Biomedical subjects

A I Cederbaum

Publications and source records attributed to A I Cederbaum.

At least 91 records · Page 5Linked to original sources

PubMed1994

Characterization of pyrazole and 4-methylpyrazole induction of cytochrome P4502E1 in rat kidney.

Pyrazole and 4-methylpyrazole induce cytochrome P4502E1 (P4502E1) in the liver. It is not known whether induction occurs in nonhepatic tissue such as kidney and lung. Rats were treated with saline, pyrazole or 4-methylpyrazole and assayed for the activity and content of P4502E1 and mRNA in liver, lung and kidney. Treatment with these agents resulted in increases in P4502E1 content as detected by immunoblots in liver and kidney, but not lung, microsomes. Oxidation of relatively specific substrates for P4502E1 was also significantly increased with liver and kidney microsomes after pyrazole or 4-methylpyrazole treatment. P4502E1 mRNA levels in liver, kidney and lung were not increased by treatment with pyrazole or 4-methylpyrazole. Associated with the induction of P4502E1 was an elevated production of reactive oxygen intermediates such as superoxide radical and H2O2 by kidney and liver, but not lung. microsomes. Lipid peroxidation induced by CCI4 was also increased with kidney microsomes after treatment with pyrazole or 4-methylpyrazole. Anti-P4502E1 IgG inhibited the increased oxidation of substrates and the increased production of H2O2 by the kidney microsomes found after treatment with pyrazole and 4-methylpyrazole. These results show that pyrazole and 4-methylpyrazole, which induce P4502E1 in liver, are also effective in inducing this enzyme in the kidney, whereas the lung is not sensitive to induction by these agents. The mechanism of induction of kidney P4502E1, similarly to that of liver, appears to reflect a post-transcriptional effect-probably stabilization of the protein against degradation.

Animals↗

PubMed1994

Generation of reactive oxygen intermediates by human liver microsomes in the presence of NADPH or NADH.

Studies were carried out to evaluate the ability of human liver microsomes to generate superoxide radical and hydrogen peroxide, and to interact with ferric chelates to produce more potent oxidizing species such as the hydroxyl radical (.OH). In the presence of either NADPH or NADH, human liver microsomes produced superoxide and H2O2 at rates about 20 to 30% of that found with rat liver microsomes. These lower rates are caused, in part, by the 3-fold lower content of total cytochrome P450 in the human liver microsomes. NADH-dependent rates were about 25 to 30% of the NADPH-dependent rates. In the presence of appropriate ferric complexes, human liver microsomes generated .OH, promoted cleavage of vicinal diols, and underwent lipid peroxidation. In contrast to results with rat liver microsomes, NADH-dependent rates of .OH production or lipid peroxidation by human liver microsomes were similar to the NADPH-dependent rates. Human liver microsomes reduced ferric ATP or ferric EDTA at nearly comparable rates with NADPH and NADH. Sensitivity of the various iron-dependent reactions to antioxidants was found to be characteristic of the particular system. These results suggest the possibility that human liver microsomes are an important source of reactive oxygen intermediates, especially under conditions of increased NADH or NADPH availability and elevated iron concentration.

Animals↗

PubMed1993

Stimulation by paraquat of microsomal and cytochrome P-450-dependent oxidation of glycerol to formaldehyde.

Glycerol can be oxidized to formaldehyde by microsomes in a reaction that is dependent on cytochrome P-450. An oxidant derived from the interaction of H2O2 with iron was responsible for oxidizing the glycerol, with P-450 suggested to be necessary to produce H2O2 and reduce non-haem iron. The effect of paraquat on formaldehyde production from glycerol and whether paraquat could replace P-450 in supporting this reaction were studied. Paraquat increased NADPH-dependent microsomal oxidation of glycerol; the stimulation was inhibited by glutathione, catalase, EDTA and desferrioxamine, but not by superoxide dismutase or hydroxyl-radical scavengers. The paraquat stimulation was also inhibited by inhibitors, substrate and ligand for P-4502E1 (pyrazole-induced P-450 isozyme), as well as by anti-(P-4502E1) IgG. These results suggest that P-450 still played an important role in glycerol oxidation, even in the presence of paraquat. Purified NADPH-cytochrome P-450 reductase did not oxidize glycerol to formaldehyde; some oxidation, however, did occur in the presence of paraquat. Reductase plus P-4502E1 oxidized glycerol, and a large stimulation was observed in the presence of paraquat. Rates in the presence of P-450, reductase and paraquat were more than additive than the sums from the reductase plus P-450 and reductase plus paraquat rates, suggesting synergistic interactions between paraquat and P-450. These results indicate that paraquat increases oxidation of glycerol to formaldehyde by microsomes and reconstituted systems, that H2O2 and iron play a role in the overall reaction, and that paraquat can substitute, in part, for P-450 in supporting oxidation of glycerol. However, cytochrome P-450 is required for elevated rates of formaldehyde production even in the presence of paraquat.

