Phenobarbital-treated Rats Research Articles

Inhibition of Ferritin-Stimulated Microsomal Production of Reactive Oxygen Intermediates by Nitric Oxide

Experiments were carried out to evaluate the effect of nitric oxide exposure on the ability of NADPH-dependent microsomal electron transfer to mobilize iron from ferritin. Such interactions could play a role in potential antioxidant actions of nitric oxide (NO). Preincubation of the microsomes from phenobarbital-treated rats with NO donors such asS-nitroso-d,l-N-acetyl penicillamine (SNAP),S-nitroso-l-glutathione, SIN-1, and DETANONOate followed by centrifugation, washing, and resuspension of the microsomes resulted in a decrease in the ferritin-dependent oxidation of 2′,7′-dichlorofluorescein diacetate (DCFDA) or ferritin-catalyzed chemiluminescence compared to microsomes pretreated with buffer. The ferritin-stimulated rate of oxidation of DCFDA or of chemiluminescence was completely restored if the microsomal preincubation with NO donors was performed in the presence of hemoglobin. In contrast to results with ferritin, ferric-stimulated oxidation of the dye was not affected by any of the tested NO donors. The microsomal oxidation of aminopyrine was inhibited after SNAP treatment, indicating that NO inhibited cytochrome P450 catalyzed activity. Inhibition of cytochrome P450 also resulted in an inhibition of microsomal production of superoxide. Similar results were obtained using microsomes from a cloned cell line which express the CYP2E1 isoform. Since superoxide is required for the mobilization of iron from ferritin by microsomes, inhibition of superoxide production as a consequence of NO interaction with cytochrome P450 is likely to be responsible for the prevention of ferritin-catalyzed formation of reactive oxygen species by NO donors. The results suggest that NO could exhibit an antioxidant capacity through its ability of decreasing the activity of iron–heme compounds, such as cytochrome P450, preventing the release of catalytically active iron from ferritin, and thus decreasing the ability to generate oxygen free radicals involved in cytotoxicity.

Substrate Enantioselective and Product Stereoselective Metabolism of Racemic 2-Hydroxy-3-methylcholanthrene by Rat Liver Microsomes

Abstract Racemic 2-hydroxy-3-methylcholanthrene (rac-2-OH-3MC) is a potent carcinogen and 2S-OH-3MC is the most abundant metabolite formed in the metabolism of 3-methylcholanthrene (3MC) by rat liver microsomes. Eighteen identifiable metabolites were formed in the metabolism of rac-2-OH-3MC by liver microsomes prepared from phenobarbital-treated rats. Metabolites were separated by sequential use of reversed-phase and normal-phase high performance liquid chromatography (HPLC) and characterized by ultraviolet-visible absorption, mass, and circular dichroism spectral, and chiral stationary phase (CSP) HPLC analyses. Identified metabolites were: 2-OH-3MC 7,8-dihydrodiol (enriched in 7R,8R enantiomer), 2-OH-3MC 9,10-dihydrodiol (2S,9R,10R:2R,9S,10S ≈ 27:73 and 2R,9R,10R:2S,9S,10S ≈ 81:19), 2-OH-3MC 11,12-dihydrodiol (2R,11R,12R:2S,11S,12S ≈ 5:95 and an essentially optically pure 2S,11R,12R enantiomer), 3MC trans-1,2-diol (1S,2S:1R,2R ≈ 68:32), 3MC cis-1,2-diol (1S,2R:1R,2S ≈ 18:82), 2-OH-3-hydroxymethyl-cholan...

