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PubMed1971

A role for phospholipids in the binding and metabolism of drugs by hepatic microsomes. Use of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulphonate.

1. The pretreatment of rat liver microsomes with phospholipase C or D decreased the N-demethylation of (+)-benzphetamine. The hydroxylation of aniline was essentially unchanged by pretreatment of microsomes with phospholipase C. 2. Some components of the microsomal mixed-function oxidase system were impaired by phospholipases. 3. The fluorescence of 1-anilinonaphthalene-8-sulphonate (ANS) was greatly enhanced by microsomes. Phospholipase C or D markedly decreased ANS-microsome fluorescence. Quantum yield of ANS-microsome fluorescence appeared to be related directly to phospholipid content of microsomes. 4. Most of the drugs studied enhanced ANS-microsome fluorescence. Warfarin, however, displaced ANS fluorescence competitively from microsomes. The latter effect was postulated as being due to warfarin competing with ANS for the cationic site on microsomal phosphatidylcholine. 5. ANS fluorescence was also increased by the presence of phospholipid micelles. The fluorescence of ANS-phosphatidylcholine micelles was modified by warfarin and (+)-benzphetamine in a manner similar to that observed with microsomes. Warfarin decrease of fluorescence was absent when ANS was bound to phosphatidic acid, which lacks a cationic site. 6. Trypsin pretreatment of microsomes did not modify ANS-microsome fluorescence, including drug-induced changes. 7. It was postulated that phospholipids have a permissive role in the metabolism of most drugs by hepatic microsomes and that the ANS probe might reflect interactions of compounds with microsomal membrane phospholipids.

Aniline Compounds↗
PubMed1988

Role of specific cytochrome P-450 isoenzymes in the regio-selective metabolism of 7,12-dimethylbenz[a]anthracene in microsomes from rats treated with phenobarbital or Sudan III.

The rates of formation of diols of dimethylbenz[a]anthracene (DMBA-3,4-diol, DMBA-5,6-diol and DMBA-8,9-diol) have been determined in hepatic microsomes prepared from untreated rats or from animals treated with phenobarbital (PB) or Sudan III. PB treatment enhanced the formation of the proximate carcinogen, DMBA-3,4-diol, and of the 5,6-diol while treatment with Sudan III suppressed the formation of DMBA-3,4-diol but greatly increased the rates of formation of the other two diols. Metyrapone, a reagent which is specific for members of the major PB-induced cytochrome P-450 subfamily (P-450-PB3), did not alter the rate of formation of the diols other than in microsomes prepared from PB-treated animals in which formation of the 5,6-diol was inhibited. Incubation of DMBA with microsomes resulted in the formation of covalent, DMBA-microsome adducts. Treatment of the animals with PB and Sudan III increased the rate of formation of DMBA-microsome adducts to a similar extent (approximately 5-fold). The formation of adducts could be inhibited by metyrapone in microsomes from untreated and PB-treated animals but the reagent had no effect on adduct formation in microsomes from Sudan III-treated animals. These observations may indicate that adduct formation in microsomes from Sudan-treated animals involves primary epoxide metabolites while in microsomes from PB-treated animals secondary metabolites are involved and these may be formed by a P-450-PB3 isoenzyme. Specific P-450 isoenzymes involved in the regioselective formation of DMBA-diols have been identified by the use of antibodies directed against specific isoenzymes. An antibody to P-450-MC1b inhibited the formation of DMBA-5,6-diol and 8,9-diol in microsomes from Sudan III-treated animals. Western blot analysis demonstrated that P-450-MC1b was induced in microsomes of animals treated with Sudan III but was not present in the other two microsomal preparations. In accord with the observations with metyrapone, anti-P-450-PB3 inhibited formation of DMBA-5,6-diol in microsomes from PB-treated animals but was without effect on the formation of other diols. Anti-P450-PB1 inhibited the formation of DMBA-3,4-diol in microsomes from PB-treated animals. Western blot analysis of microsomes from animals treated with several xenobiotics indicated a qualitative correlation between the content of P-450-PB1 and the rate at which DMBA-3,4-diol was formed.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2002

Sudan I is a potential carcinogen for humans: evidence for its metabolic activation and detoxication by human recombinant cytochrome P450 1A1 and liver microsomes.

