Liver Microsomes Of Phenobarbital-treated Rats Research Articles

Ring-oxidative biotransformation and drug interactions of propofol in the livers of rats.

Propofol, an intravenous anesthetic agent, is widely used for inducing and maintaining anesthesia during surgical procedures and for sedating intensive care unit patients. In the clinic, rapid elimination is one of the major advantages of propofol. Meanwhile, the biotransformation and drug interactions of propofol in rat livers are still little known. In this study, we evaluated the ring-oxidative metabolism of propofol in phenobarbital-treated rat livers and possible drug interactions. Administration of phenobarbital to male Wistar rats significantly increased levels of hepatic cytochrome P450 (CYP) 2B1/2 and microsomal pentoxyresorufin O-dealkylase (PROD) activity. Analyses by high-performance liquid chromatography and liquid chromatography mass spectroscopy revealed that propofol was metabolized by phenobarbital-treated rat liver microsomes into 4-hydroxypropofol. In comparison, PROD activity and 4-hydroxy-propofol production from propofol metabolism were suppressed by orphenodrine, an inhibitor of CYP2B1/2, and a polyclonal antibody against rat CYP2B1/2 protein. Furthermore, exposure of rats to propofol did not affect the basal or phenobarbital-enhanced levels of hepatic CYP2B1/2 protein. Meanwhile, propofol decreased the dealkylation of pentoxyresorufin by phenobarbital-treated rat liver microsomes in a concentration-dependent manner. Taken together, this study shows that rat hepatic CYP2B1/2 plays a critical role in the ring-oxidative metabolism of propofol into 4-hydroxypropofol, and this anesthetic agent can inhibit CYP2B1/2 activity without affecting protein synthesis.

Four new koumine metabolites in rat liver microsomes

Four new metabolites M-1 [1,2,18,19-tetradehydro-4-demethyl-3,17-epoxy-7,20(2H,19H)-cyclovobasan], M-2 [1,2,4,21,18,19-hexadehydro-4-demethyl-3,17-epoxy-7,20(2H,19H)-cyclovobasan], M-3 [1,2,18,19-tetradehydro-4-demethyl-4-formaldehyde-3,17-epoxy-7,20(2H,19H)-cyclovobasan], and M-4 [1,2,4,21,18,19-hexadehydro-4-demethyl-4-oxy-3,17-epoxy-7,20(2H,19H)-cyclovobasan] were isolated from the chloroform extract of koumine incubated with phenobarbital-treated rat liver microsomes. The structures of M-1, M-2, M-3, and M-4 were elucidated by spectroscopic methods including ESI-TOF-MS, 1D, and 2D NMR experiments. The metabolic pathway of koumine was proposed. The cytotoxic activities between koumine and its metabolites were also compared in the A549 cell line.

Antitumor activity of two gelsemine metabolites in rat liver microsomes

Gelsemine is one of the major alkaloids from Gelsemium elegans Benth., which has been used as an antitumor remedy in clinic. In this paper, metabolism of gelsemine has been investigated in vitro in phenobarbital-treated rat liver microsomes. The metabolites of gelsemine were separated and evaluated using the flash silica gel column, preparative HPLC, using NMR and MS methods. According to the spectral data, two metabolites, M1 and M2, were identified as 4-N-demethylgelsemine and 21-oxogelsemine, respectively. By the MTT method in vitro, the antitumor activities between gelsemine and its metabolites were compared in the HepG2 and HeLa cell lines. Moreover, the main metabolic pathway was further proposed.

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the Vmax/Km values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25 nM−1 min−1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a Vmax value of 6.96 nmol min−1 nmol−1 P450 and apparent Km value of 0.09 mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the Km values for the formation of fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03 mM, and Vmax values were 2.94, 6.1 and 13.8 nmol min−1 nmol−1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.

