The expression and metabolic activity of cytochrome P-450 isozymes in control and phenobarbital-induced primary cultures of rat hepatocytes

The expression and activity of the phenobarbital (PB)-inducible P-450 isozymes, P-450b and P-450e, and the major 3-methylcholanthrene (MC)-inducible form, P-450c, were studied in primary cultures of adult rat hepatocytes in T1, Leibovitz L-15 (L-15), and a modification of Waymouth 752/1 (Way) media. P-450 isozymes in initially isolated hepatocytes and control and PB-treated cultures were quantitated by Western blot analysis, and activity was determined with 7,12-dimethylbenz[a]anthracene (DMBA) as substrate. Data from the Western blot analysis correlated well with the metabolic activity toward DMBA. P-450b was consistently induced by PB in hepatocytes in T1 and to a lesser extent in Way. P-450e protein was constitutive in initially isolated cells, expressed in control cultures at a reduced level, and increased or maintained by PB in all three media. DMBA metabolite formation associated with P-450b and P-450e activity was induced by PB in hepatocytes in T1 and Way and was inhibited by antibodies to P-450b. P-450c was only infrequently expressed in freshly prepared hepatocytes, but was detected in all control and PB-treated cultures although at a much higher level in T1. Thus, the amounts of P-450 isozymes, their inducibility by PB, and their activity toward DMBA were found to be dependent on the medium. We have demonstrated enzyme induction and increased activity of the major PB-inducible isozymes in hepatocytes in T1; these are also associated with a change in the control of P-450c expression leading to enhanced constitutive expression and inducibility by phenobarbital.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Dynamics of androstenedione metabolite formation in rat liver microsomes during temporal phenobarbital induction.

1. Formation of androstenedione (AD) 7 alpha-, 16 alpha-, 16 beta- and 6 beta-hydroxymetabolites produced in rat liver microsomes and differing by the duration of phenobarbital (PB) induction (temporal induction) has been studied. 2. Formation of 7 alpha-, 16 alpha- and 6 beta-metabolite is sexually differentiated during PB-induction. 3. The most dramatical changes were observed in the 16 beta-hydroxylase activity specific for cytochrome P-450b which increased in all rat groups investigated. 4. The immunochemical method using antibodies against P-450b/e was applied to measure its content in microsomes. 5. It was shown that the microsomal level of P-450b/e correlated (r = 0.63) with a 16 beta-hydroxylase activity in a narrow range of enzyme concentrations (from 0.16 to 0.32 nmol/mg). 6. In microsomal preparations with a higher level of P-450b/e the correlation is lower (r = 0.4). 7. The dependence of the P-450b catalytic activity on the P-450b to NADPH-cytochrome P-450 reductase relation is discussed.     

Differential time course of induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by Aroclor 1254

The time course of induction of rat liver microsomal cytochromes P-450a, P-450b + P-450e, P-450c, and P-450d and epoxide hydrolase has been determined in immature male rats administered a single large dose [1500 mumol (500 mg)/kg body wt] of the polychlorinated biphenyl mixture Aroclor 1254. Differential regulation of these xenobiotic-metabolizing enzymes was indicated by their characteristic patterns of induction. The rate of induction of cytochrome P-450a and epoxide hydrolase was relatively slow, and steady-state levels of these enzymes were maintained from approximately Days 9 to 15 after Aroclor 1254 treatment. In contrast, cytochrome P-450c was maximally induced 2 days after Aroclor 1254 treatment and remained at a constant level through Day 15. Steady-state levels of cytochrome P-450d, beginning 1 week after Aroclor 1254 treatment, were preceded by a fairly rapid rate of induction and possibly by a small decline from maximal levels observed around Days 4 to 5. Like those of the other cytochrome P-450 isozymes and epoxide hydrolase, the levels of cytochromes P-450b + P-450e were constant from Day 9 to 15 after Aroclor 1254 treatment. However, an unexpected but reproducible decline (approximately 25%) in total cytochrome P-450 content observed between Days 4 and 9 after Aroclor 1254 treatment principally reflected a dramatic and totally unanticipated decrease (approximately 45%) in the level of cytochromes P-450b + P-450e. This transient decline in the level of cytochromes P-450b + P-450e was not due to an unusual effect of a mixture of polychlorinated biphenyls, since identical results were obtained with two individual congeners, namely 2,3,4,5,4'-penta- and 2,3,4,5,3',4'-hexachlorobiphenyl, that induced the same isozymes as Aroclor 1254. In contrast, when rats were treated with 2,4,5,2',4',5'-hexachlorobiphenyl, which induces cytochromes P-450a and P-450b + P-450e and epoxide hydrolase but not cytochromes P-450c or P-450d, maximal levels of cytochromes P-450b + P-450e were attained on Day 4 and no decrease was observed over the next 11 days. These results suggest that there may be an interaction in the regulation of induction of certain individual cytochrome P-450 isozymes.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms

The "major" phenobarbital (PB)-induced cytochrome P-450 species present in livers of male Sprague-Dawley rats was resolved into two catalytically active heme-protein fractions on diethylaminoethyl cellulose. The two species, P-450 PB-4 (Mr = 49,000) and P-450 PB-5 (Mr = 51,000), were purified to homogeneity, and their chromatographic, spectral, catalytic, and structural properties were compared. P-450 BP-5 eluted earlier on hydroxylapatite and exhibited a more significant cholate-induced Type I spectral shift than P-450 BP-4. Very similar substrate specificity profiles were evident when the two isozymes were reconstituted with lipid, cytochrome P-450 reductase, and cytochrome b5 for oxidative metabolism of several xenobiotics, although P-450 PB-4 exhibited a higher specific catalytic activity (greater than or equal to 5-fold) with all substrates tested. Marked differences were also observed in the sensitivities of both isozymes to several P-450 inhibitors. In addition, P-450 PB-4 was greater than or equal to 10-fold more susceptible than P-450 PB-5 to suicide inactivation by two allyl-containing compounds, allylisopropylacetamide and secobarbital, providing a possible explanation of the previously observed partial inactivation by such compounds of phenobarbital-induced P-450 activity in liver microsomes. One-dimensional peptide maps of the two isoenzymes were highly similar. Antibody raised against purified Long Evans rat liver P-450b (Thomas, P. E., Korzeniowski, D., Ryan, D., and Levin, W. (1979) Arch. Biochem. Biophys. 192, 524-532) cross-reacted with P-450 PB-4 and P-450 PB-5. NH2-terminal sequence analysis demonstrated that the first 31 residues of both PB-4 and PB-5 were identical. These sequences indicated that a highly hydrophobic terminal segment, observed previously for other P-450s as well, is followed by a cluster of basic residues, suggesting that the NH2-terminal portion of these P-450s might be involved in membrane anchoring. Although it is unclear whether P-450 PB-4 and P-450 PB-5 are separate gene products or are related by post-translational modifications, this present demonstration of closely related isozymic forms suggests the possible added complexity of microheterogeneity for this family of microsomal monooxygenases.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

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.     

Induction of specific cytochrome P-450 isozymes by methylenedioxyphenyl compounds and antagonism by 3-methylcholanthrene

Two methylenedioxyphenyl compounds, isosafrole (5-propenyl-1,3-benzodioxole) and an analog, 5-t-butyl-1,3-benzodioxole (BD), differ markedly as inducers of cytochrome P-450 isozymes in rat liver microsomes. Isosafrole is a mixed-type inducer, inducing P-450b, P-450c, and P-450d. In contrast, BD is a phenobarbital-type inducer, increasing P-450b, but producing little or no increase in P-450c or P-450d. Similarly, isosafrole increases the amount of translatable mRNA for P-450b, c and d, while BD induces only the mRNA for P-450b. Dimethylation of the methylene bridge carbon of BD to give 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBD) blocks the formation of NADPH-reduced Type III metabolite-P-450 complexes in vitro, and diminishes but does not abolish the ability of the compound to induce P-450b. Western blots of microsomes from isosafrole and BD-treated rat livers confirm that in contrast to isosafrole, BD does not induce P-450d or P-450c. However, the antibody to P-450d recognizes two new polypeptides (approximately 50K Mr) from sodium dodecyl sulfate-polyacrylamide gels of liver microsomes from BD-treated rats. These polypeptides are not observed in control, isosafrole, 3-methylcholanthrene (3-MC), or DBD-treated rats. They are intensified by coadministration of 3-MC with BD and may represent either modified isozyme-metabolite adducts or degradation products of P-450d. However, the polypeptides could not be generated in vitro by addition of BD to 3-MC-induced microsomes with NADPH under conditions which produced spectral metabolite complexes, or in a reconstituted system with P-450d. The two methylenedioxyphenyl compounds do not form stable metabolite complexes with the same P-450 isozymes. BD formed distinct spectral metabolite complexes in vitro with both P-450b and P-450c but not with P-450d in a reconstituted system. In contrast, isosafrole forms metabolite complexes with all three isozymes. Coadministration of 3-MC with BD blocked induction of P-450b by 80% and produced a similar repression of its translatable mRNA. This finding indicates that 3-MC type inducers not only induce certain cytochrome P-450 isozymes, but also repress synthesis of other isozymes.     