1-Phenylazo-2-hydroxynaphthol (Sudan I, C.I. Solvent Yellow 14) is a liver and urinary bladder carcinogen in mammals. We compared the ability of hepatic microsomal samples from different species including human to metabolize Sudan I. Comparison between experimental animals and human cytochromes P450 (CYP) is essential for the extrapolation of animal carcinogenicity data to assess human health risk. Human microsomes generated the pattern of Sudan I metabolites reproducing that formed by hepatic microsomes of rats. Using hepatic microsomes of rats pretreated with specific CYP inducers, microsomes from Baculovirus-transfected insect cells expressing recombinant human CYP enzymes, purified CYP enzymes, and selective CYP inhibitors, we found that rat CYP1A1 and recombinant human CYP1A1 are the most efficient enzymes metabolizing Sudan I. Microsomes from livers (the target of Sudan I carcinogenicity) of different human donors were used to estimate whether authentic human CYPs oxidize Sudan I. Using Western blot analysis and NH(2)-terminal sequencing, we were able to detect and quantify CYP1A1 in human hepatic microsomes. The sequence of nine amino acids of the protein band cross-reacting with antirat CYP1A1 in human microsomes, LFPISMSAT, matched the sequence of human CYP1A1 perfectly (residues 2-10). CYP1A1 expression levels varied significantly among the different human microsomes (0.04-2.4 pmol/mg protein), and constituted <0.6% of the total hepatic CYP complement. All of the human hepatic microsomal samples oxidized Sudan I to C-hydroxymetabolites. Moreover, using the nuclease P1-enhanced version of the (32)P-postlabeling assay, we found that human microsomes were competent in activating Sudan I to form adducts with DNA. The role of specific CYP enzymes in the human hepatic microsomal metabolism was investigated by correlating the CYP-catalytic activities (or CYP contents) in each microsomal sample with the levels of individual metabolites and/or Sudan I-DNA adducts formed by the same microsomes, and by examining the effects of agents that can inhibit specific CYP in Sudan I metabolism. On the basis of these studies, we attribute most of Sudan I metabolism in human microsomes to CYP1A1, but participation of CYP3A4 cannot be ruled out. These results, the first report on the metabolism of Sudan I by human CYP enzymes, strongly suggest a carcinogenic potency of this rodent carcinogen for humans.

Animals↗
PubMed1982

Influence of vitamin E and nitrogen dioxide on lipid peroxidation in rat lung and liver microsomes.

Rat lung and liver microsomes were used to examine the effects of dietary vitamin E deficiency on membrane lipid peroxidation. Microsomes from vitamin-E-deficient rats displayed increased lipid peroxidation in comparison to microsomes from vitamin-E-supplemented controls. The extent of lipid peroxidation, as determined by measurement of thiobarbituric acid reacting materials, was enhanced by addition of reduced iron and ascorbate (or NADPH). Rats fed a vitamin-E-supplemented diet and exposed to 3 ppm NO2 for 7 days did not exhibit increases in microsomal lipid peroxidation compared to air-breathing controls. However, increase were found in microsomes prepared from rats fed a vitamin-E-deficient diet and exposed to NO2. Lung microsomes from vitamin-E-fed rats contained almost 10 times as much vitamin E as liver microsomes when expressed in terms of polyunsaturated fatty acid content. The extent of lipid peroxidation was, in turn, considerably less in lung than in liver microsomes. Lipid peroxidation in lung microsomes from vitamin-E-deficient rats comparable to liver microsomes from vitamin-E-supplemented rats as was the content of vitamin E in these respective microsomal samples. A combination of vitamin E deficiency and NO2 exposure resulted in the greatest increases in lung and liver microsomal lipid peroxidation with the largest relative increases occurring in lung microsomes. An inverse relationship was found between the extent of lipid peroxidation and vitamin E content. Most of the peroxidation in lung microsomes appeared to proceed nonenzymatically whereas peroxidation in liver was largely enzymatic. Vitamin E appears to be assimilated by the lung during oxidant inhalation, but with dietary vitamin E deprivation, the margin for protection in lung may be less than in liver.

