Hepatic microsomal azoreductase activity: Reactivity of azo dye substrates is determined by their electron densities and redox potentials

We have previously established that microsomal reduction of azo dyes requires polar electron-donating ring substituents [e.g. -OH, -NH₂, - NHCH₃, or -N(CH₃)₂]. Reduction of dyes substituted exclusively with such moieties on either phenyl ring is insensitive to both O₂ and CO (I- substrates). In contrast, azoreduction of compounds containing additional electron-withdrawing substituents (e.g. -SO₃H, -COOH, -COOCH₃, and - AsO₃H₂) on the opposite (prime) phenyl ring is sensitive to both O₂ and CO (S-substrates). We have recently shown that Hammett aromatic substituent constants and redox potentials of the dyes, determined by cyclic voltammetry (CV), distinguish between I- and S-substrates. Dyes that exhibit positive Hammett σ substituent constants on 1 of the 2 rings are S-substrates, and undergo immediate quenching of their one electron-reduced intermediates upon exposure to air, as observed by CV. In contrast, dyes that have negative Hammett σ values on one or both rings are I-substrates, and their one electron-reduced intermediates are relatively stable in air. In this study, we have investigated the susceptibility to microsomal azoreduction of monosubstituted dyes, with ring substituents possessing a wide range of Hammett σ values. We have observed a direct correlation between the susceptibility of these compounds to microsomal azoreduction and the Hammett σ constant of the aromatic ring substituent, and the presence of a positive potential in the dyes observed by CV. A Hammett σ value of -0.37 or lower is required for substrate activity. Dyes with two substituents in the prime ring confirmed the previous correlation of negative and positive Hammett σ values with I- and S-substrate activities, respectively. The results suggest that binding and reduction of azo compounds by different forms of microsomal cytochrome P-450 is regulated by the electron densities and redox potentials of the dyes.