[Studies on the microsomal antigens of Schistosoma japonicum. II. Extraction, fractionation and chemical and immunological properties of microsomal antigens of adult worms].
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Animals↗
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Immunochemical study on the contributions of two molecular species of microsomal cytochrome P-450 to the metabolism of 2-acetylaminofluorene by rat liver microsomes.
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2-Acetylaminofluorene↗
Selective changes in microsomal enzymes of triacylglycerol and phosphatidylcholine synthesis in fetal and postnatal rat liver. Induction of microsomal sn-glycerol 3-phosphate and dihydroxyacetonephosphate acyltransferase activities.
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Acyltransferases↗
Microsomal metabolism of benzene to species irreversibly binding to microsomal protein and effects of modifications of this metabolism.
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Animals↗
Identification and characterization of a novel microsomal enzyme with glutathione-dependent transferase and peroxidase activities.
5-Lipoxygenase activating protein (FLAP), leukotriene-C4 (LTC4) synthase, and microsomal glutathione S-transferase II (microsomal GST-II) are all members of a common gene family that may also include microsomal GST-I. The present work describes the identification and characterization of a novel member of this family termed microsomal glutathione S-transferase III (microsomal GST-III). The open reading frame encodes a 16.5-kDa protein with a calculated pI of 10.2. Microsomal GST-III has 36, 27, 22, and 20% amino acid identity to microsomal GST-II, LTC4 synthase, microsomal GST-I, and FLAP, respectively. Microsomal GST-III also has a similar hydrophobicity pattern to FLAP, LTC4 synthase, and microsomal GST-I. Fluorescent in situ hybridization mapped microsomal GST-III to chromosomal localization 1q23. Like microsomal GST-II, microsomal GST-III has a wide tissue distribution (at the mRNA level) and is predominantly expressed in human heart, skeletal muscle, and adrenal cortex, and it is also found in brain, placenta, liver, and kidney tissues. Expression of microsomal GST-III mRNA was also detected in several glandular tissues such as pancreas, thyroid, testis, and ovary. In contrast, microsomal GST-III mRNA expression was very low (if any) in lung, thymus, and peripheral blood leukocytes. Microsomal GST-III protein was expressed in a baculovirus insect cell system, and microsomes from Sf9 cells containing either microsomal GST-II or microsomal GST-III were both found to possess glutathione-dependent peroxidase activity as shown by their ability to reduce 5-HPETE to 5-HETE in the presence of reduced glutathione. The apparent Km of 5-HPETE was determined to be approximately 7 microM for microsomal GST-II and 21 microM for microsomal GST-III. Microsomal GST-III was also found to catalyze the production of LTC4 from LTA4 and reduced glutathione. Based on these catalytic activities it is proposed that this novel membrane protein is a member of the microsomal glutathione S-transferase super family, which also includes microsomal GST-I, LTC4 synthase, FLAP, and microsomal GST-II.
Amino Acid Sequence↗
Evidence that biosynthesis of phosphatidylethanolamine, phosphatidylcholine, and triacylglycerol occurs on the cytoplasmic side of microsomal vesicles.