Animals↗
PubMed1984

Biochemical and morphological comparison of microsomal preparations from rat, quail, trout, mussel, and water flea.

Differential centrifugation methods already in use were applied to purify rat, quail, and trout liver microsomes and modified as necessary to purify microsomes from mussel digestive gland and whole water flea. All these microsomal preparations were comparatively characterized with respect to protein and RNA content, levels of markers of subcellular contaminants, ultrastructural morphology, differential spectra of cytochromes P-450 and b5, monoxygenase activity, and in vitro metabolism of p-dichlorobenzene. Yields of microsomal proteins of the tested organisms differed widely, with mussel showing the lowest yield. Very low levels of nuclear and mitochondrial contaminants were detected in all microsomal preparations, but cell membrane contaminants were clearly present in most preparations. Daphnia microsomes were significantly contaminated by plasma membranes, and hepatopancreas microsomes contained significant amounts of partially disrupted secretory granules and plasma membrane. From a qualitative standpoint differential spectra of cytochrome b5 were very similar for all the preparations, whereas cytochrome P-450 spectra were largely dependent on the microsomal preparation as well as on the assay method used. The content of cytochrome P-450 was highest for rat liver microsomes and very low or absent in Daphnia and mussel preparations; the range of cytochrome b5 contents was much narrower. Significant differences were observed among monoxygenase activities of the different preparations. In Daphnia and mussel microsomes, aniline hydroxylase was absent and benzo[a]pyrene hydroxylase activity was much lower than in rat and quail microsomes. Benzo[a]pyrene hydroxylase activity of trout liver microsomes was similar to that of rat and quail microsomes, whereas hydroxylation of substrates which in rat liver are preferentially metabolized by phenobarbital-inducible forms of cytochrome P-450 was much lower in trout microsomes.

Animals↗
PubMed1973

Heterogeneous distribution of glucose 6-phosphatase in rat liver microsomal fractions as shown by adaptation of a cytochemical technique.

1. A novel technique for the subfractionation of rat liver smooth and rough microsomal fractions according to their content of glucose 6-phosphatase is described. This technique, based on the Gomori lead histochemical procedure, involves incubation of smooth and rough microsomal fractions with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate. Control experiments, in which enzyme was assayed in the presence of various amounts of Pb(NO(3))(2) or in which microsomal fractions were reisolated after incubation with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate, showed that lead does not interfere with glucose 6-phosphatase activity. 2. Discontinuous sucrose-density-gradient centrifugation of microsomal fractions which had previously been incubated with various amounts of Pb(NO(3))(2) and glucose 6-phosphate showed that it is possible to subfractionate both smooth- and rough-microsomal fractions into several bands, owing to a differential modification of the density of the microsomal vesicles by the trapping of lead phosphate within them. 3. When the material in the bands obtained by density-gradient centrifugation of incubated microsomal fractions was assayed for glucose 6-phosphatase activity, it was found that the modification of the density of the microsomal fractions was directly related to their relative enrichment in glucose 6-phosphatase activity. Control experiments, in which microsomal fractions were incubated with Pb(NO(3))(2) and glucose 6-phosphate and then treated with EDTA, showed that the subfractionation was not due to aggregation of microsomal vesicles, lead and glucose 6-phosphate. Thus the resolution of microsomal preparations into subfractions with different glucose 6-phosphatase activities is interpreted as indicating heterogeneity of glucose 6-phosphatase distribution in the microsomal vesicles. 4. Electron micrographs of both smooth- and rough-microsomal subfractions show deposits of lead phosphate within the microsomal vesicles. The frequency and extent of these deposits correlate with the different amounts of glucose 6-phosphatase activity measured biochemically. 5. The nature of the heterogeneous distribution of glucose 6-phosphatase is discussed and the more general applicability of the technique for studying membrane fractions containing a heterogeneous distribution of phosphatases is indicated.

Animals↗
PubMed1991

Characterization of covalent binding of [14C]-2-chloro-4-acetotoluidide to microsomes of starling liver and kidney.

