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PubMed1999

Inactivation of cytochrome P-450 (CYP2E1) and carboxylesterase (hydrolase A) enzymes by vinyl carbamate in murine pulmonary microsomes.

We tested the hypothesis that vinyl carbamate (VC) is metabolized in vitro by cytochrome P-450 and carboxylesterase enzymes in murine lung. Incubations with VC and an NADPH-generating system produced a 50% decrease in N-nitrosodimethylamine (NDMA) demethylation and a corresponding loss in the amounts of immunodetectable CYP2E1. Preincubation of microsomes with a CYP2E1 inhibitory antibody or the CYP2E1-selective inhibitor diallyl sulfone (DASO2) inhibited demethylase activity; no alterations were detected upon subsequent exposure to VC. Carboxylesterase-mediated hydrolysis of p-nitrophenyl acetate was reduced by 22% in microsomes incubated with VC. Decreased carboxylesterase activity also was detected in microsomes incubated with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of hydrolase A, a carboxylesterase isozyme. No change in enzyme activity was detected when microsomes were subsequently incubated with VC. The loss in carboxylesterase activity correlated with decreased immunodetectable hydrolase A in microsomes incubated with VC, PMSF, or PMSF and VC. The reduction in VC-induced NDMA demethylase activity was increased to 85% of the control in microsomes previously incubated with PMSF, and this corresponded with a marked decrease in CYP2E1 immunoreactivity in the immunoblots. Covalent binding of VC to proteins was detected in microsomes incubated with VC and an NADPH-generating system. Binding was inhibited in microsomes preincubated with either an inhibitory CYP2E1 antibody or DASO2. In contrast, binding levels were augmented in microsomes preincubated with PMSF. These data supported VC metabolism by CYP2E1 and hydrolase A in murine lung microsomes and is consistent with involvement of CYP2E1 and hydrolase A in the activation and detoxication of VC, respectively.

Allyl Compounds↗
PubMed1992

Comparison of acyltransferase-mediated mutagenicity and nucleic acid binding of N-acetoxy-4-acetylaminobiphenyl by hepatic and bladder microsomes from rats and dogs.

Acyltransferase-mediated mutagenic and metabolic activation of N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP) by hepatic tissues of rats and dogs were compared. N-OAc-AABP was mutagenic in Salmonella typhimurium TA98 even in the absence of exogenous enzyme(s). However, supplementation with hepatic microsomes from dogs showed a dose-dependent increase in mutagenicity of N-OAc-AABP, whereas under the same conditions, rat microsomes were inactive. Incubation of liver microsomes with RNA showed that 46.4 and 11.2 nmole of [3H]N-OAc-AABP were bound/mg RNA/mg protein with dogs and rats, respectively. The hepatic microsome-mediated binding and mutagenicities of N-OAc-AABP were blocked by paraoxon, suggesting the involvement of deacetylase(s) in the activation process. Analyses of the in vitro incubates of N-OAc-AABP with rat and dog liver microsomes revealed the O-deacetylation product N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) as the major metabolite. The ratios of O-deacetylation of N-O[14C]Ac-AABP versus N-deacetylation of N-OAc-[14C]AABP for hepatic microsomes from dogs and rats were 2.9 and 7.2, respectively. The O- and N-deacetylases are also distributed in bladder tissues and their activities in comparison to the hepatic tissues were lower and amounted to 14.2 and 5.0 nmoles (O/N-deacetylation ratio 2.8) for dogs and 14.8 and 1.7 nmoles per mg protein per min (O/N-ratio of 8.7) for rats. The microsomes from bladder tissues also catalyzed the binding of [3H]N-OAc-AABP to RNA and enhanced its mutagenic response in TA98, both of which were blocked by paraoxon. The occurrence of deacetylase(s) in the target tissues of the bladder carcinogen 4-acetylaminobiphenyl (AABP) suggests that metabolic activation of some of the proximate metabolites could occur within these target organs. Furthermore, since the O-deacetylation product N-OH-AABP is relatively innocuous compared to the N-deacetylation product N-acetoxy-4-aminobiphenyl, these results imply that the refractiveness of rats for 4-aminobiphenyl or AABP-induced bladder carcinogenesis might in part be associated with the higher ratios of microsomal O/N-deacetylase activities. Thus susceptibility to arylamine or arylacetamide-induced liver and bladder carcinogenesis might be influenced by the microsomal deacetylases.

