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PubMed1983

Induction of drug-metabolizing enzymes by tricyclic antidepressants in human liver: characterization and partial resolution of cytochromes P-450.

Drug-metabolizing enzyme activities were determined in liver microsomes from six kidney-transplant donors, one tricyclic antidepressant-treated and five untreated donors. The tricyclic antidepressant treatment modifies neither the overall cytochrome P-450 content of the liver, nor enzymatic activities of 4-nitroanisole demethylase, aniline hydroxylase, epoxide hydrolase and glutathione S-transferase. Only benzphetamine and ketotifen demethylation and conjugation of bilirubin with UDP-glucuronic acid are markedly augmented (more than two-fold). Separation of the different cytochrome P-450 fractions on a DEAE cellulose column indicates a modification of the elution pattern: the fraction increased by tricyclic antidepressants is responsible for the enhanced monooxygenase activity towards benzopyrene and benzphetamine.

Adult↗
PubMed1992

Inhibition of cytochrome-P450 reductase by polyols has an electrostatic nature.

The present study was undertaken to examine the nature of the inhibitory action of glycerol on the liver microsomal monooxygenase system. In agreement with earlier observations, glycerol inhibited benzphetamine N-demethylation by liver microsomes of the phenobarbital-treated rabbit. The presence of glycerol in the medium did not affect binding of the substrate to cytochrome P450. Another polyol, ethylene glycol, was equally efficient in inhibiting benzphetamine N-demethylation. Both also inhibited reduction of rabbit cytochrome P450 LM2, cytochrome c and potassium ferricyanide by NADPH-cytochrome-P450 reductase in microsomes. Recently, we showed that the stimulation of electron transfer by increased ionic strength is due to neutralization of electrostatic interaction between NADPH-cytochrome-P450 reductase and its charged redox partners [Voznesensky, A. I. & Schenkman, J. B. (1992) J. Biol. Chem. 267, 14669-14676]. Polyols have an opposite effect to that of salt on ionic properties of a solution. They decrease the dielectric constant, thereby promoting electrostatic interactions between proteins. Addition of polyols decreased the conductivity of the medium. When rates of electron transfer to charged acceptors, cytochrome P450, cytochrome c and potassium ferricyanide, at various salt and polyol concentrations, relative to activities in 200 mM sodium phosphate, were plotted as a function of the conductivity the data for each acceptor fit on the same line. In contrast, neither alteration of ionic strength nor polyol addition affected the rate of electron transfer from NADPH-cytochrome-P450 reductase to an uncharged acceptor 1,4-benzoquinone. The data obtained is consistent with our earlier suggestion that charge repulsion limits redox interactions between rabbit cytochrome P450 LM2 and its reductase at low ionic strength, and suggest that the observed action of polyols is the result of enhancement of electrostatic interactions that inhibits electron transfer between NADPH-cytochrome-P450 reductase and its charged redox partners. In congruence with the hypothesis, the Km of rabbit cytochrome P450 LM2 for NADPH-cytochrome-P450 reductase was increased almost one order of magnitude by elevating the glycerol content from 5% to 25% (by vol.) without a change in Vmax.

Animals↗
PubMed2005

The naturally occurring cytochrome P450 (P450) 2B6 K262R mutant of P450 2B6 exhibits alterations in substrate metabolism and inactivation.

The polymorphic human cytochrome P450 (P450) 2B6 is primarily responsible for the metabolism of several clinically relevant drugs including bupropion, cyclophosphamide, propofol, and efavirenz. Although a number of single nucleotide polymorphisms have been found in the P450 2B6 gene, the influence of these variants on the metabolism of substrates and on the response to known inactivators of P450 2B6 has not been examined. We have compared the metabolism of different substrates of P450 2B6 (P450 Delta2B6) and the effects of mechanism-based inactivators with that observed with the polymorphic P450 Delta2B6 K262R in a reconstituted monooxygenase system (reconstituted system). Metabolism of bupropion by P450 Delta2B6 K262R resulted in increased production of hydroxybupropion compared with P450 Delta2B6. However, production of formaldehyde from the metabolism of benzphetamine by the P450 Delta2B6 K262R mutant was significantly less than that of the wild-type isozyme. P450 Delta2B6 K262R formed fewer benzphetamine metabolites compared with the wild type. N,N',N''-Triethylenethiophosphoramide (tTEPA) and bergamottin decreased the ability of both enzymes to hydroxylate bupropion and to O-deethylate 7-hydroxy-4-(trifluoromethyl)coumarin (7-EFC). Incubation with 17-alpha-ethynylestradiol decreased bupropion hydroxylation and 7-EFC O-deethylation with the wild-type enzyme but had no effect on the mutant. The kinetics for inactivation of the variant by tTEPA and bergamottin were determined using 7-EFC. The KI values for inactivation of the variant were significantly greater than those determined for the wild-type enzyme. These data demonstrate a functional difference between P450 Delta2B6 and the allelic variant P450 Delta2B6 K262R.

