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PubMed1983

Metabolism of benzo[c]phenanthrene by rat liver microsomes and by a purified monooxygenase system reconstituted with different isozymes of cytochrome P-450.

Metabolism of the environmental pollutant and weak carcinogen benzo[c]-phenanthrene (B[c]Ph) by rat liver microsomes and by a purified and reconstituted cytochrome P-450 system is examined. B[c]Ph proved to be one of the best polycyclic aromatic hydrocarbon substrates for rat liver microsomes. It is metabolized by microsomes from control rats and by rats treated with phenobarbital or 3-methylcholanthrene at 3.9, 4.2 and 7.8 nmol/nmol cytochrome P-450/min, respectively. Principal metabolites are dihydrodiols along with small amounts (less than 10%) of phenols. The K-region 5,6-dihydrodiol is the major metabolite and accounts for 77-89% of the total metabolites. The 3,4-dihydrodiol with a bay-region 1,2-double bond is formed in much smaller amounts and accounts for only 6-17% of the total metabolites, the highest percentage being formed by microsomes from control rats. Highly purified monooxygenase systems reconstituted with cytochrome P-450a, P-450b and P-450c and epoxide hydrolase form predominantly the 5,6-dihydrodiol (95-97% of total metabolites) and only a small percentage of the 3,4-dihydrodiol (3-5% of total metabolites). The 3,4-dihydrodiol is formed with higher enantiomeric purity by microsomes from 3-methylcholanthrene-treated rats (88%) than by microsomes from control rats (78%) or phenobarbital-treated rats (60%). In each case the (3R,4R)-enantiomer predominates. B[c]Ph 5,6-dihydrodiol formed by all three microsomal preparations is nearly racemic.

Animals↗
PubMed1983

Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins).

The metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (1-8C) was studied in hepatic microsomes of control, phenobarbitone-pretreated (PB) and 3-methylcholanthrene-pretreated (3MC) C57/BL10 mice. Phenoxazone and its ethers were hydroxylated and O-dealkylated respectively to a common metabolite, resorufin. The three categories of microsomes differed greatly in activity for the metabolism and binding of the various substrate homologues. The most rapidly metabolised substrates for control microsomes were phenoxazone and its shortest-chain ethers, for PB microsomes phenoxazone and the pentyl ether, and for 3MC microsomes the ethyl and propyl ethers. The variations in activity occurred in Vmax rather than in the apparent Km-value. All the ethers gave Type I cytochrome P-450-binding spectra. The substrates giving the largest Type I spectra were the same for all microsomes--the ethyl, propyl and butyl ethers--but the magnitudes of the spectra differed in the order 3MC- greater than PB- greater than control microsomes. Phenoxazone and resorufin gave Modified Type II cytochrome P-450-binding spectra. PB-induction was most marked for the depentylation reaction (increased 101-fold), whereas 3MC-induction was most marked for depropylation and debutylation (88- and 96-fold). The intermicrosomal differences were interpreted as reflecting the different metabolic specificities of variant forms of cytochrome P-450. Substrate lipophilicity increased with increasing ether chain length and was not a major influence on specificity. The main substrate influence on specificity was steric, due to the presence and length of the ether side chain. The preeminent effect of ether chain length was considered to be on the rate of substrate transformation rather than on substrate interaction with cytochrome P-450.

Animals↗
PubMed1987

Product inhibition of benzo[a]pyrene metabolism in uninduced rat liver microsomes: effect of diol epoxide formation.

