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PubMed1997

Cyclic ADP-ribose binds to FK506-binding protein 12.6 to release Ca2+ from islet microsomes.

Cyclic ADP-ribose (cADPR) is a second messenger for Ca2+ mobilization via the ryanodine receptor (RyR) from islet microsomes for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, FK506, an immunosuppressant that prolongs allograft survival, as well as cADPR were found to induce the release of Ca2+ from islet microsomes. After islet microsomes were treated with FK506, the Ca2+ release by cADPR from microsomes was reduced. cADPR as well as FK506 bound to FK506-binding protein 12.6 (FKBP12.6), which we also found occurs naturally in islet microsomes. When islet microsomes were treated with cADPR, FKBP12.6 dissociated from the microsomes and moved to the supernatant, releasing Ca2+ from the intracellular stores. The microsomes that were then devoid of FKBP12.6 did not show Ca2+ release by cADPR. These results strongly suggest that cADPR may be the ligand for FKBP12.6 in islet RyR and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, causing it to release Ca2+.

Adenosine Diphosphate Ribose↗
PubMed1999

Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases.

In this study a pathway for the synthesis of triacylglycerol (TAG) within the lumen of the endoplasmic reticulum has been identified, using microsomes that had been preconditioned by depleting their endogenous substrates and then fusing them with biotinylated phosphatidylserine liposomes containing CoASH and Mg(2+). Incubating these fused microsomes with tri[(3)H] oleoylglycerol and [(14)C]oleoyl-CoA yielded microsome-associated triacylglycerol, which resisted extensive washing and had a [(3)H]:[(14)C] ratio close to 2:1. The data suggest that the precursor tri[(3)H]oleoylglycerol was hydrolyzed by microsomal lipase to membrane-bound di[(3)H]oleoylglycerol and subsequently re-esterified with luminal [(14)C]oleoyl-CoA. The accumulation of TAG within the microsomes, even when overt diacylglycerol acyltransferase (DGAT I) was inactive, is consistent with the existence of a latent diacylglycerol acyltransferase (DGAT II) within the microsomal lumen. Moreover, because luminal synthesis of TAG was carnitine-dependent and markedly reduced by glybenclamide, a potent carnitine acyltransferase inhibitor, microsomal carnitine acyltransferase appears to be essential for trafficking the [(14)C]oleoyl-CoA into the microsomal lumen for subsequent incorporation into newly synthesized TAG. This study thus provides the first direct demonstration of an enzymatic process leading to the synthesis of luminal triacylglycerol, which is a major component of very low density lipoproteins.

Acyl Coenzyme A↗
PubMed1997

Lipid modifications of microsomes isolated from villus and crypt zones of bovine intestinal mucosa: relationship with fatty acid binding protein.

The present studies were conducted to examine the fatty acid composition of microsomal lipids of bovine small intestine. Microsomes and cytosol were isolated from mucosal scrapings enriched with villus and crypts cells, and the following studies were conducted to: 1) analyse fatty acids from microsomal lipids; 2) incorporate 1-14C oleic acid to microsomal lipids; and 3) bind 1-14C oleic acid to cytosolic proteins of villus and crypt zone. The results of these studies demonstrated that: (1) the major unsaturated fatty acids of microsomes were oleic (C18:1 n-9), linoleic (C18:2 n-6) and arachidonic acids (C20:4 n-6), which increased from crypt to villus tip of the bovine intestinal mucosa; (2) gel filtration chromatography indicated that the low-molecular weight cytosolic proteins obtained from superficial mucosal scrapings contained the greatest oleic acid binding activity; (3) the incorporation of 1-14C oleic acid to microsomes was higher in phospholipids, triglycerides and cholesterol esters from villus than in crypts zones; (4) the protein content of cytosol and microsomes was longer in villus zones than in crypts zones; (5) the peroxidizability index showed the highest value in villus microsomes.

Alkaline Phosphatase↗
PubMed1996

Pulmonary microsomal metabolism of benzo[a]pyrene following exposure of rats to silica.