Effects of Cytochrome P450 Antibodies on the Oxidative Demethylation of Methoxychlor Catalyzed by Rat Liver Microsomal Cytochrome P450 Isozymes: Isozyme Specificity and Alteration of Enantiotopic Selectivity

Anti-CYP2B1 added to the methoxychlor reaction medium catalyzed by phenobarbital-treated rat liver microsomes altered the enantiotopic selectivity of the reaction, in favor of the (S)-mono-O-demethylated metabolite. In the reaction with untreated liver microsomes, anti-CYP2B1 did not affect the enantiotopic selectivity. Phenobarbital induces CYP2B1 and 2B2, both of which have low enantiotopic selectivity in this reaction, and both are inhibited by anti-CYP2B1. Therefore, in the presence of anti-CYP2B1, the catalytic activity of other CYPs that exhibit high enantiotopic selectivity should predominate even in the phenobarbital-treated rat liver microsomes. The findings with anti-CYP2B1 were consistent with this notion. When anti-CYP2C6 was added to the reaction medium containing purified CYP2C6, the metabolic rates markedly decreased, whereas the enantiotopic selectivity was unaltered. However, when anti-CYP2C6 was added to reactions catalyzed by both microsomal preparations, the rates sometimes significantly increased, while there were no essential changes in the metabolite composition. Anti-CYP2C6 was also added to CYP2A1-catalyzed reactions. Our CYP2A1 preparation sometimes altered the metabolic rates and the (S) percentage in the metabolites. This CYP2A1 preparation may have been contaminated with a small amount of CYP2C6, and some other CYP(s) with low affinity to methoxychlor. CYP(s) metabolizing methoxychlor with low affinity to the substrate may be present in the microsomal preparations.

Luminol-and lucigenin-amplified chemiluminescence with rat liver microsomes: Kinetics and influence of ascorbic acid, glutathione, dimethylsulfoxide, N-t-butyl-a-phenyl-nitrone, copper-ions and a copper complex, catalase, superoxide dismutase, hexobarbital and aniline

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.

Identification of Cytochrome P450 3A1/2 as the Major P450 Isoform Responsible for the Metabolism of Fentanyl by Rat Liver Microsomes

The metabolism of fentanyl was investigated using rat liver microsomes to determine whether fentanyl is metabolized by rat liver microsomal cytochrome P450 and, if so, which isoform is responsible for the metabolism. Microsomes isolated from rats pretreated with phenobarbital were more active in metabolizing fentanyl than were microsomes from saline controls. The major metabolic pathway of fentanyl was an oxidative N-dealkylation to norfentanyl, which was detected by a gas chromatograph-mass selective detector (GC-MSD) method. The apparent Vm values for microsomes isolated from saline- and phenobarbital-treated rats were 2 and 9 nmol norfentanyl.min-1.mg-1 microsomal protein, and the apparent Km values were 32 and 47 microM, respectively. Fentanyl metabolism was inhibited by antibodies specific for CYP3A1/2, as well as by chemical inhibitors specific for CYP3A. These results indicate that CYP3A1/2 plays a major role in the oxidation of fentanyl to norfentanyl by rat liver microsomes.

Identification of Cytochrome P450 3A1/2 as the Major P450 Isoform Responsible for the Metabolism of Fentanyl by Rat Liver Microsomes

The metabolism of fentanyl was investigated using rat liver microsomes to determine whether fentanyl is metabolized by rat liver microsomal cytochrome P450 and, if so, which isoform is responsible for the metabolism.Microsomes isolated from rats pretreated with phenobarbital were more active in metabolizing fentanyl than were microsomes from saline controls. The major metabolic pathway of fentanyl was an oxidative N-dealkylation to norfentanyl, which was detected by a gas chromatograph-mass selective detector (GC-MSD) method. The apparent Vm values for microsomes isolated from saline- and phenobarbital-treated rats were 2 and 9 nmol norfentanyl centered dot min-1 centered dot mg-1 microsomal protein, and the apparent Km values were 32 and 47 micro Meter, respectively. Fentanyl metabolism was inhibited by antibodies specific for CYP3A1/2, as well as by chemical inhibitors specific for CYP3A. These results indicate that CYP3A1/2 plays a major role in the oxidation of fentanyl to norfentanyl by rat liver microsomes. (Anesth Analg 1996;82:936-41)

Neonatal Phenobarbital-Induced Persistent Alterations in Plasma Testosterone Profiles and Testicular Function

