Microsomal Mixed-function Oxidase Activity Research Articles

Differential localization of microsomal mixed function oxidase activity in periportal and perivenous hepatocytes isolated from rat liver

1. 1. The activity per mg of microsomal protein of aminopyrine N- demethylase was higher in perivenous (PV) than in periportal (PP) hepatocytes of rat, but when it was expressed per cytochrome P-450 content the difference in the activity was not significant. 2. 2. The activity of 7-ethoxycoumarin O-deethylase, when expressed per mg protein and per P-450 content, was significantly higher in PV than in PP cells. 3. 3. The activities of dimethylnitrosamine(DMNA) N-demethylase and aniline p-hydroxylase were not significantly different between two subpopulations of isolated hepatocytes when either expressed per mg protein or per P-450 content.

Effect of exogenous pyruvate on acrylamide neuropathy in rats

The protective effect of exogenous sodium pyruvate on the distal-proximal progression of experimental acrylamide neuropathy in rats was examined. Incorporation of 2% (w/w) sodium pyruvate powder in the diet of rats receiving subcutaneous injections of an aqueous solution of acrylamide (35 mg/kg/day, 5 days/week) retarded the onset and development of functional, morphological, and biochemical measures of acrylamide neuropathy. Pyruvate supplementation did not alter hexobarbital sleep time or zoxazolamine paralysis time, two in vivo measures of microsomal mixed-function oxidase activity, and the disposition of radioactivity in plasma or sciatic nerve following subcutaneous injection of [ 14C]acrylamide. Although acrylamide can interfere with energy metabolism at a variety of sites where pyruvate can rescue neurons (axons), the data of this study are consistent with our earlier hypothesis that acrylamide neuropathy may be associated with a glycolytic deficit. The exact site of pyruvate protection is unknown. Exogenous pyruvate is perhaps utilized by axons to circumvent toxin-induced glycolytic inhibition and provide chemical energy for fast axonal transport.

Cytochrome P-450 and oxidative metabolism in molluscs.

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs. 2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc. 3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)). 4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, alpha-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra. 5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes. 6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH. 7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones. 8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.

Effect of a Prudhoe Bay crude oil on hepatic and renal peroxisomal beta-oxidation and mixed-function oxidase activities in rats.

The effect of oral administration of a Prudhoe Bay crude oil (PBCO) to male rats (PBCO, 2.6 g/kg body weight, daily) for 5-12 days on hepatic and renal microsomal monooxygenase activities and peroxisomal beta-oxidation has been investigated. PBCO administration leads to liver enlargement. This is associated with induction of microsomal cytochrome P-450 levels (1.6- to 2.0-fold) and dependent mixed-function oxidase activities (7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-depentylase, representing cytochrome P-450I and cytochrome P-450IIB isoenzymes respectively, 9- to 15-fold; omega-oxidation of lauric acid representing the cytochrome P-450IVA1 isoenzyme, 1.4- to 1.5-fold) along with peroxisomal beta-oxidation (palmitoyl CoA oxidation, 2- to 5-fold). It was observed that rats exposed to PBCO showed an increase in renal microsomal cytochrome P-450 content (1.6- to 2.3-fold), cytochrome P-450I activity (5- to 8-fold) and omega-oxidation activity (1.3- to 1.4-fold). However, renal peroxisomal beta-oxidation was unaltered. Serum total triglycerides were lowered by 41-46% after PBCO exposure. These results suggest that induction of peroxisomal beta-oxidation and possibly mono-oxygenases may be related to the carcinogenic/tumorigenic potential of crude oil.

Effect of Sizofilan, an Immunomodulator, on Hepatic Microsomal Mixed-Function Oxidase Activities in Rats

Mixed-function oxidase activities of hepatic microsomal preparations from rats were examined after intraperitoneal administration of sizofilan (SPG), an immunomodulator. Repeated doses of SPG (3 mg/kg/12 hr, 4 times) depressed the hepatic cytochrome P-450 content and the activities of aminopyrine N-demeth-ylase and aniline hydroxylase.

