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 c
DNA
-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.