Daily sc injections of phenobarbital at anticonvulsant therapeutic doses for the rat (40 mg/kg) for the first 7 days of life resulted in below normal levels of serum testosterone from around birth to before puberty, normal levels during puberty and above normal levels of the androgen after puberty and in adulthood. Cluster analysis of the plasma testosterone secretory profiles obtained at 15-min intervals from phenobarbital-treated rats at 65 and 165 days of age revealed a significant increase in both the peak amplitudes and their durations resulting in a 100% increase in the amount of hormone secreted during the peak periods. In general, most of the rats (control and experimental) secreted testosterone as two large peaks, each 3 to 4 hr in duration, during the 10-hr lights-on collection period. In addition to permanently disrupting the ultradian profiles of plasma testosterone, neonatal exposure to the barbiturate altered testicular responsiveness to steroidogenic regulatory agents. That is, neonatal exposure to phenobarbital enhanced the responsiveness to exogenous hCG as measured by an above-normal increase in testosterone concentration. Moreover, phenobarbital-induced reductions in serum testosterone levels were delayed in adult rats neonatally exposed to the barbiturate. Whereas a single challenge dose of phenobarbital (1 or 10 mg/kg) reduced serum testosterone concentrations in control animals by almost 80% within 3 hr, a decline in serum androgen levels in the neonatally phenobarbital exposed males was not observed until 12 hr after the challenge dose. These results indicate that postpartum exposure to therapeutic levels of phenobarbital can permanently disrupt testosterone secretory profiles and alter pathways regulating testicular steroidogenesis.

Role of cytochrome P-450 in the stimulation of microsomal production of reactive oxygen species by ferritin

Microsomes can remove iron from ferritin by a superoxide-dependent reaction. The released iron can then catalyze formation of a variety of reactive oxygen species. Experiments were carried out to evaluate the role of cytochrome P-450 in the release of iron from ferritin, and whether induction of certain P-450 isoforms alters ferritin-dependent reactive oxygen radical production. Rats were treated with phenobarbital, 3-methylcholanthrene, 4-methylpyrazole, or saline to produce microsomes with varying P-450 content and composition. Oxidations of 2.7′-dichlorofluorescein diacetate to a fluorescent product and chemiluminescence were used as indices of production of reactive oxygen species. The extreme sensitivity of these reactions to trolox, a potent chain-breaking oxidant, indicates the involvement of lipid peroxidation products in these reactions. In the absence of ferritin, formation of reactive oxygen species was higher in microsomes from the treated rats compared to saline controls when results were expressed on a per mg protein basis but not per nmol P-450, suggesting that the increased content of total P-450 (2-fold increases) rather than the population of isoforms was responsible for ·h, increase. Superoxide dismutase had no effect on the non-ferritin catalyzed reactions. Ferritin increased production of reactive oxygen species with all the microsomal preparations; the increase by ferritin was completely prevented by superoxide dismutase. The net increase by ferritin was higher in microsomes from the treated rats compared to saline controls, but this, again, largely reflected the increased content, rather than the type of isoforms of P-450 present. Similar results were obtained with either NADPH or NADH as microsomal reductants, although NADPH was much more effective in supporting ferritin-dependent reactive oxygen formation. In microsomes from phenobarbital-treated rats, anti-CYP2B1/B2 IgG completely prevented the NADPH- and NADH-dependent increases in reactive oxygen formation produced by ferritin. Anti-cytochrome b 5 IgG produced partial inhibition of the ferritin-stimulation. These results indicate that P-450, and to a lesser extent, cytochrome b 5, play a role in the ferritin-dependent increase in formaion of reactive oxygen species with either NADPH or NADH, most likely reflecting the requirement of these enzymes for microsomal production of superoxide anion.

Free radical mediated release of histamine from rat mast cells induced by drugs of abuse

(Sorensen buffer, pH7.4, 37°C) with (a) cocaine, morphine and methadone, (b) with the oxidative enzymes, and (c) with cocaine, morphine and methadone in the presence of oxidative enzymes. The oxidative enzymes were PHS purified from ram seminal vesic1es (20 U/mg), and SIO Mix liver microsomes from phenobarbital treated rats (4 mg/mi protein). The reaction was stopped by chilling the tubes at 0-4 oe. After centrifugation, histamine and lactate dehydrogenase (LDH) were analysed in the supernatants and in the pellets (fluorimetric and spectrophotometric assays, respectively) and expressed as percent of the total histamine content. MDA was measured by a spectrophotometric assay [4].

