Monoclonal antibodies directed characterization of epidermal and hepatic cytochrome P-450 isozymes induced by skin application of therapeutic crude coal tar

A single application of crude coal tar (CCT) solution (USP) to the skin of neonatal rats was shown to induce epidermal and hepatic cytochrome P-450(P-450)-dependent monooxygenase activities. To further characterize the induction response, in this study we have utilized highly specific monoclonal antibodies (MoAb) 1-7-1, 2-66-3, and 1-98-1 directed against highly purified rat liver P-450s induced by 3-methyl-cholanthrene, phenobarbital and ethanol, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of hepatic microsomes prepared from CCT-treated animals showed a significant increase in the coomassie blue stainable proteins in the P-450 region; however, this was not evident in epidermal microsomes. Immunoblot analysis of epidermal and hepatic microsomes with MoAb 1-7-1 revealed strong immunoprecipitin bands in both tissues. MoAb 2-66-3 showed significant immunoreactivity only with hepatic microsomes. Interestingly, CCT treatment resulted in suppression of immunoreactivity with MoAb 1-98-1 in hepatic microsomes. MoAb 1-7-1 and 2-66-3 exhibited concentration-dependent inhibitory effects in aryl hydrocarbon hydroxylase and 7-ethoxycoumarin-O-deethylase activities induced by CCT application. MoAb 1-7-1 was substantially more effective in this respect. Epidermal and hepatic microsomes prepared from CCT-treated rats showed significantly greater metabolism of benzo(a)pyrene (BP). MoAb 1-7-1 and MoAb 2-66-3 inhibited BP metabolism in both the tissues. However, MoAb 1-7-1 was more inhibitory in this regard as compared to MoAb 2-66-3. These studies indicate that topical application of therapeutic CCT to the skin of neonatal rats results in induction of P-450 isozyme c in epidermis and isozymes b and c in liver, and that this induction is associated with the suppression of P-450 isozyme j in liver.     

Induction of cytochrome CYPIA1 and formation of toxic metabolites of benzo[a]pyrene by rat aorta: a possible role in atherogenesis.

Cigarette smoking is a leading risk factor for atherosclerosis. Endothelial injury may be the initial event in this process. The carcinogenic metabolites of the polycyclic aromatic hydrocarbons found in cigarette smoke tars could cause this injury. We tested this model by examining the effect of 3-methylcholanthrene administration on aortic polycyclic aromatic hydrocarbon metabolism. Immunoblotting with a monoclonal antibody (mAb 1-7-1) specific for cytochromes CYPIA1 and CYPIA2 showed that aortic microsomes from treated, but not from control, animals contained CYPIA1; the CYPIA1 was primarily in the endothelium. Aortic microsomes from induced animals metabolized benzo[a]pyrene (BaP) to the 7R,8S,9,10-tetrahydrotetrol-, 7,8-dihydrodiol-, 1,6 quinone-, 3,6 quinone-, 6,12 quinone-, 3-hydroxy-, and 9-hydroxy-BaP. mAb 1-7-1 inhibited the formation of the tetrahydrotetrol, the dihydrodiol-BaP, and the 3-hydroxy-BaP but did not inhibit the quinones or the 9-hydroxy-BaP. Arachidonic acid did not affect metabolism. These data suggest that the aortas of induced animals metabolize the BaP in cigarette smoke to carcinogenic and toxic products and that this metabolism may initiate vessel injury and lead to the accelerated atherosclerosis seen in cigarette smokers.     