Animals↗

PubMed1993

Stable expression of human cytochrome P4502E1 in HepG2 cells: characterization of catalytic activities and production of reactive oxygen intermediates.

Experiments were carried out to stably and constitutively express the coding sequence of the human cytochrome P4502E1 in HepG2, a human-hepatoma-derived cell line, by recombinant retroviral expression. Southern blot analysis showed a successful integration of a single copy of unaltered viral DNA into the genome of each transduced clone tested. Northern blot analysis showed that the transduced clones produced an RNA species which hybridized to the CYP2E1 cDNA probe. Western blot analysis using anti-human P4502E1 IgG indicated that the transduced clones produced a protein band with molecular weight of 54 000. Microsomes from transduced clones were catalytically active with p-nitrophenol, dimethylnitrosamine, aniline, and ethanol as substrates; little or no activity was found with control clones. Oxidation of p-nitrophenol was inhibited by anti-human P4502E1 IgG, diethyl dithiocarbamate, 4-methylpyrazole, and ethanol. ESR spectroscopy showed that microsomes from clone MV2E1-9 produced superoxide radical. Rates were an order of magnitude higher than that for control microsomes, most likely reflecting the loose coupling associated with P4502E1. The rate of H2O2 production by microsomes from MV2E1-9 was 2-fold greater than that of control clones. The elevated rate of H2O2 production in clone MV2E1-9 is about half the rate of superoxide radical production, suggesting that this H2O2 is largely derived from superoxide radical dismutation. Microsomal lipid peroxidation was determined using ferric-ATP as the iron catalyst. When the concentration of iron was "high" (0.025 mM), rates of production of thiobarbituric acid reactive components were identical for microsomes from MV2E1-9 and control clones. However, when the concentration of iron was lowered to 0.005 mM, control clones did not display lipid peroxidation, whereas microsomes from MV2E1-9 were reactive. This peroxidation was sensitive to antioxidants such as trolox, propyl gallate, and glutathione but not to catalase or superoxide dismutase. Rates of superoxide and H2O2 production and of lipid peroxidation were 7-20-fold higher on a per nanomole of P450 basis with clone MV2E1-9 compared to human liver microsomes, indicating that the human P4502E1 is especially reactive in production of reactive oxygen intermediates and in catalysis of lipid peroxidation.

3T3 Cells↗

PubMed1993

Stimulation of microsomal chemiluminescence by ferritin.

The ability of ferritin to catalyze rat liver microsomal chemiluminescence was determined in the absence and presence of the redox cycling agent paraquat, and with either NADPH or NADH as reductant. Microsomal chemiluminescence was used as a index of lipid peroxidation. In the absence of added ferritin, NADPH-dependent microsomal light emission was 4-fold greater than the NADH-dependent reaction, and was not sensitive to superoxide dismutase, catalase or DMSO. Ferritin stimulated NADPH-, but not NADH-dependent chemiluminescence in a time- and concentration-dependent manner. The stimulation by ferritin was completely sensitive to superoxide dismutase, but not to catalase or DMSO, suggesting the requirement for superoxide to mobilize iron from ferritin. An iron ligand was not required for the stimulation by ferritin; the addition of certain ligands such as EDTA, DETAPAC or desferrioxamine resulted in inhibition of the stimulation by ferritin. Paraquat potentiated the effect of ferritin on microsomal chemiluminescence with NADPH as cofactor and was weakly stimulatory with NADH. The potentiation by paraquat plus ferritin was prevented by superoxide dismutase and was further elevated by ligands such as ATP. Chemiluminescence proved to be a more sensitive parameter than production of thiobarbituric acid-reactive components to evaluate the stimulation of oxygen radical production by iron released from ferritin, in the absence or in the presence of paraquat.

