Three immunoidentical cytochromes P-450 from liver microsomes of phenobarbital-treated rats.

Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.     

Tissue-specific expression of rat mRNAs homologous to cytochromes P-450b and P-450e.

The tissue-specific expression of cytochrome P-450b and P-450e mRNAs was examined with synthetic 18-mer oligomer probes in the liver, lung, kidney, and testis of control and inducer pretreated adult rats. RNAs homologous to the P-450e probe were detected in trace amounts in control and 3-methylcholanthrene (MC) induced livers and at high levels in livers from phenobarbital (PB) induced animals. P-450e mRNA levels were below detection limits in the other tissues examined, regardless of pretreatment. In contrast, mRNAs homologous to the P-450b oligomer were detected at low levels in control and inducer pretreated lung and testis, and at high levels in PB induced liver. No P-450b mRNAs were detected in these assays in RNA isolates from the kidney or from control or MC pretreated liver. Solution hybridization data indicated that the rat lung contained 9-12%, and the testis, 6-9%, respectively, of the levels of P-450b mRNA measured in the PB induced liver. Results from oligo(dT)-cellulose and poly(U)-affinity experiments indicated that the hepatic mRNAs for P-450b and P-450e were present predominantly in the bound, polyadenylated fraction, whereas the homologous lung and testes P-450b mRNAs predominated in the flow-thru fractions.     

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

Thyroid hormone suppression of hepatic levels of phenobarbital-inducible P-450b and P-450e and other neonatal P-450s in hypophysectomized rats

Mechanism of developmental suppression of cytochrome P-450 (P-450) in rat livers was studied using Western blots. The contents of phenobarbital (PB)-inducible P-450b and P-450e, expressed constitutively in livers, were higher in neonate than in adult rats. The contents were also 10 approximately 50 fold higher in hypophysectomized than in intact adult male rats. Administration of L-triiodothyronine (T3, 50 micrograms/kg) or human growth hormone (4 U/kg) reversed almost completely the increased amounts of P-450b and P-450e. T3-induced suppression was also observed on two other neonatal P-450s (P-450 6 beta-1 and P-448-H), which are expressed in neonatal periods in livers. The postnatal developmental profiles of hepatic P-450b were correlated inversely with that of serum free T3 level in rats reported (Walker et al. (1980) Pediat. Res. 14, 249). These results suggest, in addition to pituitary growth hormone (Yamazoe et al. (1987) J. Biol. Chem. 262, 7423), the possible involvement of T3 on the suppressive regulation of PB-inducible and other neonatal P-450s.     

Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.     

Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

Circular dichroism spectra of purified cytochromes P-450 from rabbit liver microsomes.

Circular dichroism (CD) spectra were measured for cytochromes P-450 (P-450) purified from phenobarbital- and 3-methylcholanthrene-induced rabbit liver microsomes. No striking difference in alpha-helix content was seen between phenobarbital-induced P-450 (PB P-450) (50%), phenobarbital-induced P-448 (PB P-448) (40%) and 3-methylcholanthrene-induced P-448 (MC P-448) (45--50%) in terms of ultraviolet CD spectra. Strong negative CD spectra associated with 3-methylcholanthrene transitions for MC P-448 in the near-ultraviolet region (250--310 nm) and weaker negative CD spectra associated with Soret transitions for PBP-448 ([theta] = 50 000) and MCP-448 ([theta] = 160 000), indicated that structures of these preparations are strikingly different from each other. Reduction of P-450 and P-448 led to a remarkable decrease of the Soret CD trough, suggesting that reduction was accompanied by a striking conformational change in the vicinity of the heme. Since CO complexes of reduced P-450 and P-448 showed a CD trough and an S-shaped CD, respectively, associated with the absorption peak at 450 nm, the heme vicinities are remarkably different from each other. The CD spectra in the visible region are also discussed. It was noticed that P-420, the denatured form of P-450, exhibited no CD spectra in the Soret and visible regions.     

Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.     