Conversion of benzo[a]pyrene (BP) to BP 7,8-dihydrodiol 9,10-oxides (DE) (measured as 7,10/8,9-tetrols) by untreated (UT) rat liver microsomes is over 10 times slower than following 3-methylcholanthrene (MC) induction. Time courses have been subjected to a kinetic analysis analogous to that previously reported for metabolism by MC-induced microsomes (J. Biol. Chem., 259 (1984) 13770-13776). Competition between BP and 7,8-dihydrodiol for P-450 is the major determinant of the rate of DE formation. Glucuronidation of quinones and phenols only increases the isolated BP metabolites including DE by 40%. This indicates far less inhibition by these products than for metabolism in MC-microsomes (4-6-fold). Thus stimulation may result from a decreased quinone-mediated oxidation of metabolites. In the presence of DNA, UT-microsomes metabolize BP to approximately equal amounts of 9-phenol-4,5-oxide (9-PO) and DE/DNA adducts. Addition of uridine diphosphoglucuronic acid (UDPGA) fails to enhance modification of DNA by DE, but formation of the 9-PO adduct is reduced as a result of lower free 9-phenol levels. The kinetic characteristics of BP metabolism by UT-microsomes are highly sensitive to the presence of very small but variable amounts (2-25 pmol/mg) of the very active cytochrome P-450c, which is the predominant form in MC-microsomes. The major effect of elevated levels of P-450c is an 8-fold increase in DE formation at low concentrations of BP due to a lowering of Km (7.9-2.6 microM) and an increase in the regioselectivity for DE formation from 7,8-dihydrodiol (5-15% of total BP metabolites). The formation of DE was directly correlated with the content of P-450c (r = 0.94). The presence of increased levels of P-450c in UT-microsomes is probably due to previous exposure of the animals to environmental inducers and is minimized by controlled housing and feeding.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1989

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin.

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.

Androstenedione↗
PubMed1989

Studies on the mechanism of reduction of azo dye carcinogens by rat liver microsomal cytochrome P-450.

This laboratory has described the azoreduction of p-dimethylaminoazobenzene (1c) by rat liver microsomal cytochrome P-450. To elucidate the mechanisms involved, the reduction of structurally related azobenzenes by hepatic microsomes was investigated. High substrate reactivity was observed for 1c, its corresponding secondary (1a) and primary (1b) amines and p-hydroxyazobenzene (1d). In contrast, only negligible rates were obtained for unsubstituted azobenzene (1g), hydrazobenzene (2g), p-isopropylazobenzene (1e) and 1f, the benzoylamide derivative of 1b. These results clearly indicate that electron-donating groups, such as hydroxyl or primary, secondary and tertiary amines, are essential for binding of azo dye carcinogens to liver microsomal cytochrome P-450 and, by implication, their enzymic reduction. No inhibition of azoreduction of 1c or 1d was obtained by addition of 1e, 1g, or 2g to the reaction mixture. In the presence of hepatic microsomes, a type I binding spectrum was obtained for 1d and type II binding spectra for 1a, 1b and 1c, the reactive azo dyes. In contrast, very weak binding was observed for the unreactive compounds 1e, 1f, 1g and 2g. Thus, there is good correlation between binding and substrate reactivity. The apparent lack of binding may explain the inability of the non-reactive compounds to inhibit azoreduction. The difference in the reduction rate observed for 1g vs. 1d suggested that hydroxylation would facilitate the reduction of an otherwise non-reactive azo dye. Support for such a mechanism was obtained in two experiments. In the first, marked facilitation of azoreduction of both the inactive compounds, 2g and 2f, was seen when they were incubated with microsomes under aerobic conditions where preliminary hydroxylation can occur. In the second, azobenzene was initially incubated aerobically with microsomes from phenobarbital- or beta-naphthoflavone-induced rats. The hydroxyazobenzene formed was then readily reduced anaerobically by microsomes from untreated rats.

Animals↗
PubMed1992

Inhibition of microsome-mediated binding of benzo[a]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats.

