Hepatic microsomes were prepared from liver homogenates suspended in 0.15 M KCL. The homogenates were centrifuged at 9,000 × g for 20 min, and then the resulting supernatant fractions were centrifuged at 105,000 × g for 60 min. The resulting pellets (microsomes) were re-suspended in 0.15 M KCL. All procedures were carried out at 4 °C. CYP2E1 activity was measured by the rate of oxidation of 1 mM p-nitrophenol to p-nitrocatechol as described (Lu et al., 2005). Coumarin 7-hydroxylase (COH) is encoded by the mouse Cyp2a5 gene and its human orthologue Cyp2a6 gene, and COH activity is considered as a specific marker for catalytic activities of CYP2A5 and CYP2A6 (Su and Ding, 2004). CYP2A5 activity was measured by assessing COH with 100 μM coumarin as substrate (Kobliakov et al., 1993).
