Endogenous role of epoxide-hydratase. Development of a steroid epoxide-hydratase assay and properties of the enzyme.

A highly sensitive and rapid radiometric assay for the determination of specific epoxide hydratase activity with a steroid epoxide (16α, 17α-epoxy-1,3,5(10)-estratrien-3-ol, ‘estroxide’) has been developed. The unreacted substrate was separated from the product 1,3,5(10)-estratrien-3,16β,17α-triol by extraction into light petroleum. The product was then extracted into ethyl acetate and measured by scintillation spectrometry. Radiochromatography established that after subtraction of the blank the entire radioactivity measured in the ethyl acetate phase resulted from the product 1,3,5(10)-estratrien-3,16,17-triol, whilst high performance liquid chromatography with the four possible isomers of…

Purification of rat liver epoxide hydratase to apparent homogeneity.

Epoxide hydratase (EC 4.2.1.63) is a microsomal enzyme which catalyses the conversion of epoxides to trans-dihydrodiols. Epoxides, produced by the action of microsomal monooxygenases (EC 1.14.1.1) from aromatic and olefinic compounds, are thought to be responsible for many of the harinful effects of polycyclic hydrocarbons and related compounds. Thus epoxide hydratase, together with glutathione 9transferases, (EC 2.5.1.18) may play an important role in the removal of carcinogenic and cytotoxic metabolites (for reviews see [l-3]). It has been reported [4,5] that dihydrodiols formed from some polycyclic hydrocarbons (benz(a)anthracene and benzo(a)pyrene) are reactivated by the microsomal mono…

Large differences in metabolic activation and inactivation of chemically closely related compounds: effects of pure enzymes and enzyme induction on the mutagenicity of the twelve monomethylated benz[a]anthracenes, 7,12-dimethylbenz[a]anthracene and benz[a]anthracenes in the Ames test

Properties and amino acid composition of pure epoxide hydratase

1. Introduction Rat liver epoxide hydratase [EC 4.2.1.631 which catalyses the conversion of epoxides to trurans-dihydro- diols has been purified to apparent homogeneity as determined by three independent criteria [l] . The preparation obtained was capable of catalysing the hydration of both styrene oxide and the 4,5- (K- region)epoxide of benzo(a)pyrene [ 11. Epoxides of polycyclic hydrocarbons have been implicated as the agents responsible for the cytotoxic and carcinogenic properties of such compounds (for reviews see [2-41). A detailed knowledge of the properties of epoxide hydratase may, therefore, contribute towards an understanding of the mechanisms of cytotoxicity and carcinogenesis.…

ISOLATION OF RAT LIVER EPOXIDE HYDRATASE: PROPERTIES AND SUBSTRATE SPECIFICITY OF THE PURE ENZYME

ABSTRACT The microsomal enzyme epoxide hydratase has been purified to homogeneity as judged by electrophoretical, ultracentrifugal and immunological criteria and by C- and N-terminal analysis. The preparation procedure consisted of solubilisation using the non-ionic detergent cutscum, (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-cellulose and cellulose phosphate and hydrophobic chromatography on butyl-sepharose. The product was detergent-free, had a relatively high content of hydrophobic amino acids and tended to aggregate in aqueous solutions. The protein had a minimum molecular weight of 49,000 ± 500 with a sedimentation coefficient of S20w≃ 3. Antibodies raised against th…

Antibodies against homogeneous epoxide hydratase provide evidence for a single enzyme hydrating styrene oxide and benz(a)pyrene 4,5-oxide

THE microsomal enzyme epoxide hydratase (EC 4.2.1.63) is potentially important in the inactivation of metabolically produced epoxides which may be responsible for the mutagenic and/or carcinogenic properties of polycyclic hydrocarbons (for reviews see refs 1–3). Reports4,5 suggest that the enzyme plays a dual role in (a) producing proximate carcinogens which, after biotransformation to carcinogens by microsomal mono-oxygenase(s) are (b) inactivated by epoxide hydratase. As this enzyme can be induced6–8, activated9–10 and inhibited9–13 it should be useful in studies of the mechanism of chemical carcinogenesis: some inverse correlations have been reported between susceptibility to carcinogene…

Dihydrodiol Dehydrogenase: An Important Enzyme in Dihydrodiol-Epoxide Pathway — Mediated Benzo(A)Pyrene Mutagenicity

Benzo(a)pyrene is metabolized to two major groups of mutagenically reactive metabolites: Monofunctional epoxides and dihydrodiol-epoxides. Various monooxygenase forms catalyze the various pathways at very different rates. In metabolic situations where the contribution by dihydrodiol-epoxides is small, epoxide hydratase represents a very efficient protective system. However, in situations where the mutagenic effect is predominately due to dihydrodiol-epoxide, the effect of epoxide hydratase is complicated and weak. We have now obtained evidence that a dihydrodiol dehydrogenase represents an efficient protective system in the latter situation. The enyzme was purified to homogeneity and the pu…

Prevention of benzo(a)pyrene-induced mutagenicity by homogeneous epoxide hydratase

