Formation of N-alkylated protoporphyrin IX in the livers of mice after diethylnitrosamine treatment

Abstract

The administration of di[1-14C]ethylnitrosamine to phenobarbital-pretreated mice resulted in the formation of a radiolabelled green pigment in their livers. Green-pigment concentrations were time- and dose-dependent, maximum levels being reached 1-2 h after dosing. There was only a slight decrease in cytochrome P-450 levels and accumulation of porphyrins in the liver at this time. Dimethyl- or dipropyl-nitrosamine also caused an accumulation of similar, though not identical, compounds in the liver. The formation of green pigment was induced by pretreatment of mice with phenobarbital or 3-methylcholanthrene and was inhibited by the acute administration of pyrazole or ethanol. From the absorption spectra, the green pigment methyl esters appeared to be N-alkylporphyrins. Analysis of the diethylnitrosamine-induced green pigment by high-pressure liquid chromatography showed it to be more polar than the expected N-ethylprotoporphyrin IX, having a retention time similar to that of N-hydroxyethylprotoporphyrin IX. Desorption chemical-ionization mass spectrometry gave a protonated molecular ion, m/z 635, compatible with N-hydroxyethylprotoporphyrin IX. The presence of a free hydroxy group was demonstrated by acetylation with [1-14C]acetic anhydride. No conversion of N-ethylprotoporphyrin IX into N-hydroxyethylprotoporphyrin IX could be demonstrated in vivo or in vitro. Little or no N-ethylprotoporphyrin IX accumulated in the livers of mice given diethylnitrosamine. It was concluded that N-hydroxyethylprotoporphyrin IX is the primary reaction product between an active metabolite of diethylnitrosamine and hepatic haem.