3-Aminobenzamide: Effects on Cytochrome P450-Dependent Metabolism of Chemicals and on the Toxicity of Dichlobenil in the Olfactory Mucosa

Treatment with 3-aminobenzamide, known as an inhibitor of poly(ADP-ribose)polymerase, decreased the toxicity and covalent binding of the herbicide dichlobenil (2,6-dichlorobenzonitrile; 12 mg/kg; ip) in the mouse olfactory mucosa.In vitrostudies showed that 3-aminobenzamide markedly reduced the NADPH-dependent covalent binding of [14C]dichlobenil and the hydroxylation ofp-nitrophenol which have previously been suggested to be mediated by a common form of cytochrome P450 (P450) in rat olfactory microsomes (Eriksson and Brittebo,Chem.-Biol. Interact.94,183–196, 1995). Furthermore, 3-aminobenzamide markedly reduced the P450-dependent metabolic activation of [3H]NNK (4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone) in rat olfactory microsomes and slightly decreased the P450 2B1-dependent pentoxyresorufindealkylase activity in liver microsomes of phenobarbital-treated rats. The present results suggest that 3-aminobenzamide is also an inhibitor of P450 and that the lack of toxicity of dichlobenil in the olfactory mucosa of 3-aminobenzamide-treated mice is related to a decreased metabolic activation of dichlobenil at this site. Further experiments showed that there was no evidence for a binding of [14C]dichlobenil metabolites to calf thymus DNA or a formation of mutagenic dichlobenil metabolites in Ames’Salmonellaassay when dichlobenil was incubated in the presence of homogenates of the olfactory mucosa. Finally, analysis of proteins from olfactory microsomes incubated with [14C]dichlobenil using SDS–PAGE/fluorography revealed a binding of metabolites to all major proteins. Addition of glutathione or the P450-inhibitor metyrapone prevented the binding, suggesting the formation of relatively stable electrophilic products which can leave the activating enzyme and then unselectively bind to the major olfactory microsomal proteins.

Reductive activation of 1,1-dichloro-1-fluoroethane (HCFC-141b) by phenobarbital- and pyridine-induced rat liver microsomal cytochrome P450.

1. During anaerobic reductive incubation of liver microsomes, from either the pyridine- or phenobarbital-treated rat, with 1,1-dichloro-1-fluoroethane (HCFC-141b) in the presence of a NADPH-regenerating system, a time- and dose-dependent formation of reactive metabolites was detected as indicated by a depletion of added exogenous glutathione. 2. A statistically significant, dose-dependent loss of both cytochrome P450 and microsomal haem was also observed under these experimental conditions. Furthermore, a statistically significant decrease of p-nitrophenol hydroxylase and pentoxyresorufin O-depentylase activity was measured in microsomes from the pyridine- and phenobarbital-induced rat, respectively indicating that both P4502E1 and P4502B undergo substrate-dependent inactivation. 3. Both reactive metabolite formation and P450 inactivation were almost completely inhibited by previous bubbling of the incubation mixture with carbon monoxide, indicating that interaction of the substrate with a free and reduced P450 haem iron is required for substrate bioactivation and enzyme loss. 4. The presence in the incubation mixture of the spin-trap N-t-butyl-alpha-phenylnitrone (PBN) and the carbene trap 2,3-dimethyl-2-butene (DMB) largely prevented both glutathione depletion and P450 loss. This suggests that free radical and carbene intermediates formed by the metabolic activation of the substrate are involved in the inactivation of P450 and the loss of its prosthetic haem group.

Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates and their conjugates: a structure-activity relationship study.

