Philip Bentley

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 monooxygenases to dihydrodiol epoxides which may represent ultimate carcinogens. If this is the case epoxide hydratase could play a critical role being responsible both for forming the proximate carcinogen and removing the ultimate carcinogen. Indeed an inverse correlation between susceptibility to carcinogenesis by polycyclic hydrocarbons and inducibility of epoxide hydratase by the same agents at different developmental stages of the same animal species has been observed [6], A thorough knowledge of the properties of the enzymes involved in the metabolic activation and inactivation processes is essential for an understanding of the mechanisms of cytotoxicity and carcinogenesis by polycyclic hydrocarbons. Many research groups have studied the monooxygenases and several have recently reported a purification of cytochromes I’450 and ph4s [7-91. However, although many inhibitors, activators, and inducers of epoxide hydratase exhibiting various degrees of specificity are known [2] , pure

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 metabolites were produced. Epoxide hydratase was very efficient at reducing the mutagenic effect when benzo(a)pyrene was activated by microsomes from untreated or phenobarbital-treated mice. However, when microsomes from methylcholanthrene-treated mice were used the effect of hydratase depended upon the benzo(a)pyrene concentration. At low concentrations the mutagenicity was increased by addition of epoxide hydratase and decreased by inhibition of the hydratase. At high concentrations the reverse was true. These findings indicate that when microsomes from untreated and phenobarbital-treated mice were used the main contributors to the mutagenicity were simple epoxides (or compounds arising non-enzymically from them). The activation of dihydrodiols must, however, contribute to a significant extent when microsomes from methylcholanthrene-treated mice were used. Thus the role of epoxide hydratase was determined by the monooxygenase form present in the microsomes in the activating system.ZusammenfassungDie Rolle der Epoxidhydratase wurde untersucht in bezug auf die Mutagenität von Benzo(a)pyren. Benzo(a)pyren wurde mit Lebermikrosomen aktiviert. Mutagene wurden festgestellt anhand der Reversion der his−Salmonella typhimurium-Stämme TA 1537 und TA 98. Die beiden Stämme wurden sehr unterschiedlich durch verschiedene mutagene Benzo(a)pyren-Metabolite rückmutiert. Es zeigte sich, daß das Muster der mutagenen Metabolite, die durch Mikrosomen von Methylcholanthren-behandelten Mäusen aus Benzo(a)pyren gebildet wurden, sehr verschieden war vom Muster bei Aktivierung durch Mikrosomen von Kontroll-oder von Phenobarbital-behandelten Mäusen. Jedoch trugen in allen drei Fällen wenigstens zwei verschiedene mutagene Metabolite signifikant zur Mutagenität bei. Epoxidhydratase reduzierte sehr effektiv die Mutagenität, wenn Benzo(a)pyren durch Mikrosomen von Kontroll-oder von Phenobarbital-behandelten Mäusen aktiviert wurde. Wenn jedoch Mikrosomen von Methylcholanthren-behandelten Tieren verwendet wurden, war der Effekt der Epoxidhydratase stark von der Benzo(a)pyren-Konzentration abhängig. Bei niedriger Konzentration erhöhte Zugabe von Epoxidhydratase und erniedrigten Epoxidhydratasehemmstoffe die Mutagenität. Bei hohen Konzentrationen wurde das Umgekehrte festgestellt.Diese Befunde wurden dahingehend interpretiert, daß bei der Aktivierung mit Mikrosomen von unbehandelten und von Phenobarbital-induzierten Mäusen einfache Epoxide (oder Substanzen, die nicht-enzymatisch daraus gebildet wurden) hauptsächlich für die Mutagenität verantwortlich waren, daß dagegen Mutagene, die über Dihydrodiole gebildet wurden, bedeutend zur Mutagenität beitrugen, wenn Mikrosomen von Methylcholanthren-behandelten Mäusen verwendet wurden.Die Rolle der Epoxidhydratase, ob aktivierend oder inaktivierend, wird demnach bestimmt durch die Form der Monooxygenase, die an der Aktivierung beteiligt ist.

[46] Microsomal epoxide hydrolase

Publisher Summary Microsomal epoxide hydrolase catalyzes the conversionof epoxides to glycols. The microsomal enzyme should not be confused with another epoxide hydrolase activity, found primarily in the cytosolic fraction, which differs greatly from membrane-bound enzyme in substrate specificity and immunological properties. As with other membrane-bound enzymes, it is highly lipophilic and easily forms aggregates in solution. Its physical properties, therefore, present special problems for purification. The general scheme of purification presented in this chapter consists of solubilization from microsomes by a nonionic detergent; chromatography with siethylaminoethyl-cellulose and phosphocellulose, which effect purification on the basis of charge; chromatography with butylsepharose, which effects purification on the basis of hydrophobicity; and the removal of detergent by a second phosphocellulose step. Enzyme activity can be assayed by the conversion of radiolabeled epoxide substrates to dihydrodiols and the subsequent separation of products from substrate by simple solvent extraction. Tritiated benzo[a]pyrene 4,5-oxide and styrene 7,8- oxide can be used to monitor enzyme activity during purification. One enzyme unit is defined as the amount catalyzing the hydration of 1 pmol of styrene oxide in 1 min under specific conditions.