Experiments were performed to localize the hepatic microsomal enzymes of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol biosynthesis to the cytoplasmic or lumenal surface of microsomal vesicles. Greater than 90 percent of the activities of fatty acid-CoA ligase (EC 6.2.1.3), sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15), lysophosphatidic acid acyltransferase, diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2), and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) was inactivated by proteolysis of intact microsomal vesicles. The phosphatidic acid phosphatase (EC 3.1.3.4) was not inactivated by any of the protease tested. Under conditions employed, <5 percent of the luminal mannose-6-phosphatase (EC 3.1.3.9) activity was lost. After microsomal integrity was disrupted with detergents, protease treatment resulted in a loss of >74 percent of the mannose-6-phosphatase activity. The latency of the mannose-6-phosphatase activity was not affected by protease treatment. Mannose-6-phosphatase latency was not decreased by the presence of the assay components of several of the lipid biosynthetic activities, indicating that those components did not disrupt the microsomal vesicles. None of the lipid biosynthetic activities appeared latent. The presence of a protease-sensitive component of these biosynthetic activities on the cytoplasmic surface of microsomal vesicles, and the absence of latency for any of these biosynthetic activities suggest that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. The location of biosynthetic activities within the transverse plane of the endoplasmic reticulum is of particular interest for enzymes whose products may be either secreted or retained within the cell. Phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol account for the vast majority of hepatic glycerolipid biosynthesis. The phospholipids are utilized for hepatic membrane biogenesis and for the formation of lipoproteins, and the triacylglycerols are incorporated into lipoproteins or accumulate within the hepatocyte in certain disease states (14). The enzymes responsible for the biosynthesis of these glycerolipids (Scheme I) from fatty acids and glycerol-3P have all been localized to the microsomal subcellular fraction (12, 16, 29, 30). Microsomes are derived from the endoplasmic reticulum and are sealed vesicles which maintain proper sidedness. (11, 22). The external surface of these vesicles corresponds to the cytoplasmic surface of the endoplasmic reticulum. Macromolecules destined for secretion must pass into the lumen of the endoplasmic reticulum (5, 23). Uncharged molecules of up to approximately 600 daltons are able to enter the lumen of rat liver microsomes, but macromolecules and charged molecules of low molecular weight do not cross the vesicle membrane (10, 11). Because proteases neither cross the microsomal membrane nor destroy the permeability barrier of the microsomal vesicles, only the enzymes and proteins located on the cytoplasmic surface of microsomal vesicles are susceptible to proteolysis unless membrane integrity is disrupted (10, 11). By use of this approach, several enzymes and proteins have been localized in the transverse plane of microsomal membranes (11). With the possible exception of cytochrome P 450, all of the enzymes and proteins investigated were localized asymmetrically by the proteolysis technique (11). By studies of this type, as well as by product localization, glucose-6-phosphate (EC 3.1.3.9) has been localized to the luminal surface of microsomal vesicles (11) and of the endoplasmic reticulum (18, 19). All microsomal vesicles contain glucose-6-phosphatase (18, 19) which can effectively utilize mannose-6-P as a substrate, provided the permeability barrier of the vesicles has been disrupted to allow the substrate access to the active site located on the lumenal surface (4). An exact correspondence between mannose- 6-phosphate activity and membrane permeability to EDTA has been established (4). The latency of mannose-6-phosphatase activity provides a quantitative index of microsomal integrity (4.) Few of the microsomal enzymes in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol have been solubilized and/or purified, and little is known about the topography of these enzymes in the transverse or lateral planes of the endoplasmic reticulum. An asymmetric location of these biosynthetic enzymes on the cytoplasmic or lumenal surface of microsomal vesicles may provide a mechanism for regulation of the glycerolipids to be retained or secreted by the cell, and for the biogenesis of asymmetric phospholipid bilayers. In this paper, we report investigations on the localization of all seven microsomal enzymes (Scheme I) in the biosynthesis of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine, using the protease technique with mannose-6-phosphatase serving as luminal control activity. The latency of these lipid biosynthetic enzymes was also investigated, using the latency of mannose-6-phosphatase as an index of microsomal integrity.
Animals↗
Vitamin K dependent carboxylation of glutamate residues to gamma-carboxyglutamate in microsomes from spleen and testes: comparison with liver, lung, and kidney.
