Microsomal Mixed-function Oxidase Research Articles

The Different Genotoxicity of P-Dichlorobenzene in Mouse and Rat: Measurement of the in Vivo and in Vitro Covalent Interaction with Nucleic Acids

Twenty-two hours after i.p. injection to male Wistar rats and BALB/c mice para-dichlorobenzene (p-DCB) is bound covalently to DNA from liver, kidney, lung and stomach of mice but not of rats. DNA adducts in mouse liver are repaired in seventy-two hours. The covalent binding index value, calculated on the labelling of mouse liver DNA, classifies p-DCB as a weak initiator with an oncogenic activity lower than that of chlorobenzene. The labelling of RNA and proteins from the different organs of both species is, however, low. In vitro interaction with calf thymus DNA mediated by mouse and rat microsomes from liver and lung did occur. Binding extent was strongly reduced by addition of 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF 525-A) to the microsomal standard incubation mixture, whereas it was enhanced by adding GSH. Cytosolic fractions from kidney and lung were able to induce binding of p-DCB to DNA to a lower extent with respect to microsome-mediated binding. These results indicate that microsomal mixed function oxidase system and microsomal GSH-transferases can be involved in overall activating metabolism whereas cytosolic GSH-transferases play a minor role. This study, which is a part of a structure-activity relationship approach on benzene and its haloderivatives, provides the first evidence of genotoxicity of p-DCB in mammalian cell. It allows to partly explain variations of susceptibility of different species to hepatocarcinogenesis and of hepatotoxicity of different isomers.

The effects of cadmium, mercury and lead in vitro on hepatic microsomal mixed function oxidase and lipid peroxidation.

In this study, the authors investigated the effect of cadmium, mercury and lead in vitro on hepatic microsomal mixed function oxidase and lipid peroxidation. The results showed that these metals could inhibit the activity of hepatic microsomal aniline hydroxylase, decrease the concentration of hepatic microsomal cytochrome P-450 and increase the concentration of the inactive form of hemoprotein, P-420. Besides, they could enhance hepatic microsomal lipid peroxidation. There were marked concentration-dependent responses in these microsomal reactions. Thus, it may be suggested that cadmium, mercury and lead are capable of impairing hepatic microsomal mixed function oxidase in vitro by stimulating membrane lipid peroxidation.

Metabolic Sources of Reactive Oxygen Metabolites During Oxidant Stress and Ischemia with Reperfusion

The lung is especially sensitive to a variety of vastly different agents and conditions including hyperoxia, certain drugs and xenobiotics, particulate debris, and ischemia/reperfusion. There is a growing body of experimental data to suggest that most, if not all, of these agents or conditions mediate pulmonary injury by forming reactive O2 metabolites such as O2-., H2O2.OH, HOCl, and RNHCl. The presence mechanisms by which these different agents converge to produce free radical-mediated pulmonary injury is not entirely clear. The lung does contain several metabolic pathways that will produce large amounts of reactive O2 metabolites. For example, hyperoxia-induced pulmonary injury may be mediated by oxidants produced by both mitochondrial and microsomal electron transport. Certain drugs and xenobiotics may be metabolized by nonspecific flavoproteins found in the mitochondrial electron transport chain and associated with microsomal mixed function oxidase system to yield a variety of free radicals and oxidants. Inhalation of particulate debris will activate resident phagocytic leukocytes to produce large quantities of cytotoxic oxidants. Ischemia and reperfusion appear to produce substantial amounts of xanthine oxidase-derived oxy-radicals that recruit and activate inflammatory phagocytes to produce cytotoxic HOCl and N-chlorinated oxidants. Finally, inappropriate metabolism of arachidonate by prostaglandin synthetase in the presence of NADH (NADPH) produces a burst of O2-. The fact that the lung contains so many different metabolic avenues for oxidant and free radical production suggests that this particular organ may be the most sensitive to oxidative insult.

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.

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.

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.