Effect of sizofilan, an immunomodulator, on hepatic microsomal mixed-function oxidase activities in rats.

Mixed-function oxidase activities of hepatic microsomal preparations from rats were examined after intraperitoneal administration of sizofilan (SPG), an immunomodulator. Repeated doses of SPG (3 mg/kg/12 hr, 4 times) depressed the hepatic cytochrome P-450 content and the activities of aminopyrine N-demethylase and aniline hydroxylase.

Structural role of an unsaturated lactone in acute toxicity of rubratoxin B isolated from Penicillium rubrum

AbstractRubratoxin B is a substituted higher homolog of byssochlamic acid in which the ethyl group is replaced by a six‐membered ring containing an unsaturated lactone. Hydrogenation of the unsaturated lactone greatly reduces the acute toxixity of the compound. The acute median lethal doses of rubratoxin B and hydrogenated rubratoxin in male mice were 5.6 and 31 mg/kg, respectively. Pretreatment with phenobarbital (60 rag/kg × 4 days) or with SKF 525A (40 mg/kg) failed to significantly alter the toxicity of either compound. Histopathological examination of mice treated with rubratoxin B (3.5 mg/kg) revealed areas of hepatic zonal necrosis and marked necrosis of kidney tubules. No necrosis was observed in mice treated with hydrogenated rubratoxin (20 mg/kg). Hepatic microsomal mixed function oxidase activity was significantly reduced in mice pretreated with rubratoxin B (0.5 mg/kg × 4 days), while pretreatment with hydrogenated rubratoxin (2.5 mg/kg × 4 days) failed to depress microsomal activity. Our studies document the requirement of an alpha, beta unsaturated lactone ring for rubratoxin B toxicity. Based on the results from these studies the mechanism for rubratoxin B induced toxicity is postulated to be due to its electrophilic properties and subsequent alkylating activity.

Environmental implications of incineration of municipal solid waste and ash disposal

Owing to unsightliness and the threat of groundwater pollution, landfilling of municipal solid waste (MSW) is giving way to incineration in many communities. Environmental contamination from particulate and gaseous emissions containing heavy metals, polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzofurans (PCDF), polycyclic aromatics (PCA), acids and other compounds from such incinerators, as well as safe ash disposal, are of great concern. Concentration ranges of elements and organic toxicants in incinerator ashes, emissions and cooling waters are given. The literature is reviewed concerning the effects of incinerator operating parameters on emissions. Incinerators equipped with modern pollution control devices (electrostatic precipitators, fabric filters, dry scrubbers, spray towers) and operated at optimum temperature with sufficient oxygen, turbulence (mixing) and residence time for complete combustion appear to minimize ash, elemental, gaseous and organic emissions. Environmental aspects of MSW incineration are considered and reviewed. The presence of metals and organics in incinerator quench water and in leachates from ash disposed in landfills are reviewed, as well as their toxicity to fish. The behavior and effects of atmospheric emissions in soils and plants are discussed. Research on the effect of ash-derived PCDD and PCDF on hepatic microsomal mixed function oxidase activity and the immune system in laboratory animals is cited. The presence of metals, organics and mutagens in the incinerator workplace air and the possible effects of air-borne contaminants on inhabitants nearby is reviewed. Several studies dealing with human risk assessment of MSW incineration are cited.