A series of arylalkyl and alkyl isothiocyanates, and their glutathione, cysteine, and N-acetylcysteine conjugates were used to study their inhibitory activity toward the dealkylation of ethoxyresorufin (EROD), pentoxyresorufin (PROD), and methoxyresorufin (MROD) in liver microsomes obtained from the 3-methylcholanthrene or phenobarbital-treated rats. These reactions are predominantly mediated by cytochrome P450 (P450) isozymes 1A1 and 1A2, 2B1 and 1A2, respectively. All isothiocyanates inhibited PROD more readily than EROD. Increases in the alkyl chain length of arylalkyl isothiocyanates to C6 resulted in an increased inhibitory potency in these assays; at longer alkyl chain lengths (C8-C10) the inhibitory potency declined. The IC50s for phenethyl isothiocyanate (PEITC) were 47, 46 and 1.8 microM for EROD, MROD and PROD, respectively. Substitution of an additional phenyl group on PEITC also increased the inhibitory potency; the IC50s for 1,2-diphenylethyl isothiocyanate (1,2-DPEITC) and 2,2-diphenylethyl isothiocyanate (2,2-DPEITC) were 0.9 and 0.26 microM for EROD, and 0.045 and 0.13 microM for PROD, respectively. The relative inhibitory potency of PEITC and its conjugates was N-acetylcysteine-PEITC (PEITC-NAC) < glutathione-PEITC (PEITC-GSH) < cysteine-PEITC (PEITC-CYS) < PEITC. The observations that the parent isothiocyanates were more potent inhibitors than the conjugates suggest that dissociation of the conjugate is required for activity. Naturally occurring alkyl isothiocyanates, sulforaphane (SFO) and allyl isothiocyanate (AITC), were very weak inhibitors in the assays. These results suggest the potential of isothiocyanates as structural probes for studying P450 isozymes. In addition, the inhibitory activity of isothiocyanates for PROD correlated with the previously demonstrated tumor inhibitory potency in (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced A/J mouse lung tumor bioassays, which supports earlier findings that P450 2B1 is one of the major isozymes involved in NNK activation and that inhibition of this isozyme is an important mechanism for the chemopreventive activity of isothiocyanates.

Cytochrome P450-dependent transformations of 15R- and 15S-hydroperoxyeicosatetraenoic acids: stereoselective formation of epoxy alcohol products.

Although there are many reports of epoxy alcohol synthesis from lipoxygenase products (fatty acid hydroperoxides) in mammalian tissues, there are no well-defined examples of the stereoselective synthesis of individual epoxy alcohol diastereomers. An earlier report on the metabolism of 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE) in rat liver microsomes suggested such a specific reaction [Weiss, R. H., et al. (1987) Arch. Biochem. Biophys. 252, 334-338]. To characterize this reaction further, we set out to determine the precise structures and mechanism of biosynthesis of the epoxy alcohol products. We compared the products formed from 15R- and 15S-HPETE by hematin (a nonenzymatic reaction), by liver microsomes isolated from control and phenobarbital-treated rats, and by purified cytochrome P450 2B1. Eight epoxy alcohol isomers were identified by mass spectrometry and 1H NMR. In the hematin reaction, the major products are four epoxy alcohols with the epoxide in the trans configuration, diastereomers are formed in similar amounts, and the 15-HPETE enantiomers give indistinguishable patterns of products. By contrast, the liver microsomes and P450 2B1 enzyme form predominantly single diastereomers, and the configuration of the epoxide is dependent on the stereochemistry of the substrate. The main product formed from 15S-HPETE is 11S-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,12E- trienoic acid, and the amounts increase upon phenobarbital induction. The main products from 15R-HPETE are 11-hydroxy-14S,15R-epoxyeicosa-5Z,8Z,12E-t rienoic acid from microsomes from control rats and 13-hydroxy-14S,15R-cis-epoxyeicosa-5,8,11-trienoic acid in microsomes from phenobarbital-induced rats. The P450 2B1 enzyme gave products similar to those from the phenobarbital-induced microsomes. Analysis of an incubation using the 18O-labeled 15S-HPETE substrate demonstrated 97.6% retention of both hydroperoxy oxygens in the major product with progressively lower 18O retentions in the minor products (74-32%), possibly reflecting degrees of enzymatic control of these reactions. These results establish a precedent for the stereoselective synthesis of epoxy alcohols by mammalian cytochrome P450s.