Because some evidence suggests that there may be an increased incidence of lung cancer in silicosis and because previous studies have shown that exposure of rats to silica alters the pulmonary cytochrome P-450 system, we studied the effects of exposing rats to silica on the lung microsomal metabolism of benzo[a]pyrene (BaP). Rats were exposed to silica by intratracheal administration, lung microsomes were obtained 2 wk later from untreated and silica-treated animals, and the amounts of microsomal tissue and metabolites formed during the in vitro microsomal metabolism of BaP were measured. When the formation of BaP metabolites in equal amounts of lung microsomal tissue from the 2 treatment groups is compared, 3-OH BaP, BaP 4,5-diol, and BaP 9,10-diol are reduced by 45-70%, but the formation of BaP 7,8-diol or the BaP-quinones is not significantly altered following exposure to silica. In fact, the ratio of the BaP diols and BaP quinones, potentially toxic metabolites, to the relatively nontoxic 3-OH BaP produced by equal amounts of lung microsomal tissue is increased more than threefold following exposure of rats to silica. Since exposure of rats to silica leads to increased levels of lung microsomal protein, the amounts of BaP metabolites that could be produced by all microsomal tissue in the lungs were calculated. In silica-treated animals, the calculated total lung production of 3-OH BaP, BaP 4,5-diol, and BaP 9,10-diol tends to be increased by 1.2- to 2.0-fold, but BaP 7,8-diol and the BaP quinones are increased by 3.5-fold. These results demonstrate that exposure of rats to silica may alter the capacity of the lungs to metabolize benzo[a]pyrene, and the greatest effect seems to be enhanced accumulation of BaP 7,8-diol and the BaP quinones.

Animals↗
PubMedDate not supplied

Effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism.

The effects of microsomal enzyme inducers on thyroid hormone homeostasis and the thyroid gland are of concern. We have investigated the effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism in rats. We have shown that small increases in serum TSH can result in large increases in thyroid follicular cell proliferation. Furthermore, only those microsomal enzyme inducers that increase serum TSH--that is, phenobarbital (PB) and pregnenolone-16alpha-carbonitrile (PCN)-increase thyroid follicular cell proliferation, whereas those microsomal enzyme inducers that do not increase serum TSH--that is, 3-methylcholanthrene (3MC) and Aroclor 1254 (PCB)-do not increase thyroid follicular cell proliferation. Deiodination does not appear to be the reason why serum T3 concentrations are maintained in microsomal enzyme inducer-treated rats. We have also shown that those microsomal enzyme inducers that increase serum TSH increase T3 UDP-glucuronosyltransferase (UGT) activity, whereas those microsomal enzyme inducers that do not increase serum TSH do not increase T3 UGT activity. This finding suggests that induction of T3 glucuronidation, rather than T4 glucuronidation, mediates increases in serum TSH of microsomal enzyme inducer treated rats.

Animals↗
PubMed1959

Phospholipide turnover in microsomal membranes of the pancreas during enzyme secretion.

After incubation of pigeon pancreas slices with P(32) and isolation of various fractions by differential centrifugation the deoxycholate extract of the microsome fraction was found to account for over half of the phospholipide P and over half of the P(32) incorporated into the phospholipides. The remaining phospholipide P and P(32) were fairly evenly distributed in the nuclei, zymogen granules, mitochondria, microsomal ribonucleoprotein particles, and the soluble fraction. When enzyme secretion was stimulated with acetylcholine about two-thirds of the increment in radioactivity in the total phospholipides was found in deoxycholate soluble components of the microsome fraction. The remainder of the increment was distributed in the other fractions. This indicates that the cellular component in which the increase in phospholipide turnover occurs on stimulation of secretion is a membranous structure. Evidence is presented which indicates that the increment in radioactivity in the non-microsomal fractions on stimulation of secretion is due to contamination of these fractions with fragments of the stimulated membranous structure. The distribution of P(32) radioactivity in each of the chromatographically separated phospholipides in the various fractions from unstimulated tissue paralleled the distribution of radioactivity in the total phospholipide fraction, indicating that individual phospholipides are not concentrated in different fractions but are associated together in the membranous structures of the microsome fraction. The major proportion of the stimulation of the turnover of the individual phospholipides also occurred in the microsome fraction. The distribution of radioactivity from glycerol-1-C(14) in the total phospholipides and in the individual phospholipides in the various fractions was similar to the distribution of P(32). In the microsome fraction acetylcholine stimulated the incorporation of glycerol-1-C(14) in each phospholipide which showed a stimulation of P(32) incorporation. The significance of the turnover of phosphatides in microsomal membranes in relation to the mechanism of secretion is discussed.

Acetylcholine↗
PubMed1990

Metabolic deactivation of furylfuramide by cytochrome P450 in human and rat liver microsomes.