Vitamin K dependent carboxylation of glutamate residues to gamma-carboxyglutamate was demonstrated in proteins of spleen and testes microsomes. The rate of carboxylation in spleen microsomes was 0.9% and testes 3% of that in liver microsomes per milligram of microsomal protein. For comparison the rates of carboxylation in lung and kidney microsomes were 17 and 8%, respectively, of the rate in liver microsomes. The high rate in liver microsomes may be due to a high carboxylase level as indicated by the high rate of peptide carboxylation in liver microsomes. Protein carboxylation in liver microsomes was linear for only 15 min while carboxylation in microsomes from extrahepatic tissue persisted much longer so that the total protein carboxylation in lung microsomes was 60%, kidney 18%, testes 12%, and spleen 9% of that occurring in liver microsomes. Protein carboxylation was higher in microsomes from extrahepatic tissues of rats fed a vitamin K deficient diet as compared to animals fed a vitamin K sufficient diet. Protein carboxylation in microsomes from extrahepatic tissues was greatly stimulated by manganese ions and was dependent upon the addition of dithioerythritol. NADH could partially replace the dithiol in spleen, testes, and lung, but NADH-dependent carboxylation was relatively low in kidney and liver microsomes. Dithiol-dependent carboxylation was completely blocked by 10 microM warfarin, but NADH-dependent carboxylation was only slightly inhibited by 100 microM warfarin. Menaquinone-3 was much more active than vitamin K1 in driving carboxylation. Solubilized microsomes catalyzed the carboxylation of glutamate residues to gamma-carboxyglutamate in a pentapeptide Phe-Leu-Glu-Glu-Leu. The rate of carboxylation in lung microsomes was 22%, testes 3.3%, kidney 1.9%, and spleen 1.6% of the rate in liver microsomes.
1-Carboxyglutamic Acid↗
Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. II. Significance of binding of glutamate dehydrogenase to microsomal membrane.
1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes. 2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes. 3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin. 4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes. 5. A difference was noticed among the glutamate dehydrogenase isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor post-translational modification of enzyme molecules in the transport from the site of synthesis to mitochondria.
Animals↗
Metabolism of benzo [a] pyrene by guinea pig adrenal and hepatic microsomes.
Studies were carried out to compare the metabolism of benzo [a] pyrene (BP) by adrenal and hepatic microsomes obtained from adult male guinea pigs. Adrenal microsomes produced fluorescent metabolites (primarily phenols) approximately three to four times more rapidly than hepatic microsomes, but the differences in the rates were considerably smaller when total BP metabolism was assessed using an isotopic assay. The apparent discrepancy between the two assays is attributable to differences in the profiles of BP metabolites produced by adrenal and liver. Separation of metabolites by high pressure liquid chromatography revealed that adrenal microsomes converted BP to primarily a phenolic metabolite with a retention time identical to that of 3-hydroxy-BP. Liver microsomes, by contrast, produced approximately equal amounts of compounds co-chromatographing with 3-hydroxy-BP and BP-4,5-dihydrodiol. Small amounts of other metabolites were also produced by adrenal and hepatic microsomes. Liver microsomes catalyzed the conversion of BP to metabolites that became covalently bound to exogenous DNA. The amount of binding was dependent upon the duration of incubation and concentration of microsomal protein. Adrenal microsomes, by contrast, did not promote BP binding to DNA. Inhibition of microsomal epoxide hydratase activity with trichloropropene oxide (TCPO) blocked the formation of dihydrodiol metabolites of BP by adrenal and liver microsomes. In the presence of TCPO, liver microsomes produced large amounts of a BP metabolite co-chromatographing with BP-4,5-oxide. TCPO also increased the rate of production of DNA-binding metabolites by liver microsomes but had no effect on the formation of DNA-binding metabolites by adrenal microsomes. The results demonstrate major differences in the pathways of BP metabolism by guinea pig adrenal and hepatic microsomes. Although adrenal microsomes metabolize BP more rapidly than hepatic microsomes, far greater amounts of reactive metabolites are produced by the liver. Thus, adrenal metabolism of BP may be of little toxicological significance.
Adrenal Glands↗
Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in patas monkey lung and liver microsomes.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.
Animals↗
Identification and characterization of a novel human microsomal glutathione S-transferase with leukotriene C4 synthase activity and significant sequence identity to 5-lipoxygenase-activating protein and leukotriene C4 synthase.