Production of superoxide radical in reductive metabolism of a synthetic food-coloring agent, indigocarmine, and related compounds

Indigocarmine, which is widely used as a synthetic colouring agent for foods and cosmetics in many countries, was reduced to its leuco form and decolorized by rat liver microsomes with NADPH under anaerobic conditions. The reductase activity was enhanced in liver microsomes of phenobarbital-treated rats, and inhibited by diphenyliodonium chloride, a NADPH-cytochrome P450 reductase (P450 reductase) inhibitor, but was not inhibited by SKF 525-A or carbon monoxide. Indigocarmine reductase activity was exhibited by purified rat P450 reductase. In contrast, when indigocarmine was incubated with rat liver microsomes and NADPH under aerobic conditions, superoxide radical was produced and its production was inhibited by superoxide dismutase and diphenyliodonium chloride. When indigocarmine was incubated with purified rat P450 reductase in the presence of NADPH, superoxide radical production was enhanced 17.7-fold (similar to the enhancement of indigocarmine-reducing ability) as compared with that of rat liver microsomes. A decrease of one molecule of NADPH was accompanied with formation of about two molecules of superoxide radical. P450 reductase exhibited little reductase activity towards indigo and tetrabromoindigo, which also afforded little superoxide radical under aerobic conditions. These results indicate that indigocarmine is reduced by P450 reductase to its leuco form, and superoxide radical is produced by autoxidation of the leuco form, through a mechanism known as futile redox cycling.

Roles of human CYP2A6 and 2B6 and rat CYP2C11 and 2B1 in the 10-hydroxylation of (-)-verbenone by liver microsomes.

(-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.

Estrogenic activity of an environmental pollutant, 2-nitrofluorene, after metabolic activation by rat liver microsomes

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.

Estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes.

In this study we examined estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes. Trans-1,2-diphenylcyclobutane (TCB), cis-1,2-diphenylcyclobutane (CCB), 1,3-diphenylpropane, 2,4-diphenyl-1-butene, 2,4,6-triphenyl-1-hexene, and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin were negative in the yeast estrogen screening assay and estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, TCB exhibited estrogenic activity after incubation with liver microsomes of phenobarbital-treated rats in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Minor activity was observed when liver microsomes of untreated or 3-methylcholanthrene-treated rats were used instead of those from phenobarbital-treated rats. CCB, 1,3-diphenylpropane, and 2,4-diphenyl-1-butene also exhibited estrogenic activity after metabolic activation by liver microsomes, but the activity was lower than that of TCB. 2,4,6-Triphenyl-1-hexene and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin did not show estrogenic activity after such incubation. When TCB was incubated with liver microsomes of phenobarbital-treated rats in the presence of NADPH, three metabolites were detected by high-performance liquid chromatography (HPLC). One metabolite isolated by HPLC exhibited a significant estrogenic activity. The active metabolite was identified as trans-1-(4-hydroxyphenyl)-2-phenylcyclobutane by mass and nuclear magnetic resonance spectral analysis. These results suggest that the estrogenic activity of TCB was caused by the formation of the 4-hydroxylated metabolite.

Sex differences in the metabolism of (+)- and (-)-limonene enantiomers to carveol and perillyl alcohol derivatives by cytochrome p450 enzymes in rat liver microsomes.

(+)-Limonene is reported to cause nephropathy in male rats, but not in female rats and other species of animals including mice, rabbits, guinea pigs, and dogs. Male rats contain high levels of alpha2u-globulin in kidneys, and it has been shown that limonene and/or its metabolites are able to bind noncovalently to alpha2u-globulin, resulting in an accumulation of protein droplets in the renal tubules. In this study, we investigated whether (+)- and (-)-limonene enantiomers are differentially metabolized by liver microsomes of male and female rats. (+)- and (-)-limonene enantiomers were found to be oxidized to their respective trans-carveol (6-hydroxylation) and perillyl alcohol (7-hydroxylation) derivatives in greater amounts by liver microsomes of male rats than those of female rats. The limonene hydroxylation activities were not detected in liver microsomes of rat fetuses and were increased developmentally after birth, only in male rats. Treatment of male rats with phenobarbital significantly increased liver microsomal 6-hydroxylation activities with both enantiomers whereas beta-naphthoflavone, isosafrole, and pregnenolone 16alpha-carbonitrile did not cause such effects. Anti-P450 2C9 which cross-reacts with rat P450 2C11 inhibited limonene hydroxylations catalyzed by liver microsomes of untreated male rats, and it was also found that anti-P450 2B1 suppressed the activities catalyzed by liver microsomes of phenobarbital-treated rats. Possible roles of P450 2C11 and P450 2B1 in the limonene hydroxylation activities were supported by the experiments with purified rat liver P450s in reconstitution systems and with recombinant rat P450s in Trichoplusia ni. Our present results showing that there are sex-related differences in the oxidative metabolism of limonene enantiomers by liver microsomes may provide useful information on the basis of limonene-induced toxicities in different animal species.

