Haruko Miller

Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): Cross reactivity with 3-methylcholanthrene induced rat cytochrome P-450☆

Hybridomas were prepared from myeloma cells and spleen cells of BALB/c female mice immunized with hepatic cytochrome P-450E purified from the marine fish, Stenotomus chrysops (scup). Nine independent hybrid clones produced MAbs, either IgG1, IgG2b, or IgM, that bound to purified cytochrome P-450E in radioimmunoassay. Antibodies from one clone MAb (1-12-3), also strongly recognized rat cytochrome P-450MC-B (P-450BNF-B; P-450c). The nine antibodies inhibited reconstituted aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase of scup cytochrome P-450E to varying degrees, and inhibited AHH activity of beta-naphthoflavone-induced scup liver microsomes in a pattern similar to that in reconstitutions, indicating that cytochrome P-450E is identical to the AHH catalyst induced in this fish by beta-naphthoflavone. MAb 1-12-3 also inhibited the reconstituted AHH activity of the major BNF-induced rat isozyme. Conversely, MAb 1-7-1 to rat cytochrome P-450MC-B had little effect on AHH activity of scup cytochrome P-450E, and did not recognize cytochrome P-450E in radioimmunoassay nor in an immunoblot. Scup cytochrome P-450E and rat cytochrome P-450MC-B thus have at least one common epitope recognized by MAb 1-12-3, but the epitope recognized by Mab 1-7-1 is absent or recognized with low affinity in cytochrome P-450E. The various assays indicate that the nine MAbs against cytochrome P-450E are directed to different epitopes of the molecule. These MAbs should be useful in determining phylogenetic relationships of the BNF- or MC-inducible isozymes and their regulation by other environmental factors.

Monoclonal antibodies to phenobarbital-induced rat liver cytochrome P-450

Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified phenobarbital-induced rat liver cytochrome P-450 (PB-P-450). Hybridomas were selected in HAT medium, and the monoclonal antibodies (MAbs) produced were screened for binding to the PB-P-450 by radioimmunoassay, for immunoprecipitation of the PB-P-450, and for inhibition of PB-P-450-catalyzed enzyme activity. In two experiments, MAbs of the IgM and IgG1 were produced that bound and, in certain cases, precipitated PB-P-450. None of these MAbs, however, inhibited the PB-P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity. In two other experiments, MAbs to PB-P-450 were produced that bound, precipitated and, in several cases, strongly or completely inhibited the AHH and 7-ethoxycoumarin deethylase (ECD) activities PB-P-450. These MAbs showed no activity toward the purified 3-methylcholanthrene-induced cytochrome P-450 (MC-P-450), beta-naphthoflavone-induced cytochrome P-450 (BNF-P-450) or pregnenolone 16-alpha-carbonitrile-induced cytochrome P-450 (PCN-P-450) in respect to RIA determined binding, immunoprecipitation, or inhibition of AHH activity. One of the monoclonal antibodies, MAb 2-66-3, inhibited the AHH activity of liver microsomes from PB-treated rats by 43% but did not inhibit the AHH activity of liver microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 also inhibited ECD in microsomes from PB-treated rats by 22%. The MAb 2-66-3 showed high cross-reactivity for binding, immuno-precipitation and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver (PB-P-450LM2). Two other MAbs, 4-7-1 and 4-29-5, completely inhibited the AHH of the purified PB-P-450. MAbs to different cytochromes P-450 will be of extraordinary usefulness for a variety of studies including phenotyping of individuals, species, and tissues and for the genetic analysis of P-450s as well as for the direct assay, purification, and structure determination of various cytochromes P-450.

Preparation and characterization of monoclonal antibodies to pregnenolone 16-α-carbonitrile inducible rat liver cytochrome P-450☆