5-Lipoxygenase-activating protein (FLAP) and leukotriene C4 (LTC4) synthase, two proteins involved in leukotriene biosynthesis, have been demonstrated to be 31% identical at the amino acid level. We have recently identified and characterized a novel member of the FLAP/LTC4 synthase gene family termed microsomal glutathione S-transferase II (microsomal GST-II). The open reading frame encodes a 16.6-kDa protein with a calculated pI of 10.4. Microsomal GST-II has 33% amino acid identity to FLAP, 44% amino acid identity to LTC4 synthase, and 11% amino acid identity to the previously characterized human microsomal GST (microsomal GST-I). Microsomal GST-II also has a similar hydrophobicity pattern to FLAP, LTC4 synthase, and microsomal GST-I. Fluorescent in situ hybridization mapped microsomal GST-II to chromosomal localization 4q28-31. Microsomal GST-II has a wide tissue distribution (at the mRNA level) and was specifically expressed in human liver, spleen, skeletal muscle, heart, adrenals, pancreas, prostate, testis, fetal liver, and fetal spleen. In contrast, microsomal GST-II mRNA expression was very low (when present) in lung, brain, placenta, and bone marrow. This differs from FLAP mRNA, which was detected in lung, various organs of the immune system, and peripheral blood leukocytes, and LTC4 synthase mRNA, which could not be detected in any tissues by Northern blot analysis. Microsomal GST-II and LTC4 synthase were expressed in a baculovirus insect cell system, and microsomes from Sf9 cells containing microsomal GST-II or LTC4 synthase were both found to catalyze the production of LTC4 from LTA4 and reduced glutathione. Microsomal GST-II also catalyzed the formation of another product, displaying a conjugated triene UV absorption spectra with a maximum at 283 nm, suggesting less catalytic stereospecificity compared with LTC4 synthase. Also, the apparent Km for LTA4 was higher for microsomal GST-II (41 microM) than LTC4 synthase (7 microM). In addition, unlike LTC4 synthase, microsomal GST-II was able to catalyze the conjugation of 1-chloro-2, 4-dinitrobenzene with reduced glutathione. Therefore, it is proposed that this novel membrane protein is a member of the microsomal glutathione S-transferase family, also including LTC4 synthase, with significant sequence identities to both LTC4 synthase and FLAP.
5-Lipoxygenase-Activating Proteins↗
Metabolism of N-nitrosodialkylamines by human liver microsomes.
The metabolism of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine, N-nitrosobenzylmethylamine, and N-nitrosobutylmethylamine was investigated in incubations with human liver microsomes. All of the 16 microsomal samples studied were able to oxidize NDMA to both formaldehyde and nitrite at NDMA concentrations as low as 0.2 mM; the rates of product formation of the samples ranged from 0.18 to 2.99 nmol formaldehyde/min/mg microsomal protein (median, 0.53 nmol). At a concentration of 0.2 mM NDMA, the rates of denitrosation (nitrite formation) were 5 to 10% (median, 6.3%) those of demethylation (formaldehyde formation); the ratio of denitrosation to demethylation increased with increases in NDMA concentration, in a similar manner to rat liver microsomes. Immunoblot analysis with antibodies prepared against rat P-450ac (an acetone-inducible form of cytochrome P-450) indicated that the P-450ac [P-450j (isoniazid-inducible form)] orthologue in human liver microsomes had a slightly higher molecular weight than rat P-450ac and the amounts of P-450ac orthologue in human liver microsomes were highly correlated with NDMA demethylase activities (r = 0.971; P less than 0.001). Analysis of four selected microsomal samples showed that human liver microsomes exhibited at least three apparent Km and corresponding Vmax values for NDMA demethylase. This result, suggesting the metabolism of NDMA by different P-450 enzymes, is similar to that obtained with rat liver microsomes, even though most of the human samples had lower activities than did the rat liver microsomes. The high affinity Km values of the four human samples ranged from 27 to 48 microM (median, 35 microM), which were similar to or slightly lower than those observed in rat liver microsomes, indicating that human liver microsomes are as efficient as rat liver microsomes in the metabolism of NDMA. The human liver microsomes also catalyzed the dealkylation and denitrosation of other nitrosamines examined. The rates of product formation and the ratios of denitrosation to dealkylation varied with the structures and concentrations of the substrates as well as with the microsomal samples tested. The results indicate that human liver microsomes are capable of metabolizing N-nitrosodialkylamines via the pathways that have been established with rat liver microsomes.
Cytochrome P-450 Enzyme System↗
Kinetic characteristics of norcocaine N-hydroxylation in mouse and human liver microsomes: involvement of CYP enzymes.