Acyltransferases↗
PubMed1995

The role of vitamin E in the susceptibility of rat lung and liver microsomes to iron-stimulated peroxidation.

The production of thiobarbituric acid-reactive substances (TBA-RS) and ethane, two markers of the lipid peroxidation process, was evaluated in rat lung and liver microsomal membranes incubated in the presence of either ferrous ions or a mixture of ferric ions and ascorbate. Microsomal fractions isolated from lung tissue were more resistant than those isolated from the liver. Compared to Fe2+, the association of Fe3+/ascorbate seemed to be totally ineffective in stimulating peroxidation of lung microsomes. The fatty acid profile of lung and liver microsomal membranes could not be responsible for their different susceptibility to free radical degradation. The microsomal fraction isolated from lung showed a higher vitamin E concentration than the liver. The importance of vitamin E in protecting lung membranes was assessed by using lung and liver isolated from vitamin E-deficient and vitamin E-supplemented rats. For both lung and liver microsomal fractions an inverse relationship between vitamin E concentrations and the extent of lipid peroxidation was observed. However, although the vitamin E concentrations in lung and liver microsomes isolated from rats submitted to a vitamin E-deficient diet were not different, lung microsomes still exhibited a lower production of TBA-RS and ethane than liver. In addition to vitamin E, other factors must be involved to explain the resistance of lung microsomes to lipid peroxidation.

Animals↗
PubMed1986

The effect of substrate on the expression of activity catalyzed by cytochrome P-450: metabolism mediated by rabbit isozyme 6 in pulmonary microsomal and reconstituted monooxygenase systems.

The content of cytochrome P-450, isozyme 6, in the rabbit pulmonary microsomal fraction was estimated by immunochemical methods to be 1 to 3% of the total cytochrome P-450. Following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin, the pulmonary microsomal concentration of isozyme 6 increased 16-fold. Isozyme 6 was also detected by immunochemical methods, but not by electrophoresis and staining for protein, in preparations of isozyme 5 isolated from the pulmonary microsomal fraction of untreated rabbits. The metabolism of benzo[a]pyrene in these preparations was found to be catalyzed by isozyme 6, not by isozyme 5 as previously concluded. Cytochrome P-450, isozyme 4, was not detected in the pulmonary microsomal fraction from untreated or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rabbits. Although benzo[a]pyrene and 7-ethoxyresorufin are both substrates for isozyme 6, the pulmonary microsomal metabolism of these compounds was not increased to the same extent by treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin (about 13-fold for 7-ethoxyresorufin and less than 2-fold for BP). However, lack of agreement between increases in isozyme 6 content and activity, and between the relative increases of the activities with the two substrates, was overcome by the addition of purified NADPH-cytochrome P-450 reductase to the microsomal incubations. When alpha-naphthoflavone, at the minimum concentration required for greater than 90% inhibition of isozyme 6 catalysis, was present in the incubations, no increases in activity were obtained by the addition of purified reductase. The turnover numbers of isozyme 6 in microsomal preparations incubated with purified reductase were similar to those of the purified isozyme in a reconstituted monooxygenase system. The relevance of our results to determinations of the substrate specificities and the microsomal concentrations and activities of isozymes of cytochrome P-450 is discussed. In addition, these parameters are used to assess the extent to which the catalytic potential of isozyme 6 is expressed in the rabbit pulmonary microsomal fraction.

Animals↗
PubMed1981

A comparison of the in vitro binding of alpha-tocopherol to microsomes of lung, liver, heart and brain of the rat.

