Abstract

Acetonitrile is an organic solvent commonly used to increase the solubility of lipophilic substrates for in vitro studies. In this study, we examined its effect on four reactions (diclofenac hydroxylation, tolbutamide methyl hydroxylation, phenytoin hydroxylation, and celecoxib methyl hydroxylation) catalyzed by human liver microsomes and by the recombinant CYP2C9. In both cases, the effect of acetonitrile on activity was found to be substrate-dependent. Namely, it increased diclofenac 4′-hydroxylase and tolbutamide methyl hydroxylase activities, but decreased celecoxib methyl hydroxylase activity in a concentration-dependent manner. By comparison, hydroxylation of phenytoin was resistant to its effect. The presence of acetonitrile (3%, v/v) gave rise to a lowerK_m and a higherV_max for diclofenac hydroxylase in both liver microsomes and recombinant CYP2C9 preparations (87 and 52% increase inV_max/K_m ratio, respectively). On the other hand, the inhibitory effect of the solvent (1%, v/v) toward celecoxib hydroxylase was characterized by a decrease in V_max (human liver microsomes) or a change in both K_m andV_max (rCYP2C9), leading to 25 and 46% decrease inV_max/K_m for both systems. The results of this study underscore the need for careful evaluation of solvent effects before initiation of inhibition or cytochrome P450 reaction phenotyping studies.