Animals↗

PubMed1993

ESR studies on the production of reactive oxygen intermediates by rat liver microsomes in the presence of NADPH or NADH.

Experiments were carried out using spin-trapping ESR spectroscopy to evaluate in a quantitative and kinetic manner the production of reactive oxygen intermediates by rat liver microsomes. Comparisons between the effectiveness of NADH and that of NADPH in catalyzing microsomal generation of reactive oxygen intermediates were made. Superoxide production was determined by assaying the generation of superoxide dismutase-sensitive stable nitroxyl radicals formed from 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine. Identical spectra were produced when microsomes were incubated with NADH or NADPH; reaction rates were linear for at least 10 min of reaction and were about three- to fourfold greater with NADPH. In the presence of iron, microsomes catalyze the production of hydroxyl radical during electron transfer. Initial experiments utilizing 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) as the spin-trapping agent proved unsatisfactory for the microsomal system as adequate kinetics could not be determined in view of the rapid decay of the DMPO-OH adduct, as well as secondary adducts such as DMPO-CH3 or DMPO-hydroxyethyl (HER). The spin-trapping agent alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone (POBN) was evaluated. POBN-OH adducts were somewhat more stable but also rapidly decayed after 2 to 3 min. However, production of POBN-CH3 and POBN-HER adducts was proportional to that of microsomal protein, increased with time over a 5- to 15-min period, and then was relatively stable. The redox cycling agent paraquat increased POBN-HER adduct formation twofold. Formation of the adduct with either NADH or NADPH required an iron catalyst and did not occur in the presence of the iron chelator desferrioxamine. Ferric EDTA was most reactive in catalyzing production of the adduct, ferric DTPA was 60 to 70% as effective, and ferric ATP was 20 to 30% as effective as ferric EDTA, with both reductants. Irrespective of the iron complex, rates of POBN-HER formation with NADH were about 70% those of NADPH. Formation of the adduct was inhibited by catalase, mannitol, and GSH, but not superoxide dismutase. These experiments support the usefulness of POBN plus ethanol for kinetic studies on the production of .OH by microsomes and validate that microsomes in the presence of an iron catalyst generate .OH not only with NADPH as cofactor, but also with NADH.

Animals↗

PubMed1993

Increased NADPH- and NADH-dependent production of superoxide and hydroxyl radical by microsomes after chronic ethanol treatment.

There is increasing evidence that elevated production of reactive oxygen intermediates may contribute to the hepatotoxic actions of ethanol. Microsomes from ethanol-treated rats have been shown to generate oxygen radicals at elevated rates. Most of these studies have utilized chemical analysis techniques to assay for the production of reactive oxygen intermediates. Experiments employing the spin-trapping ESR spectroscopy technique were carried out for a more definitive characterization of production of reactive oxygen intermediates such as superoxide and hydroxyl radical (.OH) by microsomes from ethanol-fed rats and pair-fed controls, in the presence of either NADPH or NADH as the microsomal reductant. Superoxide production was determined by assaying the superoxide dismutase-sensitive generation of stable nitroxyl radical from 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine resulting from hydrogen abstraction by superoxide radical, while .OH production was determined by assaying the formation of the POBN-alpha-hydroxyethyl radical adduct resulting from addition of the hydroxyethyl radical produced by hydrogen abstraction by .OH. Microsomes from ethanol-treated rats displayed elevated rates of superoxide production with NADPH (50%) as well as with NADH (34%). With both microsomal preparations, the NADPH-dependent rates were three- to fourfold greater than the NADH-dependent rates. Microsomes from the ethanol-treated rats also displayed elevated rates of .OH production (POBN-HER adduct formation) with NADPH (74%) and NADH (52%). With both microsomal preparations, NADPH-dependent rates were equivalent to the rates with NADH. The increase in superoxide and .OH production after ethanol treatment was highly significant (n = 6, P < 0.001). Production of the POBN-HER adduct was sensitive to catalase, mannitol, and GSH, but not to superoxide dismutase. Addition of desferrioxamine caused a marked decrease in the signal intensity; the POBN-HER adduct formed under these conditions most likely reflects the previously reported radical-independent, direct oxidation of ethanol by cytochrome P450. Signal intensity in the presence of desferrioxamine was about threefold higher after ethanol treatment. These results demonstrate that production of reactive oxygen intermediates by microsomes, as determined by ESR spectroscopy, is elevated after chronic ethanol consumption, and that increases in production of reactive oxygen intermediates can be found with NADPH as well as NADH as the microsomal reductant.