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.

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.

Identification and characterization of lysosomal enzymes involved in the proteolysis of phenobarbital-inducible cytochrome P450.

We have studied lysosomal proteases capable of degrading a major form of cytochrome P450 (CYP2B1) which was purified from the liver microsomes of phenobarbital-treated rats. After incubation of CYP2B1 with extracts of triton-filled lysosomes (tritosomes), its proteolysis was measured by a quantitative immunoblot procedure. A CYP2B1 protein band with an apparent molecular mass of 53 kilodaltons (kDa) was degraded to 42-, 38-, and 29-kDa polypeptides after a short period of incubation. These proteolytic fragments disappeared on prolonged incubation. One milligram of tritosomal protein contained enough activity to hydrolyze approximately 67.3 micrograms CYP2B1 protein/min at pH 4.5. Approximately 85% of the hydrolyzing activity was localized in a soluble fraction of the tritosomes. The degradation of CYP2B1 was effectively inhibited by pepstatin A, an aspartic protease inhibitor, but not by phenylmethanesulfonyl fluoride (PMSF), o-phenanthroline and leupeptin. CYP2B1-hydrolyzing activity was coeluted with cathepsin D when the soluble fraction was chromatographed by means of Ultrogel Ac44 gel filtration. Cathepsin D, purified from rat livers, was able to degrade CYP2B1 at pH 4.5 which corresponds to the intralysosomal pH. These results indicate that cathepsin D is responsible for the major CYP2B1-hydrolyzing enzyme activity in lysosomes.

Covalent binding of ethylene dibromide and its metabolites to albumin

The present study was undertaken to determine covalent binding of [1,2- 14C]ethylene dibromide (EDB) to albumin under in vivo and in vitro conditions. For the in vivo covalent binding, 25 mg kg body weight of [1,2- 14C]EDB was given daily to male rats for 12 consecutive days and the animals were sacrificed at 24 h following the last dose. Blood was withdrawn from inferior vena cava in heparinized tubes and plasma was separated, dialyzed against ice-cold 10 mM phosphate buffer (pH 7.4) and then subjected to size-exclusion high-performance liquid chromatography (SE-HPLC). A major radioactive peak eluted at an elution volume corresponding to 65 000 dalton molecular mass was found to be associated to albumin at a level of 0.14 nmol equivalent EDB mg protein. For the in vitro covalent binding, human plasma or purified albumin was incubated with [1,2- 14C]EDB in the presence of phenobarbital-treated rat liver microsomes and NADPH-generating system for 2 h at 37°C. The 100 000 × g supernatant of the incubation mixture was dialyzed extensively and analyzed as described for the in vivo studies. Approximately 0.28 nmol equivalent EDB mg protein was found to be associated to albumin (about 2-fold higher than the in vivo binding). Binding of 14C-label to albumin under in vivo and in vitro conditions was further supported by the affinity chromatography of albumin fraction isolated by SE-HPLC. Reversed-phase HPLC analysis of pronase digest of the albumin obtained from in vitro studies indicated formation of several amino acid adducts of EDB and/or its metabolites. Structure elucidation of such amino acid adducts will be helpful in developing a relatively non-invasive method of measuring the EDB exposure.

Fate of isopropyl-iodoamphetamine (IMP) in rat liver microsomes

Para-iodoamphetamines are currently used in nuclear medicine to detect brain perfusion abnormalities with tomoscintigraphy. Little is known about their metabolism pathways in rat and humans. N-isopropyl-125I-iodoamphetamine (IMP) interactions were studied with rat liver microsomes. The first dealkylated metabolite (IAMP) at low concentration gave a type I binding complex then, with a high concentration, a very stable type II complex with oxidized cytochrome P-450 FeIII. In contrast, IMP only gave a type I binding complex in the absence of NADPH. In the presence of NADPH, IAMP and IMP produced 455 nm absorbing complexes, which were enhanced when phenobarbital-treated rat liver microsomes were used. During in vitro metabolic activation, covalent binding of IMP and IAMP on rat liver microsomal proteins was observed. This process was mixed function oxidase (MFO) dependent. The covalent binding level was higher with IAMP and was not affected by flavine oxidase inhibitors. These results confirm the interaction of IMP and IAMP with microsome proteins and cytochrome P-450 and suggest that an N-oxidation of IMP occurs after N-dealkylation. As cytochrome P-450 and dealkylated IMP (IAMP) were found in brain, cerebral metabolism in brain and evolution of activity biodistribution with the course of time can be suggested.

