Microsomal Monooxygenase Activities Research Articles

Responses of biotransformation and antioxidant enzymes in Lemna minor and Oncorhynchus mykiss exposed simultaneously to hexachlorobenzene

Effect of hexachlorobenzene (HCB), a persistent environmental contaminant, exposure on the enzymes of oxidative biotransformation and antioxidant system of the aquatic plant L. minor and fish were studied simultaneously. Furthermore, the effects of fish - plant interactions in a HCB polluted environment on the responses of hepatic biotransformation and antioxidant enzymes of fish were also investigated. The peroxidase and glutathione S-transfemse (GST) activity in HCB exposed plants were significantly high following 1, 2 and 7 days of the exposure. In general, the activity of antioxidant enzymes in plants also increased following exposure to HCB. Exposure to either HCB or plants in uncontaminated water did not cause any major changes in the hepatic microsomal monooxygenase and antioxidant enzyme activities in fish. However, significant inhibition in liver monooxygenase enzyme activities and increase in the antioxidant enzyme activities were observed in fish that were exposed to HCB in the presence of L. minor. Such observations suggests that interactions between fish and plants in a HCB contaminated environment may affect fish hepatic biotransformation and antioxidants enzymes.

Heterogenous Effects of Natural Flavonoids on Monooxygenase Activities in Human and Rat Liver Microsomes

The in vitro effects of nine flavonoids on different monooxygenase activities from human and rat liver were investigated. Flavonoids belonging to different chemical classes [flavones (chrysin, tangeretin, flavone, 5,6-benzoflavone, 7,8-benzoflavone), flavonols (quercetin), and flavanones (pinocembrin, eriodictyol, flavanone)] were chosen. Ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-debenzylase (BROD) were selected as marker activities of P450 isoenzymes involved in carcinogen activation. Human EROD activity was inhibited by all flavonoids. Flavonoids without hydroxyl groups were more effective than those with hydroxyl groups. Flavonoids with an unsaturated flavone nucleus were more inhibitory than the corresponding saturated analogues. Similar structure-activity relationships were found in microsomes from methylcholanthrene-treated rats. The effects of flavonoids on human BROD activity were quite different from those observed on EROD activity. Nonhydroxylated flavones were effective activators. Hydroxylated flavonoids showed a biphasic effect, being activators at low concentrations and inhibitors at higher concentrations. The effects of flavonoids on BROD activity were rather similar in man and control rats.

Influence of propranolol on the membrane fluidity and monooxygenase activities in rat liver microsomes

Influence of propranolol on the membrane fluidity and monooxygenase activities in rat liver microsomes

Mechanism of Synergism between the Pyrethroid Insecticide λ-Cyhalothrin and the Imidazole Fungicide Prochloraz, in the Honeybee ( Apis mellifera L.)

Ergosterol biosynthesis-inhibiting fungicides have been found to synergize the toxicity of pyrethroid insecticides to the honeybee ( Apis mellifera L.). The mechanism by which the fungicide prochloraz enhances the toxicity of the pyrethroid insecticide λ-cyhalothrin was investigated. In vitro incubations with honeybee midguts were used to study the metabolism of [ 14C]λ-cyhalothrin. The principal metabolite was identified as 4-hydroxy-3-phenoxybenzyl alcohol (4′-OH-3-PBAlc) with small amounts of 3-phenoxybenzoic acid (3-PBAc). Both are products of ester bond cleavage, but microsomal oxidation was implicated in the formation of 4′-OH-3-PBAlc. After treating midguts with prochloraz, metabolism was predominantly to 3-PBAc, with little formation of 4′-OH-3-PBAlc, strongly indicating an inhibition of microsomal monooxygenase activity. In vivo investigations showed the major metabolic products of [ 14C]λ-cyhalothrin extracted from frass of treated honeybees were 4′-OH-3-PBAlc, 2′-hydroxy-3-phenoxybenzyl alcohol, and 4′-hydroxy-3-phenoxybenzoic acid. However, when bees were simultaneously dosed with prochloraz, there was an absence of metabolites detected in the frass of bees for 16 hr post-treatment Thus, prochloraz delayed the metabolism, detoxication, and excretion of λ-cyhalothrin by inhibition of microsomal oxidation, effectively enhancing the toxicity of the pyrethroid to the honeybee.

