Alpha-oxidative metabolism of the bladder carcinogens N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine within the rat isolated bladder

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Abstract
The most widely accepted metabolic pathway leading to the formation of reactive intermediates from nitrosamines involves enzymatic hydroxylation at the carbon atom α to the nitroso moiety. All subsequent steps are non-enzymatic reactions and the final result is the stoichiometric formation of a cationic product and molecular nitrogen. Thus the amount of molecular nitrogen evolved can be used as an indicator of α-hydroxylation. The use of doubly 15N-labelled nitrosamines and the detection of 15N2 by MS makes it simpler to measure the extent of α-hydroxylation. We have studied the α-oxidation of doubly 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine (BBN) and its metabolite N-nitrosobutyl(3-carboxypropyl)amine (BCPN), two potent urinary bladder carcinogens in animals, within the target organ. Various amounts of 15N-labelled BBN ranging from 0.1 to 5 µmol were incubated at 37°C for 4 h in the isolated rat bladder and the formation of 15N2 was measured by GC-MS. 15N2 production was linear up to 1 µmol and represented ∼0.1% of the substrate incubated. Time-course experiments showed that 15N-labelled production was linear over a 6 h incubation period, ranging from 2.16 ± 0.05 to 4.55 ± 0.33 nmol/mg urothelial cell protein. 15N-labelled BCPN (1–5 µmol) was also incubated within the rat isolated bladder. 15N2 production from BCPN was ∼10 times less than that from BBN. The results indicate that, though to a lower extent, the target organ activates 15N-labelled BBN and BCPN through the α-hydroxylation pathway.

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