Alleles↗
PubMed2003

Threonine-205 in the F helix of p450 2B1 contributes to androgen 16 beta-hydroxylation activity and mechanism-based inactivation.

Four mutants of Thr-205 in cytochrome p450 2B1 were constructed and expressed in Escherichia coli. The Ser-, Ala-, and Val-mutants displayed stable reduced CO difference spectra and were able to metabolize 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, androstenedione, and benzphetamine. The Arg-mutant displayed an unstable reduced CO difference spectrum at 450 nm, was concomitantly converted to a denatured form with a peak at 422 nm, and showed no catalytic activity with any of the four substrates tested. The Ser-mutant displayed activity and metabolite profiles for testosterone and androstenedione similar to those of the wild-type p450 2B1 (WT). Substitution of Thr-205 with Ala or Val markedly suppressed the 16 beta-hydroxylation activity but exhibited little effect on the 16 alpha-hydroxylation activity for testosterone and androstenedione. Because 16 beta-hydroxylation activity of androgens is a specific p450 2B subfamily marker and residue 205 is located in the F helix, which forms the ceiling of the active site, we postulate that the gamma-hydroxyl side chain of Thr may play an important role in directing the 16 beta-face of testosterone and androstenedione toward the active site. Surprisingly, the Val-mutant retained full activity for benzphetamine demethylation. When mechanism-based inactivators for p450 2B1 were used to evaluate the susceptibility to inactivation, the Val-mutant was resistant to inactivation by 17 alpha-ethynylestradiol and less sensitive to inactivation by 2-ethynylnaphthalene compared with the WT enzyme. Our results demonstrate the importance of Thr-205 in determining substrate specificity and product formation as well as in influencing the susceptibility of p450 2B1 to mechanism-based inactivators.

Androgens↗
PubMedDate not supplied

Enhanced arsenite-induced hepatic morphological and biochemical changes in phenobarbital-pretreated rats.

Changes in liver morphology and biochemistry have been assessed 16 hr after a sc injection of sodium arsenite [As(III), 75 mumol/kg] to control and phenobarbital (PB)-pretreated (80 mg/kg, ip daily for 3 days) adult male Wistar rats. As(III) administration to PB-pretreated rats [PB + As(III)] caused hydropic degeneration, total loss of glycogen, necrosis in some centrilobular zones, and an increase in lipid vacuoles around the periportal area. Electron microscopy showed an increased number of vacuoles and autophagosomes containing organelle-like material. There was a 30% decrease in total hepatic cytochrome P-450 (CYP450). O-dealkylation of ethoxy- and pentoxyresorufin and N-demethylation of benzphetamine decreased to 42, 32, and 30% of control values, respectively. 5-Aminolevulinic acid dehydrase decreased 25% from controls, and metal chelatase activities decreased to 25% of the PB-treated group. Injection of As(III) alone resulted in a mild increase in lipid-containing vacuoles around the periportal zone, a moderate loss of glycogen in midzonal areas, and, by electron microscopy, a dilatation of the bile canaliculi and an increase of the number of myelin-like structures. CYP450 content and the O-dealkylation of ethoxy- and pentoxyresorufin and N-demethylation of benzphetamine all decreased between 30 and 50%. These results demonstrate the greater susceptibility of the liver to injury following PB with compounds not requiring metabolic activation. The metabolic basis of these changes are unclear but may result from an increased demand for metabolic energy due to PB induction and decreased adenosine triphosphate synthesis caused by arsenic.

Animals↗
PubMed1997

No linkage of the cytochrome P-450IIE1 (CYP2E1) C1/C2 polymorphism to schizophrenia.