Comparative effects of antithrombitic and antimycotic N-substituted imidazoles on rat hepatic microsomal steroid and xenobiotic hydroxylases in vitro

N-Substituted imidazoles have been shown to be potent inhibitors of microsomal mixedfunction oxidase activities in vitro and in vivo. In the present study the effects of two antithrombitic (dazmegrel and dazoxiben) and four antimycotic (ketoconazole, econazole, miconazole and clotrimazole) imidazoles on microsomal cytochrome P-450-mediated steroid and xenobiotic hydroxylases were studied in vitro. Despite the presence of the N-substituted imidazole moity, the antithrombitic agents were essentially non-potent as inhibitors of all of the oxidase activities evaluated. In contrast, the antimycotic drugs were potent inhibitory compounds. Binding studies revealed that all six imidazoles elicited type II optical difference spectra and exhibited relatively high affinity for ferricytochrome P450 in microsomal suspensions ( K s range 0.26–0.73 μM for the antimycotic agents and 6.5 μM and 21 μM for dazmegrel and dazoxiben, respectively). The structural feature that the antithrombitic compounds share is a carboxylate function so that, at physiological pH, less than 1% of the drug would be present in the unionised form. This functionality is absent from the structures of the antimycotic agents which possess much greater hydrophobic character. Even though the antithrombitic imidazoles elicit type II binding interactions of quite high affinity it would appear from this study that significant inhibition potency does not necessarily follow. The present findings also suggest that interesting differences exist between the active site binding regions in the cytochrome P-450 that catalyse thomboxane synthetase activity and those involved in microsomal drug oxidation. Inhibitor hydrophobicity is clearly an important factor in the inhibition of microsomal cytochromes P-450 whereas effective thromboxane synthetase inhibitors may be quite hydrophilic at physiological pH.

Influence of Excessive Ascorbic Acid Dose on Liver Microsomal Mixed Function Oxidase System in Guinea Pigs

In the first experiment, guinea pigs were fed diets containing graded levels of ascorbic acid (AsA) from deficient to excess (0, 6, 60, 600, 6, 000, and 12, 000mg/kg of diet) for 15 days after pre-feeding with a low AsA diet (60mg/kg) for one week. Pentobarbital sleeping time measured on day 9 was shortened in the 600- to 12, 000-mg AsA groups as compared with the two AsA deficient (0 and 6mg) and 60-mg AsA groups. Liver microsomal cytochrome P-450 content increased with a dose-response relation to the dietary AsA level except in the excess AsA group (12, 000mg/kg). However, cytochrome b5 content and NADPH-cytochrome c reductase activity showed no significant response. Activities of aminopyrine N-demethylase and aniline hydroxylase showed the same response as seen in cytochrome P-450 content, but to a lesser extent. In the second experiment, guinea pigs were fed the diet containing the normal level of or excess AsA (300 or 24, 000mg/kg of diet, respectively) for 4, 9 or 15 days after a pre-feeding with the normal AsA diet for one week. No significant differences in the time courses of those responses described above were shown between the two AsA groups. In conclusion, the liver microsomal mixed function oxidase activity did not change even transiently during a short period after administration of a large excess of AsA such as 24, 000mg/kg of diet, and 300mg AsA/ kg of diet is enough to ensure normal levels of the liver microsomal mixed function oxidase system in guinea pigs.

Effect of vitamin A on rat hepatic mixed-function oxidases, glutathione transferase activity and generations of oxygen radicals.

Rat hepatic microsomal mixed-function oxidase activities were not significantly affected by vitamin A deficiency. Similarly cytosolic glutathione S-transferase and glutathione reductase activities as well as total glutathione levels were unaffected by the vitamin A status. Induction of the mixed-function oxidases by 3-methylcholanthrene or phenobarbitone was independent of the vitamin A status. No significant differences in microsomal chemiluminescence, before and following challenge with tertiary butyl hydroperoxide, were evident between the vitamin-A-deficient animals and those maintained on vitamin-A-supplemented diets. The present findings indicate that the protective action of vitamin A against chemical carcinogens is unlikely to involve modulation of the enzyme systems responsible for their metabolism.

Generation of superoxide by the microsomal mixed-function oxidase system.