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. We report here the results of initial investigations with the pure enzyme. 2. Materials and methods Anhydrous hydrazine was obtained from Pierce Chem. Co., USA. Carboxypeptidases A and B were isopropylphosphate treated preparations and were obtained from Worthington Biochemical Co. Carboxy- peptidase Y (from Yeast) and Carboxypeptidase P (from Penicillium) were kindly donated by Dr R. Hayashi from Kyoto University, Japan, and Dr E. Ichishima, Tokyo Noko University, Japan. Epoxide hydratase activity was assayed under the conditions 296 described in detail [5], specifically in the absence of Tween 80. 2.1. Amino acid analysis Samples containing 250 ,ug protein were dialysed against 0.2 M borate buffer, pH 9.0, free ammonia was removed by placing samples in a boiling water bath for 5 min and samples were dried in vacua over H2S04. Samples were hydrolysed under reduced pressure at 108 + 1°C [6] in 1 ml of twice distilled 5.7 N HCl for 24 h and 144 h in evacuated sealed tubes. The hydrolysates were dried under vacuum with a rotary evaporator at 80°C. The dried residues were divided into five portions and one portion (50 pg) was applied to one column. The analysis of the hydrolysates was carried out with a Beckman amino acid analyser model 4255, equipped with a 5 cm column (for basic amino acids) and 50 cm column (for acidic and neutral amino acids) at a flow rate of 30 ml/h according to Spackmann et al. [7]. Trypto- phan was analysed using 5% thioglycolic acid [8]. Tyrosine and tryptophan residues were also determined spectrophotometrically in 0.1 N NaOH according to Goodwin Morton [9]. Cyst(e)ine methionine residues were also determined after performic acid oxidation of the protein [lo] N-terminal analysis was performed using [‘“Cl - dinitrofluorobenzene [ 1 l] and the Edman degrada- tion methods [ 121. C-terminal analysis was performed by hydrazinolysis [ 131 and by carboxypeptidase

Specificity of human, rat and mouse skin epoxide hydratase towards K-region epoxides of polycyclic hydrocarbons

Abstract Epoxide hydratase activity has been measured in microsomal fractions of skin from mouse, rat and humans. The skin enzyme was able to hydrate all epoxides tested. The specific enzyme activities decreased in the order human > mouse > rat. The relative activity towards K-region epoxides of various polycyclic hydrocarbons in skin microsomal fractions from all three species decreased in the order phenanthrene 9,10-oxide > benz(a)anthracene 5,6-oxide ≃ benzo(a)pyrene 4,5-oxide ≃ 7-methylbenz(a)anthracene 5,6-oxide > 3-methylcholanthrene 11,12-oxide > dibenz(a,h)anthracene 5,6-oxide. The activity of epoxide hydratase in human skin microsomal fractions showed little pH dependence and was inhibited by small molecular weight inhibitors in a manner similar to that of the liver microsomal enzyme. Interindividual variation of epoxide hydratase activity in skin microsomal fractions from six human subjects was considerable, namely from 175 to 447 pmoles benzo(a)pyrene 4,5-dihydrodiol/min per mg protein. This variation was not due to skin disease or treatment and had no apparent correlation with age or sex. A possible correlation with the part of the body from which the skin sample was taken could not be excluded since the activity in skin samples from the abdomen seemed lower than that in samples from leg or breast.

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 importance of epoxides among other reactive metabolites is at present unknown. With respect to the model compound used in this study, benzo[a]pyrene, our previous studies had shown that the 4,5- (K-region-) epoxide metabolite was a potent mutagen for the frameshift-sensitive Salmonella strains TA 1537 and TA 1538 (28), that the premutagenic hydrocarbon required a NADPH-supported microsomal monooxygenase system to become mutagenically active, and that the mutagenic response was potentiated by the presence of epoxide hydratase inhibitors at concentrations where no interference with other systems has been observed (28). Yet no conclusion could be reached whether the relative contribution of epoxide metabolites to the overall muta-genic effect of bioactivated benzo[a]pyrene was of any significance since the potentiation of the mutagenic effect by epoxide hydratase inhibitors could simply mean that blocking this pathway led to an accumulation of epoxides, making them important in this situation, while in absence of such inhibitors their contribution to the overall mutagenic effect may have been negligible.