The in vitro binding of alpha-tocopherol to microsomes of lung, liver, heart and brain of the rat was studied with the insoluble tocopherol ligand presented as a complex with bovine serum albumin. Under these conditions, all microsomes showed nonsaturable binding of alpha-tocopherol and the amount bound to microsomes was linearly proportional to the concentration of albumin-complexed tocopherol. Increasing the amount of alpha-tocopherol bound to microsomes in this manner reduced the extent of lipid peroxidation induced by added ferrous iron. The apparent affinities of the microsomes for alpha-tocopherol, as indicated by the amount bound at a given concentration of albumin-complexed tocopherol, decreased in the order brain greater than liver approximately equal to heart greater than lung. The differences in affinity did not correlate with total fatty acid content (r = -0.39), total unsaturated fatty acid content (r = -0.26), or with the content of fatty acids containing two or more double bonds (r = -0.01). A high positive correlation was found with the content of fatty acids containing three or more double bonds (r = +0.96). Since lung microsomes contain approx. 6-times the tocopherol levels of liver and brain and about twice that of heart microsomes, these results show that the in vivo levels of microsomal tocopherol do not reflect microsomal affinity for this biological antioxidant.

Animals↗
PubMed1984

Metabolism of 4-chlorobiphenyl by guinea pig adrenocortical and hepatic microsomes.

Studies were carried out to determine if 4-chlorobiphenyl (4-CB) was a substrate for adrenal monooxygenases and to compare its interactions with adrenal and hepatic microsomal enzymes. Addition of 4-CB to guinea pig adrenal microsomes produced a typical type I spectral change, indicative of binding to cytochrome(s) P-450 and similar to that seen in hepatic microsomal preparations. The activities of several adrenal and hepatic microsomal monooxygenases were decreased by 4-CB in vitro. High pressure liquid chromatographic analyses revealed that both adrenal and hepatic microsomes, in the presence of NADPH, converted 4-CB to a major metabolite which eluted with a retention time identical to that of 4-chloro-4'-biphenylol (4'-OH-4-CB). The identity of 4'-OH-4-CB was confirmed by mass spectrometry. The maximal rate of 4-CB metabolism was greater in adrenal, compared with liver microsomes, but 4-CB had a higher affinity for hepatic than for adrenal enzymes. The rate of adrenal 4-CB metabolism was four to five times greater in microsomes derived from the inner cortical zone (zona reticularis) than those from the outer zones (zona fasciculata and zona glomerulosa). Hepatic microsomes also converted 4-CB to a minor metabolite whose production was blocked by epoxide hydrolase inhibitors, suggesting it might be a diol. 4-CB metabolism was not demonstrable in adrenal mitochondrial preparations. The results indicate that chlorinated biphenyls can serve as substrates for adrenal microsomal monooxygenases, suggesting that local activation may contribute to their adrenocortical toxicity.

Adrenal Cortex↗
PubMed1995

Metabolic activation of the olfactory toxicant, dichlobenil, in rat olfactory microsomes: comparative studies with p-nitrophenol.

The tissue-specific toxicity of the herbicide, dichlobenil (2,6-dichlorobenzonitrile), in the olfactory mucosa is related to a cytochrome P450 (P450)-dependent metabolism, depletion of glutathione and covalent binding of metabolites. Pretreatment of mice with diethyldithiocarbamate (DEDTC) protected against the dichlobenil-induced necrosis. Addition of DEDTC abolished the covalent binding of [14C]-dichlobenil to rat olfactory microsomes, whereas P4502E1-substrates such as ethanol, acetone or p-nitrophenol (NP) had no effect. The NP-hydroxylation in olfactory microsomes was > 6 times higher than that in liver microsomes and was markedly decreased following addition of dichlobenil, DEDTC or metyrapone. In liver microsomes of acetone-treated rats the NP-hydroxylation was markedly decreased following addition of DEDTC, whereas metyrapone and dichlobenil had no effect. In acetone-treated rats, the NP-hydroxylation and the metabolic activation of [14C]-dichlobenil in olfactory microsomes were decreased to 50 and 73% of untreated controls, respectively, whereas in liver microsomes these activities increased > 6 and 3.5-fold, respectively. An antibody to P4502E1 had no effect on the NP-hydroxylation or metabolic activation of [14C]-dichlobenil in olfactory microsomes, whereas the NP-hydroxylation in liver microsomes of acetone-treated rats was markedly decreased. In conclusion, the results do not support a major role for P4502E1 in the metabolic activation of dichlobenil or hydroxylation of NP in rat olfactory microsomes and suggest that these catalytic activities in the olfactory mucosa may represent a common form of P450.

