Challenging the specificity of chlorzoxazone and 4-nitrophenol as marker substrates for CYP2E1 activity in Supersomes™.

Cytochrome P450 2E1 (CYP2E1) plays a pivotal role in the metabolism and detoxification of low molecular-weight xenobiotics, as well as in their bioactivation into toxic and DNA-reactive intermediates. Prominent examples include drugs and chemicals like acetaminophen and N-nitrosamines. Chlorzoxazone and 4-nitrophenol are widely used as probe substrates to assess CYP2E1 activity, however, their specificity remains controversial. This study aimed to reevaluate the selectivity of chlorzoxazone and 4-nitrophenol toward CYP2E1 using recombinant Supersomes™ expressing individual human CYP isoforms. Incubations were performed with chlorzoxazone and 4-nitrophenol at 100 and 500 µM, followed by quantification of 6-hydroxychlorzoxazone and 4-nitrocatechol via UPLC-MS/MS. Chlorzoxazone turnover was detected across nearly all major liver CYP isoforms, with CYP1A1 showing the highest contribution (39%), followed by CYP2D6 (14%), CYP1A2 (10%), and CYP2E1 (10%). Similarly, 4-nitrophenol hydroxylation was not limited to CYP2E1, with significant contributions from CYP1A2 (20%), CYP2A6 (19%), and CYP2D6 (16%), while CYP2E1 accounted for 26% of total activity. In conclusion, our results underscore the limited selectivity of chlorzoxazone and 4-nitrophenol and highlight the need for more specific substrates for accurate CYP2E1 enzyme characterization.