In this study, we have characterized the covalent binding of [14C]-2-chloro-4-acetotoluidide (CAT) radioactivity to microsomes of starling liver and kidney. The maximal velocity (Vmax) of covalent binding and apparent Michaelis constant (Km) for both tissues were similar. The Vmax for liver and kidney were 52.8 and 68.9 pmol/min/mg protein, and the apparent Kms were 0.54 and 0.87 mM, respectively. The covalent binding of radioactivity to heat-denatured microsomes of liver and kidney was reduced by 62% and 15%, respectively. Incubation at 0 degrees C reduced the binding by 80% to liver and 70% to kidney microsomes. Absence of nicotinamide adenine dinucleotide phosphate (NADP) and molecular O2 reduced the binding to liver microsomes by 36 and 53%, as opposed to 28% increase and 26% decrease in binding to kidney microsomes, respectively. Inducers of cytochrome P450 monooxygenase (P450), phenobarbital, and 3-methylcholanthrene (3-MC), had opposite effects on the covalent binding of [14C]-CAT radioactivity to hepatic and renal microsomes. Phenobarbital increased the binding to hepatic microsomes by 100% and had no effect on binding to renal microsomes. 3-MC, on the other hand, increased the binding to kidney microsomes by threefold and had no effect on the binding to hepatic microsomes. SKF 525A, an inhibitor of P450, inhibited the binding to hepatic microsomes by 60% at 0.5 mM but failed to have any effect on binding to renal microsomes. alpha-Naphthoflavone, another inhibitor of P450, had no effect on the covalent binding of [14C]-CAT radioactivity to microsomes of either tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A new proposal for the mechanism of cyclosporine A nephrotoxicity. Inhibition of renal microsomal protein chain elongation following in vivo cyclosporine A.

In this paper, we report experiments examining the effect of cyclosporine A on "run-off" translation in microsomes isolated from tissues of Sprague-Dawley rats. In microsomes isolated from rat brain, kidney and thymus, cyclosporine A added in vitro in concentrations of up to 100 micrograms/ml did not reduce [3H]L-leucine incorporation relative to controls. A small dose-dependent reduction in [3H]leucine incorporation was observed in microsomes isolated from rat liver when cyclosporine A was added in high concentrations (5 and 6% at 25 and 100 micrograms/ml). However, when cyclosporine A was injected at 50 mg/kg/day for 10 days, [3H]L-leucine incorporation was inhibited 99.9% in microsomes isolated from kidney. The oral administration of cyclosporine A at 50 mg/kg/day for 6-10 days produced a 75% inhibition of incorporation by isolated renal microsomes. These changes were observed in the absence of measurable reductions in "run-off" transcription measured as [3H]UTP incorporation by renal nuclei exposed to cyclosporine A in concentrations of up to 100 micrograms/ml in vitro or isolated from animals given oral cyclosporine A at 50 mg/kg/day for 6 days. Cross-over experiments were performed using microsomes and microsomal supernatant fractions (cell saps) from tissues of animals treated with cyclosporine A and control vehicle. Renal cell sap from cyclosporine A treated animals inhibited [3H]L-leucine incorporation by microsomes isolated from the kidneys or other tissues of animals treated with control vehicle. These experiments demonstrated that a translation inhibitor was present in the cell sap of cyclosporine A treated animals which could directly block translation elongation in microsomes from control animals. When renal cell sap from both control and cyclosporine A treated animals was added to control microsomes, inhibition was still prominent, suggesting the presence of an inhibitor rather than the absence of an elongation factor. Oral administration of cyclosporine A at 50 mg/kg/day for 6 days depressed renal microsomal [3H]L-leucine incorporation equally in male and female rats to 25% of control. The dose-response relationship for microsomal protein synthesis inhibition after 6 days of oral cyclosporine A administration was: 5 mg/kg, 73.7% of control; 10 mg/kg, 64.1% of control; 25 mg/kg, 54.9% of control and 50 mg/kg, 24.1% of control. Renal microsomal protein synthesis following oral cyclosporine A at 50 mg/kg/day was reduced to 54% of control by day 2 and was maximally inhibited at 25-30% of control by day 4.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. I. Ring- and N-hydroxylations of N-2-fluorenylacetamide.