Interspecies differences in mixed function oxidase activity in birds: Relationship between feeding habits, detoxication activities and organochlorine accumulation

The relationship between feeding habits and interspecies differences in the detoxication ability of the mixed function oxidase (MFO) system was investigated in birds. The role of MFO (particularly aldrin epoxidase activity) in the detoxication/bioaccumulation of organochlorines was also investigated. Euriphagic (yellow-legged herring gull ( Larus cachinnans), black-headed gull ( Larus ridibundus), jackdaw ( Corvus monedula), magpie ( Pica pica) and stenophagic (cormorant ( Phalacrocorax carbo), coot ( Fulica atra) and sparrow ( Passer italiae)) species were collected in northern and central Italy. The following liver microsomal monooxygenase activities were measured: aldrin epoxidase, ethoxyresorufin dealkylation (EROD), benzyloxyresorufin dealkylation (BROD) and pentoxyresorufin dealkylation (PROD). NADPH-and NADH-cytochrome c reductase (NAD(P)H-CYT-CRED) and NADH-ferricyanide reductase (NADH-FERRIRED) activities were also measured in the liver microsomal fraction. Glutathione concentration (GSH) was measured in the liver cytosolic fraction. As a marker of liver damage γ-glutamyl transpeptidase (GGTP) activity was assayed in the serum. The residues of organochlorines (HCB, pp′DDTs and PCBs) were determined in muscle samples. Omnivorous species, particularly the yellow-legged herring gull, had the highest aldrin epoxidase activities. In the yellow-legged herring gull the activity was approximately twice as high as in the jackdaw ( p < 0·05), black-headed gull ( p < 0.05) and coot ( p < 0·05). Values five times lower were detected in the specialist fish-eater, the cormorant ( p < 0·001). The lowest values of aldrin epoxidase activity were detected in the sparrow. Feeding habits were found to be related to evolutionary interspecies differences in MFO activity. A significant statistical correlation ( r = 0·656) was found between the ‘omnivore index’ and MFO detoxication activity expressed as aldrin epoxidation. Organochlorines, and particularly PCBs, were higher in cormorants and yellow-legged herring gulls from a polluted lagoon than in the other species analysed (black-headed gull p < 0·01; sparrow p < 0·001; jackdaw p < 0·01; coot p < 0·001). Levels of pp′DDE were much higher in the cormorant and sparrow than in the other birds. Knowledge of the different species-specific detoxication abilities was found to be a useful tool for the identification of species potentially at risk in environments with high pollution by chlorinated hydrocarbons, or in general contaminants metabolized by the MFO system.

The protective effects of eugenol on carbon tetrachloride induced hepatotoxicity in rats.