Benzo(a)pyrene and benz(a)anthrancene which, in contrast to the K-region epoxides benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, are not mutagenic to Salmonella typhimurium TA 1537 in the absence of mammalian enzyme preparations, were activated by liver microsomes from C3H mice, which had not received any pretreatment, to mutagens reverting this tester strain to histidine prototrophy. Addition of epoxide hydratase inhibitors greatly increased this mutagenicity and addition of pure epoxide hydratase reduced it by more than 95% down to the range of spontaneous mutations as observed in absence of any added mutagen. This demonstrates that the metabolic pathway responsible for the mut…

Epoxide Hydratase: Purification to Apparent Homogeneity as a Specific Probe for the Relative Importance of Epoxides among Other Reactive Metabolites

Aromatic and olefinic compounds can be metabolized by microsomal monooxygenases to epoxides which chemically represent electrophilic species (for reviews, see refs. 1–5). Spontaneous binding of such epoxides to DNA, RNA, and protein has been observed (6–10). Accordingly, such metabolites have been suggested and, in some instances, shown to disturb the normal functions of cells, leading to such effects as mutagenesis (11–14), malignant transformation (15–19), or cell necrosis (20). However, aromatic and olefinic compounds are biotransformed to a vast array of metabolites (cf. refs. 21–27), possibly including a considerable number of reactive metabolites other than epoxides. The relative impo…

Endogenous Role of Microsomal Epoxide Hydrolase

The specific activities of microsomal epoxide hydrolase with 16α,17α-epoxyandrosten-3-one (androstene oxide) as substrate were measured in various metabolically important and in various steroidogenic organs of the male and female rat and compared with the activities of 16α,17α-epoxyestratrienol (estroxide) and benzo[a]pyrene 4,5-oxide. Androstene oxide was an exceptionally good substrate. The specific activities differed widely between organs but the ratio of the activities towards these substrates was constant in all organs investigated. The ratios compared to benzo[a]pyrene 4,5-oxide were 2.5 for estroxide, and 8.6 for androstene oxide. The ontogenetic development of specific epoxide hydr…

DRUG-DRUG INTERACTIONS VIA INHIBITION OF MICROSOMAL ENZYMES INVOLVED IN METABOLISM OF EPOXIDES PRODUCED BY MICROSOMAL MONOOXYGENASE

SUMMARY Benzo(a)pyrene was activated by liver microsomes to mutagens detected by the reversion of histidine dependent Salmonella typhimurium TA 1537. Using pure epoxide hydratase or epoxide hydratase inhibitors, comparing animal species with high and low epoxide hydratase activity, or inducing monooxygenase activity, it was shown that epoxide hydratase was a critical enzyme for the inactivation of these mutagens. Many clinically used drugs are metabolized to epoxides. Epoxides are not necessarily mutagenic, but since epoxide hydratase has a very low substrate specificity, such epoxides may competitively inhibit the hydration of mutagenic epoxides, as demonstrated in the present study for th…

Dual role of epoxide hydratase in both activation and inactivation of benzo(a)pyrene.

The effect of epoxide hydratase upon the mutagenicity of benzo(a)pyrene was investigated using two Salmonella typhimurium strains (TA 1537 and TA 98). These two bacterial strains were found to differ characteristically in their susceptibility to different mutagens biologically produced from benzo(a)pyrene providing a diagnostic tool to investigate which types of mutagenic metabolites were produced in various metabolic situations. The results showed that the pattern of mutagenic metabolites produced by microsomes from methylcholanthrene-treated mice was very different from that produced by microsomes from phenobarbital-treated or untreated mice. However in all cases at least two mutagenic me…

Reduction of benzo(a)pyrene mutagenicity by dihydrodiol dehydrogenase

THE enigma of how inert chemicals can exert potent mutagenic, carcinogenic, allergenic and cytotoxic effects has been much debated. It has been learned that such compounds are metabolically converted to chemically reactive species1. In the case of aromatic or olefinic compounds, monooxygenases located in the membranes of the cell can transform these compounds into epoxides2–5 which by virtue of electrophilic reactivity can bind chemically to cellular macromolecules such as DNA, RNA and proteins, thereby disturbing biochemical control mechanisms and leading to the above mentioned toxic effects. The same membranes in which such epoxides are produced possess an enzyme, epoxide hydratase, which…

Epoxides derived from various polycyclic hydrocarbons as substrates of homogeneous and microsome-bound epoxide hydratase. A general assay and kinetic properties.

A general assay for epoxide hydratase using epoxides derived from polycyclic aromatic hydrocarbons as substrates is described. Addition of dimethylsulphoxide to the incubation mixture after incubation allowed unreacted epoxide and its phenolic by-product to be extracted into light petroleum whilst the product dihydrodiol remained in the aqueous phase. The product was then extracted into ethyl acetate and estimated radiochemically. This assay gave low extraction blanks (0.8-3.8%) when six K-region epoxides of polycyclic hydrocarbons were used, with high recoveries of the corresponding dihydrodiol in the ethyl acetate phase (65-89%). Radiochromatograms demonstrated that all the radioactivity …