Alcoholism↗

PubMed1993

Combined effects of streptozotocin-induced diabetes plus 4-methylpyrazole treatment on rat liver cytochrome P4502E1.

The content and activity of cytochrome P4502E1 is increased in the diabetic state, primarily due to stabilization of the P4502E1 mRNA. Chemical inducers such as 4-methylpyrazole (4MP) increase P4502E1 content by stabilization of the protein. Experiments were carried out to evaluate the combined effects of 4MP and streptozotocin-induced diabetes on P4502E1 protein, catalytic activity and mRNA levels. Immunoblots showed an elevated content of P4502E1 after treatment with 4MP or streptozotocin, which was further increased when the two treatments were combined. Similarly, catalytic activity with effective substrates for P4502E1 was increased by the two separate treatments, and further increased by combined treatment. In all treatment groups, catalytic activity was strongly inhibited by antibody against P4502E1. The content of P4502E1 and catalytic activity in the 4MP plus streptozotocin group appeared to be additive of the values for the separate treatments. P4502E1 mRNA levels were elevated by the streptozotocin treatment but not by 4MP treatment; combined treatment with both inducers did not elevate P4502E1 mRNA levels beyond the increase produced in the diabetic state. CCl4 decreased cellular viability in hepatocytes from streptozotocin- or 4MP-treated rats, and increased toxicity was found after treatment with both inducers. These results contrast the mechanisms of induction of P4502E1 by streptozotocin and 4MP, and suggest that each individual mechanism is maintained when the two inducers are administered such that effects on P4502E1 protein and catalytic activity, but not mRNA, are additive of values found for each inducer alone. The diabetic state may be associated with increased sensitivity to toxins which are activated by P4502E1, especially if chemical inducers similar to 4MP, e.g., ethanol, isoniazid are also present.

Animals↗

PubMed1993

Requirement for iron for the production of hydroxyl radicals by rat liver quinone reductase.

NADPH-quinone reductase catalyzes the two-electron reduction of quinones such as menadione, and generally is considered to play a protective role against quinone-mediated toxicity. Recent studies have shown that reactive oxygen intermediates may be produced during metabolism of quinones by quinone reductase. Experiments were carried out to evaluate the effect of iron complexes on production of hydroxyl radical (.OH) when menadione was oxidized by a rat liver cytosolic fraction. Menadione-stimulated H2O2 production when added to the cytosol; dicoumarol, a potent inhibitor of quinone reductase, completely blocked this stimulation. Results were identical with either NADH or NADPH as reductant. In the absence of added iron, .OH, assessed as oxidation of chemical scavengers, was not produced. Various ferric chelates, added to the cytosol in the absence of menadione, did not catalyze .OH production. However, .OH was produced in the presence of menadione with all ferric complexes evaluated except for ferric-desferrioxamine. Catalase, competitive scavengers and GSH inhibited .OH production, as did dicoumarol. Superoxide dismutase inhibited with ferric-ATP, ferric-citrate, ferric-histidine or ferric ammonium sulfate as iron catalysts, but had no effect with ferric-EDTA or ferric-diethylenetriamine penta-acetic acid. Reduction of the ferric complexes was increased by menadione. NADH and NADPH were equally effective as cofactor for all these reactions. Metabolism of menadione in the presence of iron complexes caused inactivation of enzymes present in the cytosolic fraction such as glutamine synthetase and lactic dehydrogenase. These results indicate that metabolism of menadione by quinone reductase can lead to the production of .OH in the presence of various ferric catalysts.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1993

Ethanol consumption by the nursing mother induces cytochrome P-4502E1 in neonatal rat liver.