Isoelectric focusing of cytochrome P450: Isolation of six phenobarbital-inducible rat liver microsomal isoenzymes

A procedure for the isolation of native proteins from membranes by isoelectric focusing is described. It was used to resolve into six components the major fraction of cytochrome P450, obtained from liver microsomes of phenobarbital-treated rats, after chromatography on DE-52 cellulose. When eluted from the gel, these proteins are in a native form as shown by (a) the light absorption spectra of the Soret region of their reduced carbonyl derivatives, all characterized by maxima around 450 nm, and (b) their enzymatic activities toward three different substrates. Characterization by a monoclonal antibody and partial sequence analysis of tryptic peptides reveal that three of the IEF-purified proteins have P450IIB1 character, whereas the other three are related to P450IIB2.

Effects of added protein (bovine serum albumin) on the rate and enantiotopic selectivity of oxidative O-demethylation of methoxychlor in rat liver microsomes

The addition of bovine serum albumin (BSA) to rat liver microsomal suspensions during the O-demethylation of methoxychlor (1,1-bis(4-methoxyphenyl)-2,2,2-trichloroethane) produced a good substrate concentration-reaction rate relationship, and allowed us to determine the rate parameters for this reaction. The apparent K m became larger when the albumin content was increased, whereas V max was not changed. The enantiotopic selectivity of the reaction dramatically changed when the substrate concentration was increased in the absence of BSA in phenobarbital-treated rat liver microsomes.

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)

A Mutant Rat Strain Defficient in Induction of a Phenobarbital-Inducible Form of Cytochrome P-450 in Liver Microsomes1

Two phenobarbital-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(PB-4), were purified from the liver microsomes of phenobarbital-treated rats and identified with P-450b and P-450e, respectively. It was found, however, that the content of P-450(PB-4) in the liver microsomes of a strain of SD rat, Qdj:SD, was very low even after phenobarbital-induction. The levels of the mRNAs for P-450(PB-1) and P-450(PB-4) were separately determined using Northern blot hybridization with specific oligonucleotide probes. It was found that the level of P-450(PB-4) mRNA in the livers of phenobarbital-treated Qdj:SD rats was much lower than that of phenobarbital-treated Slc:SD rats. Slc:SD rats are widely used in Japanese laboratories. Genetic analysis using the crossbred animals between Qdj:SD and Slc:SD rats showed that the low expression of P-450(PB-4) in Qdj:SD rats is a recessive trait and is caused by a single gene mutation. However, no difference in the 5' flanking region in P-450(PB-4) gene was found between Qdj:SD rats and Slc:SD rats.

The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of nadph-cytochrome P-450 reductase

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-448 1 and MC P-448 2) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-448 1 and MC P-448 2 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-448 1 or MC P448 2. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-448 1 or MC P-448 2 was added to the system containing PB P-450 and limited amount of the reductase. The apparent K m of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-448 1 or MC P-448 2. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.

Reduction kinetics of purified rat liver cytochrome P-450. Evidence for a sequential reaction mechanism dependent on the hemoprotein spin state

The anaerobic reduction kinetics of purified rat liver ferric cytochrome P-450 from phenobarbital-treated rat liver microsomes, reconstituted with saturating NADPH-cytochrome P-450 reductase, have been investigated and were shown not to be monophasic. From experiments correlating changes in the rate of fast-phase reduction with the spin state of the heme iron existing at preequilibrium, data were obtained consistent with a model for spin-state control of cytochrome P-450 reduction wherein the high-spin form of the hemoprotein is more rapidly reduced than the low-spin form. In addition, the temperature dependence of the reduction process in the presence of the substrate benzphetamine was studied. From the results obtained it is suggested that the endothermic nature of the low- to high-spin transition largely accounts for the apparent activation energy observed for the reduction of high-spin cytochrome P-450 being relatively temperature insensitive when compared to the rate constant for reduction of the membrane-bound form of the hemoprotein.