Identification of catechol and hydroquinone metabolites of 4-monochlorobiphenyl.

Polychlorinated biphenyls (PCBs) may be metabolically activated to electrophiles, which bind to proteins and nucleic acids. One activation scheme involves the formation of reactive arene oxide intermediates during cytochrome P450-catalyzed hydroxylation. We propose a second activation pathway whereby PCB catechol and hydroquinone metabolites may be oxidized to reactive semiquinones and/or quinones. By employing 4-monochlorobiphenyl (4-MCB) as a model substrate and liver microsomes from rats treated with phenobarbital and 3-methyl-cholanthrene, five monol and three diol metabolites were identified. The major metabolite was 4-chloro-4'-monohydroxybiphenyl, followed by, in decreasing order, 4-chloro-3',4'-dihydroxybiphenyl, unknown B (a monol), 4-chloro-2',3'-dihydroxybiphenyl, 4-chloro-3'-hydroxybiphenyl, 4-chloro-2',5'-dihydroxybiphenyl, unknown A (a monol), and 4-chloro-2'-monohydroxybiphenyl. A trace of a dihydrodiol was detected by GC/MS. To elucidate the source of the diols, 4-MCB and the synthetic monol metabolites 4-chloro-2'-/-3'-/-4'-monohydroxybiphenyls were each employed as substrates in incubations with microsomes from rats treated with phenobarbital, 3-methylcholanthrene, or both inducers. The three diol metabolites were all produced from 4-MCB in incubations with microsomes from 3-methylcholanthrene-treated rats, but incubations with microsomes from phenobarbital-treated rats did not yield detectable amounts of 4-chloro-2',3'-dihydroxybiphenyl. 4-Chloro-2',3'-dihydroxybiphenyl was only found as a product of 4-chloro-2'-monohydroxybiphenyl. The 4-chloro-2',5'-dihydroxybiphenyl was found in extracts of incubations with 4-chloro-2'- and -3'-monohydroxybiphenyls, while the 4-chloro-3',4'-dihydroxybiphenyl was the only product found from 4-chloro-3'- and -4'-monohydroxybiphenyls. No other chlorinated diols were detected by GC/MS. These data suggest that the major route of biosynthesis of the diols was via a second hydroxylation step and not aromatization of dihydrodiols derived from primary arene oxides. We propose a scheme for the in vitro synthesis of the catechol and hydroquinone metabolites, which may be precursors for electrophilic semiquinone or quinone products with the potential for cytotoxic and genotoxic effects.

Production of a Form-Specific, Inhibitory Antibody Against Rat Cytochrome P450 2B1 Using a Synthetic Peptide Antigen against a Putative Substrate Binding Site

Rat cytochrome P450 2B1 antipeptide antibodies were produced by immunizing rabbits with a synthetic peptide antigen. The anti-CYP2B1 IgG obtained did not cross-react with CYP2B2, which has 97% identity in primary sequence of CYP2B1. This result demonstrates that a difference of 2 amino acid residues among 12 is sufficient to produce a form-specific antibody. The CYP2B1 antipeptide IgG inhibited pentoxyresorufin O-dealkylase activity of microsomes obtained from phenobarbital-treated rats in a dose-dependent manner, whereas it did not inhibit ethoxyresorufin O-deethylase activity of microsomes obtained from 3-methylcholanthrene-treated rats. These results suggest that the selected amino acid sequence, which coincides with one of the substrate binding sites of Pseudomonas putida CYP101A (P450cam) and one of the putative substrate binding sites of CYP2B2, is located on the surface of the CYP2B1 molecule, as opposed to inside the molecule or in the lipid bilayer of microsomes.