Cytochrome P-4502E1 (P-4502E1) is not present in fetal rat liver because activation of the gene occurs shortly after birth. Ethanol is an inducer of P-4502E1 in adult rats. Studies were carried out to evaluate whether transplacental induction of P-4502E1 by ethanol can occur after oral consumption of ethanol by the pregnant mother. Because ethanol can be excreted in breast milk, the possible induction of P-4502E1 in neonatal liver when ethanol was consumed during the gestational and neonatal period by the mother was also determined. Pregnant rats received control or an ethanol-containing liquid diet starting on the 9th day of gestation and were killed on the 17th day or 21st day, of gestation or allowed to deliver. The rats continued on their respective diets for the first 2 weeks of the neonatal period. P-4502E1 messenger RNA (mRNA), protein or catalytic activity was not detectable in fetal liver and was not induced in the fetuses from the ethanol-consuming mothers. Transplacental induction of P-4502E1 by ethanol did not occur in this model. Induction by ethanol of P-4502E1 protein and catalytic activity but not mRNA occurred in maternal liver. P-4502E1 mRNA, protein and catalytic activity were detected shortly after birth and increased over the 2-week neonatal period. The P-4502E1 content and oxidation of p-nitrophenol or dimethylnitrosamine by hepatic microsomes from neonates of mothers consuming the ethanol diet were increased 2- to 3-fold compared with controls however, P-4502E1 mRNA levels were not elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Drinking↗

PubMed1993

Induction of liver cytochrome P4502E1 by pyrazole and 4-methylpyrazole in neonatal rats.

Cytochrome P4502E1 (P4502E1) is not present in fetal rat liver; activation of the gene occurs within hours after birth. In adult rats, chemical inducers increase P4502E1 levels largely by a post-transcriptional type of mechanism. Experiments were carried out to evaluate how soon after birth chemicals such as pyrazole or 4-methylpyrazole (MP) can induce P4502E1 and whether the mechanism for induction at these early developmental stages, during active transcription, is different from that found in adults. No P4502E1 was found in fetal liver; in liver microsomes from saline control rats, there was a progressive increase in P4502E1 levels and oxidation of dimethylnitrosamine every 2 days after birth, with maximal levels 8 to 14 days after birth. Injecting pyrazole and MP on day 0 and day 1 after birth, resulted in 2- to 4-fold increases (compared to saline control values) in P4502E1 content and oxidation of dimethylnitrosamine in liver microsomes isolated from 2-day-old pups. This extent of increase by treatment with pyrazole or MP over saline control values was similar to that found when pups were treated for 2 days with the inducers on days 2, 4, 6, 8, 12 and 19 after birth. Northern blot analysis indicated a progressive increase in P4502E1 mRNA levels, reaching a maximum at about 8 days after birth for saline-treated pups. Pyrazole or MP did not increase P4502E1 mRNA levels over values for the saline controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Expression of a catalytically active human cytochrome P-4502E1 in Escherichia coli.

The cDNA coding for human cytochrome P-4502E1 has been expressed in Escherichia coli by placing it under the control of the isopropylthiogalactoside inducible trc promoter. Production of P4502E1 was demonstrated by immunoblots and by catalytic activity with dimethylnitrosamine as substrate. Modifications which favor expression in E. coli were made within the first seven codons. This resulted in approx. a 2- to 2.5-fold increase in P4502E1 isolated from the bacterial membranes as detected by immunoblots and catalytic activity. CO-reduced difference spectra of the modified P4502E1 revealed a peak at 452 nm, which is characteristic of hepatic P4502E1, and a molecular weight of 54 kDa. A partially purified preparation of recombinant P450 protein was active with dimethylnitrosamine, a substrate specific for this isozyme, when reconstituted with purified rat liver NADPH-cytochrome P-450 oxidoreductase. This activity was enhanced in the presence of cytochrome b5 and inhibited by the addition of antibody to the P4502E1 purified from pyrazole-treated rats. E. coli were capable of oxidizing p-nitrophenol when transformed with vector containing the human P4502E1 cDNA but not with vector alone. This in vivo metabolism of p-nitrophenol was increased 2-fold when the modified P4502E1 cDNA was used, which corresponds to the increase in P4502E1 content. Expression of human P4502E1 in E. coli appears to be an attractive system for producing large amounts of this isozyme, and for studies on the toxicological properties and structure-function relationship of the human P4502E1.

