted in a 2.7-fold increase in the 15N 2 produced from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9. No effect was observed with urothelial cell homogenates. Acetone treatment had no effect on 15N 2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9, but raised 15N 2 production by urothelial cell homogenates 1.8 times. Although the liver has a greater capacity than the bladder for activating the 15N-labelled nitrosamines studied, the target organ can metabolize bladder carcinogens, thus increasing the possibility of a local toxic effect. Moreover, the distribution of P-450 isozymes might be different in the bladder and this could affect the metabolism of nitrosamines reportedly formed in the human bladder in some pathological conditions.