Glucuronidation of 4-((Hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone, a Metabolically Activated Form of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, by Phenobarbital-Treated Rats

In the rat, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung tumors independent of the route of administration. To exert its carcinogenic potential, NNK must be metabolically activated. Like most nitrosamines NNK is activated by α-hydroxylation. The striking tissue specificity of tumor induction by nitrosamines has been primarily attributed to the efficient α-hydroxylation of a particular nitrosamine by its target tissue. Two other factors which may contribute to this are the following: the relative capacity of different tissues to detoxify the α-hydroxynitrosamine and the preferential uptake of the active metabolite by the target tissue. In the present study we report the characterization of the O-glucuronide of 4-((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone (α-hydroxymethylNNK-Gluc). The formation of this glucuronide could either serve as a detoxification pathway or provide a stable transport form of the α-hydroxylated metabolite. In addition, the metabolism of NNK to a glucuronide of the α-hydroxynitrosamine provides the first definitive evidence for the formation of α-hydroxymethylNNK. α-HydroxymethylNNK-Gluc was present in the urine of rats treated with phenobarbital (PB) and NNK. It was also formed by hepatocytes from PB-treated rats, accounting for 4% of the total metabolites in the media following incubation with 1 μM NNK. The data that support the identity of this metabolite as α-hydroxymethylNNK-Gluc are as follows. (1) Incubation of this metabolite with β-glucuronidase resulted in the quantitative release of 4-hydroxy-1-(3-pyridyl)-l-butanone (HPB), the decomposition product of α-hydroxymethylNNK. (2) This glucuronide was detected by radioflow HPLC analysis when NNK which was tritium labeled in either the pyridine ring or the methyl group was used. Therefore, it contains both these functional groups. (3) When hepatocyte media was analyzed for this glucuronide (molecular weight 399) using LC/MS with selected ion monitoring in the positive ion mode, a peak that was sensitive to β-glucuronidase treatment, with m/z (M + H), was detected at the correct retention time. LC/MS/MS analysis of this peak with selection of m/z 400 generated daughter ions of m/z 206, 176, 148, and 106. This fragmentation is consistent with this metabolite being α-hydroxymethylNNK-Gluc.