We investigated, using PCR-SSCP analysis, the relationship between schizophrenia and the polymorphism of d-benzphetamine N-demethylase (cytochrome P-450j or CYP2E1), which metabolizes psychotropic substances such as d-benzphetamine and alcohols. Among 41 patients with schizophrenia, no statistically significant change in the frequency of the mutant (C2) allele relative to in controls was found, and no novel structural mutation in the CYP2E1 gene, which would be expected to alter the CYP2E1 protein, was found. This could be explained by no linkage of the CYP2E1 gene (mutations in the exon 1-9, and C1/C2 polymorphism) to schizophrenia.

Adult↗
PubMed1995

Hair analysis for drugs of abuse. X. Effect of physicochemical properties of drugs on the incorporation rates into hair.

To determine the mechanism involved, the incorporation rate (ICR) of drugs into hair was compared to melanin affinity, lipophilicity and membrane permeability. The following 20 drugs were tested; amphetamine, methamphetamine, p-hydroxyamphetamine, p-hydroxymethamphetamine, cocaine, benzoylecgonine, ecgonine methyl ester, morphine, 6-acetylmorphine, phencyclidine, 1-(1-phenyl)-piperidinyl-cyclohexanol, methylenedioxyamphetamine, methylenedioxymethamphetamine, methoxyphenamine, O-desmethyl methoxyphenamine, benzphetamine, norbenzphetamine, deprenyl, lysergic acid diethylamide (LSD) and 11-nortetrahydrocannabinol-9-carboxylic acid (THCA). Their ICRs were represented as the ratios of the drug concentrations in rat hair to AUCs (the areas under the concentration vs. time curves) in rat plasma. Cocaine had the highest incorporation rate understood and there was a 3600 fold difference between the ICR of cocaine and that of THCA, the lowest drug. The melanin affinity of these drugs was determined by incubating a test solution with melanin at 36 degrees C in the dark for 2 h. After incubation and centrifugation, the drug concentration in the filtrate was determined by GC/MS or LC. The drug most affinitive to melanin was cocaine, followed by benzphetamine, phencyclidine, methylenedioxymethamphetamine and LSD. The correlation coefficient between ICR and melanin affinity of the 20 drugs was 0.947 (0.949 excluding THCA). Lipophilicity was calculated from the retention times of HPLC according to Kaliszan's method. Although the correlation coefficient between ICR and lipophilicity was very low (0.201), it rose to 0.770 by removing only THCA. The combination of melanin affinity and lipophilicity brought about a higher correlation (0.979) with the ICRs. Our data also suggested that the higher ICRs of basic drugs than neutral or acidic ones are strongly related to the membrane permeability of the drug based on the pH gradient between blood (pH 7.4) and hair matrix (acidic).

Animals↗
PubMed1991

Site-selective oxidation of strychnine by phenobarbital inducible cytochrome P-450.

The metabolism of strychnine was studied using liver microsomes of rats treated with phenobarbital or 3-methylcholanthrene (MC). The phenobarbital-treatment resulted in 7.9-fold and 4.8-fold increases in 2-hydroxylation and N-oxidation of strychnine, respectively. The formation of 16-hydroxystrychnine, strychnine 21,22-epoxide and 22-hydroxystrychnine was induced about 2-fold. MC-treatment resulted in only 1.4-fold induction of each oxidation activity. In addition, strychnine 2-hydroxylation activity was markedly induced in liver microsomes of phenobarbital-treated mice, guinea pigs, rabbits and dogs (2.5-10.5-fold). Induction of N-oxidation activity was also higher than that of the three other oxidation activities. A reconstituted system of strychnine metabolism using cytochrome P-450 isozymes, P-450I (P450IIB1) and P-450II (P450IIB2), purified from liver microsomes of phenobarbital-treated rats showed significantly high and selective activities towards 2-hydroxylation and N-oxidation of strychnine. This characteristic metabolism of strychnine appears to occur in common with interspecies P450IIB gene subfamily. The pH Optima of 2-hydroxylation and N-oxidation of strychnine were between 8.4 and 8.6 in the microsomes of phenobarbital-treated rats, while that of N-demethylation of benzphetamine, a typical substrate of phenobarbital-inducible cytochrome P-450, was between 7.4 and 7.6. In a reconstituted system with P-450I, strychnine oxidation was little affected by pH change, while benzphetamine N-demethylation activity was decreased in the alkaline side. Among several oxidations tested, only ethylmorphine N-demethylation underwent the same pH effect as did strychnine oxidation with the microsomes and reconstituted system.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Effect of co-administration of interferon inducer, polyriboinosinic acid-polyribocytidylic acid, with SKF 525-A on hepatic drug metabolizing enzymes of rats.