7-HETE, 10-HETE, and 13-HETE Are Major Products of NADPH-Dependent Arachidonic Acid Metabolism in Rat Liver Microsomes: Analysis of Their Stereochemistry, and the Stereochemistry of Their Acid-Catalyzed Rearrangement

A complex profile of metabolites of [ 14C]arachidonic acid are formed in NADPH-dependent reactions in liver microsomes from phenobarbital-treated rats. The products were resolved by HPLC and analyzed initially with an on-line diode array detector set to monitor 205, 220, 235, and 270 nm. In addition to the epoxyeicosatrienoic acids and their diol hydrolysis products, prominent hydroxyeicosatetraenoic acids (HETE) metabolites were identified as omega-hydroxy derivatives and three bis-allylic products, 7-HETE, 10-HETE, and 13-HETE. The formation of 13-HETE was reported by Oliw et al. ( Arch. Biochem. Biophys. 300, 434-439 (1993)). Structures of the new products were established by GC MS, and for 10-HETE, by comparison to authentic synthetic material. Chiral column analysis indicated that 7-HETE and 10-HETE were essentially racemic. The 13-HETE resolved into a 40:60 ratio of 13S:13R. The bis-allylic HETEs readily undergo rearrangement to conjugated diene-containing HETEs in mild acidic conditions (1% acetic acid). Using [ 18O]13-HETE we observed 69% retention of the labeled oxygen in the 11- and 15-HETE arrangement products. Furthermore, steric analysis of products from acid treated 10-HETE and 13-HETE enantiomers revealed partial retention of chirality (80:20 ratio of enantiomers for methyl esters, and 60:40 ratio for free acids) in the course of mild acid-catalyzed rearrangement. This represents a novel reaction of this class of arachidonic acid metabolites of cytochromes P450.

Isoflurane and cytochrome b5 stimulation of 2-chloro-1,1-difluoroethene metabolism by reconstituted rat CYP2B1 and CYP2C6

Isoflurane stimulates the metabolism of 2-chloro-1,1-difluoroethene (CDE) in liver microsomes from phenobarbital-treated rats or rabbits. The P450 isozymes involved and the mechanism by which such stimulation occurs have not been clarified. The present study examined the effects of isoflurane ami cytochrome b 5 on CDE metabolism in reconstituted systems containing purified rat CYP2B1 orCYP2C6. Under similar incubation conditions, CYP2B1 defluorinated CDE at approximately five times the rate of CYP2C6. Isoflurane was a potent stimulator of CDE metabolism, increasing it nearly 5-fold when catalyzed by CYP2B1, but only 2-fold when catalyzed by CYP2C6. Isoflurane had no stimulatory effect on benzphetamine metabolism by CYP2B1 or CYP2C6. Cytochrome b 5 was not required for isoflurane-facilitated CDE metabolism; however, the addition of cytochrome b 5 to CYP2B1 increased CDE metabolism 71 and 44%, in the absence and presence of isoflurane, respectively. In reconstituted CYP2B1, isoflurane generated a type I difference spectrum of approximately twice the magnitude of CDE and stimulated NADPH consumption more so than CDE. The same quantity of NADPH was consumed when CDE was present with isoflurane as compared with isoflurane alone. These data support the hypothesis that isoflurane stimulates CDE metabolism by a mechanism involving increased P450 reduction via direct isoflurane interaction with P450.

Melatonin administration prevents lipopolysaccharide‐induced oxidative damage in phenobarbital‐treated animals