Metabolic deactivation of furylfuramide by human and rat liver microsomal cytochrome P450 enzymes has been investigated in a system measuring induction of umu gene expression response in Salmonella typhimurium TA1535/pSK1002. Both human and rat liver microsomes catalyzed the metabolism of furylfuramide to inactive form(s) that are incapable of inducing umu gene expression in the tester strain. The reaction required an NADPH-generating system and molecular oxygen and was inhibited by carbon monoxide, suggesting that a cytochrome P450-linked mono-oxygenase system is prerequisite for the deactivation reaction. With liver microsomes from variously pretreated rats, 3-methylcholanthrene was found to be a powerful inducer for the furylfuramide-metabolizing activity, and antibodies raised against rat P450IA1(BNF-B, c) and P450IA2(ISF-G, d) inhibited the microsomal activity. Human liver microsomal furylfuramide-metabolizing activity was also inhibited significantly by anti-P450IA2 IgG but weakly by anti-P450IA1 IgG. In liver microsomes prepared from seven different human samples, the activities of deactivation of furylfuramide were found to correlate with the amounts of immunoreactive protein related to rat P450IA2 and with the monooxygenase activities of metabolic activation of 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ) and of ethoxyresorufin O-deethylation. These results suggest that P450IA1 and P450IA2 in rats, and P450PA (IA2, the phenacetin O-deethylase and ortholog of rat P450IA2) in humans are the major enzymes involved in the deactivation of furylfuramide in liver microsomes. The metabolic studies involving HPLC analysis of products followed by spectrophotometric examination have also suggested that furylfuramide can be degraded very rapidly through the aerobic metabolism by liver microsomes.

Animals↗
PubMed1990

Microsomally-mediated denitrosation of nitrosoguanidinium compounds.

A major metabolic fate of 1-methyl-2-nitro-1-nitrosoguanidine (MNNG) and nitrosocimetidine (NC) in rodents is denitrosation to generate the unmodified, parent guanidinium compound. MNNG is a potent, locally-acting carcinogen. NC is the nitrosated derivative of cimetidine, an important clinical drug administered orally for the treatment of stomach ulcers. Contrary to expectations based on the results of various short-term in vitro tests for carcinogenic potential, NC is not a carcinogen when administered to rats or mice. Rat liver microsomal enzymes have been found to be capable of catalyzing the denitrosation of MNNG, NC and an NC analog, 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG) in an NADPH-dependent reaction. The denitrosated guanidinium compound generated accounts for 50-70% of the nitroso compound metabolized in a microsomal incubate; nitrite is generated with a yield which represents 40-60% of the guanidinium compound produced. The cytochrome P450 inhibitors metyrapone, n-octylamine, 1-n-hexylimidazole and ellipticine inhibit the conversion of CyanoDMNG to 1,3-dimethyl-2-cyanoguanidine (Cyano-DMG) and nitrite. Microsomal NADPH-cytochrome c reductase activity is not perturbed by this series of organic compound inhibitors. Diethyl maleate at high concentrations weakly stimulates the reaction. The rates of production of the CyanoDMNG degradation products CyanoDMG, nitrite and nitrate are markedly diminished in nitrogen-saturated and in carbon dioxide-saturated microsomal incubates. Preincubating microsomes for 1 h at 37 degrees C prior to substrate and NADPH addition has no effect on the denitrosation activity. Kinetic analysis of the conversion of CyanoDMNG to CyanoDMG indicates a Km of 1.0 mM and a Vmax of 2.7 nmol/min/mg protein. Microsomes isolated from rats pretreated with the cytochrome P450 inducers pyrazole or phenobarbital show enhanced denitrosation activity. The denitrosation capacity of hamster liver microsomes is similar to that observed for rat microsomes.

Aminopyrine↗
PubMed1991

Roles of different cytochrome P450 enzymes in bioactivation of the potent hepatocarcinogen 3-methoxy-4-aminoazobenzene by rat and human liver microsomes.