Our earlier studies in vitro have shown that eugenol inhibits liver microsomal monooxygenase activities and carbon tetrachloride (CCl4)-induced lipid peroxidation (Free Rad. Res. 20, 253-266, 1994). The objective of the present investigation was to study the in vivo protective effect of eugenol against CCl4 toxicity. Eugenol (5 or 25 mg/kg body wt) given orally for 3 consecutive days did not alter the levels of serum glutamic oxalacetic transaminase (SGOT), microsomal enzymes such as cytochrome P450 reductase, glucose-6-phosphatase (G-6-Pase) xenobiotic-metabolizing enzymes (aminopyrine-N-demethylase, N-nitrosodimethylamine-demethylase and ethoxyresorufin-O-deethylase) and liver histology. Doses of eugenol (5 or 25 mg/kg) administered intragastrically to each rat on three consecutive days i.e. 48 hr, 24 hr and 30 min before a single oral dose of CCl4 (2.5 ml/kg body wt) prevented the rise in SGOT level without appreciable improvement in morphological changes in liver. Eugenol pretreatment also did not influence the decrease in microsomal cytochrome P450 content, G-6-Pase and xenobiotic-metabolizing enzymes brought about by CCl4. Since eugenol is metabolized and cleared rapidly from the body, the dose schedule was modified in another experiment. Eugenol (0.2, 1.0, 5.0 or 25 mg/kg) when given thrice orally i.e. prior to (-1 hr) along with (0 hr) and after (+3 hr) the i.p. administration of CCl4 (0.4 ml/kg) prevented significantly the rise in SGOT activity as well as liver necrosis. The protective effect was more evident at 1 mg and 5 mg eugenol doses. However, the decrease in microsomal G-6-Pase activity by CCl4 treatment was not prevented by eugenol suggesting that the damage to endoplasmic reticulum is not protected. The protective effect of eugenol against CCl4 induced hepatotoxicity is more evident when it is given concurrently or soon after rather than much before CCl4 treatment.

Induction of cytochrome P450 by toluene

At least six cytochrome P450 ( P450) isoenzymes, including CYP1A1/2, CYP2A1, CYP2B1/2, CYP2C6, CYP2C11 and CYP2E1, are involved in the metabolism of toluene in rat liver. Toluene exposure induces CYP1A1/2, CYP2B1/2, CYP2E1 and CYP3A1, but decreases CYP2C11/6 and CYP2A1 in adult males. Both sex and age influence the induction of P450s by toluene: in general, the inductive effect is more prominent in younger than in older animals; in males than in females. Neonatal exposure to toluene causes significant changes in liver microsomal P450 dependent monooxygenase activities during the early stage of life, whereas the enects on the rats of more than 3 weeks of age are small. Although structurally related chemicals of toluene also influence similar hepatic P450 isoenzymes, the degree of CYP2B1/2 induction increases, whilst that of CYP2E1 decreases with increasing molecular weight and aliphatic moieties. Unlike liver, exposure to toluene does not influence the distribution of pulmonary or renal microsomal P450-related enzyme activity in rats. In humans, occupational exposure to toluene is so low that it could not lead to the induction of P450. However, the induction may be seen in toluene sniffers who are exposed to high concentrations.

Herbicide-Resistant Tobacco Plants Expressing the Fused Enzyme between Rat Cytochrome P4501A1 (CYP1A1) and Yeast NADPH-Cytochrome P450 Oxidoreductase

Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase were produced. The expression plasmid pGFC2 for the fused enzyme was constructed by insertion of the corresponding cDNA into the expression vector pNG01 under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase gene terminator. The fused enzyme cDNA was integrated into tobacco genomes by Agrobacterium infection techniques. In transgenic tobacco plants, the fused enzyme protein was localized primarily in the microsomal fraction. The microsomal monooxygenase activities were approximately 10 times higher toward both 7-ethoxycoumarin and benzo[a]pyrene than in the control plant. The transgenic plants also showed resistance to the herbicide chlortoluron.

Microsomal metabolism of N, N-diethylacetamide and N, N-dimethylacetamide and their effects on drug-metabolizing enzymes of rat liver