We determined ring- and N-hydroxylations of a systemic mammary gland carcinogen, N-2-fluorenylacetamide (2-FAA), by microsomal fractions of liver and mammary gland of female rats and the effects of in vivo and/or in vitro modifiers of these oxidations. Pretreatment of lactating rats with 3-methylcholanthrene (3-MC) or beta-naphthoflavone (beta-NF) and non-lactating (50-day old virgin) rats with beta-NF showed similar effects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAA by hepatic microsomes was increased manyfold and the formation of 1-hydroxy-2-FAA was induced. In mammary gland microsomes, the formation of 3-, 5- and 7-hydroxy-2-FAA was likewise increased, but of 9-hydroxy-2-FAA was unaffected. Only mammary microsomes of lactating rats had capacity for N-hydroxylation which was increased approximately 3 times by pretreatment of rats with 3-MC or beta-NF. All of the induced increases of metabolites of 2-FAA in hepatic and mammary microsomes were inhibited by 0.1 mM alpha-naphthoflavone (alpha-NF) in vitro. Pretreatment of non-lactating rats with phenobarbital increased only the formation of 7-hydroxy-2-FAA in hepatic microsomes which was further stimulated by alpha-NF in vitro. The latter also stimulated the formation of 7- and 9- hydroxy-2-FAA by hepatic microsomes of the uninduced rats, but had no effects in mammary microsomes, in which 9-hydroxy-2-FAA was a major metabolite. Hence, the data showed qualitative and quantitative differences between lactating and non-lactating rats in metabolism of 2-FAA by mammary microsomes which may result from differences in the levels (e.g., of cytochrome P-450) and activities of microsomal enzymes determined herein. In hepatic microsomes of these rats, differences in quantities of metabolites of 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in corn oil-treated rats only. The solvent (methanol or acetone) used for addition of 2-FAA to the incubation mixtures altered quantitatively the metabolite profiles in hepatic and mammary microsomes of 3-MC or beta-NF treated rats. The formations of 1- and 3- or 5- and 7-hydroxy-2-FAA were greater in the presence of acetone or methanol, respectively. The results of this study suggest that the formation of phenolic and N-hydroxy metabolites of 2-FAA in both hepatic and mammary microsomes of lactating rats is catalyzed by similar form(s) of cytochrome P-450 induced by pretreatment with 3-MC or beta-NF.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Acetylaminofluorene↗
PubMed1985

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase.

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.

Acid Anhydride Hydrolases↗
PubMed1984

Influence of vitamin E and selenium on glutathione-dependent protection against microsomal lipid peroxidation.

A GSH-dependent microsomal protein which inhibits lipid peroxidation has been described [R. F. Burk, Biochim. biophys. Acta 757, 21 (1983)]. Studies of its mechanism indicate that it scavenges free radicals. Vitamin E (alpha-tocopherol) and selenium are micronutrients which protect against lipid peroxidation. The effect of nutritional deficiencies of these substances on the GSH-dependent protection against rat liver microsomal lipid peroxidation was studied to determine whether GSH, selenium and alpha-tocopherol function through separate or shared mechanisms. In the ascorbate-iron microsomal lipid peroxidation system, there is a 1-3 min lag phase before lipid peroxidation begins. The length of the lag correlated well (r = 0.87) with the microsomal alpha-tocopherol content as measured by high pressure liquid chromatography. Thus, the selenium-deficient microsomes, which had a shorter lag than controls, had a somewhat lower alpha-tocopherol content. The vitamin E-deficient microsomes, which had no detectable alpha-tocopherol, had the shortest lag, but a distinct lag was present. Addition of 0.1 mM GSH to control microsomes prolonged the lag by 270%. In selenium-deficient and vitamin E-deficient microsomes, which had shorter initial lags, GSH addition caused 345 and 280% increases respectively. This suggests that the function of the GSH-dependent protective mechanism is unimpaired in these deficiencies. Trypsin digestion of microsomes, which abolished the lag completely and destroyed the GSH-dependent protection, had no effect on microsomal alpha-tocopherol content, however. These experiments illustrate the importance of two defenses against microsomal lipid peroxidation: the GSH-dependent protein which is responsible for the existence of the lag, and alpha-tocopherol which affects the length of the lag. They suggest that these defenses function separately to prevent peroxidation of membrane polyunsaturated fatty acids. Selenium appears to affect microsomal alpha-tocopherol content but to have no other effect on the microsomal lipid peroxidation system.