The potent hepatocarcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) has been reported to be bioactivated to mutagenic intermediates by rat liver microsomal cytochrome P450 (P450) and to be a selective inducer of rat P450IA2. In this study we have further investigated the roles of individual rat and human P450 enzymes in the bioactivation of this hepatocarcinogen in a Salmonella typhimurium TA1535/pSK1002 system where umu response is indicative of DNA damage. 3-MeO-AAB was found to be bioactivated by liver microsomal enzymes from rats and humans in this assay system. The liver microsomal activities are increased by pretreatment of rats with various P450 inducers such as phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX), acetone, ethanol, isoniazid (INH), diphenylhydantoin and valproic acid, and can be inhibited considerably by SKF-525A and metyrapone. alpha-Naphthoflavone (ANF) is also an inhibitor for the reaction catalyzed in BNF-treated rats, but stimulated the microsomal activity in DEX-treated rats. Evidence has also been obtained that specific antibodies raised against P450IIB1, P450IA1 or IA2, P450IIE1, and P450IIIA2 inhibited the activation in liver microsomes from rats pretreated with PB, BNF, INH and DEX respectively, suggesting the possible roles of several P450 enzymes in the bioactivation of 3-MeO-AAB. The results obtained with reconstituted monooxygenase systems containing various rat P450 enzymes are highly supportive of this conclusion. Human liver microsomal activation of 3-MeO-AAB was also inhibited to various extents by antibodies raised against P450IA2, P450MP, P450IIE1 and P450IIIA4. In a reconstituted system containing purified forms of human P450, P450IA2 was the most active in catalyzing 3-MeO-AAB, followed by P450IIIA4 and P450MP. ANF, a known activator of P450IIIA-catalyzed reactions, caused an increase in activation of 3-MeO-AAB in human liver microsomal and P450IIIA4- and P450MP-containing reconstituted systems. From these results it is concluded that multiple P450 enzymes in rat and human liver microsomes are involved in the bioactivation of 3-MeO-AAB, regardless of its selective induction of the rat P450IA2 gene.

Animals↗
PubMed1992

Stereoselective metabolism of (-)-benzo[a]pyrene-7,8-diol by human lung microsomes and peripheral blood lymphocytes: effect of smoking.

Benzo[a]pyrene (B[a]P)-tetrols formed after stereoselective cytochrome P450-dependent metabolism from (-)-trans-7,8-dihydroxy-7,8- dihydrobenzo[a]pyrene [(-)-B[a]P-7, 8-diol] by lung microsomes (n = 19) and peripheral blood lymphocytes (n = 13) from lung cancer patients were measured, and the effect of smoking explored. B[a]P-tetrols were quantified by an HPLC/fluorescence assay with a detection limit of approximately 300 attomol, after incubation with peripheral blood lymphocytes or microsomes from lung cancer patients who were current cigarette smokers, ex-smokers and non-smokers. In lymphocytes from these subjects, high, medium and low metabolic activities respectively for (-)-B[a]P-7,8-diol to tetrol conversion were found, but there was no statistically significant difference between smokers, ex-smokers and non-smokers. When the B[a]P-tetrol formation by human lung microsomes was measured, recent smokers had 4- to 7-fold higher (P = 0.04) metabolic activity than ex-smokers and non-smokers. The mean lung microsomal arylhydrocarbon hydroxylase (AHH) activity was three times higher in smokers than in non-smokers and was undetectable in ex-smokers. AHH activity was correlated with tetrol formation in the same lung microsomal samples (r = 0.62, P less than 0.01 in smokers; and r = 0.67, P less than 0.01 in all subjects). When subjects were grouped according to smoking habits, however, no correlation was detected between mean tetrol formation by lung microsomes and that of lymphocytes. Thus, lymphocytes cannot serve as a surrogate for lung microsomes concerning the pulmonary metabolism of (-)-B[a]P-7,8-diol. The much higher B[a]P-tetrol formation observed in lung microsomes from smokers is in accord with a reported higher pulmonary AHH activity, cytochrome P450IA level, and CYP1A1 gene expression in recent tobacco smokers.

Adolescent↗
PubMed1995

Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse and rat liver microsomes.