The protective effect of melatonin on lipopolysaccharide (LPS)-induced oxidative damage in phenobarbital-treated rats was measured using the following parameters: changes in total glutathione (tGSH) concentration, levels of oxidized glutathione (GSSG), the activity of the antioxidant enzyme glutathione peroxidase (GSH-PX) in both brain and liver, and the content of cytochrome P450 reductase in liver. Melatonin was injected intraperitoneally (ip, 4mg/kg BW) every hour for 4 h after LPS administration; control animals received 4 injections of diluent. LPS was given (ip, 4 mg/kg) 6 h before the animals were killed. Prior to the LPS injection, animals were pretreated with phenobarbital (PB), a stimulator of cytochrome P450 reductase, at a dose 80 mg/kg BW ip for 3 consecutive days. One group of animals received LPS together with Nw-nitro-L-arginine methyl ester (L-NAME), a blocker of nitric oxide synthase (NOS) (for 4 days given in drinking water at a concentration of 50 mM). In liver, PB, in all groups, increased significantly both the concentration of tGSH and the activity of GSH-PX. When the animals were injected with LPS the levels of tGSH and GSSG were significantly higher compared with other groups while melatonin and L-NAME significantly enhanced tGSH when compared with that in the LPS-treated rats. Melatonin alone reduced GSSG levels and enhanced the activity of GSH-PX in LPS-treated animals. Additionally, LPS diminished the content of cytochrome P450 reductase with this effect being largely prevented by L-NAME administration. Melatonin did not change the content of P450 either in PB- or LPS-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloramphenicol oxamylethanolamine as an end product of chloramphenicol metabolism in rat and humans: evidence for the formation of a phospholipid adduct.

Chloramphenicol (CP) has been implicated as, although not proven to be, a causative agent of aplastic anemia in humans. Recent studies from our laboratory have presented evidence that CP-oxamylethanolamine was an end product of CP biotransformation in birds. Because this novel metabolic pathway has never been reported in other species, we have now expanded these investigations to rat and humans. [3H]CP was administered po (10 mg/kg) to adult male Wistar rats and to a human volunteer. Urine was collected and analyzed by HPLC and GC-MS for CP metabolite determination. In rat, the two most important metabolites in 0-24 h urine were CP-base and CP-acetylarylamine which together accounted for about 50% of the ingested radioactivity. The remainder was due to unchanged CP, CP-oxamic acid, CP-alcohol, CP-glucuronide, and CP-oxamylethanolamine. The presence of these end products was also demonstrated in man. CP-oxamylethanolamine represented 0.74% and 1.37% of the ingested radioactivity in rat and human urine samples, respectively. CP-oxamylethanolamine formation was confirmed in vitro with isolated rat hepatocytes, suggesting the involvement of liver in the production of this metabolite. The origin of CP-oxamylethanolamine has been investigated with the use of hepatic liver microsomes from phenobarbital-treated rats. The incubation of [3H]CP with this subcellular fraction led to the binding of a radiolabeled compound to the microsomal lipids, whereas no binding occurred when CP-oxamic acid was incubated with the microsomes. Enzymatic hydrolysis of the microsome lipid fraction with phospholipase D from Streptomyces chromofuscus released CP-oxamylethanolamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucuronidation of 4-((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone, a metabolically activated form of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, by phenobarbital-treated rats.

In the rat, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung tumors independent of the route of administration. To exert its carcinogenic potential, NNK must be metabolically activated. Like most nitrosamines NNK is activated by alpha-hydroxylation. The striking tissue specificity of tumor induction by nitrosamines has been primarily attributed to the efficient alpha-hydroxylation of a particular nitrosamine by its target tissue. Two other factors which may contribute to this are the following: the relative capacity of different tissues to detoxify the alpha-hydroxynitrosamine and the preferential uptake of the active metabolite by the target tissue. In the present study we report the characterization of the O-glucuronide of 4-((hydroxymethyl)nitrosamino)-1-(3-pyridyl)-1-butanone (alpha-hydroxymethylNNK-Gluc). The formation of this glucuronide could either serve as a detoxification pathway or provide a stable transport form of the alpha-hydroxylated metabolite. In addition, the metabolism of NNK to a glucuronide of the alpha-hydroxynitrosamine provides the first definitive evidence for the formation of alpha-hydroxymethylNNK. alpha-HydroxymethylNNK-Gluc was present in the urine of rats treated with phenobarbital (PB) and NNK. It was also formed by hepatocytes from PB-treated rats, accounting for 4% of the total metabolites in the media following incubation with 1 microM NNK. The data that support the identity of this metabolite as alpha-hydroxymethylNNK-Gluc are as follows. (1) Incubation of this metabolite with beta-glucuronidase resulted in the quantitative release of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), the decomposition product of alpha-hydroxymethylNNK.(ABSTRACT TRUNCATED AT 250 WORDS)