The protective effect of SKF 525-A on the suppression of cytochrome P-450 content and monooxygenase activities by treatment with CoCl2 and polyriboinosinic acid-polyribocytidylic acid [poly(I.C] was compared as a part of studies of suppression of drug metabolizing enzymes by interferon inducers. Induction of heme oxygenase activity by CoCl2 and poly (I.C) was not altered by simultaneous treatment with SKF 525-A. Depression of cytochrome P-450 content and benzphetamine N-demethylase activity by treatment with CoCl2 was prevented by co-treatment with SKF 525-A. This effect was explained by the prevention of release of heme from cytochrome P-450 by forming metabolic intermediate complexes with metabolites of SKF 525-A. On the other hand, poly(I.C) significantly suppressed P-450 content and benzphetamine N-demethylase and benzo [a] pyrene hydroxylase activities, even under simultaneous treatment with SKF 525-A. This inhibition by poly (I.C) was accompanied by weak staining of proteins corresponding to cytochrome P-450 in SDS gel electrophoresis. In addition, the activity of non-heme enzyme, 4-hydroxybiphenyl glucuronyltransferase, was suppressed by treatment with poly (I.C) but not by CoCl2-treatment. These findings strongly suggested that, unlike CoCl2, poly (I.C) suppressed cytochrome P-450 content and monooxygenase activities due to decreased synthesis or increased degradation of the apoprotein of cytochrome P-450 with slight contribution of the induced heme oxygenase.

Animals↗
PubMed1991

Comparison of the effects of piperine administered intragastrically and intraperitoneally on the liver and liver mixed-function oxidases in rats.

Piperine, a major pungent constituent of black and red peppers, was administered to rats intragastrically and intraperitoneally to study whether it alters the activities of hepatic mixed-function oxidases (MFO) and serum enzymes as specific markers of hepatotoxicity. An intragastric dose of 100 mg/kg of piperine to adult, male Sprague-Dawley rats caused an increase in hepatic microsomal cytochrome P-450 and cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h following treatment. On the other hand, a 10 mg/kg dose given i.p. exhibited no effect on the activities of the aforementioned parameters of the hepatic drug-metabolizing enzyme system. However, when the intragastric and intraperitoneal doses were increased to 800 mg/kg and 100 mg/kg, respectively, the black pepper alkaloid produced a significant decrease in the levels of cytochrome P-450, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h after treatment. None of the treatments significantly elevated the activities of serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and isocitrate dehydrogenase (ICD), suggesting that piperine is not a hepatotoxic agent.

Alkaloids↗
PubMed1976

Anorectic drugs: use in general practice.

The treatment of obesity is one of the major measures available today in the field of preventive medicine. In particular, the coronary epidemic of Western civilisation would be halted, and most cases of maturity-onset diabetes prevented, if obesity were to be treated effectively. Anorectic drugs act mainly on the satiety centre in the hypothalamus to produce anorexia. They also have various metabolic effects involving fat and carbohydrate metabolism, but many of these may be secondary to loss of weight. Most of the drugs are related directly or indirectly to amphetamine and in addition act by increasing general physical activity. Anorectic drugs tend to lose their effect after some months, and part of this reduction in effect may be due to chemical alterations produced by the drugs in the brain. All the drugs, with the exception of fenfluramine, have a stimulant effect on the central nervous system in some individuals, resulting in restlessness and nervousness, irritability and insomnia. Fenfluramine commonly produces drowsiness in normal doses, but has stimulant effects with overdosage. Dexamphetamine, phenmetrazine and benzphetamine all tend to cause euphoria and the risk of addiction is therefore considerable. Euphoria occasionally occurs with diethylpropion, phentermine and chlorphentermine, but to a much lesser extent. Side-effects also occur due to sympathetic stimulation and gastro-intestinal irritation. These side-effects may cause some individuals to stop taking the drug, but are never serious or dangerous. Drug interactions may occur with monoamine oxidase inhibitors and to a clinically unimportant extent, with antihypertensive drugs. The anorectic drugs have a very definite part to play in the treatment of obesity, mainly for those individuals who have altered their eating habits but have come to a plateau of weight which they find difficult to get below. The drugs are best given in a long-acting form and can safely be continued as long as weight loss persists, provided that the clinician exercises careful supervision. Dexamphetamine, phenmetrazine and benzphetamine should rarely be used because of the danger of addiction, and chlorphentermine is potentially hazardous for long-term use. Diethylpropion emerges as the drug of first choice, as fenfluramine has a tendency to cause depression and has a higher incidence of side-effects. Fenfluramine is mainly useful for people who are especially tense and for obese maturity-onset diabetics who have been unable to lose weight with the biguanides. Mazindol and phentermine appear to be useful as alternative drugs.