This study was undertaken to investigate: (1) the effect of N, N-diethylacetamide (DEAC) and N, N-dimethylacetamide (DMAC) administration to rats on drug-metabolizing enzymes in the liver; (2) the in vitro dealkylation of DEAC and DMAC by hepatic microsomes from rats treated with various P450 inducers and purified P450 (2B1 and 2E1). DEAC administration at doses of 100–300 mg/kg i.p. for 3 days mostly induced P450 2B1/2-associated hepatic microsomal monooxygenase activities (pentoxyresorufin O-depenthylase and the 16 β-testosterone hydroxylase) and its own dealkylation (DEAC deethylase activity). P4502E1-linked monooxygenase activities, such as aniline and p-nitrophenol hydroxylases, were not affected. DEAC treatment increased the amount of P4502B1/2 in microsomes in a dose-dependent manner, but depressed the amount of P-4502C11 as assayed by western blotting. DMAC treatment did not alter any microsomal monooxygenases or phase II enzymatic activity. The oxidative metabolism of DEAC and DMAC with control and induced microsomes resulted in the dealkylation of these solvents, giving rise to acetaldehyde and formaldehyde, respectively. The kinetic parameters for these N-dealkylations were investigated. It was found that phenobarbital-, dexamethasone- and DEAC-induced microsomes deethylated DEAC with a V max ∼3-fold of control-, ethanol- or β-naphtoflavone-induced microsomes, although with a similar affinity; ethanol- or acetone- induced microsomes demethylated DMAC with a V max higher than that of control microsomes. In a reconstituted system, the purified P4502B1 dealkylated DEAC, but not DMAC, at the rate of 6.2 nmol/min/nmol P450, whereas purified P4502E1 dealkylated DMAC, but not DEAC, at the rate of 7.9 nmol/min/nmol P450. Oxidation of DEAC and DMAC were markedly inhibited in microsomes from DEAC-treated rats by anti-P4502B1 IgG and in microsomes from acetone-treated rats by anti-P4502El IgG, respectively. These results indicate that DMAC and DEAC are predominantly oxidated by different P450 isozymes and that only DEAC, when administered to rat, is capable of altering the expression of the hepatic P450 system. This latter feature could be related to the higher toxicity reported for DEAC.

In vivo administration of H 2 blockers, cimetidine and ranitidine, reduced the contents of the cytochrome P450IID (CYP2D) subfamily and their activities in rat liver microsomes

1. 1. The effects of in vivo administration of H 2 blockers, cimetidine and ranitidine (0.6 mmol/kg body weight/day, for 5 days), on several P450 isozymes, the P450IID (CYP2D) subfamily, and their monooxygenase activities in rat liver microsomes were investigated. 2. 2. In vivo administration of cimetidine and ranitidine decreased the contents of P450 isozymes and the activities of P450-linked monooxygenase systems; i.e., benzphetamine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarine O-deethylase, debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase. 3. 3. The inhibitory effect on the enzymatic activities of the P450IID (CYP2D)-linked monooxygenase systems was studied by Western blot analysis with serum containing antiCYP2D6 IgG, i.e., LKM1 autoantibody. The amount of P450IID (CYP2D) in liver microsomes decreased more remarkably in the group administered ranitidine or cimetidine in vivo than in controls. 4. 4. The effects of cimetidine and ranitidine on the activities of the P450IID (CYP2D)-linked monooxygenase systems were investigated in vitro. The activities of debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase were inhibited in vitro by cimetidine or ranitidine at a higher concentration than that on in vivo administration of either H 2 blocker. 5. 5. The kinetic parameters for cimetidine or ranitidine as to the activities of debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase in liver microsomes were determined by means of Lineweaver-Burk plots. 6. 6. The suppressive effects of cimetidine and ranitidine on the activities of the P450IID (CYP2D)-linked monooxygenase systems in vivo were found to be due to a decrease of the content of the P450IID (CYP2D) protein.

Use of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as fluorescent substrates for rainbow trout hepatic microsomes after treatment with various inducers

