Janeric Seidegård

Trans-stilbene oxide: A new type of iducer of drug-metabolizing enzymes

Abstract Changes in hepatic drug-metabolizing enzymes after interperitoneal administration of trans -stilbene oxide to rats have been investigated. This treatment increases the specific content of cytochrome P -450 over 2-fold, the specific activity of epoxide hydratase about 7-fold, and the specific activities of glutathione S -transferases 3- to 4-fold. On the other hand, enzyme markers for the soluble cytoplasm, lysosomes, the plasma membrane, and the inner mitochondrial membrane were not affected. The time course and the dose response of the changes brought about by trans -stilbene oxide have been characterized. trans -Stilbene oxide seems to be a different type of inducer of drug-metabolizing enzymes than either phenobarbital or methylcholanthrene.

Induction of durg-metabolizing systems and related enzymes with metabolites and structural analogues of stilbene

trans-Stilbene oxide has been found to be a new type of inducer of drug-metabolizing systems. In order to identify the true inducer and to determine the structural requirements for induction, rats were treated with metabolites and structural analogues of stilbene. Subsequently, hepatic levels of cytochrome P-450, microsomal epoxide hydrolase, and cytoplasmic glutathione S-transferase were assayed. All three enzymes were induced by cis- and trans-stilbene and cis- and trans-stilbene oxide. In addition, epoxide hydrolase and glutathione S-transferase activities were induced by benzoin and benzil. In contrast, the diols and benzoic acid had little, if any, effect. The main conclusions drawn from these findings are that: (1) trans-stilbene oxide itself seems to be the inducer of drug-metabolizing enzymes; and (2) benzil is more selective as an inducer of epoxide hydrolase than is trans-stilbene oxide. Attempts to induce epoxide hydrolase with other structural analogues of stilbene led to the following conclusions: (1) two phenyl rings are required for induction; (2) the induction is not as great if the rings are substituted or one of the ring carbon atoms is replaced by a nitrogen; (3) a carbon bridge between the phenyl groups generally results in a greater induction, especially if the bridge contains an epoxy group or one or two keto groups.

The topology of epoxide hydratase and benzpyrene monooxygenase in the endoplasmic reticulum of rat liver.

The distributions of benzpyrene monooxygenase and epoxide hydratase in subfractions of liver microsomes from control and from phenobarbital- and methylcholanthrene-treated rats have been investigated. The specific activities of these enzymes in rough and smooth microsomes from control and phenobarbital-treated animals are approximately the same, whereas after methylcholanthrene treatment benzpyrene monooxygenase is four times higher and epoxide hydratase twice as high in the rough vesicles. Further subfractionation of rough and smooth microsomes by rate differential centrifugation revealed the distributions of both enzymes among microsomal vesicles to be highly heterogeneous. Comparison of these distributions leads to the conclusion that the benzpyrene monooxygenase system and epoxide hydratase may form a complex of unique stoichiometry in the membrane of microsomes from control rats, but that such a complex is not consistent with the distributions obtained after methylcholanthrene induction. Studies with proteases and the nonpenetrating chemical reagent diazobenzene sulfonate suggest that epoxide hydratase may be buried deeply in the hydrophobic phase of the membrane of the hepatic endoplasmic reticulum.

Induction of hepatic cytosolic DT diaphorase in rats treated with trans—stilbene oxide

The flavoprotein DT diaphorase (NAD(P)H:oxidoreductase, EC 1.6.99.2) is a highly dicoumarolsensitive enzyme catalyzing the oxidation of NADH and NADPH by various quinones and redox dyes [l-3]. The physiological electron acceptor for DT diaphorase has not yet been established. However, it has been suggested that the enzyme serves as a quinone reductase in connection with conjugation of hydroquinones during detoxification, as well as in biosynthetic processes such as the vitamin K-dependent y-carboxylation of glutamyl residues in prothrombin synthesis (reviewed [3]). Treatment of rats with polycyclic aromatic hydrocarbons, potent inducers of the aryl hydrocarbon monooxygenase system [4,5], is known to increase hepatic DT diaphorase [6-l 11. The induction of DT diaphorase is due to an increase in the amount of the enzyme [ 121. The coinduction of DT diaphorase and aryl hydrocarbon monooxygenase [3,9] has drawn attention to a possible linkage between the genetic regulation of these activities. truns8tilbene oxide has been suggested to represent a new class of inducers of drug-metabolizing enzymes in the rat [ 13-161. This compound preferentially induces epoxide hydratase [ 13-161 and glutathione S-transferases [ 161, whereas cytochrome P450 linked monooxygenase reactions are only moderately affected [ 13-171. Because of the speci-

