Page 200 - The Toxicology of Fishes

180 The Toxicology of Fishes

cis–stilbene oxide
O H OH
H H
microsomal EH H
HO

trans–stilbene oxide
H O H OH
cytosolic EH
H H OH

FIGURE 4.5 Reactions catalyzed by epoxide hydrolases.

Hydrolysis
Epoxide Hydrolase
Overview
Xenobiotic epoxides and arene oxides are usually formed by cytochrome P450-dependent oxygenation
of a double bond or an aromatic ring. Due to the strain of the three-membered oxirane ring, they readily
react with cellular nucleophiles such as water, glutathione, or nucleophilic centers in DNA bases. The
function of the epoxide hydrolase (EH) group of enzymes is to catalyze the addition of water to an
epoxide or arene oxide. Epoxide hydrolase enzymes are considered part of a larger class of hydrolytic
enzymes, including esterases, proteases, dehalogenases, and lipases (Beetham et al., 1995). Studies with
mammalian enzymes have shown that two major epoxide hydrolase enzymes utilize xenobiotic epoxides
as substrates. These are the cytosolic enzyme that utilizes trans-epoxides as substrates and the microsomal
enzyme that prefers cis-epoxides and arene oxides. In both cases, the products are trans-dihydrodiols
(Hammock and Hasagawa, 1983) (see Figure 4.5). In a given animal, no evidence indicates multiple
forms of the major microsomal or cytosolic epoxide hydrolase.
Microsomal epoxide hydrolase is of particular importance for arene oxides produced by the action of
CYP on polycyclic aromatic hydrocarbons. For most arene oxides, conversion to the dihydrodiol results
in detoxification of the PAHs. In some cases, however, epoxide hydrolase plays a role in the formation
of reactive diol epoxide metabolites; for example, conversion of benzo(a)pyrene-7,8-oxide to the 7,8-
dihydrodiol is part of the pathway leading to the ultimate carcinogen, (+)-anti-benzo(a)pyrene-7,8-
dihydrodiol-9,10-oxide.
The preferred substrates for study of microsomal epoxide hydrolase activity are cis-stilbene oxide
(shown in Figure 4.5) and benzo(a)pyrene-4,5-oxide, both of which are commercially available in
radiolabeled form. The earliest studies of this enzyme were conducted with racemic styrene oxide, but
this epoxide was thought to be less definitive in measuring microsomal epoxide hydrolase activity than
a true cis-epoxide. Regardless of substrate, the method most commonly used to measure epoxide
hydrolase activity was to incubate the radiolabeled epoxide or arene oxide with microsomes at pH 9
and then measure the amount of product formed. The pH optimum of microsomal EH activity in most
species that have been examined was 8.5 to 9.5 (Balk et al., 1980; James et al., 1979). For some substrates
(e.g., styrene oxide and cis-stilbene oxide), unreacted substrate was separated from product by extraction
(Gill et al., 1983; James et al., 2004) and for others, such as benzo(a)pyrene-4,5-oxide, by chromatog-
raphy (Jerina and Dansette, 1977). Other methods have also been developed, such as gas chromatography,
spectrophotometric, and fluorimetric methods (Dansette et al., 1976; Westkaemper and Hanzlik, 1981),
although these tend to be of lower sensitivity than the radiochemical methods. In all methods, an important
consideration is to keep the reaction pH 6 or higher at all steps, as epoxides undergo spontaneous
hydrolysis at acidic pH.