1,3-Butadiene is carcinogenic in B6C3F1 mice and Sprague-Dawley rats, and has been classified as a probable human carcinogen. The genetic basis for butadiene carcinogenicity is likely mediated by its metabolite, 1,2:3,4-diepoxybutane (BDE). Oxidation of butadiene to 1,2-epoxy-3-butene (BMO) and further activation to BDE is catalysed by cytochrome P450 (CYP) isozymes. The production of BMO from butadiene is mediated by CYP2E1 and, at high butadiene concentrations, by CYP2A6. The purpose of the present study was to identify which human CYP isozymes have the ability to oxidize BMO to BDE, and to determine the extent to which this reaction occurs in B6C3F1 mouse, Sprague-Dawley rat, and human liver microsomes. Of the human cDNA-expressed CYP isozymes tested, only CYP2E1 formed detectable concentrations of BDE at 80 microM BMO. CYP2E1 and CYP3A4 were active at 5.0 mM BMO. Interindividual and interspecies variation in the initial rate of oxidation of 80 microM BMO to BDE was determined using 10 samples of human liver microsomes and single pooled samples from rats and mice. Those experiments revealed a 60-fold variation in activity among 10 human liver samples (range: 0.005-0.324 nmol/mg protein/min). Rates of BMO oxidation for mouse and rat liver microsomes were 0.473 and 0.166 nmol/mg protein/min, respectively. Apparent kinetic constants for the oxidation of BMO to BDE by four human microsomal preparations, and pooled samples from mice and rats were estimated from detailed investigations of BMO oxidation at various BMO substrate concentrations. Apparent Km for the human liver samples ranged from 0.304-0.880 mM, and Vmax values ranged from 0.38 to 1.2 nmol/mg protein/min. The apparent values of Km and Vmax for mouse liver microsomes were 0.141 +/- 0.007 mM (mean +/- SE) and 1.303 +/- 0.141 nmol/mg protein/min, respectively. For rat liver microsomes, apparent Km and Vmax were 0.145 +/- 0.036 mM and 0.408 +/- 0.031 nmol/mg protein/min, respectively. Measured rates of BDE formation correlated well with CYP2E1 protein concentrations in the human microsome samples. These results implicate human CYP2E1 as a hepatic isoform responsible for the oxidation of BMO to BDE at low concentrations of BMO. Moreover, our in vitro results reveal that microsomes prepared from human, rat and mouse liver possess the ability to form BDE from BMO. Previous in vitro results suggest that following exposure to butadiene more BMO would probably be present in mice than in rats or humans. Thus, in mice more BMO would be available for activation to BDE.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Evidence for cytochrome P450 2A6 and 3A4 as major catalysts for N'-nitrosonornicotine alpha-hydroxylation by human liver microsomes.

The tobacco specific carcinogen N'-nitrosonornicotine (NNN), is believed to be a causative agent for esophageal cancer in smokers. NNN requires metabolic activation to exert its carcinogenic potential. Metabolism occurs through cytochrome P450 (P450) catalyzed 2'- and 5'-hydroxylation, which generates unstable metabolites that decompose to 4-hydroxy-1-(3-pyridyl)-1-butanone ('keto alcohol') and 4-hydroxy-4-(3-pyridyl)butanal, respectively. The latter cyclyzes to 5-(3-pyridyl)-2-hydroxytetrahydrofuran ('lactol'). 2'-Hydroxylation of NNN is believed to be the pathway critical for esophogeal NNN carcinogenesis in the rat. The ability of human liver microsomes and expressed human P450s to metabolize [5-(3)H]NNN to keto alcohol and lactol was determined by reverse phase HPLC with radioflow detection. At low NNN concentrations, 11 human liver microsomes metabolized NNN primarily by 5'-hydroxylation to lactol. This reaction was strongly correlated (r = 0.92) with coumarin 7-hydroxylation, suggesting that NNN 5'-hydroxylation is catalyzed mainly by P450 2A6. 2'-Hydroxylation of NNN by human liver microsomes correlated with 6beta-hydroxylation of testosterone, a P450 3A4-specific activity (r = 0.94). The relative rates of 2'- and 5'-hydroxylation by human P450s 2A6, 2E1, 2D6 and 3A4 expressed in Sf9 cells by the baculovirus-insect cell expression system, and human P450 3A4 produced by stable expression in Chinese hamster ovary cells, were determined. Human P450 2A6 metabolized 1 microM NNN exclusively by 5'-hydroxylation. The rate of lactol formation was 317 pmol/min per nmol P450. Human P450s 2E1 and 2D6 also metabolized NNN only to lactol, but at much lower rates, 0.4 and 0.8 pmol/min per nmol of P450 respectively. In contrast, the metabolism of NNN by expressed human P450 3A4 was specific for keto alcohol formation. The Km for 5'-hydroxylation by baculovirus-expressed P450 2A6 was 2.1 microM, and k(cat) was 953 pmol/min per nmol of P450. The Km for lactol formation by human liver microsomes containing high levels of P450 2A6, was 5 microM . Human liver microsomes exhibited a Km of 312 microM for keto alcohol formation. Coumarin, 8-methoxypsoralen (P450 2A6 inhibitors), and anti-2A6 monoclonal antibody were strong inhibitors of NNN-derived lactol formation in human liver microsomes. Troleandomycin, an inhibitor of P450 3A4, effectively inhibited the metabolism of NNN to keto alcohol by human liver microsomes. These results are consistent with P450 2A6 mediated 5'-hydroxylation and P450 3A4 mediated 2'-hydroxylation of NNN in human liver microsomes.