Acetone↗
PubMed1987

Lipid alterations in liver and kidney induced by normobaric hyperoxia: correlations with changes in microsomal membrane fluidity.

The effects of normobaric hyperoxia on both microsomal membrane fluidity and mechanism of phospholipid synthesis in rabbit liver and kidney have been studied. Hyperoxia induces in both organs an impairment of de novo synthesis of glycerolipids which could be due to an inactivation of acyltransferase activities involved in the initial formation of phosphatidic acid. The ability to replace phospholipid fatty acids by reacylation mechanism decreases slightly in the hyperoxic kidney, while it does not change in the hyperoxic liver. Concerning the effect of high arterial pO2 on microsomal membrane fluidity, the hyperoxic liver shows a more fluid environment within the membrane core of microsomes; however, no difference was shown in that of microsomal membrane core of hyperoxic kidney. An insight into the lipid composition of microsomes indicates that liver microsomal membranes have lower cholesterol content and higher unsaturation degree of phospholipid fatty acids, whereas hyperoxic kidney microsomes become more saturated and did not show any difference in their cholesterol content. In both hyperoxic liver and kidney microsomes, phospholipid content decreases in agreement with the depression of phosphatidic acid biosynthesis. These results are discussed in relation to the values of microsomal membrane microviscosity obtained.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1997

Metabolism of the cytochrome P450 mechanism-based inhibitor N-benzyl-1-aminobenzotriazole to products that covalently bind with protein in guinea pig liver and lung microsomes: comparative study with 1-aminobenzotriazole.

The metabolism and covalent binding of radioactivity to microsomal protein of the cytochrome P450 (P450) mechanism-based inhibitors 1-amino-[14C]-2,3-benzotriazole ([14C]ABT) and two radiolabeled forms of N-benzyl-1-aminobenzotriazole (BBT), N-benzyl-1-amino-[14C]-2,3-benzotriazole ([14C]-2,3-BBT) and [14C]-N-7-benzyl-1-aminobenzotriazole ([14C]-7-BBT), were examined in hepatic or pulmonary microsomes from untreated and phenobarbital (PB)- or beta-naphthoflavone (betaNF)-induced guinea pigs. [14C]-2,3-BBT and [14C]-7-BBT were converted to multiple metabolites including ABT, benzotriazole, benzaldehyde, 2- or 3-hydroxy-BBT, and 4-hydroxy-BBT by hepatic microsomes, while [14C]ABT, whose primary metabolite was benzotriazole, underwent little biotransformation. Neither ABT nor BBT was extensively metabolized by pulmonary microsomes. Hepatic microsomes from betaNF (vs PB)-treated guinea pigs metabolized [14C]ABT, [14C]-2,3-BBT, and [14C]-7-BBT more extensively. The degree of NADPH-dependent covalent binding of [14C]-2,3-BBT- or [14C]-7-BBT-derived radioactivity (1.0 nmol/mg of protein) was higher than that of [14C]ABT (0.3-0.8 nmol/mg of protein) in hepatic microsomes, especially those from PB-induced animals. Covalent binding per nmol of P450 in pulmonary microsomes was 3-4-fold higher with [14C]-2,3-BBT (2.9 nmol/nmol of P450) than with [14C]-7-BBT (1.0 nmol/nmol of P450), whereas in hepatic microsomes from PB- or betaNF-treated animals the ratio of binding with the two forms of BBT was approximately 1:1. [14C]-2,3-BBT- and [14C]-7-BBT-derived radioactivity was covalently bound to proteins that migrated in the molecular weight region corresponding to P450 on SDS-PAGE following incubation with NADPH. These data indicate that BBT is metabolized to at least two reactive compounds capable of covalent modification of protein and/or a single reactive product is formed which contains both the benzo ring (of benzotriazole) and the benzyl carbon atom (of the N-benzyl group); that P450 apoprotein modification may be an important mechanism of inactivation of pulmonary and hepatic P450 by BBT; and that hepatic microsomes from betaNF-induced guinea pigs generate more metabolites that do not act as mechanism-based P450 inhibitors from BBT than do those from PB-induced animals.