The first step in the oxidative metabolism of cocaine is N-demethylation to norcocaine, which is further N-hydroxylated to more toxic N-hydroxynorcocaine. In this study we examined the kinetics of norcocaine N-hydroxylation mediated by cytochrome P450 (CYP) in mouse and human liver microsomes. N-hydroxynorcocaine was identified by analytical HPLC-MS after incubation of norcocaine with mouse liver microsomes in the presence of NADPH. In mouse liver microsomes, there was no apparent difference in Km values for norcocaine N-hydroxylation between male and female microsomes, while the Vmax rate was approximately two times higher in female than in male microsomes (34+/-10 v. 16+/-4 pmol/min per mg protein). The Km value for norcocaine N-hydroxylation in human liver microsomes was approximately three times higher than that observed in comparable incubations using mouse liver microsomes, whereas the Vmax rate was ten times lower. Both cocaine and norcocaine induced type I difference spectra upon interaction with CYP in mouse liver microsomes. In contrast, in human microsomes both type I and type II spectra were recorded. In the 0.01 to 1 mM concentration range, cocaine and norcocaine inhibited mouse microsomal testosterone 6alpha-, 7alpha- and 16alpha-hydroxylation reactions by 20% to 30%. Testosterone 6beta- and 15alpha-hydroxylations were blocked by 60% and 50%, respectively, by 1 mM norcocaine, while only 40% inhibition was obtained with 1 mM cocaine. Coumarin 7-hydroxylation and pentoxyresorufin O-deethylation were inhibited by 50% by 1 and 0.4 mM norcocaine, respectively. In contrast, 10 and 2 mM cocaine, respectively, were needed to obtain the same degrees of inhibition. In human liver microsomes, 1 mM norcocaine and cocaine blocked testosterone 6beta-hydroxylase by 60% and 40%, respectively. Coumarin 7-hydroxylation was inhibited by only 30% by norcocaine (5.4 mM) and cocaine (10 mM). Norcocaine N-hydroxylation in mouse and human liver microsomes was blocked by 30% and 60%, respectively, by alpha-naphthoflavone (0.1 mM). The reaction was inhibited by 30-40% by metyrapone, cimetidine and gestodene at a concentration of 1 mM in mouse microsomes, while in human microsomes, 70% inhibition was obtained with 1 mM metyrapone and cimetidine. Taken together, these results indicate that (1) norcocaine N-hydroxylation is at least partly a CYP-mediated reaction, (2) the rate of reaction is considerably lower in human liver microsomes than in mouse liver microsomes and (3) several CYP subfamilies including 1A, 2A, 3A and possibly 2B may contribute to the formation of N-hydroxynorcocaine.
Animals↗
Involvement of CYP3A1, 2B1, and 2E1 in C-8 hydroxylation and CYP 1A2 and flavin-containing monooxygenase in N-demethylation of caffeine; identified by using inducer treated rat liver microsomes that are characterized with testosterone metabolic patterns.
