Alcohol dehydrogenase-dependent reduction of 2-nitrosoflurene and rearrangement of N-hydroxy-2-aminofluorene.

Abstract

Various C-nitroso compounds are intermediates of arylamine oxidation or nitroarene reduction. Reductive metabolism of C-nitroso compounds to their corresponding hydroxylamines is a necessary step in the activation of these compounds to mutagenic end points. In this study, 2-nitrosofluorene (2-NOF) has been investigated as an aldehyde substrate analogue for horse liver alcohol dehydrogenase (HLADH). The reaction products are assigned on the basis of their UV/visible spectra and coelution with authentic standards in reversed-phase HPLC. The direct product of 2-NOF reduction is N-hydroxy-2-aminofluorene (N-OH-2-AF), which undergoes further reduction to 2-aminofluorene (2-AF) and rearrangement to 1- and 3-hydroxy-2-aminofluorene (1- and 3-OH-2-AF). The formation of these products is potently inhibited by pyrazole indicating the involvement of active-site zinc ion in the role of a Lewis acid catalyst. It is suggested that the rearrangement reaction occurs via an inner-sphere N-OH 2AF-Zn2+...E-coenzyme complex following the elimination of the hydroxyl group from the N-OH-2-AF intermediate and the hydrolysis of the fluorenyl nitrenium-derived carbocations to yield the hydroxy 2-AF products. Herein ADH is identified as a C-nitroso-reducing enzyme which must be considered in the mutagenic sequelae of nitro and nitrosoarenes.