Animals↗
PubMed2004

Non-enzymatic and enzymatic lipid peroxidation of microsomes and nuclei obtained from rat liver.

The present study investigates in a experimental system in vitro the relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 degrees C. Approximately 50-55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH.

Animals↗
PubMed1990

Biotransformation of aflatoxin B1 in rabbit lung and liver microsomes.

Aflatoxin B1 (AFB1) is a potent hepatotoxic and hepatocarcinogenic mycotoxin that requires bioactivation to AFB1-2,3-oxide for activity. In addition to epoxidation, microsomal monooxygenases biotransform AFB1 to the less toxic metabolites, aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1). The lung is at risk from AFB1 both via inhalation and via the circulation. In the present study, we have characterized rabbit lung and liver microsomal AFB1-DNA binding (an index of AFB1-2,3-oxide formation), AFM1 formation and AFQ1 formation. Vmax values for AFB1-DNA binding were not different between lung and liver when expressed per mg microsomal protein (1.06 +/- 0.13 and 2.12 +/- 1.30 nmol/mg/h for lung and liver respectively), but lung values were greater than liver when expressed per nmol cytochrome P450 (3.64 +/- 0.31 and 1.29 +/- 0.70 nmol/nmol P450/h for lung and liver respectively). Km values for this reaction were not different between lung and liver. Vmax values for AFM1 formation in liver microsomes were greater than in lung when expressed per mg protein, but not when expressed per nmol P450. No differences were detected for the Km for AFM1 formation between lung and liver microsomes. For AFQ1 formation, no differences were detected between Vmax values of lung and liver, regardless of whether results were expressed per mg protein or per nmol P450, while the Km for AFQ1 formation was lower in liver. SKF-525A inhibited these reactions by 63-74% in lung microsomes and 90-96% in liver microsomes. These results indicate that the lung is capable of activating AFB1, and that rabbit lung microsomes contain high activity for this reaction. Furthermore, little AFM1 and AFQ1 are formed in lung microsomes, leading to minimal shunting of AFB1 from the activation pathway.

Aflatoxin B1↗
PubMed1986

Effect of dietary zinc on endogenous free radical production in rat lung microsomes.

The objective of this study was to investigate the effect of dietary zinc on endogenous production of free radicals in lung and liver microsomes. Male weanling rats were fed a zinc-deficient basal diet containing less than 1.1 ppm zinc, or were pair-fed or fed ad libitum a zinc-adequate diet supplemented with 100 ppm zinc. The isolated microsomes (100,000 X g precipitate) of lung and liver were incubated with 0.1 M PBN (spin trap) and 0.3 mM NADPH (cofactor) at 37 degrees C for 1.0 h. A carbon-centered free radical (aN = 16.0 G, aH beta = 3.4 G) was trapped in both lung and liver microsomes. There was a significant increase in the concentration of carbon-centered free radicals generated in lung microsomes in animals fed a zinc-deficient diet. Dietary zinc status did not significantly affect the concentration of free radicals in liver microsomes. The amount of free radicals generated is proportional to microsomal protein concentration and is linear with protein concentration between 5 and 20 mg per milliliter of incubate. The free radicals formed in the microsomal system were dependent on the presence of NADPH. Carbon monoxide inhibited 40-50% of the free radical production in both lung and liver microsomes. The results suggest that dietary zinc deficiency stimulates the production of endogenous free radicals in rat lung microsomes by an NADPH- and cytochrome P-450-dependent system.

Animals↗
PubMed1976

UDP glucuronosyltransferase and mixed function oxidase activity in microsomes prepared by differential centrifugation and calcium aggregation.