Enzymic hydration of benzene oxide: Assay and properties

Abstract A radiometric assay for epoxide hydratase using [ 14 C]benzene oxide as substrate has been developed. The reaction product trans -1,2-[ 14 C]dihydroxy-1,2-dihydrobenzene (benzene dihydrodiol) was separated from the other components by simple extraction of the unreacted substrate and phenol (a rearrangement product) into a mixture of light petroleum and diethyl ether followed by extraction of the benzene dihydrodiol into ethyl acetate. The product was then estimated by scintillation counting. Using this assay the enzymic hydration of benzene oxide and the possible existence of a microsomal epoxide hydratase with a greater specificity toward benzene oxide were reinvestigated. The sequence of activities of microsomes from various organs was liver > kidney > lung > skin, the pH optimum of enzymic benzene oxide hydration was about pH 9.0, which is similar to that of styrene oxide hydration and both activities were equally stable when liver microsomal fractions were stored. The effect of low molecular weight inhibitors upon the hydration of styrene and benzene oxide by liver microsomes was similar in some cases and dissimilar in others. However, all the dissimilarities could be explained without recourse to the hypothesis of the existence of a separate benzene oxide hydratase. During enzyme purification studies the activity toward benzene oxide was inhibited by the detergent used (cutscum) but was recovered when the detergent was removed. Solubilization without significant loss of activity was successful using sodium cholate. This allowed immunoprecipitation studies, which were performed using monospecific antiserum raised against homogeneous epoxide hydratase. The dose-response curves of the extent of precipitation of activity with increasing amounts of added antiserum were indistinguishable for benzene oxide and styrene oxide as substrate. At high antiserum concentrations precipitation was complete with both substrates. The findings, taken together, indicate the presence in rat liver microsomes of a single epoxide hydratase catalyzing the hydration of both styrene and benzene oxide or the presence of enzymes so closely related that these cannot be distinguished by any of the criteria tested.

Influence of carbamazepine 10, 11-oxide on drug metabolizing enzymes☆

Induction studies with carbamazepine 10,11-oxide (carbamazepine oxide) were carried out with male Sprague-Dawley rats. After 3 and 7 days intraperitoneal application of carbamazepine oxide, the amount of cytochrome P-450 and the activities of monooxygenases and epoxide hydratase in liver microsomes and the glutathione S-transferase activity in the 100,000 g supernatant fraction were measured. After 3 days of pretreatment with carbamazepine oxide, the specific monooxygenase activity with 7-ethoxycoumarin as substrate was induced by 115 per cent. The specific activity of epoxide hydratase measured with styrene oxide was 46 per cent higher than in the controls. The maximal induction of glutathione 5-transferase activity was 57 per cent. The amount of cytochrome P-450 in rat liver microsomes was not affected by carbamazepine oxide. The maximal induction of all enzyme activities measured was reached after three days of application. Seven days treatment with carbamazepine oxide resulted in no further induction. Inhibition of epoxide metabolizing enzymes by carbamazepine oxide was also investigated. The enzymatic activities of epoxide hydratase and glutathione S-transferases were measured in the absence and after addition of carbamazepine oxide in vitro. In order to allow the use of substrate concentrations far below Km, the epoxide hydratase was measured with a sensitive radiometric assay using 14C-styrene oxide of a high specific radioactivity. Under all conditions studied, carbamazepine oxide showed no significant inhibition of the epoxide hydratase. The glutathione S-transferse activity was measured in the 100,000 g supernatant fraction of rat liver homogenate with 1-chloro-2, 4-dinitrobenzene and glutathione as substrates. Under all conditions tested, only the highest concentration of carbamazepine epoxide (1.0 mM) at unphysiologically low glutathione concentrations (0.25 mM, 0.10 mM) resulted in a borderline significant (P < 0.05) inhibition of maximally 19 per cent.

A highly sensitive assay for epoxide hydrolase using an endogenous epoxide as substrate: 16α, 17α-epoxyandrost-4-en-3-one

Abstract A highly sensitive and convenient assay for epoxide hydrolase, which uses a tritiated endogenous steroid epoxide (16α, 17α-[17-3H]epoxyandrost-4-en-3-one) as substrate, is described. The method involves a simple extraction procedure for separating the product of the enzymatic reaction (16β,17α-dihydroxyandrost-4-en-3-one) from the incubation mixture and unreacted epoxide. Quantitation of epoxide hydrolase activity is achieved by scintillation photometry of the tritium-labeled diol product. The assay is very sensitive because of (a) the low blank value (0.2% of added radioactivity, because nonenzymaic hydrolysis of the substrate could not be detected and a very low amount of radioactive substrate and/or impurities are left in the final sample) and (b) the V of the enzyme toward androstene oxide (55 nmol diol/min/mg protein) and the resulting low amount of protein required per incubation. These features and the simplicity of the method make this assay widely applicable and valuable for the determination of epoxide hydrolase activity in tissue samples of low mass or low activity. Additionally, the assay is very important for the investigation of the physiological role of epoxide hydrolase, since it uses an endogenous substrate of the enzyme.

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 pure enyzme added to systems generating mutagenically active benzo(a)pyrene metabolites by the presence of various monooxygenase forms. In situations where epoxide hydratase had only weak effects, dihydrodiol dehydrogenase afforded efficient protection and vice-versa.