Induction of cytochrome P-450, cytochrome b-5, nadph-cytochrome c reductase and change of cytochrome P-450 isozymes with long-term trichloroethylene treatment

Several reports have described the effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system (MFOS). These studies suggest that repeated TCE administration induces MFOS, especially cytochrome P-450 and NADPH-cytochrome c reductase. However, it is uncertain what isozymes are induced by TCE treatment, and it is not clear how microsomal enzymes or cytochrome P-450 isozymes are altered when TCE is administered for a duration longer than 28 days. We investigated the changes of MFOS by long-term TCE treatment. Male Wistar rats were injected TCE, 1.0 k/kg body weight once a day for 5 continuous days or 2.0 g/kg body weight twice a week for 15 days. The mean body weight of the rats treated with TCE for 15 weeks was slightly, but not significantly, less than that of the control rats. Relative liver weights (liver wt/body wt) of the TCE-treated group were however significantly larger (21 %) than those of the control group. The weights of the other organs were not changed by long-term TCE treatment. Trichloroethylene treatments for 5 days and 15 weeks caused significant increases in microsomal protein, cytochrome P-450, cytochrome b-5 and NADPH-cytochrome c reductase. TCE treatments produced an increase in a polypeptide band at 52 000 molecular weight range observed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This increase is similar to, but less pronounced than that induced by phenobarbital (PB) treatment. There were no remarkable changes at 56 000 molecular weight range where a band appeared after the treatment with 3-methylcholanthrene (MC). It is likely that the induction of cytochrome P-450 by TCE is relatively similar to that by PB.

Induction of the rat hepatic microsomal mixed-function oxidases by two aza-arenes: A comparison with their non-heterocyclic analogues

The ability of the aza-aromatic polycyclic aromatic hydrocarbons 10-azobenz( a)pyrene and benz( a) acridine to induce the rat hepatic microsomal mixed-function oxidases was compared to that of their non-heterocyclic analogues benz( a) pyrene and benz( a) anthracene respectively. All four hydro-carbons markedly increased the O-deethylations of ethoxyresorufln and ethoxycoumarin, the non-heterocyclic analogues being the more potent. A more modest increase was seen in the O-dealkylation of pentoxyresorufin. All four hydrocarbons induced proteins recognised by antibodies to cytochrome P-450aIAI but no increase was seen when antibodies to cytochrome P-450IIB1 were employed. The metabolic activation of benz( a)pyrene and Glu-P-1 to mutagenic intermediates in the Ames test was enhanced by all pretreatments. It is concluded that the aza-aromatic polycyclic hydrocarbons, like their non-heterocyclic analogues, selectively induce the cytochrome P-450I family of proteins.

Evidence for metabolism of 3-methylindole by prostaglandin H synthase and mixed-function oxidases in goat lung and liver microsomes

3-Methylindole (3MI) is the causative agent of a naturally occurring lung disease in cattle. The effects of 3MI on the prostaglandin H synthase (PHS) and mixed-function oxidase (MFO) systems in lung and liver microsomes were investigated. Addition of 3MI in goat lung microsomes resulted in a pronounced increase in PHS activity as indicated by both the initial rate and total oxygen consumption. The effect of 3MI on PHS activity was dependent on arachidonic acid and inhibited by indomethacin. PHS was capable of activating [ 14C]3MI to a reactive intermediate as indicated by the covalent binding of 3MI to microsomal protein. [ 14C]3MI was converted to water-soluble 3MI metabolites in the PHS system. Biosynthesis of total prostaglandins was enhanced 69% with 0.5 mM 3MI. PHS-catalyzed co- oxidation of 3MI was shown to be independent of the MFO-catalyzed metabolism of 3MI. PHS and MFO enzyme activities were found to exist in both goat lung and liver microsomes. PHS activity was significantly higher in lung microsomes than in liver microsomes. Pulmonary MFO activity, unlike hepatic MFO activity, was enhanced with addition of 3MI. This indicates a tissue selectivity for the metabolism of 3MI. Lung microsomal PHS and MFO systems were capable of activating 3MI to a greater extent than liver microsomes. Thus, the combined effects of PHS and MFO systems in the activation of 3MI, the selectivity of pulmonary MFO to metabolize 3MI, and the capacity for altered prostaglandin biosynthesis in lung tissue may explain the tissue specificity of 3MI-induced toxicity.