Various fluorescent substrates have been used as specific indicators of induction or activity of different cytochrome P450 isozymes in both fish and mammalian species. In an attempt to identify additional definitive fluorescent substrates for use in fish, we examined a series of 7-alkoxyphenoxazones, 7-alkoxycoumarins and 7-alkoxyquinolines as substrates in O-dealkylation assays with hepatic microsomes from rainbow trout ( Oncorhynchus mykiss). Microsomes were prepared after 48 hr of treatment with β-naphthoflavone (β-NF), pregnenolone-16α-carbonitrile (PCN), phenobarbital (PB), isosafrole (ISF), or dexamethasone (DEX). Total P450 spectra were obtained, and spectral binding studies were performed. Microsomal O-dealkylation rates were greater after ISF treatment than after β-NF treatment for 7-methoxy-, 7-ethoxy-, 7-propoxy- and 7-benzyloxyphenoxazones but not for 7-butoxyphenoxazone. DEX treatment resulted in a significant elevation of pentoxyphenoxazone metabolism (about a 144-fold increase) compared with microsomes induced by β-NF (11-fold) and ISF (37-fold). The rates of dealkylation of the alkoxyphenoxazones by ISF-treated microsomes occurred in the following order: methoxy > ethoxy > propoxy > benzxyloxy > butoxy > pentoxy. When β-NF-treated microsomes were used, the 7-alkoxyphenoxazones were metabolized as follows: methoxy > ethoxy > propoxy > butoxy > benzyloxy ≈ pentoxy while the order of metabolism of the 7-alkoxycoumarins was: ethoxy ⪢ butoxy > propoxy ≈ methoxy > benzyloxy > pentoxy. None of the other treatments significantly increased the rate of metabolism of any of the alkoxycoumarins. Treatment with β-NF did not significantly elevate the rate of metabolism of any of the alkoxyquinolines. DEX treatment produced significant elevations in the rate of metabolism of benzyloxy-, ethoxy-, and butoxy- ≈ pentoxy- ≈ propoxyquinoline, in that order. ISF treatment significantly elevated the rate of metabolism of benzyloxy-, methoxy- and butoxyquinoline, in that order. These results suggest that some of these new fluorescent substrates can be used to characterize induction of rainbow trout hepatic microsomal monooxygenase activity by ISF and DEX, in addition to the commonly used ethoxyphenoxazone and ethoxycoumarin for the characterization of induction by β-NF or other 3-methylcholanthrene-type P450 inducers. Distinction between ISF-type and β-NF-type inducers in rainbow trout hepatic microsomes may best be made using 7-methoxycoumarin as a substrate. Distinction between ISF-type and DEX-type inducers and between β-NF-type and DEX-type inducers may best be made using 7-methoxy-phenoxazone as a substrate. With β-NF induction 7-methoxycoumarin, with ISF induction 7-methoxy-phenoxazone, and with DEX induction 7-ethoxyquinoline were metabolized to the greatest extent compared with controls and all other substrates tested.

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes.

The involvement of cytochrome b5 in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b5. Anti-b5 antiserum inhibited the reduction of cytochrome b5 by NADH-cytochrome b5 reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b5 is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b5. The results suggest that cytochrome b5 involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved.

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.

Effects of Oral Administration of Acitretin on Rat Liver Microsomal Phospholipids, P-450 Content and Monooxygenase Activities

Doses of 3 and 10 mg/kg/day acitretin were orally administered to female Wistar rats over a period of 6 weeks. Phospholipid classes, P-450 content, aminopyrine-N-demethylase (ADM) and 7-ethoxyresorufin-O-deethylase (7-ERO-D) activities were determined in the liver microsomes of the treated animals. Both dosages caused statistically significant alterations in rat liver microsomal phospholipid composition which may be associated with changes in the metabolic activity, ionic transport, cell-cell interaction and other processes of the hepatic cellular components. Furthermore, statistically significant alterations of P-450 isozyme activities were induced by both dosages. Our data suggest a possible interaction of acitretin with other drugs and endogenous substances metabolized by these enzyme systems.