The in vitro and in vivo effects of selected natural flavonoids (flavone, flavanone, tangeretin, quercetin, chrysin) on the microsome-catalysed binding of [3H]benzo[a]pyrene to calf thymus DNA were investigated and compared with those of two synthetic flavonoids, 7,8-benzoflavone and 5,6-benzoflavone. In vitro addition of these flavonoids (0.1 mM) to an incubation system containing hepatic microsomes prepared from Aroclor 1254-pretreated rats strongly inhibited BaP-DNA adduct formation (72-89%). The incubation of BaP with hepatic microsomes prepared from animals fed 0.3% quercetin, tangeretin and 7,8-benzoflavone for 2 weeks also resulted in less effective binding of BaP metabolites to added DNA, than with microsomes from untreated rats. Other tested compounds, chrysin, flavone, flavanone and 5,6-benzoflavone showed no or little effect. The influence of flavonoid pretreatment on hepatic microsomal enzymes involved in BaP metabolism has also been examined. Aryl hydrocarbon hydroxylase activity was moderately increased (1.5-1.8-fold) in microsomes prepared from rats fed flavone, tangeretin, 7,8-benzoflavone and 5,6-benzo-flavone. Epoxide hydrolase activity was enhanced by 7,8-benzoflavone (1,6-fold), and by flavone and flavanone (5-fold). These results confirm that flavonoids, in vitro, are potent inhibitors of carcinogen-DNA binding. Oral administration of 0.3% flavonoids alters the properties of liver microsomes, resulting in the decreased ability of BaP metabolites to bind DNA.

Animals↗
PubMed1984

Glycerophosphate acylation by microsomes and mitochondria of normal and dystrophic human muscle.

The incorporation of [14C]glycerophosphate into phosphatidic acid, diacylglycerol, triacylglycerol and phosphatidylcholine by microsomes and mitochondria prepared from normal and dystrophic human muscle incubated in vitro in the presence of 0.3 mmol/l CDP-choline was measured. In mitochondria only phosphatidic acid and diacylglycerol are labelled; the rate of incorporation into these two compounds showed no difference between dystrophic and normal mitochondria. In dystrophic microsomes the incorporation into phosphatidic acid was delayed and decreased. No incorporation of glycerol into diacylglycerol, phosphatidylcholine and triacylglycerol could be measured. Thus in dystrophic muscle microsomes only PA was labelled during an incubation of up to 45 min. In both types of microsomes the concentration of endogenous free fatty acids and diacylglycerol was nearly identical. The level of phosphatidylcholine was 186 and 79 nmol/mg microsomal protein in normal and dystrophic muscle microsomes, respectively. Whether the results could be explained as secondary changes was discussed. Despite some unsolved problems the conclusion was drawn that a better explanation of the results is to assume a primary defect involving microsomal-bound phosphatidic acid phosphohydrolase and possibly glycerol-P-acyltransferases.

Acylation↗
PubMed1989

Testosterone metabolism by microsomal cytochrome P-450 in liver of rats treated with some inducers.

1. The stereoselective hydroxylation of testosterone by microsomal cytochrome P-450 and the changes in level of components participated in the microsomal electron transport system were observed in the microsomes induced unique P-450 isozymes. 2. Flavone- and hesperetin-inducible P-450 catalyzed the hydroxylation of testosterone more effectively than other chemicals-inducible ones. 3. The P-450 in all the microsomal preparations tested most rapidly oxidized testosterone to 6 beta-monohydroxy form. 4. Particularly, MC- and BNF-inducible P-450 showed high stereoselectivity on C6-position of testosterone, and PB-, flavone- and hesperetin-inducible one showed that on C2-position of this compound, respectively. 5. This specificity of two flavonoid-inducible P-450 for the formation of 2 alpha- and 2 beta-epimer of monohydroxytestosterone was opposite to each other. 6. The content of P-450 and the activity of NADPH-cytochrome P-450 reductase were high in PB-, MC- and BNF-microsomes, whereas NADH-cytochrome b5 reductase activity was high in two flavonoid-microsomes and the content of cytochrome b5 was not changed except the PB-treated rats. 7. It is suggested that the increasing activities of testosterone hydroxylases in flavonoid-microsomes seems to be closely related to NADH-cytochrome b5 reductase.

Animals↗
PubMed1984

High affinity progesterone binding sites of human uterine microsomal membranes.

Microsomal membranes sedimented at 40 000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [3H]progesterone at 4 degrees C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable (Kd approximately 4 X 10(-9) M, concentration of binding sites 400-900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17 alpha-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0-20.0% (R.A. of progesterone 100%). 5 alpha-Dihydroprogesterone and 5 alpha-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.