Effects of emulsified perfluorochemicals on liver cytochromes P-450 in rats

1. The effects of intravenous (i.v.) injection of various perfluorochemical (PFC) emulsions or different fractions of the non-ionic poloxamer surfactant, Pluronic F-68, have been studied separately in male and female rats. 2. Injection of 10 ml/kg body wt of either Fluosol-DA 20% (F-DA) or a novel perfluorodecalin emulsion containing a C-16 oil additive in male rats increased liver weight up to 7 days later; no corresponding effect occurred in response to injection of Oxypherol (FC-43). 3. Liver weight was also increased in female rats at 72 hr after injection of the novel emulsion but this was less pronounced than in males; liver weight in female rats was unchanged in response to injection of either F-DA or FC-43. 4. Mean liver microsomal cytochromes P-450 concentrations in male rats were increased 2-3 fold at 72 hr after injection of either F-DA or the novel emulsion with a less pronounced increase also seen at 7 days in animals receiving the novel emulsion. No significant alterations in cytochrome concentration occurred in response to injection of FC-43 or either commercial grade or purified pluronic solution. 5. Liver cytochromes P-450 concentrations in female rats were unaffected by any of the experimental treatments. 6. These results show that injection of a single low dose of emulsified PFCs into male rats can increase hepatic microsomal cytochromes P-450 concentration but the response is highly variable, depending on composition of emulsion injected.     

Phenobarbital induction of rat liver cytochromes P-450b and P-450e. Quantitation of specific RNAs by hybridization to synthetic oligodeoxyribonucleotide probes

Cytochromes P-450b and P-450e are extremely homologous and immunochemically indistinguishable proteins that are coordinately induced by phenobarbital in rat liver. To assess the effect of phenobarbital on mRNA levels for each of these hemoproteins we performed solution hybridization and Northern blot experiments with synthetic oligodeoxynucleotide probes of defined sequence. Our data demonstrate that phenobarbital administration to rats resulted in marked increases in levels of hepatic mRNA for both cytochrome P-450b and cytochrome P-450e, with a 4- to 5-fold greater accumulation of P-450b mRNA vis à vis P-450e mRNA. The level of hepatic mRNA increased from less than 3 molecules/cell of each mRNA in untreated rats, to 630 and 130 molecules/cell for P-450b and P-450e, respectively, in phenobarbital-treated rats. Data obtained in Northern blot hybridization experiments demonstrated that the size of the mRNAs for each protein were identical, being approximately 1800 bases in length.     

Differential stereoselectivity on metabolism of triphenylene by cytochromes P-450 in liver microsomes from 3-methylcholanthrene- and phenobarbital-treated rats

Metabolism of triphenylene by liver microsomes from control, phenobarbital(PB)-treated rats and 3-methylcholanthrene(MC)-treated rats as well as by a purified system reconstituted with cytochrome P-450c in the absence or presence of purified microsomal epoxide hydrolase was examined. Control microsomes metabolized triphenylene at a rate of 1.2 nmol/nmol of cytochrome P-450/min. Treatment of rats with PB or MC resulted in a 40% reduction and a 3-fold enhancement in the rate of metabolism, respectively. Metabolites consisted of the trans-1,2-dihydrodiol as well as 1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the dihydrodiol predominated (70 to 92%) under all incubation conditions. Incubation of racemic triphenylene 1,2-oxide with microsomal epoxide hydrolase produced dihydrodiol which was highly enriched (80%) in the (-)-1R,2R-enantiomer. Experiments with 18O-enriched water showed that attack of water was exclusively at the allylic 2-position of the arene oxide, indicating that the 1R,2S-enantiomer of the oxide was preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that liver microsomes from MC-treated rats produced almost exclusively (greater than 90%) the 1R,2S-enantiomer of triphenylene 1,2-oxide whereas liver microsomes from PB-treated rats formed racemic oxide. The optically active oxide has a half-life for racemization of only approximately 20 s under the incubation conditions. This study may represent the first attempt to address stereochemical consequences of a rapidly racemizing intermediary metabolite.     