Animals↗
PubMed1983

The effect of enzyme induction on the stereoselective metabolism of optically pure (-)1R,2R- and (+)1S,2S-dihydroxy-1,2-dihydrobenz-[a]anthracenes to vicinal 1,2-dihydrodiol 3,4-epoxides by rat liver microsomes.

The optically pure (-) and (+)trans-1,2-dihydroxy-1,2-dihydrobenz[a]anthracenes (BA trans-1,2-dihydrodiol) were obtained through the resolution of their diastereomeric di(-)menthoxyacetates by normal-phase h.p.l.c., followed with base-catalyzed hydrolysis. The (-)-BA trans-1,2-dihydrobiol has been determined to have 1R,2R absolute stereochemistry by exciton chirality method. Each of the enantiomeric and racemic BA trans-1,2-dihydrodiol was incubated with liver microsomes from untreated, phenobarbital (PB)-, and 3-methylcholanthrene (MC)-treated male Sprague-Dawley rats. The racemic and enantiomeric BA trans-1,2-dihydrodiols were each metabolized to two 1,2,3,4-tetrahydroxy-1,2,3,4-tetrahydrobenz[a]anthracenes (BA 1,2,3,4-tetrol) derived from the hydrolysis of BA trans-1,2-dihydrodiol anti-3,4-epoxide (the 3,4-epoxy oxygen is trans to the 1-hydroxyl group) and syn-3,4-epoxide (the 3,4-epoxy oxygen is cis to the 1-hydroxyl group), respectively. All the BA 1,2,3,4-tetrols were identified by comparing the reversed-phase h.p.l.c. retention times of tetrols and their vicinal acetonides with the hydrolysis products of the chemically synthesized BA trans-1,2-dihydrodiol anti-3,4-epoxide, BA trans-1,2-dihydrodiol syn-3,4-epoxide, BA trans-3,4-dihydrobiol anti-1,2-epoxide, and BA trans-3,4-dihydrodiol syn-1,2-epoxide, respectively, by their modes of forming vicinal acetonides, and by ultraviolet absorption and mass spectral analyses. From the metabolism of (-)-BA trans-1,2-dihydrodiol, a BA trans-1,2-dihydrodiol anti-3,4-epoxide was the major product formed by liver microsomes from MC-treated rats whereas a BA trans-1,2-dihydrodiol syn-3,4-epoxide was the major product formed by liver microsomes from either untreated or PB-treated rats. In contrast, BA trans-1,2-dihydrodiol syn-3,4-epoxide was the major product formed from the metabolism of (+)-BA trans-1,2-dihydrodiol by all three rat liver microsomal preparations. Liver microsomes from PB-treated rats were found to catalyze the metabolism of both the racemic and the enantiomeric BA trans-1,2-dihydrodiols at a rate higher than those by liver microsomes from untreated and MC-treated rats. All BA 1,2,3,4-tetrol metabolites were found to be optically active by circular dichroism spectral analysis. The results indicate that the 'bay-region' BA trans-1,2-dihydrodiol is metabolized by rat liver microsomes predominantly at the vicinal 3,4-double bond and that each enantiomeric BA trans-1,2-dihydrodiol is metabolized to a pair of diastereomeric BA trans-1,2-dihydrodiol 3,4-epoxides with varying degrees of stereoselectivity depending on the constitutive forms of cytochrome P-450 in the rat liver microsomal preparations.

Animals↗
PubMed1984

Metabolic processing of 2-acetylaminofluorene by microsomes and six highly purified cytochrome P-450 forms from rabbit liver.