Binding, Competitive↗
PubMed1988

Hepatic microsomal metabolism of androst-4-ene-3,17-dione: relative importance of ring hydroxylation and aromatization in control and induced rat liver.

The purpose of these studies was to determine whether oestrogen production is a quantitatively important pathway in the hepatic microsomal metabolism of androst-4-ene-3,17-dione. The effects of the enzyme inducing agents phenobarbitone and beta-naphthoflavone on microsomal cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation and aromatization was investigated in the rat in vitro. In microsomal fractions from untreated rats the ratio of hydroxylated products to aromatized (oestrogenic) metabolites was 33:1. Phenobarbitone pretreatment of rats increased total hydroxylation by about 20% but did not change the ratio of hydroxylated to aromatized products (27:1). In contrast, beta-naphthoflavone induction decreased total hydroxylation to about 35% of control but did not affect total aromatization. Thus the ratio of hydroxylation to aromatization was significantly lower than in control microsomes (17:1). The principal aromatized products were oestriol and 2-hydroxyoestradiol-17 beta, with oestradiol-17 beta and its 4-hydroxy metabolite as minor products; no oestrone was observed. In further studies of the microsomal metabolism of oestrone, the major product was oestradiol-17 beta whereas hydroxylated metabolites were only minor products. Oestradiol-17 beta, in contrast, was hydroxylated to a considerable extent. These findings suggest that oestrone is a better substrate for the microsomal 17 beta-oxidoreductase than it is for cytochrome P-450. It therefore appears likely that any oestrone formed from the aromatization of androst-4-ene-3,17-dione would be readily converted to oestradiol-17 beta which, in turn, is subject to cytochrome P-450-mediated hydroxylation. Although the liver is a site of C19-steroid aromatization, it appears unlikely that this organ could contribute significantly to serum oestrogen levels since microsomal hydroxylases are readily able to convert aromatized products to biologically inactive metabolites.

Androstenedione↗
PubMed1991

Arylamine activation following chronic ethanol ingestion by rats: studies on the liver S9, microsomal and cytosolic fractions and comparison with Aroclor 1254 pretreatment.

That enzyme fractions derived from animals chronically fed alcohol can alter the metabolism of carcinogenic xenobiotic compounds has been documented. To further understand this relationship the mutagenicity of 3 aromatic amines was determined in the Ames test, employing activation systems derived from rats maintained on an alcohol-containing liquid diet, an isocaloric control liquid diet or Aroclor 1254-pretreated animals fed standard laboratory chow. Depending upon protein and substrate concentrations, S9 from ethanol-fed rats was 30-50% less efficient than S9 from pair-fed rats in activating arylamines (2-aminofluorene, 2-aminoanthracene and 2-acetylaminofluorene) to mutagens in Salmonella typhimurium TA98 and TA100. Cytosolic fractions from ethanol-fed animals always resulted in greater arylamine activation than that of controls whereas the opposite was true of the microsomal compartment in which the ethanol-treated group was consistently less active than the controls. The cytosolic N-acetyltransferase activities with respect to 2 different substrates, isoniazid and 2-aminofluorene, were unaffected by ethanol consumption, indicating that this activity probably does not account for the different activation profiles exhibited by the ethanol and pair-fed cytosolic systems. Both the cytosolic and microsomal compartments are required for maximal expression of the mutagenicity of each arylamine however, each compartment can activate arylamines independently of the other. Reconstituting cytosol with microsomes from ethanol- and pair-fed rats, but not Aroclor-pretreated rats, resulted in a synergistic activation of the aromatic amines and displayed an effect similar to that of S9. Compared to Aroclor pretreatment and pair-fed controls, microsomes from ethanol-fed rats displayed the least capacity for activating any of the arylamines to mutagens. Microsomes from Aroclor-pretreated rats accounted for at least 80% of the S9-mediated activation of each of the arylamines to mutagenic metabolites which was in marked contrast to the contribution of the microsomal fractions to the S9 activity in the ethanol- (5-20% of S9 activity) and pair-fed systems (22-30% of S9 activity). The data indicate that 2 opposing reactions occur in S9, a cytosolic activity that augments and a microsomal activity that attenuates the mutagenicity of arylamines. Both activities are modified by ethanol consumption and Aroclor pretreatment.