Caffeine (CA) is oxidized by rat liver microsomal enzymes to theobromine (TB), paraxanthine (PX), and theophylline (TP) by N-demethylation and to trimethylurate (TMU) by C-8 hydroxylation, In order to identify the specific enzymes responsible for productions of these primary CA metabolites, liver microsomes enriched with various isoforms of cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) are prepared by pretreatment of rats with several inducers. The specific increases in various CYP or FMO activities are identified with the diagnostic testosterone metabolic patterns or the thiobenzamide S-oxidation assay. They are then employed to metabolize the CA. Liver microsomes isolated from rats pretreated with phenobarbital (PB-microsomes) did not have increased FMO activity but had increased activities for hydroxylating the testosterone at 6 beta-(CYP3A1), 16 beta-(CYP2B1), and 2 beta-(CYP3A1) positions. This PB-microsomes had increased activity for TMU production from CA (result of C-8 hydroxylation). Liver microsomes isolated from rats pretreated with acetone (AC-microsomes) had a normal level of FMO activity but had enhanced rates of 6 beta-(CYP3A1) and 2 beta-(CYP3A1) hydroxylations of testosterone. The AC-microsomes again had increased activity for production of TMU. Similarly, the liver microsomes isolated from rats pretreated with dexamethasone (DEX-microsomes) had a normal level of FMO activity but had enhanced rates of forming 6 beta-and 2 beta-hydroxytestosterone (Cyp3A1) as well as androstenedione (CYP3A1). The DEX-microsomes again had increased activity for production of TMU only. Liver microsomes isolated from rats pretreated with 3-methylcholanthrene (MC-microsomes), however, had increased FMO activity and also enhanced rates of forming the 7 alpha-(CYP1A1/2, and 2A1), 6 beta-(CYP3A1), and 2 beta-(CYP3A1) hydroxytestosterone. The MC-microsomes had increased activity for producing all of the four primary metabolites of CA, i.e. the N-demethylation metabolites like TB, PX. and TP, as well as the C-8 hydroxylation metabolite TMU. By the process of association of the obtained results, liver microsomes with increased contents of CYP2B1, 3A1, and 2E1 could catalyze the C-8 hydroxylation at an increased rate producing increased amount of TMU. Increased productions of CA N-demethylation metabolites (TB, PX, and TP) are, however, catalyzed by the increased activities of CYP1A2 and FMO which are associated uniquely with the MC-microsomes.
Animals↗
Drug-induction decreases the mobility of cytochrome P-450 in rat liver microsomes: protein rotation study.
Effect of drug-induction on the rotation of cytochrome P-450 and on lipid fluidity in rat liver microsomes was examined. Rotational diffusion of cytochrome P-450 was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. Microsomal lipid fluidity was measured by observing fluorescence anisotropy of DPH incorporated in the lipid bilayer. The absorption anisotropy decayed within 2 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the drug-induction with PB, MC, and PCB when compared with non-induced CON-microsomes. The observed values for the normalized time-independent anisotropy r(infinity)/r(0) are r(infinity)/r(0) = 0.41 (CON-microsomes), 0.54 (PB-microsomes), 0.52 (MC-microsomes), and 0.57 (PCB-microsomes). The average rotational relaxation time phi = 580-690 microseconds was almost unchanged over all microsomes presently examined. A significantly high value of r(infinity)/r(0) = 0.41-0.57 implies the co-existence of mobile and immobile populations of cytochrome P-450. Based on the assumption that the heme tilts about 55 degrees from the membrane plane for all species of P-450s besides P-450PB, 59% (CON-microsomes), 46% (PB-microsomes), 48% (MC-microsomes), and 43% (PCB-microsomes), respectively, of the cytochrome P-450 in microsomes is calculated to be mobile. Upon drug-induction the microsomal membrane was fluidized to some extent as judged by the steady-state fluorescence anisotropy of 0.156 for CON-microsomes and 0.139-0.148 for drug-induced microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates.
The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were found with different effectors. Activation of epoxide hydrolase activity towards styrene oxide by clotrimazole was found to be uncompetitive with the substrate and highly structure specific. On the other hand, inhibition of epoxide hydrolase activity towards androstene oxide by clotrimazole was found to be competitive in microsomes. It is concluded that the marked effects of these four modulators on microsomal epoxide hydrolase activity are due to an interaction with the enzyme protein itself, but that the presence of total microsomal phospholipids allows the maximal expression leading to similar degrees of modulation as those observed in intact microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)
Aldehydes↗
Alteration of aflatoxin B1 metabolic profiles and reduction of aflatoxin B1 mutagenicity by hepatic microsomes of rats fed butylated hydroxyanisole.