The microsomal mixed-function oxidase (MFO) system catalyzes the oxidation of numerous endogenous and exogenous compounds using NADPH as its electron donor. During its oxidation of drug substrates and even in the absence of substrates, this electron transport system has been demonstrated to produce O2·, H2O2, and excess H2O. We have investigated the ability of several purified forms of cytochrome P450 to produce O2· when incubated with purified NADPH cytochrome P450 reductase. Addition of cytochrome P450 isozymes to the purified reductase resulted in increased rates of O2· formation, assessed using either EPR spin trapping techniques or acetylated cytochrome c reduction. Conditions necessary for optimal rates of O2· production were similar to those previously shown to be necessary for optimal reconstitution of MFO activity (phospholipid, detergent-solubilized reductase, cytochrome P450:reductase ratios of greater than 3:1). Cytochrome P450d addition to incubations of reductase resulted in greater rates of O2· production than either cytochrome P450c or P450b, especially at equimolar concentrations of cytochrome P450 isozymes and reductase. The contribution of cytochrome P450d to microsomal O2· generation was addressed by investigating O2· production by microsomes isolated from isosafrole-treated rats.

Effects of pregnenolone-16 α - carbonitrile on drug metabolizing enzymes in hypophysectomized female rats

Treatment of intact and hypophysectomized female rats with pregnenolone-16α-carbonitrile (PCN) resulted in a significant increase in hepatic aryl hydrocarbon hydroxylase (AHH) activity. However, the total cytochrome P-450 concentration, as measured by CO difference spectra, was increased to a greater extent in hypophysectomized rats than in intact rats. Total cytochrome P-450 was found to be 0.82 ± 0.16 vs 2.43 ± 0.31 nmoles/mg protein for control and PCN-treated hypophysectomized rats, respectively, and 0.68 ± 0.23 vs 1.28 ± 0.05 nmoles/mg protein for control and PCN-treated intact rats respectively. The concentration of metyrapone complex in microsomes from intact control and PCN-treated rats was found to be 0.4 ± 0.11 vs 1.88 ± 0.23 M respectively. Treatment of hypophysectomized rats with PCN resulted in an approximate 10-fold increase in the concentration of the metyrapone complex (0.42 ± 0.15 M for control and 4.46 ± 0.44 M for PCN-treated). Microsomal NADPH and NADH cytochrome c reductase activities were also altered by PCN-treatment. Aminopyrine demethylase activity was stimulated approximately three-fold by PCN treatment in both intact and hypophysectomized rats. Benzphetamine demethylase activity was not significantly affected by PCN treatment. The results of these studies suggest that the absence of the pituitary gland can markedly influence PCN induction of cytochrome P-450 in the liver in female rats. PCN also differentially affects microsomal mixed-function oxidase activities associated with drug and xenobiotic metabolism.

Changes in rat hepatic microsomal mixed function oxidase activity following exposure to halothane under various oxygen concentrations

This study demonstrates that the exposure of phenobarbitone-treated rats to halothane at an oxygen concentration of either 10% or 14% results in marked decreases in cytochrome P-450 content and aminopyrine deraethylase activity in animals sacrificed from 1 to 48 hr post-exposure. The alterations observed in the hepatic mixed function oxidase system were accompanied by increases in serum alanine aminotransferase (ALT), ornithine carbamyl transferase (OCT) and changes in liver pathology. However, the minor changes in cytochrome P-450 content and aminopyrine demethylase activity observed following exposure of enzyme-induced rats to halothane under normoxic conditions (i.e. 21% oxygen) were not of a sufficient magnitude to lead to hepatic cell necrosis. Halothane administration in the absence of phenobarbitone pretreatment (i.e. 21% oxygen) or during hypoxia alone (i.e either 10% or 14% oxygen) did not result in any systematic changes in the parameters assayed. The results suggest that cytochrome P-450 may catalyse its own inactivation by virtue of greater free radical production under conditions which favour the non-oxygen dependent metabolism of halothane. The impairment in microsomal function as evidenced by decreases in cytochrome P-450 and aminopyrine demethylase activity are considered to occur as a primary consequence of the reductive metabolism of halothane. Data are presented which support the concept of the initiation of hepatic damage occurring during the period of anaesthesia with halothane.