2-Acetylaminofluorene↗
PubMed1991

Interaction with microsomal lipid as a major factor responsible for S9-mediated inhibition of 1,8-dinitropyrene mutagenicity.

1,8-Dinitropyrene (1,8-DNP), present in polluted air, is a rodent carcinogen and a potent, direct-acting mutagen in salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of mammalian hepatic S9 or microsomes. We demonstrate that at least a substantial part of this effect is attributable to non-enzymatic processes. The microsomal-dependent inhibition was unaffected by omission of an NADPH-generating system or when the cytochrome P-450 inhibitor, SKF-525A, or the cytochrome P-448 inhibitor, ellipticine, was incorporated in the metabolic activation system, suggesting that mixed function oxidases are not involved. Heat inactivation partially decreased the ability of induced S9 to reduce DNP mutagenicity. Substitution of S9 with a similar concentration of bovine serum albumin did not affect DNP activity. Thus non-specific binding to microsomal protein is not involved. However, when lipids, derived from uninduced microsomes, were added to incubations of DNP and S. typhimurium TA98, mutagenicity was decreased. Furthermore, substitution of microsomal lipids with a suspension of phosphatidylcholine (PC), a major lipid constituent of microsomes, affected DNP mutagenicity similarly. An increase in PC concentration resulted in a greater inhibitory effect. The reduction in DNP mutagenicity observed with microsomal lipids or with PC was less than that detected with uninduced S9, whilst the mutagenicity of 2-nitrofluorene was reduced to an approximately equal extent by lipids and S9. This phenomenon may be responsible for the S9-mediated detoxification of other mutagenic nitroaromatic compounds and may have important implications for mutagenicity testing.

Air Pollutants↗
PubMed1986

3 beta-Hydroxysteroid dehydrogenase in human placental microsomes and mitochondria: co-solubilization of androstene and pregnene activities.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) was solubilized from human term-placental microsomes and mitochondria using the non-ionic detergent, polyoxyethylene 20 cetyl ether (BrijR-58). Electron photomicrographs showed microsomes and mitochondria well disrupted by the detergent. The pregnene (C-21) and androstene (C-19) activities co-solubilized over a range (0.04-0.44) of BrijR-58/protein (B/P) concentration ratios (w/w). Optimal solubilization of the C-19 and C-21 activities were 63.3 +/- 2.6% (mean +/- SEM) from mitochondria (B/P ratio 0.37) and 71.8 +/- 2.1% from microsomes (B/P ratio 0.34). Detergent treatment of microsomes and mitochondria--varying time (5-90 min, pH 7.4) or varying pH (6.0-7.8, 90 min)--yielded C-19 activities identical with C-21 activities. The C-21/C-19 specific activity ratios of 3 beta-HSD in particulate, solubilized and chromatographed preparations were 2.28 +/- 0.16 (mean +/- SEM) for mitochondria and 1.97 +/- 0.07 for microsomes. 3 beta-HSD molecular weight estimates were 208,000 (microsomes) and 220,000 (mitochondria). These studies argue that a single protein is responsible for both the C-19 and C-21 activities of 3 beta-HSD and that this protein is the same in microsomes and mitochondria.

3-Hydroxysteroid Dehydrogenases↗
PubMed1989

The effect of inducers of mixed-function oxidases on hepatic microsome-mediated aflatoxin B1 transformation in C3H/10T1/2 cells.