Microsomes were prepared from the liver, kidney and lung of phenobarbital or 20-methylcholanthrene treated and control rats with the conventional ultracentrifugation and calcium aggregation methods. The two methods were compared as to the yield of microsomal protein, amount of cytochrome P-450/448 and activity of UDP-GLUCURONOSYLTRANSFERASE, BENZOPYRENE HYDROXYLASE AND P-NITROANISOLE O-demethylase. The absolute amount of cytochrome P-450/448 (nmol/g wet weight), as well as the enzymatic activities dependent on it (nmol produced/g wet weight) did not differ significantly in any tissue of either treated or control animals nor did that of UDPglucuronosyltransferase. However, the ultracentrifugation method resulted in a slightly smaller yield of the hepatic microsomal protein and a correspondingly higher yield of cytochrome P-450/448 per mg protein as well as higher specific enzymatic activities of both the consecutive drug biotransformation reactions studied. The specific activity of UDPglucuronosyltransferase in digitonin treated microsomes was twice as high in the conventional microsomes as in the calcium aggregated microsomes; no differences was found in the trypsin treated microsomes. The specific activity of the hepatic benzpyrene hydroxylase of the benzpyrene treated animals in the calcium harvested microsomes was 55 per cent of that in the ultracentrifugated microsomes.

Animals↗
PubMed1998

Microsomal metabolism of carbamazepine and oxcarbazepine in liver and placenta.

Metabolism of both carbamazepine (CBZ) and oxcarbazepine (OCBZ) were catalyzed by human liver microsomes and microsomes from livers of CBZ-induced or non-induced C57BL/6 mice. Human placental microsomes metabolized only OCBZ. Mouse liver microsomes metabolized CBZ to carbamazepine-10,11-epoxide (CBZ-E), 10-hydroxy-10,11-dihydro-carbamazepine (10-OH-CBZ), 3hydroxy-carbamazepine (3-OH-CBZ), 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine (10,11-D) and to an unidentified metabolite. CBZ-pretreatment of mice increased both ethoxyresorufin O-deethylase activity in the liver and the amount of CBZ-E in microsomal incubations regardless of the age of mice. Human liver microsomes catalyzed the formation of CBZ to 9-hydroxymethyl-10-carbamoyl acridan (9-AC) in addition to CBZ-E, 3-OH-CBZ and 10-OH-CBZ. OCBZ was metabolized to its active metabolite in all incubations. An unknown metabolite was also present in some of the incubations. Human liver microsomes catalyzed only minute covalent binding of CBZ and OCBZ to DNA. Binding of OCBZ was, however, one order of magnitude greater than binding of CBZ. Human placental microsomes from the mothers on CBZ therapy did not catalyze CBZ metabolism. The same microsomes catalyzed OCBZ metabolism to 10-OH-CBZ and to an unknown metabolite. These results indicate autoinduction in CBZ metabolism in mouse liver. Due to the higher binding of OCBZ than CBZ to DNA in vitro, further studies on the potential mutagenicity of OCBZ may be warranted.

Animals↗
PubMed1994

Determination of flavin-containing monooxygenase activity in rat brain microsomes with benzydamine N-oxidation.

The activity of flavin-containing monooxygenase (FMO) in rat brain microsomes was measured by fluorometrical determination of benzydamine (BZY) N-oxygenation with HPLC. The apparent Km value for the formation of BZY N-oxide from BZY by brain microsomes was similar to that by hepatic microsomal fraction or purified FMO, but the Vmax value for brain microsomes was about one-hundredth of that for hepatic microsomes. BZY N-oxygenation activity by brain microsomes was at a maximum near pH 8.5, slightly more acidic than the optimum pH for liver FMO. BZY N-oxygenation activity was inhibited completely by heat inactivation and markedly in the presence of 1 mM thiourea, but slightly in the presence of 1 mM SKF-525A, and it was only barely activated in the presence of 5 mM n-octylamine, a positive effector of liver FMO. The addition of rabbit antisera raised against rat liver FMO resulted in 30% inhibition of BZY N-oxygenation by solubilized brain microsomes. Compared with microsomes from five different brain regions, the activity was highest in microsomes of olfactory bulbs. These results show that the activity of FMO in rat brain is distinctly determined by BZY N-oxygenation.