A review of new approaches to assessing hepatic function in animals.

Although a multitude of effective liver function tests are available for use in animals, a variety of modifications of currently used tests have been recently reported. In addition, newer procedures now used in human medicine may also provide unique insights into assessing and detecting acquired hepatic disorders in animals. Examples of new procedures are: assessing microsomal mixed function oxidase activity by plasma caffeine clearance; estimating the extent of active hepatic fibrogenesis through serum procollagen-III peptide levels; determining hepatic blood flow and functional mass by the galactose elimination capacity; detecting primary hepatocellular cancer through serum or urinary ligandin levels; and to estimate the liver's maximal capacity to excrete indocyanine green independent of blood flow. In evaluating drugs as to their hepatotoxicity, function tests should be included in the liver profile which measure specific metabolic alterations unique to the compound under study.

Inhibitory and inducing effects of denzimol on carbamazepine metabolism in the rat.

In vivo and in vitro alterations in carbamazepine (CBZ) metabolism and the extent of enzyme induction of the hepatic cytochrome P-450 system after chronic oral denzimol to rats were evaluated. No effect on drug-metabolizing enzymes was detected for this new anticonvulsant drug at a dose of 15 mg/kg, which is just above the anticonvulsive dose. At higher doses (60 mg/kg) denzimol significantly raised the hepatic cytochrome P-450 content, enhanced CBZ clearance and tend to shorten its elimination t1/2 and that of its active metabolite. These results, combined with those of a previous study showing impairment of CBZ metabolism after single doses of denzimol, suggest that the drug may have either inductive or inhibitory effects on microsomal mixed-function oxidase activity in the rat, depending on the dose and schedule of treatment.

Induction of rat hepatic mixed function oxidases by aromatic amines and its relationship to their bioactivation to mutagens

Hepatic microsomal mixed-function oxidase activities were determined in rats pretreated with the aromatic amines 2-aminoanthracene, 2-naphthylamine or 4-aminobiphenyl. All three amines stimulated the O-deethylation of ethoxyresorufin (cytochromes P-448) but none had any effect on the p-hydroxylation of analine. 2-Aminoanthracene and 4-aminobiphenyl also stimulated the NADPH-dependent reduction of cytochrome c and 2-naphthylamine inhibited the N-demethylation of benzphetamine. Hepatic preparations from animals pretreated with 2-aminoanthracene were more efficient in converting this carcinogen to mutagens while in contrast pretreatment with Aroclor 1254 caused a marked decrease in mutagenicity. 4-Aminobiphenyl also enhanced its own activation but Aroclor-pretreated preparations were the most effective. The latter preparations were also more efficient than controls in activating 2-naphthylamine to mutagens. It is concluded that 4-aminobiphenyl and 2-aminoanthracene enhance their own activation at least partly, by inducing the synthesis of cytochromes P-448.

New heterocyclic modifiers of oxidative drug metabolism—I: 6-substituted-2-aminobenzothiazoles