Adipose Tissue↗
PubMed1986

Comparison of cytochrome P-450 content and activities in liver microsomes of seven animal species, including man.

The cytochrome P-450 content found in human livers obtained post mortem was between 0.21-0.42 nmol/mg protein. The kinetic parameters of the mono-oxygenase activities--Km and Vmax--were determined in liver microsomes for N-demethylation (aminopyrine, benzphetamine, ethylmorphine), O-demethylation (4-nitroanisole), O-deethylation (7-ethoxycoumarin) and hydroxylation (benzo[a]pyrene), in an attempt to establish an inter-species comparison between man and the six animal species studied. The four substrates studied (aminopyrine, benzphetamine, ethylmorphine, benzo[a]pyrene) were shown to be less active in humans than the male rat, which is the most commonly used model. However, other animal species, such as the female Sprague-Dawley rat and the pig, are much more similar to man. From a procedural point of view, the optimal substrate concentrations vary from one experimental species to another. Due to the apparent Km observed, for example, the activities of the guinea-pig require a higher substrate concentration.

Adult↗
PubMed1989

Induction of cytochrome P-450 isozymes by chromanamine derivatives in rat liver.

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24 h after the last treatment. 2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene. 3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes. 4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes. 5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30 min after treatment with the drug.

Animals↗
PubMed1989

Cytochrome P450 monooxygenases in crustaceans.

1. The hepatopancreas is the major site of cytochrome P450-dependent xenobiotic monooxygenation in crustacean species, but the presence of monooxygenase inhibitors in hepatopancreas microsomes and cytosol from many decapod species has impeded in vitro studies. Cytochrome P450 and monooxygenase activities have been reported in other crustacean organs including the antennal gland (green gland) and stomach. 2. NADPH cytochrome c reductase activity is often very low (typically less than 10 nmol cytochrome c reduced/min per mg microsomal protein) in hepatopancreas microsomes from crustacean species. NADPH cytochrome P450 reductase activity has not yet been detected in crustacean hepatopancreas microsomes. 3. The cytochrome P450 present in hepatopancreas of several crab species and the spiny lobster has been resolved into several fractions by chromatography on DEAE-cellulose. One form of cytochrome P450 from spiny lobster has been purified to 12 +/- 2 nmol/mg protein. 4. Reconstitution studies with spiny lobster hepatopancreas P450 have shown that the vertebrate sex steroids, progesterone and testosterone, are excellent substrates, whereas ecdysone--the crustacean molting hormone--is not a substrate. Activity was found with several xenobiotic substrates including benzphetamine, aminopyrine, benzo(a)pyrene, ethyl-, benzyl- and pentyl-phenoxazones and ethoxycoumarin. Highest activities (greater than 50 nmol/min per nmol P450) were found for N-demethylation of benzphetamine and aminopyrine. 5. The ability of agents which induce vertebrate cytochrome P450 to induce cytochrome P450 in crustaceans is still unclear. Some studies indicate that polycyclic aromatic hydrocarbons, but not phenobarbital-type inducers, could induce cytochrome P450 in crustaceans, whereas other studies showed no effect of either inducer type. Crustaceans are not as sensitive as fish to induction of P450 and monooxygenase activity.

Animals↗
PubMed1991

Comparison of the effects of inducers of cytochrome P450 on Mongolian gerbil and rat hepatic microsomal monooxygenase activities.