Amino Acid Sequence↗

PubMed1992

Inhibition of rat liver microsomal lipid peroxidation by boldine.

The alkaloid boldine, found in the leaves and bark of boldo, was an effective inhibitor of rat liver microsomal lipid peroxidation under a variety of conditions. The following systems all displayed a similar sensitivity to boldine: non-enzymatic peroxidation initiated by ferrous ammonium sulfate; iron-dependent peroxidation produced by ferric-ATP with either NADPH or NADH as cofactor; organic hydroperoxide-catalyzed peroxidation; and carbon tetrachloride plus NADPH-dependent peroxidation. Boldine inhibited the excess oxygen uptake associated with microsomal lipid peroxidation. Thus, boldine was effective in inhibiting iron-dependent and iron-independent microsomal lipid peroxidation, with 50% inhibition occurring at a concentration of about 0.015 mM. Boldine did not appear to react efficiently with superoxide radical or hydrogen peroxide, but was effective in competing for hydroxyl radicals with chemical scavengers. Concentrations of boldine which produced nearly total inhibition of lipid peroxidation had no effect on microsomal mixed-function oxidase activity nor did boldine appear to direct electrons from NADPH-cytochrome P450 reductase away from cytochrome P450. Boldine completely protected microsomal mixed-function oxidase activity against inactivation produced by lipid peroxidation. The effectiveness of boldine as an anti-oxidant under various conditions, and its low toxicity, suggest that this alkaloid may be an attractive agent for further evaluation as a clinically useful anti-oxidant.

Adenosine Triphosphate↗

PubMed1992

Stimulation of microsomal production of reactive oxygen intermediates by rifamycin SV: effect of ferric complexes and comparisons between NADPH and NADH.

Rifamycins are antibacterial antibiotics which are especially useful for the treatment of tuberculosis. Reactive oxygen intermediates are produced in the presence of rifamycin SV and metals such as copper or manganese. Experiments were carried out to evaluate the interaction of rifamycin SV with rat liver microsomes to catalyze the production of reactive oxygen species. At a concentration of 1 mM, rifamycin SV increased microsomal production of superoxide with NADPH as cofactor 3-fold, and with NADH as reductant by more than 5-fold. Rifamycin SV increased rates of H2O2 production by the microsomes twofold with NADPH, and 4- to 8-fold with NADH. In the presence of various iron complexes, microsomes generated hydroxyl radical-like (.OH) species. Rifamycin SV had no effect on NADPH-dependent microsomal .OH production, irrespective of the iron chelate. A striking stimulation of .OH production was found with NADH as the reductant, ranging from 2- to 4-fold with catalyst such as ferric-EDTA and ferric-DTPA to more than 10-fold with ferric-ATP, -citrate, or -histidine. Catalase and competitive .OH scavengers lowered rates of .OH production (chemical scavenger oxidation) and prevented the stimulation by rifamycin. Superoxide dismutase had no effect on the NADH-dependent rifamycin stimulation of .OH production with ferric-EDTA or -DTPA, but was inhibitory with the other ferric complexes. In contrast to the stimulatory effects on production of O2-., H2O2, and .OH, rifamycin SV was a potent inhibitor of microsomal lipid peroxidation. These results show that rifamycin SV stimulates microsomal production of reactive oxygen intermediates, and in contrast to results with other redox cycling agents, is especially effective with NADH as the microsomal reductant. These interactions may contribute to the hepatotoxicity associated with use of rifamycin, and, since alcohol metabolism increases NADH availability, play a role in the elevated toxic actions of rifamycin plus alcohol.

Animals↗

PubMed1992

Time course characterization of the induction of cytochrome P-450 2E1 by pyrazole and 4-methylpyrazole.