Animals↗
PubMed1980

Paraquat-stimulated binding of dopa to liver and lung microsomal protein.

1. 3H-Dopa is converted by lung and liver microsomes to a reactive intermediate which binds covalently to lung and liver microsomal protein. 2. Binding of radioactivity from 3H-dopa to lung and liver microsomes was decreased by superoxide dismutase to the level observed with boiled microsomes. 3. Paraquat (5 mM) caused a 133% increase in binding of radioactivity from 3H-dopa with lung microsomes compared with 224% increase with liver microsomes. However, superoxide dismutase decreased the binding by 38% and 50% with lung and liver microsomes respectively. 4. Benzoate and 1,4-diazobicyclo[2,2,2]octane in the absence and presence of paraquat had no effect on the binding of radioactivity from 3H-dopa. 5. Glutathione and ascorbic acid in the absence and presence of paraquat decreased the binding to below the level obtained with boiled microsomes. 6. The soluble fraction when present in the physiological ratio to microsomal protein decreased the binding, in the presence of paraquat, to the level of the control.

Animals↗
PubMedDate not supplied

Organ and species differences in microsomal activation of methyldopa.

The covalent binding of 3H-methyldopa to microsomal protein in the presence of NADHP and oxygen was studied in various microsomal preparations. Rat and mouse liver microsomes showed high binding, hamster and guinea pig liver microsomes gave intermediate values, whereas no binding was seen with rabbit liver microsomes. No activation of methyldopa was detected with kidney microsomes. Lung microsomes from rats, guinea pigs, and rabbits were quite active with respect to methyldopa binding, and the reactions were totally blocked by superoxide dismutase. There was no sex difference in the binding of methyldopa in liver microsomes from adult rats. No methyldopa activation could be detected in fetal liver microsomes, whereas a rapid increase in activity to above adult levels occurred during the first 2 days after birth.

Aging↗
PubMed1992

Characterization of human lung microsomal cytochrome P-450 1A1 and its role in the oxidation of chemical carcinogens.

Rat and human lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic activities towards several xenobiotic chemicals, including procarcinogens, in different microsomal preparations. Rat lung microsomal P-450s were more active than the human P-450s in catalyzing most of the monooxygenation reactions. Human lung microsomal P-450 was solubilized and purified. Human lung microsomes contain approximately 10 pmol of P-450/mg of protein, on the basis of Fe2+.CO versus Fe2+ difference spectra of the eluates obtained from an octylamino-agarose column. The partially purified P-450 preparations from two human lung microsomal samples showed high activities for the conversion of both (+)- and (-)-isomers of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to genotoxic products. After DEAE-cellulose column chromatography, a partially purified P-450 fraction containing polypeptides of Mr 52,000 and 58,000 was obtained from the early fraction of the octylamino-agarose column eluate, and an electrophoretically homogeneous protein having a molecular weight of approximately 52,000 was recovered from a latter fraction. The amino-terminal amino acid sequences of the two peptides in the earlier fraction were determined; neither polypeptide appears to resemble any known P-450 protein. The protein from the latter octylamino-agarose fraction was immunoreactive with anti-rat P-450 1A2 and anti-human P-450 1A2 but not with antibodies raised against other P-450 enzymes or autoimmune antibodies that specifically recognize human P-450 1A2. A tryptic peptide was isolated from the preparation, and the amino acid sequence matched that of human P-450 1A1 perfectly (residues 31-48) but not that of human P-450 1A2. All of nine human lung microsomal samples examined contained proteins that were immunoreactive with rabbit anti-rat P-450 1A2 and catalyzed the activation of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene. The activities could be inhibited by rabbit anti-rat P-450 1A2 and, to a lesser extent, by anti-rat P-450 1A1. The addition of 7,8-benzoflavone caused inhibition or stimulation, depending upon the particular human lung microsomal preparation. Thus, this work clearly shows that human lung microsomes contain at least two major P-450 enzymes; human P-450 1A1 is present in lungs and can actually catalyze the activation of environmental procarcinogens, including polycyclic aromatic hydrocarbons.

Animals↗