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J Seidegård

Publications and source records attributed to J Seidegård.

54 records · Page 3Linked to original sources
PubMed1984

Characterization of soluble glutathione transferase activity in resting mononuclear leukocytes from human blood.

Glutathione transferase activity in the soluble fraction of resting human mononuclear leukocytes was measured and characterized using [3H] trans-stilbene oxide as a substrate. Because of the low activity of this enzyme in these cells, a published assay procedure developed for rodent liver was slightly modified to improve its sensitivity: the substrate was highly radiolabeled (2 Ci/mmole) and carefully purified, and the incubation time was extended to 30-60 min. The activity measured was linear with cell density up to at least 6 million cells. Soluble glutathione transferase activity measured in this manner has a pH optimum around 7.4 and an optimal temperature of 40 degrees. This activity could be measured in lymphocytes, monocytes, granulocytes, erythrocytes and platelets, but not in plasma. From these measurements it could be calculated that lymphocytes account for somewhat more than half of the total activity in the mononuclear leukocyte fraction and that monocytes account for the rest. The intraindividual variation in soluble glutathione transferase activity towards trans-stilbene oxide in the mononuclear leukocyte fraction from different subjects was only about 10%, whereas the interindividual variation in this same activity was 15-fold. An explanation for this relatively large interindividual variation is now being sought.

Benzopyrene Hydroxylase↗
PubMed1984

Induction of cytosolic glutathione transferase and microsomal epoxide hydrolase activities in extrahepatic organs of the rat by phenobarbital, 3-methylcholanthrene and trans-stilbene oxide.

The effects of treating male Sprague-Dawley rats with phenobarbital, 3-methylcholanthrene or trans-stilbene oxide on cytosolic glutathione transferase and microsomal epoxide hydrolase activities in the liver, intestine, kidney, lung, testis, adrenal, spleen, heart and brain have been investigated. Studies on the time-courses of induction in liver demonstrate that these are complete after five days' treatment at the doses used. Phenobarbital induces both cytosolic glutathione transferase and microsomal epoxide hydrolase activities significantly only in liver and intestine. 3-Methylcholanthrene induces these activities in liver only. Trans-Stilbene oxide induces both of these activities in liver and kidney, and cytosolic glutathione transferase activity in adrenal as well.

Animals↗
PubMed1984

Measurement and characterization of membrane-bound and soluble epoxide hydrolase activities in resting mononuclear leukocytes from human blood.

Membrane-bound and soluble epoxide hydrolase activities in the mononuclear cell fraction from human blood have been characterized using cis- and trans-stilbene oxides as substrates, respectively. Because of the low activities in these cells, it was necessary to modify assay procedures developed for rat and mouse liver in the following ways: (a) the substrates were relatively highly labeled (2 Ci/mmol) and carefully purified; (b) the incubation time was extended to 45 to 60 min, during which period the activities were linear; (c) as many as 6 million cells were used for a single assay, which was also within the linear range of the procedure. The membrane-bound epoxide hydrolase characterized in this manner has an apparent Vmax of 7.26 pmol product formed per min per 10(7) cells and an apparent Km of 9.96 microM. The pH optimum was observed to be around 9.8. The dependence of this activity on temperature showed its optimum at 40 degrees. The soluble epoxide hydrolase activity has an apparent Vmax of about 8.26 pmol product formed per min per 10(7) cells, an apparent Km of 1.63 microM, a pH optimum of 6.2 to 6.8, and a temperature optimum at 60 degrees. Using these techniques, these activities have also been determined in other blood components, i.e., lymphocytes, monocytes, granulocytes, erythrocytes, platelets, and plasma. Lymphocytes account for most of the epoxide hydrolase activity towards cis-stilbene oxide, and all of the activity towards trans-stilbene oxide is in the human mononuclear cell fractions. Different substances known to affect rodent epoxide hydrolases were tested for their effects on the human mononuclear blood cell activities. Interestingly, 1,1,1-trichloropropane 2,3-epoxide, a potent inhibitor of liver microsomal epoxide hydrolase in different species including rat, mouse, and human, had little or no effect on the membrane-bound activity measured here. However, cyclohexene oxide inhibits this membrane-bound activity 60%. The soluble epoxide hydrolase is inhibited to 90% of control levels by chalcone epoxide. The membrane-bound and soluble epoxide hydrolase activities determined in 27 subjects varied from 8.2 to 18.5 and from 3.5 to 17.0 pmol product formed per min per 10(7) cells, respectively. The mean coefficient of intraindividual variation, determined with three subjects measured four times each over the course of 18 days, was approximately 10% for both enzyme activities.