Cytochrome P-450 (P-450) 2E1 is under transcriptional and post-transcriptional control. Well-defined time courses were carried out to compare the effect of pyrazole and 4-methylpyrazole on catalytic activities, apo-P-450 2E1 levels and mRNA levels to evaluate whether induction of P-450 2E1 is preceded by altered mRNA levels. Two days of treatment with pyrazole or three days of treatment with 4-methylpyrazole resulted in significant induction of P-450 2E1, as assessed by Western blots and by oxidation of dimethylnitrosamine or p-nitrophenol. No changes in mRNA levels were detected with either inducer. Within 2 h of the second treatment with pyrazole, maximal induction of P-450 2E1 was observed, however, a 8-12 h time-dependent period was required after the third treatment with 4-methylpyrazole for maximal induction. Irrespective of the time period, increased catalytic activity and P-450 2E1 appears to reflect a post-transcriptional mechanism. A single treatment with 4-methylpyrazole increased P-450 2B1/B2 levels and oxidation of pentoxyresorufin about 2- to 3-fold. No change in mRNA levels for 2B1/B2 was observed. Although significant, the induction of 2B1/B2 by 4-methylpyrazole is more than an order of magnitude less than that by phenobarbital. Pyrazole did not induce 2B1/B2. It appears that, similar to acetone and ethanol, 4-methylpyrazole may increase several P-450 isozymes, whereas pyrazole is more specific for induction of P-450 2E1.

Animals↗

PubMed1992

Effect of phenobarbital and 3-methylcholanthrene treatment on NADPH- and NADH-dependent production of reactive oxygen intermediates by rat liver nuclei.

The effect of inducing the rat liver nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene on NADPH- and NADH-dependent production of reactive oxygen intermediates was evaluated. The inducing agents produced a 2-fold increase in cytochrome P-450, a 50 to 70% increase in NADPH-cytochrome c reductase activity, and a 20 to 30% increase in NADH-cytochrome c reductase activity. Associated with these increases was a corresponding increase in NADPH- and NADH-dependent production of hydroxyl radical (.OH)-like species and of H2O2. Rates of .OH production were inhibited by catalase and partially sensitive to superoxide dismutase. The increase in nuclear production of .OH-like species after drug treatment appears to be due a corresponding increase in H2O2 generation. In contrast to H2O2 and .OH generation, production of thiobarbituric acid-reactive material by nuclei was not increased by the phenobarbital or 3-methylcholanthrene treatment. Redox cycling agents such as menadione and paraquat increased oxygen radical generation to similar extents in the control and the induced nuclei. These results indicate that induction of the nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene can result in a subsequent increase in production of reactive oxygen intermediates in the presence of either NADPH or NADH.

Animals↗

PubMed1992

Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.

Animals↗

PubMed1992

Presence of functionally active cytochrome P-450IIE1 in the plasma membrane of rat hepatocytes.

Recent experiments have described the presence of cytochrome P-450 and certain P-450 isozymes in the plasma membrane of rat liver. Experiments were carried out to evaluate whether cytochrome P-450IIE1 was present in the plasma membrane fraction of livers from control rats and rats treated with 4-methylpyrazole, which induces this isozyme. Using immunofluorescence, fluorescence was detected at the surface of intact hepatocytes that were initially incubated with anti-P-450IIE1 IgG, but not preimmune IgG, followed by incubating with goat antirabbit IgG conjugated with either fluorescein or rhodamine. The fluorescence appeared to be uniformly distributed across the entire surface. Intense intracellular staining could be observed when the hepatocytes were permeabilized by acetone treatment. Similar results were obtained with control hepatocytes; however, the fluorescence intensity was considerably less than that shown by the induced hepatocytes. Hepatocytes isolated from the pericentral zone of the liver acinus displayed more intense fluorescence at the surface than did hepatocytes from the periportal zone. Purified plasma membranes oxidized dimethylnitrosamine to formaldehyde at rates that were 14% to 30% that of the microsomes, which exceeds the 3% contamination of the plasma membranes by microsomes as assessed by glucose-6-phosphatase activity. Immunoblots of the plasma membranes revealed the presence of a single band, whose intensity of staining was 14% to 26% that of the microsomes. Oxidation of dimethylnitrosamine and immunoblot intensity were about twofold greater with plasma membrane fractions from 4-methylpyrazole-treated rats than controls. These results suggest the presence of inducible, functionally active P-450IIE1 in the plasma membrane, which may be of toxicological significance in view of the preferential metabolism of a variety of hepatotoxins and carcinogens and the elevated production of reactive oxygen intermediates by this isozyme.

Animals↗