Degradation of rat hepatic cytochrome P-450 heme by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine to irreversibly bound protein adducts

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) (a structural analog of the dihydropyridine Ca2+ antagonists) to untreated, phenobarbital-, or dexamethasone-pretreated rats results in time-dependent losses of hepatic cytochrome P-450 content. Functional markers for various cytochrome P-450 isozymes have permitted the identification of P-450h, P-450 PB-1/k, and P-450p as the isozymes inactivated preferentially by the drug. DDEP-mediated cytochrome P-450 destruction may be reproduced in vitro, is most prominent after pretreatment of rats with dexamethasone, pregnenolone 16 alpha-carbonitrile or phenobarbital, and is blocked by triacetyloleandomycin. These findings together with the observation that DDEP markedly inactivates hepatic 2 beta- and 6 beta-testosterone hydroxylase and erythromycin N-demethylase tend to indict the steroid-inducible P-450p isozyme as a key protagonist in this event. The precise mechanism of such DDEP-mediated P-450p heme destruction is unclear, but involves prosthetic heme alkylation of the apocytochrome at its active site in what appears to be a novel mechanism-based "suicide" inactivation. Such inactivation appears to involve fragmentation of the heme to reactive metabolites that irreversibly bind to the protein, but the chemical structure of the heme-protein adducts is yet to be established. Intriguingly, such DDEP-mediated P-450p destruction in vivo also results in accelerated loss of immunochemically detectable apocytochrome P-450p. It remains to be determined whether or not this loss is due to enhanced proteolysis triggered by the structural modification of the apocytochrome.     

Comparison of the inducing effect of triphenyldioxan, bis-(dichloropyridyloxy)benzene and phenobarbital on the liver monooxygenase

The induction by triphenyldioxane (TPD) of cytochrome P-450 in rat liver microsomes was studied. It was demonstrated that TPD injection in a single dose (10 mg/kg of body mass) is associated with a marked induction of cytochromes P-450 b/e (cytochrome PB-forms) in rat liver microsomes and a significant increase in the benzphetamine-N-demethylase activity typical of cytochrome P-450b. In other words, TPD is a potent inducer of PB-type, the inducing effect being attained by an injection of a single dose of TPD which is by one order of magnitude less than that of phenobarbital. It can be assumed that this compound shows a high affinity for the hypothetical receptor responsible for cytochrome P-450b synthesis. It was shown also that TPD does not induce the monooxygenase system of mouse liver, whereas 1,4-bis[2-(dichloropyridyloxy)]benzene (DPB) is a potent inducer of PB-type in mice, being fairly ineffective in rats. Hence, the species-specific effect of TPD and DPB appears to be opposite.     

Monoclonal antibody directed isolation and amino-terminal sequence analysis of phenobarbital induced rat liver cytochromes P-450

Cytochrome P-450 was isolated from liver microsomes of phenobarbital treated rats by an essentially single step immunopurification with a monoclonal antibody (MAb). The amino terminal sequence of the isolated cytochrome P-450 displayed a microheterogeneity of isozymes related to previously identified phenobarbital induced forms, indicating that each of these isozymes possess the MAb-specific epitope. This monoclonal antibody-based approach to isolation and subsequent identification of cytochrome P-450 may serve to classify different isozymes by their content of epitopes that bind to different MAbs.     

Multiple constitutive but phenobarbital-inducible rat hepatic nuclear RNA species homologous to a novel cytochrome P-450 cDNA

A cDNA clone, pPB8, representing partial information for a phenobarbital-inducible rat hepatic cytochrome P-450, immunochemically related to cytochrome P-450b and/or P-450e, hybridized to multiple hepatic nuclear RNA species. In addition to the 3.7 +/- 0.2 kb mRNA encoding this novel cytochrome P-450 isozyme, pPB8 hybridized to nuclear RNAs of 4.9 +/- 0.3, 5.4, 5.7 +/- 0.2, and 6.3 +/- 0.1 kb. These nuclear RNAs were constitutively expressed and were inducible to various extents by phenobarbital administration. The time course of induction of these nuclear RNA components suggested product-precursor relationships. A "full-length" cDNA clone, pPB8/7, synthesized from poly(A)+ RNA homologous to pPB8, detected two mRNA species of 4.6 and 1.8 kb. The 4.6 kb nuclear RNA was inducible by 3-methylcholanthrene, Aroclor 1254, and phenobarbital, while the 1.8 kb nuclear RNA was not appreciably affected. It is suggested that pPB8 and pPB8/7 were synthesized from distinct mRNAs that share homology in their 3' regions.     