Blood Platelets↗
PubMed1982

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.

Animals↗
PubMed1982

Preparation and characterization of subcellular fractions from the liver of the Northern Pike, Esox lucius.

The present study was designed to prepare and characterize subcellular fractions from the liver of the Northern pike (Esox lucius), with special emphasis on the preparation of microsomal fractions suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation, as well as the recovery of different organelles, was determined using both enzyme markers and morphological examination with the electron microscope. Attempts were also made to increase the recovery of fragments of the endoplasmic reticulum in the microsomal fraction. Finally, the subcellular distribution of several drug-metabolizing enzymes (cytochrome P-450, benzpyrene monoxygenase, epoxide hydrolase and glutathione transferases) were determined. With the exception of the subcellular distribution of epoxide hydrolase, the results obtained here resemble closely those reported fo rat liver and the microsomal fraction prepared is highly suitable for further studies of drug metabolism in pike liver.

Animals↗
PubMed1982

The effect of trans-stilbene oxide and other structurally related inducers of drug-metabolizing enzymes on glucuronidation.

Administration of trans-stilbene oxide, and new type of inducer of drug-metabolizing enzymes, to rats was found to increase hepatic microsomal UDP-glucuronyl transferase activity with both p-nitrophenol and chloramphenicol as substrate. In Triton X-100 activated microsomes the increase with p-nitrophenol as substrate was to approx. 250% of the control value, while the corresponding value for chloramphenicol was about 600%. These observations indicate that trans-stilbene oxide causes a mixed type 'induction' of UDP-glucuronyl transferase(s), i.e., changes in activity which resemble both those seen after induction with phenobarbital and after treatment with 3-methylcholanthrene. We have also shown that the activity of UDP-glucose dehydrogenase, the enzyme which produces UDP-glucuronic acid, is increased to about 300% of the control after administration of trans-stilbene oxide. The time course of this increase and of the return to control activity after cessation of treatment, the dose-response of this increase and the structural features of the trans-stilbene oxide molecule which are essential for the increase have all been examined. The other two enzymes involved in the conversion of glucose 6-phosphate to UDP-glucuronic acid, namely, phosphoglucomutase and UDP-glucose pyrophosphorylase, were found to be only slightly affected (a 30-60% increase) by treatment with trans-stilbene oxide. After induction with trans-stilbene oxide the hepatic level of UDP-glucuronic acid was unchanged.

Animals↗
PubMed1981

Induction of drug-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.

Animals↗
PubMed1980

Benzil, a potent activator of microsomal epoxide hydrolase in vitro.

Benzil was found to be a very potent activator of microsomal epoxide hydrolase activity (measured with styrene oxide as substrate) in vitro. The activating effect was uncompetitive and benzil causes approximately ninefold increases in both the apparent V and the apparent Km of the enzyme(s). The half-maximal effect on activity was obtained as a 0.3 mM concentration of benzil. The activating effect obtained with benzil was found to be very specific, since a variety of structurally related compounds had little or no effect on microsomal epoxide hydrolase activity. In order to obtain indications for the existence of more than one microsomal epoxide hydrolase the effect of benzil on this activity from rats induced with phenobarbital, 3-methylcholanthrene, 2-acetylaminofluorene, trans-stilbene oxide, and benzil was tested. The differences observed were minor.

Aldehydes↗
PubMed1978

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.

Animals↗
PubMed1977

Characterization of rat lung epoxide (styrene oxide) hydrase with a modified radioactive assay of improved sensitivity.

The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.

Alcohols↗