Immunochemical characterization of cytochrome P-450 isozymes responsible for benzene oxidation in the rat liver

The contribution of cytochrome P-450 isozymes to benzene metabolismin liver microsomes from fed, fasted, pyrazole-, pbenobarbital(PB)- and ethanol-treated rats and in respective isocaloriccontrols was investigated using monoclonal antibodies (mAbs).Clone 1-7-1 mAb did not inhibit benzene metabolism, whereasclone 2-66-3 inhibited only in PB-induced microsomes at a highconcentration of benzene (6.26 mM), and clone 1-91-3 mAb inhibitedbenzene metabolism in all cases. The degree of inhibition wasas follows: fed isocaloric control PB < fasted < pyrazole ethanol. The pattern of inhibition was similar with clone 1-91-3for low (0.23 mM) and high concentrations of benzene, exceptin PB-induced mkrosomes. Western blot analysis showed that clone1-7-1 mAb did not bind any liver mkrosomal protein in the regionof cytochrome P-450s, whereas with clone 2-66-3 a clear-cutband was seen only in liver microsomes from PB-treated rats,with clone 1-98-1, a band was detected in mkrosomes from alltreated groups, in the following order: PB = isocaloric control< fed < fasted < pyrazole < ethanol. These resultsindicate that (i) cytochromes P-450b,e and P-450J contributeto benzene metabolism in rat liver; (ii) the former has a lowaffinity to benzene and is induced by PB; and (iii) P-450J hasa high affinity to benzene and is induced by 1-day fasting,pyrazole and ethanol, but decreased by PB treatment.     

Development of a human lymphoblastoid cell line constitutively expressing human CYP1A1 cDNA: substrate specificity with model substrates and promutagens

AHH-1 TK+/–cell derivatives were developed that stablyexpress human CYP1A1 cDNA, and an AHH-1 TK+/–derivativeexpressing higher levels of CYP1A2 cDNA in extrachromosomalvectors which confer resistance to I-histidinol. The CYP1A1-expressingcell lines, designated h1A1 and h1A1v2, differ by containingone and two CYP1A1 cDNA expression units per vector. The CYP1A2-expressingcell line, designated h1A2v2, also has two CYP1A2 cDNA expressionunits per vector. Microsomes prepared from CYP1A1 cDNA expressingcells exhibit high, constitutive levels of 7-ethoxyresorufindeethylase (EROD), 7-ethoxycoumarin deethylase (ECD), 7-ethoxy-4-trifluoromethylcoumarindeethylase (EFCD), benzo[a]-pyrene hydroxylase (BPH) activitiesand spectrally quantifiable cytochrome P450. Kinetic comparisonsbetween cDNA-expressed CYP1A1 and CYP1A2 indicate that CYP1A1is more active than CYP1A2 for EROD, ECD, EFCD and BPH. CYP1A2was more active than CYP1A1 for acetanilide hydroxylation andactivation of aflatoxin B₁ (AFB₁). The mutagenicity of selectedpromutagens were examined in h1A1 cells and control cells. Relativeto control cells, the h1A1 cell line exhibits increased sensitivityto the mutagenicity of benzo[a]pyrene, cyclopenta[c,d]pyrene,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and AFB₁.     

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.     

A monoclonal antibody to rat liver cytochrome P450 IIC11 strongly and regiospecifically inhibits constitutive benzo[a]pyrene metabolism and DNA binding