Hybridomas were prepared from mouse myeloma cells and spleen cells derived from BALBc female mice immunized with purified rat hepatic pregnenolone 16-α-carbonitrile (PCN) induced cytochrome P-450 2a/PCN-E. The monoclonal antibodies (MAbs) thus obtained were screened for binding to the purified P-450 2a/PCN-E by radioimmunoassay. Eleven independent hybrid clones produced MAbs, each of which was of a single mouse immunoglobulin subclass of the IgG1, IgG2a or IgG2b type. Each of the MAbs produced by the eleven individual hybrid clones bound strongly to P-450 2a/PCN-E as assessed by radioimmunoassay and immunoprecipitation of P-450 2a/PCN-E in Ouchterlony double-immunodiffusion plates. Of the eleven MAbs, three also bound strongly to the phenobarbital-inducible rat liver cytochrome P-450 PB-4. Thus, two classes of MAbs were obtained, one class specific for P-450 2a/PCN-E and a second class that bound to both PCN- and phenobarbitalinducible P-450 forms. The reactivities of one MAb from each class toward eight highly purified rat hepatic cytochromes P-450 were examined using solid phase enzyme-linked immunosorbent analyses. The MAb designated C2 was found to be specific for P-450 2a/PCN-E and did not cross-react with seven other P-450 forms. This MAb was shown to be an effective probe for monitoring, by Western blotting, the induction of microsomal P-450 2a/PCN-E by PCN and phenobarbital. The MAb designated C1 reacted both with P-450 2a/PCN-E and with the two major phenobarbital-inducible P-450 forms, PB-4 and PB-5. None of the MAbs was inhibitory towards P-450 2a/PCN-E-dependent aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, ethoxycoumarin O-deethylase or ethylmorphine N-demethylase activity, indicating that the epitopes recognized by these MAbs are not directly associated with catalytic activity. The strong reactivities of three of the MAbs with both P-450 2a/PCN-E and P-450s PB-4 and PB-5 indicate that these two structurally quite different cytochrome P-450 families share at least one common epitope. These new MAbs are additions to our library of MAbs to different cytochromes P-450 and should help further our understanding of the relationship of cytochrome P-450 phenotype and multiplicity to inter-individual differences in drug and carcinogen metabolism and sensitivity.

Hydroxylation of benzo[a]pyrene and binding of (-)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450 : Effect of 7,8-benzoflavone, butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsomes from untreated, phenobarbital-treated and 3- methylcholanthrene-treated adult rabbits with respect to benzo(a)pyrene hydroxylation and the acti- vation of (-)truns-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene(( -)~ans-7,8-diol) to DNA-binding metab- olites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phenobarbital led to induction of P-450LMz and a concomitant 3-fold enhancement in microsomal benzo(a)pyrene hydroxylase activity, whereas the conversion of (-)rrans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM2 exhibited the highest activity and speci- ficity toward benzo(a)pyrene and the lowest activity toward (-)rrun.s-7,8-diol. Conversely, P-4SOLM4 was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or p- naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (-)trans- 7,8-diol but not of benzo(a)pyrene. Homogeneous P-450LM4 preferentially catalyzed the oxygenation of (-)tran.s-7,d in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM4 and LM2 with benzo(a)pyrene and (-)trans-7,8-dial, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8_benzoflavone, butylated hydroxy- toluene inhibited benzo(a)pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo(n)pyrene and its 7,8_dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver micro- somes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LMz and LM4, respectively.

Induction of rat liver microsomal and nuclear cytochrome p-450 by dietary 2-acetylaminofluorene and butylated hydroxytoluene

The influence of dietary 2-acetylaminofluorene (AAF) on the cytochrome P-450 content of rat liver microsomal and nuclear fractions was immunochemically probed with monoclonal and polyclonal antibodies to cytochromes P-450c and P-450d. Cytochrome P-450d but not P-450c was immunodetected in microsomes, nuclear envelopes, and nuclei from untreated rats. The levels of both cytochromes P-450c and P-450d were elevated after a diet of either 0.1% AAF for 1 week or 0.05% AAF for 3 weeks. However, the level of cytochrome P-450c relative to P-450d was lower after the more prolonged AAF feeding. Supplementation of AAF-containing diets with 0.3% butylated hydroxytoluene (BHT), which affords protection against AAF hepatocarcinogenesis in high-fat fed rats, protected and/or induced total (spectral) nuclear envelope cytochrome P-450 content. Immunochemical studies of liver fractions showed that BHT enhanced the AAF-dependent induction of cytochrome P-450c, but not of P-450d. This was a concerted effect of AAF + BHT since dietary BHT by itself did not affect the levels of cytochrome P-450c or P-450d as compared to control rats. Since 1- to 3-week dietary AAF had little effect on total (spectral analyses) microsomal cytochrome P-450 but markedly reduced total P-450 in nuclear envelopes, the coordinated induction of specific cytochrome P-450s in the different fractions suggests selective induction and depression of different forms of cytochrome P-450 and provides additional evidence for independent regulation of the drug-metabolizing system in nuclear envelope and microsomes. In addition, these results suggest that regulation of cytochrome P-450 may play a crucial role in the nutritional modulation of AAF hepatocarcinogenesis.