1. Basal cytochrome P450 content (nmol/mg protein) was higher in gerbil (1.10 +/- 0.01) than in rat (0.81 +/- 0.05) hepatic microsomes. Pretreatment of gerbils with phenobarbitone and beta-naphthoflavone increased P450 contents by 200% and 60% respectively. 2. 7-Ethoxycoumarin O-deethylase, coumarin 7-hydroxylase and 4-nitrophenol hydroxylase activities were generally higher in gerbil liver microsomes, whereas erythromycin N-demethylase, and 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylase activities were higher in rat microsomes. Microsomal benzphetamine N-demethylase activities were similar in both species. 3. Induction of specific cytochrome P450 isozymes increased similar monooxygenase activities of rat and gerbil microsomes. Phenobarbitone, beta-naphthoflavone, isoniazid and pregnenolone 16 alpha-carbonitrile principally increased benzphetamine N-demethylase, 7-ethoxyresorufin O-deethylase, 4-nitrophenol hydroxylase and erythromycin N-demethylase activities respectively. 4. Constitutive 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylase activities were markedly lower in gerbil microsomes compared with rat microsomes, and pretreatment of gerbils with cytochrome P450 inducers did not significantly increase these activities. 5. Hepatic microsomal coumarin 7-hydroxylase activities were approximately 30-200 times greater (depending on the inducer) in the gerbil than in rat. The gerbil, due to is high coumarin 7-hydroxylase activity, would appear to be a more appropriate species than rat for investigations of coumarin metabolism and toxicity relevant to humans.

Animals↗
PubMed1984

Experimental induction of chronic aflatoxicosis in chickens by purified aflatoxin B1 and its reversal by activated charcoal, phenobarbital, and reduced glutathione.

Aflatoxin B1 (AFB1) caused dose-dependent reductions in weight gain and feed consumption when day-old Hubbard X Hubbard broiler type chicks were maintained on a diet contaminated with either 0, 2.5, 5, or 10 ppm purified AFB1 for 8 weeks. Although changes in these parameters were detected at the 2.5 and 5 ppm, the most profound changes were evident at 10 ppm contamination. The concentration of cytochrome P-450 in hepatic microsomes, measured at the end of 8 weeks, also showed dose-dependent decreases. Cytochrome P-450 content in chickens receiving 2.5, 5, and 10 ppm AFB1 was 16, 28, and 65%, respectively, less than the control. Microsomal benzphetamine N-demethylase activity was not inhibited by 2.5 or 5 ppm, but ingestion of 10 ppm AFB1 reduced its activity by more than 40%. Serum glutamic oxalacetic transaminase (SGOT) levels of chickens receiving 10 ppm AFB1 increased by more than 100%, indicating substantial liver damage. However, birds simultaneously receiving 10 ppm AFB1 and activated charcoal (.1% in the feed) or either reduced glutathione (.05%) or phenobarbital (.05%, given intermittently) in their drinking water showed a trend of improvement in feed consumption (less than 10% reversal) and weight gain (less than 28% reversal) over the birds receiving 10 ppm AFB1 alone. The results also indicate that the simultaneous presence of these agents with AFB1 considerably prevented the inhibitory effect of AFB1 on the microsomal cytochrome P-450 and benzphetamine N-demethylase activity. Furthermore, these agents were able to provide moderate protection against AFB1-induced liver injury manifested by elevation of SGOT activity.

Aflatoxin B1↗
PubMed1975

Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.

Anaerobiosis↗
PubMed1992

Benzodiazepine metabolism in ethanol-treated male rats: use of pair-fed and age-matched controls.

The effects of chronic moderate (15%) ethanol consumption and ageing on rat hepatic cytochrome P450 monooxygenase activities were examined using diazepam, nordazepam, d-benzphetamine, erythromycin, ethylmorphine and nitrosodimethylamine (NDMA) as substrates. In addition, the effects of moderate ethanol alone on the oxidation of metoprolol, morphine and temazepam were examined. Cytochrome P450 specific content increased significantly only in the 6-week ethanol-treated rats, and no changes in percentage liver to body weights were apparent in any of the ethanol-treated animals compared with pair-fed controls. Only cytochrome P450IIIA enzyme activities displayed age-related decreases, these being identified in the pair-fed animals. C3-hydroxylation of diazepam and nordazepam (36% of controls) and N-demethylation of erythromycin and ethylmorphine (58% and 64% of controls) were decreased in 6-week ethanol-treated animals, these effects being less pronounced in the 12, 24 and 48-week ethanol-treated groups. The decrease seen for diazepam and d-benzphetamine N-demethylation caused by ethanol consumption was approximately 80% of control groups for the duration of the treatment. NDMA and morphine N-demethylations were increased to 120% of control activities and metoprolol alpha-hydroxylase was increased to 140% of control activities at 6 weeks, whilst metoprolol O-demethylase activity remained unaltered. NDMA N-demethylase activity showed a two-fold induction at 24 and 48 weeks of ethanol treatment, compared with corresponding pair-fed control groups. These results support previous findings from this laboratory showing that the same or similar P450IIIA family isozymes are involved in the C3-hydroxylation of diazepam and nordazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