Effect of cadmium on hepatic microsomal monooxygenase activities in guinea pigs with low and high ascorbic acid intake

The influence of in vivo administration of cadmium on hepatic microsomal cytochrome P-450 and cytochrome b 5 content, aniline hydroxylase, p-nitroanisole O-demethylase, and NADPH-cytochrome c reductase activities was investigated in guinea pigs with low (2 mg/animal/day) and high (100 mg/animal/day) ascorbic acid intake. Male guinea pigs received 10 mg Cd/L drinking water for 5 and 12 weeks. Cadmium administration resulted in significant decreases in cytochrome P-450 content and NADPH-cytochrome c reductase activity in the group of guinea pigs with low ascorbic acid intake, while the decreases were less evident in guinea pigs with high ascorbic acid intake. The activities of aniline hydroxylase and O-demethylase were not apparently changed by Cd administration, except for the group of guinea pigs with high ascorbic acid intake in 12th week of cadmium treatment, when increased activities were observed. These findings suggest that hepatotoxic effect of cadmium manifested by inhibition of hepatic microsomal metabolism may be in part beneficially moderated by enhanced ascorbic acid intake.

Effect of vitamin E dietary intake on in vitro activation of aflatoxin B1

The molecular mechanism of action of vitamin E on mammalian cells remains to be elucidated. In this study, vitamin E dietary intake was assessed for its effects on the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin E dietary intake and aflatoxin B1 (AFB1) genotoxicity measured in vitro. Thus AFB1 induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to effect of vitamin E dietary intake on hepatic microsomal P-450 content and specific activities involved in AFB1 metabolism. Rats were fed ad libitum a diet containing 0, 0.05, 0.5 or 5 IU of α-tocopherol for 8 weeks. Modulation of vitamin E level in postmitochondrial and microsomal fractions resulted in nutritional effects. Cytochrome P-450 content was not modified by the level of vitamin E in the diet. The microsomal P-450 activities, P-450 IIB1 and IIIA, were decreased in the deficient group to −35% and −16%, respectively, as compared with control diet (0.05 IU). Diet supplemented with 0.5 IU of vitamin E increased P-450 IIB and IIIA activities (+28% and +37%, respectively) whereas a diet highly supplemented in vitamin E (5 IU) reduced these specific P-450 activities. Lipid peroxidation, estimated by the formation of thiobarbituric acid reactive products, increased in the dietary vitamin E free diet (+20%) and strongly decreased in the supplemented group (−99%). This study establishes that in vivo, dietary vitamin E protects directly membrane against damage induced by lipid peroxidation and indirectly hepatic microsomal monooxygenase activities. However, vitamin E accumulation seems to alter membrane structure and function. The nutritional effect of vitamin E on hepatic microsomal cytochrome P-450 activities modified the AFB1 genotoxicity measured in vitro.

Microsomal cytochrome P450 1A1 dependent monooxygenase activity in guinea pig heart: induction, inhibition, and increased activity by addition of exogenous NADPH–cytochrome P450 reductase

Characterization of cytochrome P450 1A1 dependent monooxygenases in guinea pig heart revealed low rates of 7-ethoxyresorufin O-deethylation, which are markedly increased (20-fold) by treatment with beta-naphthoflavone, a polycyclic aromatic hydrocarbon. Both 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation were found to be approximately 4-fold higher in microsomes prepared from the ventricle than the atrium of beta-naphthoflavone-induced guinea pigs. The low rates of 7-ethoxyresorufin O-deethylation in cardiac microsomes were due, at least in part, to a deficiency of the flavoprotein NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase dramatically increased 7-ethoxyresorufin O-deethylation in cardiac microsomes of guinea pigs, before and after treatment with beta-naphthoflavone. N-Benzyl-1-aminobenzotriazole, a suicide substrate of cytochrome P450 1A1 in guinea pig, was able to inhibit almost all of the 7-ethoxyresorufin O-deethylase and 7-methoxyresorufin O-demethylase activities in polycyclic aromatic hydrocarbon induced guinea pig heart (88 and 71%, respectively), suggesting that cytochrome P450 1A1 coupled to NADPH--cytochrome P450 reductase in these microsomes inactivates itself by a suicidal mechanism. Addition of alpha-naphthoflavone, an inhibitor of cytochrome P450 1A isozymes, to cardiac microsomes from beta-naphthoflavone-induced guinea pigs resulted in greater than 95% inhibition of 7-ethoxyresorufin O-deethylase activity. The biological significance of these low levels of cytochrome P450 1A1 monooxygenase activity in guinea pig heart and their induction by polycyclic aromatic hydrocarbons are not currently understood.