The potent hepatotoxin and hepatocarcinogen aflatoxin B1 (AFB1) is metabolized by different forms of cytochrome P450 associated with the hepatic mixed-function oxidase system. C3H/10T1/2(10T1/2) cells, which have limited inherent capacity to metabolize AFB1, were treated with AFB1 in the presence of hepatic microsomes isolated from chemically treated rats to investigate the effects of the induction of specific cytochromes P450 on AFB1-mediated toxicity and transformation. Relative to uninduced microsomes, phenobarbital (PB) treatment induced AFB1-DNA binding (essentially representing the formation of AFB1-8,9-oxide bound to DNA) 3.2-fold, while pretreatment with Aroclor 1254, 3-methylcholanthrene (3-MC), or 5,6-benzoflavone (beta-NF) preferentially induced aflatoxin M1 (AFM1) formation from 2- to 5-fold. 10T1/2 cells were exposed to a multiple treatment regimen with 4 microM AFB1 and hepatic microsomes from uninduced, PB-, Aroclor 1254-, 3-MC-, or beta-NF-treated rats; respective cumulative toxicities of approximately 90, 95, 70, 60, and 40% control (no microsomes) values resulted. An enhanced AFB1 transformation response correlated with the increasing toxicities observed for the different treatments, with uninduced or PB-induced microsomes yielding approximately four foci/dish, while treatments with Aroclor 1254-, 3-MC-, or beta-NF-induced microsomes resulted in only one to two foci/dish. These results demonstrate that AFB1 is a complete carcinogen in the 10T1/2 system when repetitive incubations are used in conjunction with an appropriate hepatic microsomal activation system. These data also correlate the induction of AFB1-4-hydroxylase with a decrease in AFB1-mediated toxicity and transformation of 10T1/2 cells, and support the hypothesis that the Phase I metabolic conversion of AFB1 to AFM1 in the liver represents an effective detoxification pathway per se.

Aflatoxin B1↗
PubMed1992

Alterations in lipid peroxidation, antioxidant enzymes, and carcinogen metabolism in liver microsomes of vitamin E-deficient trout and rat.

Feeding rainbow trout for 16 weeks a diet in which the levels of vitamin E were reduced 70-fold resulted in marked depletion (18-fold) of vitamin E levels in liver microsomes from these fish. The susceptibility of hepatic microsomes to lipid peroxidation in vitro and the levels of plasma and liver microsomal lipid hydroperoxides generated in vivo were markedly elevated in vitamin E-depleted trout. No appreciable alterations were observed in the liver microsomal cytochrome P450-dependent mixed-function oxidase system or in the fatty acid composition of trout liver microsomal membranes. Livers from rats fed a vitamin E-deficient diet for 10 weeks also had significantly lower levels of microsomal vitamin E. In addition, total cytochrome P450 levels were depressed (15%) and cytosolic glutathione was enhanced (40%) in livers from rats fed the vitamin E-depleted diet. Covalent binding of [3H]-(+)-benzo[a]pyrene-7,8-dihydrodiol to exogenous DNA in vitro was enhanced with liver microsomes from vitamin E-deficient trout and these fish were much more sensitive to the acute toxicity of this carcinogenic polycyclic aromatic hydrocarbon. These results indicate that trout may be a useful model for studying the significance of peroxidative pathways in carcinogenesis and their manipulation by dietary antioxidants.

Animals↗
PubMed1986

Arrhenius plots of membrane-bound enzymes of mitochondria and microsomes in the brain cortex of developing and old rats.

To study changes in lipid-protein-interaction and fluidity in mitochondrial and microsomal membranes of brain during development and aging, the Arrhenius plots of marker enzymes for mitochondrial inner and outer membranes as well as those of microsomal membranes were compared at different ages. The enzymes were, beta-hydroxybutyrate dehydrogenase (BDH) for the inner mitochondrial membrane and rotenone-insensitive-NADH-cytochrome c reductase (Mit-NADH-CytR) for the outer membrane; also antimycin-insensitive-NADH-cytochrome c reductase (Micr-NADH-CytR) and NADPH-cytochrome c reductase for the microsomal membranes. The ages studied were, 1, 5, 30, 60 days postnatal and 2 years. In the microsomes, the plots of NADH-cytochrome c reductase were found to be biphasic at all ages except in the newborn where no break temperature was observed. The activation energy of this enzyme in the physiological range of temperature was found to be high in the newborn, declining with brain maturation. The plot of this enzyme in the old rat showed no difference when compared with the mature. The specific activity of the enzyme, however, was markedly reduced in the old brain microsomal fraction. In contrast, comparison of the plots of microsomal NADPH-cytochrome c reductase at the various ages revealed no break temperature and very similar energies of activation. For the inner mitochondrial membrane beta-hydroxybutyrate dehydrogenase, the Arrhenius plots were generally biphasic at all ages studied with a break temperature of about 20 degrees C. However, the newborn plot was only barely biphasic showing a high energy of activation in the physiological range of temperature. In contrast, for the outer membrane NADH-cytochrome c reductase, the newborn plots were definitely biphasic, exhibiting low activation energy above the breaks. There was also a significant decline in the break temperature with brain development. No significant differences in the plots of this enzyme were found between the old and mature brain cortex. It is concluded that the enzymes of inner and outer mitochondrial membranes as well as the oxidative enzymes of microsomal membranes may show differential patterns of change in lipid-protein-interaction during development and aging, the changes being more marked in development than in aging.

