CYP2E1 activity was determined by hydroxylation of p-nitrophenol to 4-nitrocatechol as described [12]. Briefly, a 50-μl reaction mixture containing 0.8 mM p-nitrophenol and 2.0 mM β-NADP (Sigma Aldrich, St. Louis, MO) was added to 50 μl of microsomal protein on ice in 1.5-ml microcentrifuge tubes. Blank tubes of identical compositions were prepared except that 20% TCA was added prior to the reaction mixture on ice. After 1 h incubation at 37 °C in a water bath shaker, addition of 30 μl of 20% TCA terminated the reactions. Samples were centrifuged for 10 min at 14,000 rpm, and to the supernatants was added 10 μl of 10 N NaOH for color development. Absorbance was read at 540 nm in the 96-well Vmax kinetic reader (Molecular Devices, Sunnyvale, CA). Using 4-nitrocatechol as internal standard, CYP2E1 activity was calculated and expressed as nanomoles per milligram protein.
