Metabolism of 2-Nitrosofluorene and p-Nitrosophenol by Horse Liver Alcohol Dehydrogenase (HLADH)

Abstract

Reductive metabolism of C-nitroso substrates to their corresponding hydroxyl amines is thought to be requisite to the activation of these compounds to mutagenic products. We have studied 2-nitrosofluorene (2-NOF) and p-nitrosophenol (p-NSP) as aldehyde substrate analogs for HLADH. 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 inhibited by pyrazole, indicating the involvement of active-site zinc ion in the role of a Lewis acid. It is suggested that the rearrangement reaction occurs via elimination of the hydroxyl group from the N-OH-2-AF and hydrolysis of the fluorenyl nitrenium-derived carbocations to yield the hydroxy 2-AF products. HLADH-dependent metabolism of p-NSP results in formation of p-aminophenol, which appears to proceed at pH above 6 via a spectral intermediate at ∽255 nm, but does not at pH below 6. It is suggested that the reduction of p-NSP proceeds via two-step reduction involving concomitant dehydration of p-hydroxylaminophenol to p-benzoquinoneimine. At pH above 6 the reduction of p-benzoquinoneimine to p-aminophenol is rate-limiting.

Key Words:

• alcohol dehydrogenase
• 2-nitrosofluorene
• p-nitrosophenol.