Induction of hepatic mixed-function oxidases by permethrin and cypermethrin in chicks fed vitamin A-deficient diet

The effect of permethrin and cypermethrin on the induction of hepatic mixed-function oxidases was studied in chicks fed a vitamin A-deficient diet. Oral administration of permethrin at 10, 50, 100, and 200 mg/kg for 6 consecutive days induced microsomal protein, cytochrome P-450, and NADPH-cytochrome c reductase in dose-dependent manner. Treatment with cypermethrin at a dose of 200 mg/kg body weight induced NADPH-cytochrome c reductase and cytochrome b 5. However both insecticides had no effect on the glutathione S-transferase activity. Permethrin and cypermethrin appear to be weak inducers of hepatic microsomal mixed-function oxidases in chicks fed vitamin A-deficient diet.

The Detection of Reactive Metabolites Generated by the Microsomal Mixed Function Oxidase System

It is recognised that the cytotoxicity of a number of xenobiotics is mediated through the production of reactive metabolites (1). Given this, and the current emphasis on the development of in vitro tests for assessment of xenobiotic cytotoxicity (2), it is of considerable interest to develop systems for the detection in vitro of cytotoxic reactive metabolites. Indeed, it would seem appropriate to employ a limited battery of tests for detection of xenobiotic-mediated cytotoxicity, some of which would be dedicated to assessment of the role of metabolism in such toxicity. The aim of this review is to identify and discuss the approaches currently being employed to this end, and to suggest some possible future trends.

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.

Effect of caffeine on the hepatic microsomal mixed function oxidase system during phenobarbital and benzo[ a] pyrene treatment in rats

Simultaneous administration of caffeine (100 mg/kg, i.p., 3 days) and phenobarbital (80 mg/kg, i.p., 3 days) to adult male rats resulted in a significant decrease in hepatic cytochrome P-450 and acetanilide hydroxylase activity, compared to phenobarbital administration alone. While simultaneous administration of caffeine and benzo[ a]pyrene (20 mg/kg, i.p., 2 days) increased acetanilide hydroxylase, compared to benzo[ a]pyrene administration, no change was seen in the cytochrome P-450 concentration. In vitro addition of 2.5 mM caffeine to microsomal incubations from untreated, phenobarbital- and benzo[ a]pyrene-treated rats inhibited aminopyrine N-demethylase activity. No significant difference was seen in the extent of aminopyrine N-demethylase inhibition due to the in vitro addition of caffeine to microsomes from untreated or phenobarbital-treated rats, whereas inhibition in microsomes from benzo[ a]pyrene-treated rats was greater.

Effects of bile duct obstruction and decompression on hepatic microsomal mixed function oxidase system in rats

The effects of biliary obstruction and drainage on the hepatic microsomal mixed function oxidase system were studied in rats. Bile duct obstruction produced a significant reduction in the hepatic cytochrome P-450 dependent mixed function oxidase system. After release of the bile duct obstruction, the reduction in microsomal enzymes was practically reversible; however, the process of recovery was slow and differed with the microsomal enzymes in question. Increases in cytochrome b 5 content and NADH-cytochrome b 5 reductase activity were slower than increases in cytochrome P-450 content and NADPH-cytochrome c reductase activity. Aniline hydroxylase activity increased more rapidly and corresponded to cytochrome P-450 contents more so than did the aminopyrine demethylase activity. After the release of bile duct obstruction, however, the bile acids which had accumulated in the liver during cholestasis were reduced rapidly, to a normal range. These results suggest that there is a discrepancy between reductions in hepatic bile acids and those in the hepatic microsomal mixed function oxidase system after biliary decompression.

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.

Induction of the rat hepatic microsomal mixed-function oxidases by 3 imidazole-containing antifungal agents: selectivity for the cytochrome P-450IIB and P-450III families of cytochromes P-450