Effect of administering butylated hydroxyanisole (BHA) on the metabolism of aflatoxin B1 (AFB1) and production of mutagenic metabolites have been compared with those of phenobarbital (PB) and 3-methylcholanthrene (MC) administration in rat liver microsomes. Male Sprague-Dawley rats were treated with these inducers and liver microsomes were isolated. These microsomes were used to metabolize AFB1 and to produce mutagenic metabolites. Results showed that normal rat liver were able to metabolize AFB1 quite actively and produced large amounts of AFB-8,9-epoxide (appearing as the AFB-8,9-dihydrodiol-Tris complex). Upon incubations of normal rat liver microsomes with increasing concentrations of AFB1, a steep dose-related increases of mutagenicity was observed in the Ames test. The PB-microsomes had an increased ability to metabolize AFB1 and particularly the rate for the production of the weakly mutagenic AFQ1 metabolite was markedly increased. Conversely, PB-microsomes had a moderate decrease in its ability to form the strongly mutagenic of AFB-8,9-epoxide metabolite. However, the ability of PB-microsomes to form mutagenic metabolites from AFB1 was somewhat greater than that of the control-microsomes. The MC-microsomes had an increased ability to metabolize AFB1 also. However, instead of the weakly mutagenic AFQ1 metabolite seen with the PB-microsomes, large amounts of the strongly mutagenic AFM1 metabolite was formed. Although AFM1 is not known to be a direct mutagen, it was highly mutagenic upon activation with microsomes. The very steep dose-related increases of mutagenicity and appearance of bacterial toxicity at relatively lower doses of AFB1 may have been caused by the secondary metabolic activation. The ability of BHA-microsomes to metabolize AFB1 was decreased. Among the metabolites produced by the BHA-microsomes, the non-mutagenic AFB2a was formed in significantly increased amounts but the toxic AFB-8,9-epoxide was produced only in much reduced amounts. The AFB2a was not mutagenic even after metabolic activation with microsomes. When increasing concentrations of AFB1 was incubated with BHA-microsomes, a very mild dose-related increases of mutagenicity was observed and the occurence of toxic effects on bacterial growth appeared only at high doses of AFB1. This may have been due both to the reduced rate of overall AFB1 metabolism and to the decreased formation of the highly mutagenic AFB-8,9-epoxide but an increased formation of the non-mutagenic AFB2a metabolite by the BHA-microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)
Aflatoxin B1↗
Induction and suppression of hepatic and extrahepatic microsomal foreign-compound-metabolizing enzyme systems by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on a number of hepatic and extrahepatic foreign-compound-metabolizing enzyme systems in microsomes from rats, rabbits and guinea pigs were investigated. Following TCDD treatment, the N-demethylation of benzphetamine, aminopyrine and ethylmorphine was suppressed in hepatic microsomes from male but not from female rats. However, both cytochrome P-450 and benzpyrene hydroxylase were significantly stimulated in hepatic microsomes from both male and female rats at doses as small as 1 mug TCDD/kg body weight. The inductive effect on rat hepatic microsomal enzymes was considerably more persistent than the suppressive effect. Following a single oral dose of 25 mug TCDD/kg weight, benzpyrene hydroxylase of male rat liver microsomes remained significantly elevated for 73 days but the suppression of benzphetamine N-demethylase had gone after 35 days. The induction of benzpyrene hydroxylase in male rat liver microsomes by TCDD was independent of the age of the rat and the levels to which this enzyme was increased was similar in male rats of all ages. However, the suppression of benzphetamine N-demethylase in male rat liver microsomes was age related: the suppression was seen only in adult animals and in the very young (10 days old) the enzyme was actually induced by TCDD. Inductive effects appeared in both smooth and rough-surfaced hepatic microsomes from male rats but the suppression of N-demethylation occurred only in the smooth-surfaced microsomes (SER). In microsomes from extrahepatic tissues of the rat, induction of mixed-function oxidases (MFOs) by TCDD occurred only in the kidney. However, UDPglucuronyltransferase was induced in microsomes from lung, kidney, intestine and brain but not testes. The response in the rabbit and guinea pig to TCDD differed considerably from that in the rat. Benzpyrene hydroxylase was unaffected in hepatic microsomes from the guinea pig and actually suppressed in microsomes from rabbit liver. Benzphetamine N-demethylase was also suppressed in rabbit liver microsomes. Glucuronyl-transferase was unaffected by TCDD in microsomes from liver, lung or kidney of the rabbit and guinea pig. The only lung enzyme responsive to TCDD was biphenyl 4-hydroxylase of the rabbit and guinea pig. Suppression was not observed in any of the extraheptic tissues studied and may be confined to only certain hepatic systems.
Animals↗