The effect of trans-stilbene oxide and other structurally related inducers of drug-metabolizing enzymes on the pentose phosphate pathway and other enzymes of carbohydrate metabolism

trans-Stilbene oxide has been found earlier to be a new type of inducer of drug-metabolizing systems. Here we demonstrate that treatment of rats with this xenobiotic results in an increase in the activity of the cytosolic glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, the first and third enzymes in th pentose phosphate pathway, to 350% and 170% of the control values, respectively. At the time microsomal glucose 6-phosphate dehydrogenase activity was unaffected by administration of trans-stilbene oxide or benzil. The time course and dose-response of the increases in glucose 6-phosphate and 6-phosphogluconate dehydrogenase activities have been characterized. The activities of ribulose 5-phosphate 3-epimerase and ribose 5-phosphate activities have been characterized. The activities of ribulose 5-phosphate 3-epimerase and ribose 5-phosphate ketol isomerase, enzymes further along in the pentose phosphate pathway, were not significantly affected by trans-stilbene oxide or benzil. An investigation of the effect of treating rats with different metabolites of stilbene and with other structurally related compounds on hepatic cytosolic glucose 6-phosphate dehydrogenase activity revealed the structural features which are important for increasing this activity. Finally, it was found that administration of trans-stilbene oxide did not affect the activities of glucokinase and phosphoglucose isomerase, the two glycolytic enzymes which can produce glucose 6-phosphate, the link between glycolysis and the pentose phosphate shunt.

EPOXIDE HYDRATASE IN RAT LUNG

Publisher Summary This chapter describes a study to analyze epoxide hydratase in rat lung. The procedure used for measuring epoxide hydratase activity was essentially that developed for Oesch for liver microsomes. Radioactive styrene oxide is incubated with the microsomes, and the remaining substrate is then selectively extracted into petroleum ether. Finally, the styrene glycol product is extracted into ethyl acetate, and scintillation counting is performed. However, this procedure had to be modified in three ways to make it sensitive enough for routine use with rat lung preparations. The substrate is purified before use. The volume of the incubation mixture is scaled down fourfold, and the incubation time is lengthened 9–12-fold. These changes increase the sensitivity of the assay procedure 75–150-fold, thus providing a reliable and convenient method for characterizing epoxide hydratase in tissues where its activity is quite low. Because polycyclic hydrocarbons appear to cause cancer much more easily in lung than in liver, and because epoxide hydratase may play an important role in the etiology of such cancer, it is of interest to compare the properties of epoxide hydratase in these two organs.

Benzil, a Selective Inducer and a Potent in Vitro Activator of Microsomal Epoxide Hydrolase

It is now well known that many chemical carcinogens, including the polycyclic hydrocarbons, must be metabolized to reactive intermediates before they can exert their biological effectsl. It is believed that both the carcinogenicity and mutagenicity of polycyclic hydrocarbons results from the covalent binding of such intermediates to protein, RNA, and/or DNA. In the case of benzo(a)pyrene the diol epoxide 7,8-dihydro-7,8dihydroxybenzo(a)pyrene-9,10-epoxide may well be the ultimate carcinogen.

DRUG-METABOLIZING SYSTEMS IN THE LIVER OF THE NORTHERN PIKE, ESOX LUCIUS

NADPH-cytochrome P-450 reductase, cytochrome P-450, benzpyrene monooxygenase, epoxide hydratase, and glutathione S-transferase activities in the liver of the Northern pike ( Esox lucius ) have been measured and partially characterized.