Administration of the imidazole antifungal agents ketoconazole, miconazole and clotrimazole gave rise to increases in the microsomal cytochrome P-450 levels and the NADPH-dependent reduction of cytochrome c. Clotrimazole, and to a much lesser extent miconazole and ketoconazole, stimulated the dealkylation of pentoxyresorufin. All 3 agents gave rise to small, but significant increases in the O-deethylations of ethoxycoumarin and ethoxyresorufin. The antifungal-induced O-deethylation of ethoxycoumarin was much more sensitive to inhibition by metyrapone rather than by α-naphthoflavone. The binding of metyrapone to reduced microsomes was enhanced by treatment of animals with the 3 antifungal agents, clotrimazole being clearly the most potent. Immunoquantitation of cytochrome P-450 proteins using an ELISA procedure and employing anti-cytochrome P-450c ( P-450IA1, P-448 low spin) and P-450b ( P-450IIB1) antisera revealed that clotrimazole and miconazole, but not ketoconazole, induced the levels of phenobarbital-induced cytochromes P-450, while none of the antifungal agents increased the levels of cytochrome of P-448 proteins. Similar results were obtained using Western blots employing the above antibodies. On SDS-polyacrylamide gel electrophoresis microsomes derived from animals pretreated with clotrimazole showed intensification of a band at 51 kDa which was identified by Western blotting as the PCN-inducible form of cytochrome P-450 (cytochrome P-450p, P-450III family). Similar, but less pronounced intensification was seen with microsomes from animals pretreated with miconazole and ketoconazole. Furthermore, microsomes from clotrimazole- and ketoconazole-treated animals interacted with erythromycin to yield type I spectra. It is concluded that the imidazole-containing agents clotrimazole and miconazole, and to a much lesser extent ketoconazole, are potent inducers of the rat hepatic microsomal mixed-function oxidases, displaying selectivity towards the P-450IIB (phenobarbital-inducible) and P-450III (PCN-inducible) families of cytochrome P-450 proteins.

Clonidine effects on disposition of xenobiotics in rats: inhibited elimination of flow-limited but not extraction-limited agents.

1. The alpha 2-adrenoceptor agonist, clonidine, reduces the hepatobiliary clearance of the anionic dye, sulphobromophthalein (BSP) in rodents. We now compare the effects of clonidine on BSP elimination with its effects on disposition of compounds which are metabolized by hepatic microsomal mixed function oxidases. 2. BSP, 100 mg kg-1 was administered i.v. to rats at 4 h after s.c. saline or clonidine, 0.2 mg kg-1. Thirty min later, plasma BSP levels were 121.4 +/- 2.25 micrograms ml-1 in saline-treated rats, while in clonidine-treated rats they were 631.5 +/- 141.0 micrograms ml-1. Clonidine raised hepatic BSP levels from 256.0 +/- 28.9 micrograms g-1 tissue to 568.5 +/- 86.5 micrograms g-1. 3. Acute administration of clonidine (0.2 mg kg-1 s.c.) or repeated clonidine dosing (0.2 mg kg-1, s.c. twice daily for 10 days) did not affect the disposition of intravenously administered [14C]-antipyrine (15 mg kg-1). 4. Activities of the P450 mixed function oxidase enzymes, aniline hydroxylase and aminopyrine N-demethylase, were identical in liver microsomes from saline-treated rats and in microsomes from rats given single or multiple s.c. doses of clonidine (0.2 mg kg-1). 5. Addition of clonidine or other 2-substituted imidazoles at concentrations up to 2 microM did not affect the activities of aniline hydroxylase or of aminopyrine N-demethylase in suspensions of rat liver microsomes. Other substituted imidazoles, including cimetidine, clotrimazole and metronidazole, at concentrations of 0.2 microM or higher, inhibited the activities of these microsomal enzymes. 6. Clonidine slowed BSP elimination, which is probably hepatic blood flow-limited, but not the extraction-limited elimination of antipyrine, which is metabolized by hepatic microsomal enzymes.

Involvement of glutathione in the reduction of captan-induced in vivo inhibition of monooxygenases and liver toxicity in the rat.

If given orally captan is relatively nontoxic, but it can be extremely toxic after parenteral exposure. Therefore, a single i.p. dose of captan (20 mg/kg) was given to male Sprague-Dawley rats and its effect on liver microsomal mixed function oxidases and certain serum enzymes (SDH, SGPT and SGOT) was studied. The single dose of captan caused marked depression of microsomal cytochrome P-450 and the activity of benzphetamine N-demethylase and aniline hydroxylase, and moderate elevation of the serum enzymes indicative of liver damage. However, reduced glutathione (100 mg/kg, i.p.) given prior to captan, appears to decrease the liver toxicity as measured by reduced inhibition of the microsomal enzymes and elevation of serum enzymes activity. The results suggest that glutathione and other compounds containing sulfhydryl groups may protect the subjects from captan-induced liver toxicity.