Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450

Abstract

The effects of methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied in vitro on nine individual, cDNAexpressed cytochrome P-450 activities (phenacetin O-deethylase for CYP1A1 and CYP1A2, coumarin 7-hydroxylase for CYP2A6, testosterone 6beta-hydroxylase for CYP3A4, 7-ethoxy-4-trifluoromethylcoumarin deethylase for CYP2B6, paclitaxel 6alpha-hydroxylase for CYP2C8, diclofenac 4'-hydroxylase for CYP2C9, S-mephenytoin 4-hydroxylase for CYP2C19, and (+/-)-bufuralol 1'-hydroxylase for CYP2D6) in commercially available human lymphoblastoid microsomes. These data show that specific solvents have enzyme-selective effects on P-450 activities. Methanol did not substantially inhibit (</=10%) any of the activities at 0.3%, but did inhibit CYP1A1, CYP2B6, CYP2C9, and CYP2D6 by 12 to 26% at 1%. In contrast, 0.1% ethanol inhibited CYP1A1, CYP2B6, and CYP2C19 by 20 to 30%. Ethanol at 1% did not inhibit CYP1A2, CYP3A4, CYP2C8, and CYP2C9. DMSO inhibited CYP3A4, CYP2C19, and CYP2D6 by 15 to 25% at 0.1%. However, DMSO had little effect on CYP1A2, CYP2A6, and CYP2C8. Acetonitrile, like methanol, did not inhibit any P-450 activity at 0.3% solvent except for CYP1A1 (26%) and CYP2B6 (13%). At 1%, acetonitrile decreased activities of CYP1A1 and CYP2B6 by 40 to 60%, and inhibited CYP2A6, CYP3A4, CYP2C19, and CYP2D6 activity by 10 to 20%. Acetonitrile also increased CYP2C9 activity by 10 to 15% above control values at 1 to 3% solvent. Excluding solubility considerations, methanol and acetonitrile appear to be the most suitable solvents for the introduction of substances to cytochrome P-450 incubations for in vitro metabolism studies.