Human Liver Cytochrome P-450 Related to a Rat Acetone-Inducible, Nitrosamine-Metabolizing Cytochrome P-450: Identification and Isolation

A monoclonal antibody (MAb) to a rat acetone-inducible and nitrosamine-metabolizing form of microsomal cytochrome P-450, P-450ac, detected a related P-450 in human liver microsomes by both immunoblot and competitive radioimmunoassay. This MAb was also used to immunopurify microsomal cytochromes P-450 from both human liver and acetone-treated rats; these were electrophoretically homogeneous with apparent molecular weights of 56,200 and 53,000 daltons, respectively. The structures of the cytochromes P-450 were compared by peptide mapping and amino-terminal sequence analyses. They differed in their peptide maps but displayed amino-terminal sequence similarity in their first 19 residues. This report thus demonstrates the utility of MAbs to rat cytochromes P-450 for detection, identification and structural characterization of human P-450s.

Synthetic peptide antigens elicit monoclonal and polyclonal antibodies to cytochrome P450 IA2

Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and polyclonal antibodies. Antisera to both peptides reacted with rat IA2 but not the structurally similar IA1 form as determined by enzyme-linked immunosorbent assay. However, antisera to both peptides detected both rat IA2 and IA1 on immunoblots. In addition immunoblots of human liver microsomes revealed that both antisera recognized human IA2, but not IA1. Monoclonal antibodies generated against one of the peptides recognized rat IA2 and IA1 but did not detect human IA2. These results demonstrate the utility of anti-peptide antisera as a practical approach for the generation of P450 specific antibodies.

Variation in Inducibility of Cytochrome P-450c and Aryl Hydrocarbon Hydroxylase in Rat Liver, Lung, Kidney, Pancreas and Nasopharynx

The effect of 3-methylcholanthrene (MC) treatment on the cytochrome P-450c content of various rat tissues was examined by measuring the level of immunodetectable P-450c in conjunction with its aryl hydrocarbon hydroxylase (AHH) activity. Immunoblots revealed that P-450d was induced in the nasopharynx and pancreas in addition to its previously reported induction in the liver, lung and kidney. In contrast to P-450c, induction of the immunorelated P-450d was observed only in the liver. The specific content of immunodetected P-450c in the tissue homogenates decreased in the order: liver, nasopharynx, pancreas, lung, kidney. The corresponding AHH activities decreased in the order: liver, kidney, lung, nasopharynx, pancreas. The ratio of AHH activity to P-450c content varied widely among tissues: ratios of 37.2:1.7:0.47:0.04:0.02 were obtained for the kidney, liver, lung, nasopharynx and pancreas, respectively. The absence of a direct relationship between the levels of AHH and P-450c indicates that the extrahepatic activity may partially derive from P-450 forms other than P-450c and/or the specific activity of P-450c varies among different tissues.

Phenotyping Cytochromes P450 with Monoclonal Antibodies

Monoclonal antibodies (MAbs) to cytochrome P-450 isozymes can be used to phenotype tissues for epitope-specific cytochrome P-450 content. MAbs that inhibit specific cytochrome P-450 dependent drug or carcinogen reactions are useful tools for quantitative measurement of the individual or classes of cytochromes P-450 that catalyze these reactions. This method has been applied successfully to animal as well as human tissues. Radioimmunoassays based on MAbs have been developed and provide a rapid and efficient means for detecting cytochromes P-450 independent of functional enzyme activity. In addition, MAbs coupled to a Sepharose support can be used to immunopurify cytochromes P-450 in a procedure that is more rapid and efficient than conventional purification schemes. MAbs add a new dimension to analyses of cytochrome P-450 multiplicity and will find numerous applications in elucidation of the relationship between cytochrome P-450 phenotype and carcinogen or drug metabolism.