Animals↗
PubMed1975

Interactions of heme with hepatic microsomal mono-oxygenase. Effect on benzpyrene hydroxylation.

The addition of heme (1-10 muM) to liver microsomes from phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated male rats increased the rate of benzpyrene (BP) hydroxylation by about 20-40%. On the other hand, protoporphyrin IX caused only inhibition of BP hydroxylation. There was no increase of enzymatic activity by heme when solubilized preparations of liver microsomes were used. This suggested the possibility that an apo-cytochrome P-450 was present in intact microsomes. Higher concentrations of heme inhibited BP hydroxylation by either intact or solubilized microsomes. The inhibition by heme with solubilized microsomal preparations was noncompetitive, "mixed-type". However, with intact microsomes, the lack of linearity, precluded the determination of the type of inhibition. To examine possible effects of heme on the binding of BP to microsomal cytochrome P-450, the spectrum elicited by the addition of BP to microsomes was obtained in the presence or absence of added heme. The addition of heme to liver microsomes produced a marked increase in the trough (419-420 nm) of the difference spectrum formed by the subsequent addition of BP. These findings would suggest that heme increased the binding of BP to microsomes. However, the possibility that BP merely displaces the bound heme of the microsomes to yield, as expected, a trough at 413-416 nm (the addition of heme to microsomes yields a peak of 413-416 nm, unpublished) cannot be ruled out. Nevertheless, independent of our understanding of the mechanism involved in the spectral interactions between heme and BP with liver microsomes it is clear that an effect at their binding site(s) must have been elicited by the presence of both compounds.

Animals↗
PubMed1988

Metabolic activation of alpha-naphthoflavone by 2,3,7,8-tetrachlorodibenzodioxin-induced rat liver microsomes.

Previous studies in our laboratory had demonstrated that addition of alpha-naphthoflavone (ANF) to lymphocytes from smokers or polychlorinated biphenyls (PCB)s-exposed individuals caused an increase in sister chromatid exchange (SCE) frequency whereas lymphocytes from controls were relatively unaffected. In order to investigate the mechanism responsible, metabolism of ANF by uninduced and 2,3,7,8-tetrachlorodibenzodioxin (TCDD)-induced microsomes was studied as a function of microsomal protein concentration and incubation time. Nonpolar metabolites were analyzed and the amount of conjugated (polar) and protein-bound metabolites determined. The initial ANF-metabolism rate was 10-fold higher in TCDD-induced microsomes (4.9 +/- 0.6 nmol/min per mg TCDD-induced microsomal protein vs. 0.5 +/- 0.2 nmol/min per mg uninduced microsomal protein) than in uninduced microsomes. Moreover, uninduced microsomes no longer metabolize ANF after 30-40 min while TCDD-induced microsomes metabolize ANF for longer than 2 h or until all the ANF is gone. In addition to the metabolites formed by uninduced microsomes [7,8-dihydro-7,8-dihydroxy-ANF (7,8-dihydrodiol); 5,6-dihydro-5,6-dihydroxy-ANF (5,6-dihydrodiol); 5,6-oxide-ANF and 6-hydroxy-ANF], TCDD-induced microsomes from unidentified metabolites. When TCDD-induced microsomes and 40 microM ANF were added to Chinese hamster ovary (CHO) cells, we found a correlation between the concentration of 5,6-oxide-ANF and clastogenicity to CHO cells. However, purified 5,6-oxide-ANF did not induce SCEs in CHO cells in the absence or presence of TCDD-induced microsomes. However, a minor metabolite (identified as the 9,10-dihydro-9,10-dihydroxy-ANF by acid dehydration) formed with TCDD-induced microsomes produces clastogenicity in CHO cells. These data indicate that a minor metabolite of ANF is a potent clastogen which suggests that this metabolite may be responsible for the ANF-mediated increases in SCE frequency in lymphocytes from smokers or PCB-exposed individuals.