Induction of renal cytochrome P-450 in hepatic microsomes of diabetic rats

We purified two forms of cytochrome P-450 which was induced in hepatic microsomes of diabetic male rates treated with streptozotocin. One of these corresponded to P-450j. The other form, designated P450 DM-2, had a minimum molecular weight 53000 and a CO-reduced absorption maximum at 452 nm. The P450 DM-2 efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was not efficient in metabolizing aminopyrine, 7-ethoxycoumarin, aniline, N-nitrosodimethylamine, or testosterone. The NH2-terminal sequence of P450 DM-2 was identical to that of P450 K-5, the major renal cytochrome P-450. Both forms gave very similar electrophoretic patterns of proteolytic digests. P450 DM-2 and P450 K-5 are closely related forms.     

Use of monoclonal antibody probes against rat hepatic cytochromes P-450c and P-450d to detect immunochemically related isozymes in liver microsomes from different species

Nine distinct monoclonal antibodies raised against purified rat liver cytochrome P-450c react with six different epitopes on the antigen, and one of these epitopes is shared by cytochrome P-450d. None of these monoclonal antibodies recognize seven other purified rat liver isozymes (cytochromes P-450a, b, and e-i) or other proteins in the cytochrome P-450 region of "Western blots" of liver microsomes. Each of the monoclonal antibodies was used to probe "Western blots" of liver microsomes from untreated, or 3-methylcholanthrene-, or isosafrole-treated animals to determine if laboratory animals other than rats possess isozymes immunochemically related to cytochromes P-450c and P-450d. Two protein-staining bands immunorelated to cytochromes P-450c and P-450d were observed in all animals treated with 3-methylcholanthrene (rabbit, hamster, guinea pig, and C57BL/6J mouse) except the DBA/2J mouse, where no polypeptide immunorelated to cytochrome P-450c was detected. The conservation of the number of rat cytochrome P-450c epitopes among these species varied from as few as two (guinea pig) to as many as five epitopes (C57BL/6J mouse and rabbit). The relative mobility in sodium dodecyl sulfate-gels of polypeptides immunorelated to cytochromes P-450c and P-450d was similar in all species examined except the guinea pig, where the polypeptide related to cytochrome P-450c had a smaller Mr than cytochrome P-450d. With the use of both monoclonal and polyclonal antibodies, we were able to establish that purified rabbit cytochromes P-450 LM4 and P-450 LM6 are immunorelated to rat cytochromes P-450d and P-450c, respectively.     

Purification and characterization of cytochromes P-450 from liver microsomes of 3-methylcholanthrene-treated crab-eating monkeys

Two forms of cytochrome P-450 (P-450MC1 and P-450MC2) were purified from liver microsomes of crab-eating monkeys (Macaca irus) treated with 3-methylcholanthrene (MC). Monkey P-450MC1 preparation had a specific content of 14.0 nmol/mg protein and showed a main protein band with a minimum molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monkey P-450MC2 preparation had a specific content of 12.1 nmol/mg protein and a minimum molecular weight of 54,000. The carbon monoxide-reduced difference spectral peaks of monkey P-450MC1 and P-450MC2 were at 448 and 447 nm, respectively. In the reconstituted system, monkey P-450MC2 had high activities for benzo[a]pyrene 3-hydroxylation and 7-ethoxycoumarin O-deethylation. Monkey P-450MC1 had low activities toward these two substrates and a high activity for benzphetamine N-demethylation. Monkey P-450MC1 and P-450MC2 were detected by immunoblotting using an antibody prepared against rat cytochrome P-450c, which is a major form of cytochrome P-450 in liver microsomes of MC-treated rats. These results suggested that the molecular properties of cytochrome P-450 in liver microsomes of crab-eating monkeys treated with MC are similar to those in rats.     

