Discovery of Metabolic Cross-Coupling in Phenol-Arylamine Mixtures by Cytochrome P450 via Combined Computational and Experimental Approaches

This study unveils a novel cytochrome P450 (P450)-mediated
metabolic
pathway that drives cross-coupling between diverse phenol and arylamine
pollutants. Phenols and arylamines constitute a large part of industrial
chemicals, pharmaceuticals, and personal care products, yet the biotransformation
of phenol-arylamine pollutant mixtures remains largely unexplored.
Density functional theory calculations revealed that the rate-limiting
rebound barriers of phenoxy and arylamino radicals formed through
O–H/N–H abstraction by the P450 catalytic oxidant, Compound
I, facilitate their dissociation from the heme, creating thermodynamically
favorable conditions for subsequent nonrebound cross-coupling reactions.
These proposed hybrid products were systematically identified and
quantified using various mass spectrometry technologies across multiple
biological systems, including human liver microsomes, recombinant
human CYP3A4, mice, and zebrafish. From a physical organic chemistry
perspective, the widespread occurrence of cross-coupling is driven
by sufficient lifetime of phenoxy and arylamino radicals due to spin
delocalization, and their concentration gradient sustained by persistent
radical effects. Notably, yeast two-hybrid assays demonstrated that
the phenol-arylamine hybrids exhibited supra-additive estrogenic activity;
for instance, the bisphenol A–sulfamethoxazole dimer with a
hydrolytic half-life of 11.6 days displayed approximately 130-fold
higher estrogenic activity than that of bisphenol A. The mechanism’s
prevalence suggests an unrecognized bioactive pathway in chemical
cocktails, thereby necessitating consideration of metabolism-driven
reactive interactions in mixtures.