Regulation of cytochrome P-450b and P-450e mRNA expression in the developing rat. Hybridization to synthetic oligodeoxyribonucleotide probes

We conducted solution hybridization and Northern blot experiments utilizing synthetic 18'-mer oligodeoxyribonucleotides complementary to two major rat hepatic phenobarbital-inducible cytochrome P-450s, P-450b and P-450e, to assess their mRNA expression during rat development. At all ages studied, with one exception (i.e. in day 22 neonates), P-450b mRNA was not detected in control animals. However, traces of P-450e message were observed in control animals on day 22 and persisted to adulthood. Phenobarbital pretreatment caused marked increases in hepatic mRNA for both P-450s as early as 22 days after conception. No increases were observed in RNA isolated from phenobarbital-pretreated day 10 or 19 rats. In general, the inducible levels of P-450b and P-450e mRNA increased as a function of age. The age-dependent increase in responsiveness to phenobarbital was associated with an age-dependent decrease in the ratio of P-450b to P-450e mRNA levels. The levels of P-450b/P-450e varied from a ratio of 19 at day 22 of development to a ratio of 5 at day 62 of development. Maximal levels of phenobarbital-induced hepatic RNA for both isozymes occurred 24 days after birth (day 46 of development), at which time P-450b and P-450e mRNAs accumulated to levels 2.4- and 1.8-fold greater, respectively, than levels found in comparably induced adult rat liver. Northern blot analyses indicated that the major mRNA species hybridizing to either the P-450b or P-450e oligomers in all age groups studied was approximately 1.8 kilobases.     

Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes

Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive Leydig cells was found to undergo significant time-of-day variation with a peak time of 0000 hours (light phase from 0800 to 2000 hours). Injection of cadinenes (300 mg/kg per day intraperitoneally at 48 and 96 h before sacrifice) induced PB-P-450 isozyme but did not induce MC-P-448 isozyme. The induction of PB-P-450 isozyme by cadinenes was time dependent, and the early dark phase (2000 and 0000 hours) was most sensitive. These results suggest that temporal variation of cytochrome P-450 isozymes is one of the important physiological variations in detoxification and activation of various xenobiotics and chemicals in the testis.     

Regulation of cytochrome P-450p by phenobarbital and phenobarbital-like inducers in adult rat hepatocytes in primary monolayer culture and in vivo

Treatment of rats with phenobarbital increases the hepatic concentration of P-450p, a form of cytochrome P-450 believed to be controlled primarily by a mechanism that stereospecifically recognizes glucocorticoids like dexamethasone and anti-glucocorticoids like pregnenolone-16 alpha-carbonitrile [Schuetz, E.G., & Guzelian, P.S. (1984) J. Biol. Chem. 259, 2007]. To test the possibility that phenobarbital induces P-450p indirectly by increasing the availability of endogenous glucocorticoids in the liver, we added phenobarbital and phenobarbital-like inducers to primary monolayer cultures of adult rat hepatocytes incubated in serum-free medium without glucocorticoids and found stimulated de novo synthesis of P-450p measured as increased incorporation of [3H]leucine into immunoprecipitable P-450p protein. With some of the inducers, notably the organochlorine pesticides chlordane and trans-nonachlor, there was a greater accumulation of P-450p measured on quantitative immunoblots than could be accounted for by the increase in P-450p synthesis. "Pulse-chase" experiments confirmed that these compounds significantly lengthen the half-life of P-450p up to 60 h as compared to the values in control (11 h) or dexamethasone-treated (10 h) cultures. Treatment of rats with chlordane, trans-nonachlor, or other cyclodiene organochlorine pesticides confirmed that these agents increase the concentration of P-450p in liver microsomes analyzed on immunoblots of two-dimensional electrophoretic gels. The time courses of induction in trans-nonachlor-treated rats of P-450p protein and of P-450PB proteins induced by phenobarbital were similar as were the amounts of P-450PB mRNA and P-450p mRNA measured by hybridization to cloned cDNA probes. However, analysis of structure-activity relationships among polychlorinated biphenyls revealed that isomers with two ortho chlorinated positions maximally induced P-450PB whereas isomers with three and four ortho chlorines maximally induced P-450p in rats and in hepatocyte culture, respectively. We conclude that P-450p is induced by the phenobarbital class of inducers through direct contact with the hepatocytes involving decreased degradation of the protein and stimulation of its synthesis in a manner similar but not identical with that of P-450PB.     

Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.