Aging↗
PubMed1994

In vitro and in vivo inhibition of microsomal lipid peroxidation by MA-631.

Excess free radicals are linked to many diseases, including aging, atherosclerosis, and cancer. Previously, we have shown that MA-631 (a complex herbal mixture) inhibits human low-density lipoprotein (LDL) oxidation and may play a role in prevention of atherosclerosis. In this study we further evaluated the in vivo and in vitro antioxidant activity of MA-631. Both the alcoholic and aqueous extracts of MA-631 inhibited enzymatic- and nonenzymatic-induced rat liver microsomal lipid peroxidation in a concentration-dependent manner. The thiobarbituric acid-reactive substances (TBARS) values (nmol malondialdehyde (MDA)/mg microsomal protein) were 1.43 +/- 0.18 for microsomes alone (baseline for enzymatic system), 19.63 +/- 2.50 for microsomes + reduced nicotinamide adenine dinucleotide phosphate (NADPH) (oxidation without inhibitor), 9.89 +/- 1.41 for heated microsomes (baseline for nonenzymatic system), and 27.15 +/- 0.08 for microsomes + ascorbate (oxidation without inhibitor). The concentrations (micrograms/2 ml) of MA-631 which produced 50% inhibition (IC50) of enzymatic- and non-enzymatic-induced lipid peroxidation were 15.2 +/- 2.0 and 17.0 +/- 2.6, respectively, for the aqueous extract, and 4.3 +/- 0.8 and 6.4 +/- 1.2, respectively, for the alcoholic extract. A 2% MA-631 (w:w) supplemented diet fed to rats for three weeks inhibited in vivo, toluene-induced microsomal lipid peroxidation in the brain, kidney, liver, and heart. These results imply that MA-631 may be useful in the prevention of free radical-linked diseases.

Animals↗
PubMed1982

Lysosomal and microsomal beta-glucuronidase of monkey brain. Differential elution characteristics from con A-sepharose and neutral sugar composition.

Microsomal and lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) of monkey brain were differentially eluted from Con A-Sepharose when subjected to chromatography and linear gradient elution with methyl alpha-glucoside at 28+/-1 degree C. The lysosomal enzyme was eluted as a sharp peak in the first few fractions, while the microsomal enzyme was eluted as a broad peak extending over several fractions. This differential pattern of elution was dependent only on the temperature of elution and the concentration of methyl alpha-glucoside used. The lysosomal and microsomal glucuronidases were purified to apparent homogeneity and their neutral sugar analysed. Both of them contained glucose, mannose and fucose but the microsomal enzyme contained about 3-times as much of all these sugars as the lysosomal enzyme. Sodium periodate treatment of the microsomal enzyme resulted in a shift in its elution pattern, similar to the lysosomal enzyme when subjected to Con A-Sepharose chromatography. The content of neutral sugars and the structural features of the oligosaccharide units in the microsomal glucuronidase might be responsible for its elution pattern. A processing of the carbohydrate units of the microsomal glucuronidase might be envisaged to take place if it were to act as a precursor of the lysosomal glucuronidase.

Animals↗