A series of 6-substituted-2-aminobenzothiazoles(2-AB) was synthesized and evaluated as in vitro inhibitors of microsomal mixed-function oxidase activity (as aminopyrine N-demethylase) from phenobarbitone-induced rat liver. Using physiochemical parameters and multiple regression analysis a quantitative structure-activity relationship (QSAR) was derived in which 82% of the data variance was accounted for in terms of the hydrophobic character of the inhibitor and the molar refractivity of the 2-AB 6-substituent. In contrast, literature equations derived from earlier studies with heterocyclic systems possessing non-polar substituents underestimated by up to an order of magnitude the potency of the present compounds. Kinetic studies revealed that 6- n-propoxy-2-AB, one of the more potent compounds, was a pure competitive inhibitor of aminopyrine N-demethylase activity ( K i = 60 μM from Dixon analysis), suggesting that the binding of substrate and inhibitor is mutually exclusive at the cytochrome P-450 active site. Binding studies indicated that most 2-AB derivatives elicited mixed type I/reverse type I optical difference spectra in phenobarbitone-induced microsomes. The overlap of these components resulted in non-linear double reciprocal plots of the spectral titrations and precluded the determination of binding parameters. In contrast, the more potent inhibitors (the 6-propoxy and 6-butoxy derivatives of 2-AB) were type I ligands with quite high affinity for ferric cytochrome P-450. Although no quantitative relationship was apparent between inhibition and spectral binding affinity a good correlation ( r = 0.93) was observed between inhibition potency (I 50) and the capacity of ten 2-AB derivatives to prevent substrate (aminopyrine) binding to cytochrome P-450. These findings suggest that 2-AB derivatives may inhibit microsomal oxidation via a direct competitive effect on substrate binding to cytochrome P-450. The present study also demonstrates that substitution of heterocyclic systems with hydrophilic groups does not necessarily produce weak inhibitors of mixed-function oxidase activity, and that extrapolation of existing QSAR equations to new inhibitor series must be interpreted with caution.

Toxicological significance of hepatic responses to furylfuramide(AF-2) in the rat

In male rats fed furylfuramide [2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, AF-2] at a dietary level of 0.1%, a marked increase in the liver weight was observed with a concomitant reduction of microsomal mixed-function oxidase activity. To clarify the significance of these hepatic responses to furylfuramide, a series of biochemical parameters that reflect hepatotoxicity was measured. The most significant elevations were seen in hepatic glycogen and glucose 6-phosphate dehydrogenase levels over 29-day feeding interval. A marked decrease was observed in mixed-function oxidases and glucose 6-phosphatase activities. Moderate decreases were seen in several other parameters, especially in the early phase of feeding. Measurement of serum parameters showed significant elevation in cholesterol and a transient increase in serum glutamic pyruvic transminase. A comparative study demonstrated small differences between furylfuramide and carbon tetrachloride in their effects on hepatic parameters. The furylfuramide-induced changes appear to be reversible within 14 days after withdrawal of furylfuramide from the diet. Reduction of mixed-function oxidase activity by furylfuramide might be caused partly by the elevation of heme degrading enzyme. The results indicate a dysfunction of drug-metabolizing activity and acute and slight toxication of the liver by furylfuramide.

Induction of renal and hepatic mixed function oxiases in the hamster and guinea pig

A marked species differences exists in the induction of renal and hepatic mixed function oxidase (MFO) activity between rats and rabbits. However, little is known about MFO induction in these organs from other laboratory animals. Male Golden Syrian hamsters and male Hartley guinea pigs were administered phenobarbital (PB) or β-napthoflavone (BNF) at 70 and 40 mg/ kg, respectively, as daily i.p. injections for 4 days. Polybrominated biphenyl (PBB) (Firemaster BP-6) was given as a single i.p. injection (50 mg/kg). Hamster hepatic microsomal ethoxyresorufin- O-deethylase (EROD) and benzphetamine- N-demethylase (BPND) were selectively induced by BMF and PB, respectively. PBB administration induced both hamster hepatic EROD and BPND. In contrast, hepatic microsomal MFO activity from the guinea pig was inducible by PB, PBB and BMF. Renal microsomal MFO activity in both species was inducible by BNF and PBB as arylhydrocarbon hydroxylase and EROD were induced approximately 10-fold. On the other hand, hamster BPND was induced by PB whereas guinea pig MFO activity was unaffected. Total renal cytochrome P-450 content was not affected by any of these inducers in either species. These data demonstrate selective patterns of induction in both hamster and guinea pig liver and kidney suggesting the involvementof multiple forms of cytochrome P-450.