Spectrophotometric Analysis of Human CYP2E1-Catalyzed p-Nitrophenol Hydroxylation

CYP2E1 is expressed in adult (1 , 2 ) and fetal (3 ) human liver in addition to extrahepatic tissues such as lung and placenta (4 ). Treatment of primary cultures of human hepatocytes with ethanol induces CYP2E1 protein (5 ) and this is consistent with the finding that hepatic CYP2El protein (6 ) and mRNA (7 ) amounts are increased in alcoholics. Although only a few drugs (e.g., acetaminophen [8 ]) have been identified as substrates for CYP2E1, many low molecular weight procarcinogens are activated by this P450 (9 ). Chlorzoxazone 6-hydroxylation (19 –12 ), N-nitrosodimethylamine N-demethylation (11 , 13 , 14 ), and p-nitrophenol hydroxylation (12 , 14 ) reactions can be used to measure the catalytic activity of cDNA-expressed CYP2El. Each of these activities is also frequently used as an enzyme-selective catalytic monitor for human hepatic CYP2El (see Notes 1 and 2 ). Experiments with inhibitory antiCYP2E1 antibodies and CYP2E1-selective chemical inhibitors suggest that CYP2E1 contributes extensively to these activities in human liver microsomes (9 , 15 –18 ). Recently, lauric acid 11 -hydroxylation was identified as an alternative probe for human hepatic CY2El (19 ). However, an advantage of using p-nitrophenol hydroxylation to measure CYP2E1 activity is that the formation of p-nitrocatechol can be measured by a simple spectrophotometric assay, although high-performance liquid chromatographic (HPLC) assays have also been developed to quantify thep-nitrocatechol metabolite (20 , 21 ). This chapter describes a modification of a spectrophotometric method (22 ) for the determination of p-nitrophenol hydroxylase activity.