Animals↗
PubMed1988

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes.

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.

Animals↗
PubMed1984

Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide.

Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.

Acrylic Resins↗
PubMed1983

A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities.

Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.

Acyltransferases↗
PubMed2005

Induction of radical scavenging ability and suppression of lipid peroxidation in rat liver microsomes following whole-body, low-dose X-irradiation.

PURPOSE: To investigate changes in radical scavenging ability and lipid peroxidation in liver microsomal membranes and cooperative suppression of lipid peroxidation by microsomal and cytosolic radical scavengers, 24 h after whole-body, low-dose X-irradiation of rats. MATERIALS AND METHODS: Male Wistar rats were irradiated with 1-50 cGy of X-rays. Liver microsomal radical scavenging ability was determined using the trapping ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical. Microsomal alpha-tocopherol (Vit.E) content was determined using an electrochemical detector. Microsomal glutathione peroxidase (GPx) activity was determined as the consuming rate of NADPH. Microsomal lipid peroxidation was determined by the thiobarbituric acid method. RESULTS: Low molecular weight radical scavenging ability of rat liver microsomes increased 24 h after whole-body, low-dose X-irradiation when alpha-tocopherol was included, showing a maximum level at 5-10 cGy. Microsomal GPx activity also increased 24 h after 5 cGy irradiation. The lipid peroxidation level in microsomes decreased, showing a maximal suppression at 5 cGy. High-dose irradiation-induced microsomal lipid peroxidation was strongly suppressed cooperatively by microsomal and cytosolic antioxidants induced by low-dose irradiation. CONCLUSION: Low doses of radiation induce increases in liver microsomal antioxidants, which in turn result in enhanced suppression of microsomal lipid peroxidation cooperatively with cytosolic antioxidants induced by low-dose irradiation.

Animals↗
PubMed2003

Metabolism of chrysene by brown bullhead liver microsomes.

We have investigated the regio- and stereoselective metabolism of chrysene, a four-ring symmetrical carcinogenic polycyclic aromatic hydrocarbon (PAH), by the liver microsomes of brown bullhead (Ameriurus nebulosus), a bottom-dwelling fish species. The liver microsomes from untreated and 3-methylcholanthrene (3-MC)-treated brown bullheads metabolized chrysene at the rate of 30.1 and 82.2 pmol/mg protein/min, respectively. Benzo-ring diols (1,2-diol and 3,4-diol) were the major chrysene metabolites formed by liver microsomes from control and 3-MC-treated fish. However, the control microsomes produced a considerably higher proportion of chrysene 1,2-diol (benzo-ring diol with a bay region double bond) plus 1-hydroxychrysene, than 3,4-diol plus 3-hydroxychrysene, indicating that these microsomes are selective in attacking the 1,2- position of the benzo-ring. On the other hand, 3-MC-induced microsomes did not show such a regioselectivity in the metabolism of chrysene. Control bullhead liver microsomes, compared to control rat liver microsomes, produced a considerably higher proportion of chrysene 1,2-diol, the putative proximate carcinogenic metabolite of chrysene. Like rat liver microsomes, bullhead liver microsomes produced only trace amounts of the K-region diol. Chrysene 1,2-diol and 3,4-diol formed by the liver microsomes from both control and 3-MC-treated bullheads consisted predominantly of their R,R-enantiomers. Chrysene is metabolized by bullhead liver microsomal enzymes to its benzo-ring diols with a relatively lower degree of stereoselectivity compared to benzo[a]pyrene (a five-ring PAH), but with a higher degree of stereoselectivity compared to phenanthrene (a three-ring PAH). The data of this study, together with those from our previous studies with phenanthrene, benzo[a]pyrene and dibenzo[a,l]pyrene (a six-ring PAH), indicate that the regioselectivity in the metabolism of PAHs by brown bullhead and rainbow trout liver microsomes does not vary greatly with the size and shape of the molecule, whereas the degree of stereoselectivity in the metabolism of PAHs to benzo-ring dihydrodiols does.

Animals↗