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.2.2. The maximum pH of the reaction in the liver microsomes was 7.6.3.3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined.4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG.7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

Effects of aging and long-term caloric restriction on hepatic microsomal monooxygenases in female fischer 344 rats: Alterations in basal cytochrome P-450 catalytic activities

The effects of aging and caloric restriction on uninduced levels of hepatic microsomal cytochrome P-450s and specific P-450-related catalytic activities were evaluated in female Fischer 344 rats. Microsomes were isolated from livers of ad libitum (AL) fed rats 1, 3, 6, 16 and 26 months of age, and from calorie-restricted (CR) rats 6, 16 and 26 months of age. The recovery of microsomal protein was higher in CR than AL rats at 6, 16 and 26 months of age. Between ages 3 and 26 months there was a gradual decline in recovered microsomal P-450, but the differences between successive age categories were not significant. A different trend was observed for total cytochrome P-450 per liver. AL rats showed a 2-fold decline in P-450/liver between 6 and 26 months of age, while P-450/liver of corresponding CR rats increased 1.7-fold. Neither cytochrome b5 nor NADPH cytochrome C reductase varied consistently as a function of increased age, with all age groups showing greater reductase activity for CR rats than for AL rats. Phenobarbital (PB) and methylcholenthrene (MC) inducible cytochrome P-450 catalytic activities (pentoxy [PROD] and ethoxy [EROD] resorufin O-deethylases) exhibited similar age-related alterations that were distinctly different from the two other enzymes examined. Both PROD and EROD showed dramatic declines in activity between 1 and 3 months of age, with a slight increase in activity at 6 months and maintenance of the activity levels through 26 months. Benzphetamine N-demethylase (BND) and aniline p-hydroxylase (APH) activities were also altered as a function of increased age, with CR rats exhibiting increased isozyme activity above that seen for aging AL rats. Calorie restriction appears to have a modulating effect, compensating for an age-related decline of hepatic microsomal monooxygenase activity in female Fischer 344 rats.

Effects of D-limonene on hepatic microsomal monooxygenase activity and paracetamol-induced glutathione depletion in mouse.

1. D-Limonene, a monoterpenoid constituent of citrus fruit oil, blocks tumour induction by chemical carcinogens in laboratory animals, apparently by preventing bioactivation of procarcinogens and by enhancing conjugation of proximal carcinogenic metabolites. 2. Inhibitory effects of D-limonene were measured in vitro using cytochrome P450 isoform-specific substrates. D-Limonene inhibited p-nitrophenol hydroxylase (pNP) activity in vitro in liver microsomes from acetone-, phenobarbital (PB)- and beta-naphthoflavone (BNF)-treated mouse, and 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes from PB- and BNF-treated mouse. p-Nitrophenol and ethoxyresorufin are substrates for cytochromes P2E1 and P1A1, respectively. No inhibition of benzphetamine (BNZP) or aminopyrine (AP) demethylases by D-limonene was observed. 3. EROD, BNZP and AP activities in liver microsomes were increased 18 h after i.p. administration of D-limonene to acetone-induced mouse, while pNP activity was unchanged. The immunodetectable protein level of cytochrome P2B1 in non-acetone treated mouse was increased 18 h after D-limonene, with no differences in P2E1 or P1A1. 4. Acute D-limonene did not protect against paracetamol (acetaminophen)-induced depletion of liver reduced glutathione (GSH). A prolonged paracetamol challenge (0.6% diet for 10 days) elevated liver cytosolic GSH-S-transferase activity (GST) two-fold and decreased liver GSH to 46% of control values. Dietary D-limonene (1.0% diet for 10 days) maintained liver GSH concentrations at 92% of control values in the paracetamol-challenged mouse without altering GST activity. D-Limonene also increased liver GSH concentration (23%) in mouse fed 1.0% D-limonene alone.