Kinetic analysis of oxidative metabolism of 2-acetylaminofluorene (AAF) was studied in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced microsomes and with six highly purified cytochrome P-450 isoenzymes from rabbit liver. Kinetic parameters were defined for 7-, 5-, 3-, 1- and N-hydroxylations of AAF. 7-Hydroxylation was best defined by a two enzyme system, displaying a high affinity and relatively low capacity and a low affinity high capacity components in both control and TCDD induced microsomes. All the purified cytochrome P-450 isoenzymes were capable of catalyzing the 7-hydroxylation of AAF and, with the exception of form 4, this was the only oxidation on the AAF molecule catalyzed by these forms. It is probable that forms 1, 4 and 6 accounted for a substantial part (greater than or equal to 25%) of total metabolic capacity corresponding to the high affinity component of 7-hydroxylation, whereas forms 3b and 3c accounted for less than 5% of the metabolic capacity displayed by the low affinity component in control microsomes. However, forms 4 and 6 could account for greater than 90% of the metabolic capacity of the high affinity component of 7-hydroxylation in TCDD microsomes, whereas the form(s) responsible for the metabolic capacity of the low affinity component were not identified. Each of the 1-, 3-, 5- and N-hydroxylations were best defined by a single enzyme system in both control and TCDD microsomes (3- and 5-hydroxylations could not be defined in TCDD microsomes). Close agreements were found between the apparent Km for N-hydroxylation in control, TCDD induced microsomes and with form 4. alpha-Naphthoflavone inhibited AAF N-hydroxylation to a similar extent in control and TCDD microsomes and in form 4. These date indicate that: a subpopulation of cytochrome P-450 isoenzymes, which includes all the purified P-450 forms tested in the present study, is solely involved in detoxification (i.e., 7-hydroxylation) of AAF, and as such probably behave as a functional unit in vivo; modulation of cytochrome P-450 content by inducers such as TCDD results in emergence of relatively few cytochrome P-450 isoenzymes that can account for most of the oxidative metabolism of AAF; and a single cytochrome P-450 isoenzyme (i.e., form 4) is responsible for catalyzing N-hydroxylation of AAF, the first and the obligatory step in the metabolic activation of this carcinogen.

2-Acetylaminofluorene↗
PubMed1988

Increases in cytochrome P-450 mediated 17 beta-estradiol 2-hydroxylase activity in rat liver microsomes after both acute administration and subchronic administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a two-stage hepatocarcinogenesis model.

Administration of single doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10 micrograms/kg) increased estradiol 2-hydroxylase (E2OHase) activity approximately 2-fold in liver microsomes of female rats but had no effect on E2OHase activity in hepatic microsomes of male rats. In contrast, TCDD increased P-450d (an enzyme which has a high turnover number for E2OHase in a reconstituted enzyme system) 10- to 20-fold in livers of both male and female rats. The discrepancy between the increases in P-450d and E2OHase activity in liver microsomes of TCDD-induced rats was abolished by the addition of exogenous purified P-450 reductase to the microsomal assays for E2OHase, suggesting that reductase was limiting in the in vitro assays. When E2OHase activity was assayed in the presence of exogenous reductase, TCDD increased E2OHase 2-fold and 4-fold respectively in liver microsomes of male and female rates. Antibody to P-450d completely inhibited the increase in E2OHase activity in liver microsomes of TCDD-treated male and female rats, but had little effect on E2OHase activity in liver microsomes of untreated male or female rats. These data indicate that P-450d is responsible for the increase in E2OHase activity in TCDD-treated rats, but other P-450 isozymes are responsible for constitutive E2OHase activity. Biweekly administration of 1.4 micrograms/kg of TCDD for 30 weeks as a potential promoter of hepatocarcinogenesis increased the volume of the liver occupied by gamma-glutamyl transpeptidase (GGT)-positive foci in livers of female rats given a necrogenic dose of diethylnitrosamine (DEN) (200 mg/kg) as the initiator. Biweekly doses of 0.14-1.4 micrograms/kg TCDD in this model also increased P-450d (7-fold) and E2OHase activity maximally (4-fold) in DEN-initiated rats. Moreover, initiation with DEN substantially enhanced the effects of the low dose of TCDD on both hepatic microsomal P-450d and E2OHase activity.

Animals↗
PubMed1990

Mutagenicity of oxidized microsomal metabolites of 1-nitropyrene in Chinese hamster ovary cells.