Purification and characterization of six cytochromes P-450 from hepatic microsomes of immature female rats

Six rat hepatic cytochromes P-450, named P-450IF-1-6, were purified from hepatic microsomes of immature female rats by high-performance liquid chromatography with anion-exchange, cation-exchange, and hydroxylapatite columns. The purified forms, except for P-450IF-4, gave a single protein-staining band on SDS-polyacrylamide gel electrophoresis, with a minimum molecular weight of 50,000 for P-450IF-1, 49,000 for P-450IF-2, 47,000 for P-450IF-3, 53,500 for P-450IF-5, and 54,000 for P-450IF-6. The CO-reduced spectral maximum of these forms was at 450 nm for P-450IF-1, 448 nm for P-450IF-2, 451 nm for P-450IF-3, 449 nm for P-450IF-4, 449 nm for P-450IF-5, and 450 nm for P-450IF-6. All of these cytochromes had the low-spin state of heme in the oxidized form. P-450IF-4 had high metabolic activity for both benzphetamine and 7-ethoxycoumarin. P-450IF-5 had moderate activity toward 7-ethoxycoumarin. P-450IF-3 catalyzed the hydroxylation of testosterone at the 7 alpha-position effectively, but the other forms did not hydroxylate testosterone. Analysis of the NH2-terminal sequence showed that P-450IF-1, 2, 3, 5, and 6 differed structurally from each other. The sequences of P-450IF-1 and IF-2 were somewhat homologous, but the NH2-terminal sequences of the other forms were all different. Based on these results, we concluded that P-450IF-1 corresponded to one of the phenobarbital-inducible forms in male rat liver. P-450IF-2 was a female-specific form and its concentration was low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors involved in the regulation of the levels and activities of rat liver cytochromes P-450

Methods have been developed for the purification of eight rat liver microsomal cytochrome P-450 (P-450) isoenzymes. Another rat P-450, responsible for the metabolism of the genetic polymorphism prototype debrisoquine, has also been partially purified from rat liver. Six P-450s have been purified to electrophoretic homogeneity from human liver preparations. The rat and human P-450s can be quantified in crude samples using 'immunoblotting' methods coupled with peroxidase visualization. A study on the effects of a family of polybrominated biphenyl congeners led to the conclusion that the levels of all of the rat P-450s considered above are under some degree of independent regulation. In monolayer culture, different P-450s show different stabilities and levels of several are selectively regulated by various media components. Studies with the eight isolated rat P-450s indicate that the iron spin state, oxidation-reduction potential (Fe3+/Fe2+ couple), and catalytic activity towards substrates are not related to each other. The major function of phospholipid in reconstituted P-450/NADPH-P-450 reductase systems is the facilitation of formation of a complex of the two proteins. Studies on the regioselective hydroxylation of warfarin have been used to develop an order of binding affinity of the different P-450s for NADPH-P-450 reductase.     

Cytochrome P-450 isozyme LM3b from rabbit liver microsomes. Induction by triacetyloleandomycin purification and characterization

Oral administration of triacetyloleandomycin (TAO), 1 mmol/kg/day for 7 days to mature male New Zealand White rabbit results in a significant increase in the content of liver microsomal cytochrome P-450. This increase is accompanied by the occurrence on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microsomes of a strong band in the zone of electrophoretic mobility associated with the LM3 isozymes and the stimulation of a number of monooxygenase activities of these microsomes including aminopyrine, chlorcyclizine, TAO, and erythromycin demethylation as well as 2-OH-estradiol and 6 beta OH-testosterone hydroxylation. Cytochrome P-450 LM3 (TAO) from these liver microsomes, purified to electrophoretic homogeneity, had Mr = 52,000 as determined by calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison with isozymes LM3a, LM3b, and LM3c isolated from control animals, by a number of criteria including spectral data, amino acid content, NH2-terminal sequence analysis, peptide mapping, immunological properties, and monooxygenase activities of reconstituted system, indicated that isozymes LM3 (TAO) and LM3b are very similar, if not identical, proteins. We conclude that TAO must be considered as a new type of inducer of microsomal cytochrome P-450 from rabbit liver.