1-Nitropyrene, a tumorigenic environmental pollutant, is mutagenic in Chinese hamster ovary (CHO) cells in the presence of a liver homogenate 9000 g supernatant fraction (S9). The metabolic pathways involved in this response were studied by comparing the mutagenicities at the hypoxanthine-guanine phosphoribosyl transferase locus of 1-nitropyrene, some oxidized microsomal metabolites of 1-nitropyrene, and related compounds. In the absence of S9, pyrene 4,5-oxide and 6-hydroxy-1-nitropyrene displayed the highest mutagenicities, followed by 1-nitropyrene 9,10-oxide and 1-nitropyrene 4,5-oxide; 3- and 8-hydroxy-1-nitropyrene were weaker mutagens, while pyrene and 1-nitropyrene were essentially without activity. With S9, the order of mutagenic potency was 1-nitropyrene 4,5-oxide greater than 6-hydroxy-1-nitropyrene approximately 1-nitropyrene 9,10-oxide greater than 1-nitropyrene approximately 3-hydroxy-1-nitropyrene approximately 8-hydroxy-1-nitropyrene greater than pyrene approximately pyrene 4,5-oxide, with the latter two compounds being essentially inactive. Inclusion of the epoxide hydrolase inhibitor 1,2-epoxy-3,3,3-trichloropropane during the S9-mediated treatment of CHO cells with 1-nitropyrene increased mutation induction 5-fold. Also, liver microsomes prepared from guinea-pigs treated with Aroclor 1254 mediated a stronger mutagenic response with 1-nitropyrene than microsomes from Aroclor-treated rats. 1-Nitropyrene was essentially non-mutagenic in the presence of microsomes from untreated and phenobarbital-treated rats. Examination of the 1-nitropyrene metabolites produced during the microsomal incubations indicated that Aroclor-induced guinea-pig microsomes yielded substantial amounts of 1-nitropyrene 4,5-dihydrodiol, while Aroclor-induced rat microsomes produced 6-fold more 6- and 8-hydroxy-1-nitropyrene than phenobarbital microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Biosynthesis and localization of rat liver microsomal carboxyesterase E1.

One of the microsomal carboxyesterases, carboxyesterase E1, was purified from rat liver to homogeneity. Carboxyesterase E1 is a glycoprotein of high mannose type, and is composed of three identical subunits of 59 kDa each. It is very similar to "esterase pI 6.0" described by Menthein et al. (Arch. Biochem. Biophys. 200, 547-559 (1980)) in molecular weight, amino acid composition, and enzymic activities. Carboxyesterase E1 was found to be evenly distributed between rough and smooth microsomes. The content of the enzyme in microsomes was about 1.5% of total microsomal protein. It was exclusively located on the luminal side of microsomes, and was not detected immunologically in Golgi fractions or serum. In vitro translation of rat liver RNA by reticulocyte lysate showed that carboxyesterase E1 was synthesized preferentially on the bound ribosomes, as a precursor peptide larger than the peptide of the mature enzyme. Carboxyesterase E1 was solubilized from microsomes by treatment with low concentrations of detergents. However, it was not released from microsomes by treatment with a synthetic peptide which made the microsomal membrane permeable to soluble protein molecules. Carboxyesterase E1 is not a soluble luminal protein, and seems to be bound to the luminal surface of the membrane.

Amino Acids↗
PubMed1995

Contribution of peroxidation products to oxidative inactivation of rat liver microsomal glucose-6-phosphatase.

Exposure of rat liver microsomes to ascorbic acid/Fe(2+) caused decreases in the membrane-bound glucose-6-phosphate (G-6-Pase) activity and the protein thiols after a short lag period (4 min). Under the same conditions, the production of thiobarbituric acid-reactive substances and fluorescent products was also initiated from 4 min after the start of the treatment, although conjugated diene was formed immediately on incubation of the microsomes with ascorbic acid/Fe(2+). After centrifugation of the treated microsomes, the fluorescent products and the enzyme activity remained in the membrane fraction. The results of kinetic studies of the enzyme activity indicated that ascorbic acid/Fe(2+)-induced inhibition of the enzyme activity is mainly due to an increased Km value for the substrate. A decreased activity of the microsomal G-6-Pase was also observed when the microsomes were incubated with aldehydes such as malondialdehyde, n-heptaldehyde, acetaldehyde, and trans-2-nonenal. However, loss of protein thiols was detected only upon treatment of the microsomes with trans-2-nonenal. Glucose-6-phosphate (G-6-P)effectively prevented ascorbic acid/Fe(2+)- or trans-2-nonenal-induced inhibition of the enzyme activity, but the substrate failed to protect the protein thiols in both systems. The results of fluorescence anisotropy measurements of diphenylhexatriene-labeled microsomes suggested that changes in the lipid dynamics are not directly related to peroxidation- mediated inhibition of the enzyme activity. Based on these results, a possible reason for the inhibition of the microsomal G-6-Pase activity associated with ascorbic acid/Fe(2+) treatment is discussed.

Aldehydes↗