The monoclonal antibody MAb 1-68-11, prepared to constitutive cytochrome P450 IIC11 (2c/RLM5) from male Sprague-Dawley rat liver, was used to study the contribution of the class of cytochrome P450s epitopically related to P450 IIC11 to the regiospecific metabolism of benzo[a]pyrene (BP) and its binding to DNA. The effect of MAb 1-68-11 was determined on the conversion of BP to BP-9,10-dihydrodiol, BP-7,8-dihydrodiol, BP-4,5-dihydrodiol, BP phenols, and BP quinones, and on the P450-dependent DNA binding catalyzed by P450 in microsomes from uninduced male and female Wistar and Sprague-Dawley rat livers, as well as 3-methylcholanthrene- and phenobarbital (PB)-induced male Wistar rat livers. In liver microsomes from untreated male rats, MAb 1-68-11 inhibited BP-9,10-dihydrodiol formation by 80%; in liver microsomes from untreated female rats, the inhibition was 100%. BP-7,8-dihydrodiol formation was inhibited from 38 to 77% in microsomes from males and 50% in those from females. In microsomes from PB-induced rats, inhibition of the 9,10-dihydrodiol and the 7,8-dihydrodiol was 90% and 73%, respectively, whereas BP-4,5-dihydrodiol formation was enhanced 80%. In microsomes from 3-methylcholanthrene-treated rats, no inhibition of MAb 1-68-11 was observed on either the metabolism of BP or its binding to DNA. In contrast, the binding of BP to DNA was completely inhibited by MAb 1-68-11 in microsomes from uninduced male Wistar rats and 70% in PB-induced microsomes. 32P-postlabeling analysis showed that formation of the major stable adduct, BP diol epoxide bound at C-10 to the 2-amino of deoxyguanosine, was strongly inhibited in uninduced and PB-induced microsomes. Formation of the major labile BP-DNA adduct 7-(benzo[a]pyren-6-yl) guanine (BP-N7Gua) was inhibited about 60% in microsomes from untreated male Wistar rats. These results show that MAb 1-68-11 regiospecifically inhibits cytochrome P450 IIC11 and epitopically related P450s that metabolize BP at the 7,8 and 9,10 positions. MAb 1-68-11 also inhibits enzyme-catalyzed binding of BP to DNA in the specific formation of BP-N7Gua and adducts detected by the 32P-postlabeling technique.     

The metabolism of 1-nitropyrene by human cytochromes P450

The metabolism of [3H]1-nitropyrene by specific forms of human cytochrome P450 was investigated in vitro using Vaccinia virus expression of P450 cDNAs in HepG2 cells. Cell lysates were injected individually with recombinant Vaccinia virus containing human P450 cDNA (P450 IA2, IIA3, IIB7, IIC8, IIC9, IID6, IIE1, IIF1, IIIA3, IIIA4, IIIA5 AND IVB1). Only IIIA3 and IIIA4 demonstrated significant activity in the C-oxidation of 1-nitropyrene. The principal metabolite from both P450 forms was 1-nitropyren-3-ol, produced in at least 4-fold greater abundance than the mixture of 1-nitropyren-6-ol and 1-nitropyren-8-ol, or the K-region dihydrodiols. This is in contrast to the metabolism in many species where 6-ol and 8-ol formation predominate over 3-ol formation. In fact, in rats and rabbits, P450 forms quite distinct from the IIIA P450s catalyze the majority of the metabolism of this pollutant. This is the first demonstration of the role of individual human P450 forms in the metabolism of a representative chemical in this important class of environmental pollutants. The importance of these observations in the overall carcinogenic risk of humans to these chemicals remains to be established. These studies furthermore establish a marked species difference in the metabolism of nitrated polycyclic aromatic hydrocarbons.     

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

Hybridomas were prepared from mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified rat hepatic pregnenolone 16-alpha-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 phenobarbital-inducible 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 ethymorphine 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.     

A monoclonal antibody against cytochrome P-450 enhances mutagen activation of N-nitrosodimethylamine by mouse liver S9: studies on the mode of action

The effect of the monoclonal antibody MAb 2-66-3, directed againstthe major rat liver phenobarbital (PB)-induced cytochrome P-450(P-450), on the S9-mediated mutagenicity of N-nitrosodimethylamine(DMN) in Salmonella typhimurium strain TA1530 was studied usingliver S9 from PB-treated mice. This MAb enhanced {small tilde}2-fold S9-mediated mutagenicity of DMN but inhibited both itsN-demethylation and N-denitrosation by 50%. Thus MAb-mediatedenhancement of DMN mutagenesis does not result from alteredactivationl inactivation pathways, both known to involve P-450isozymes. DMSO, a hydroxyl radical (HO⁺) scavenger and desferrioxamine,an inhibitor of HO⁺-dependent reactions, quenched the MAb-mediatedenhancement of DMN mutagenesis, implicating the HO⁺-dependentactivation of DMN to mutagenic species. As a mechanism, we proposethat the binding of this MAb to P-450 isozyme implicated inDMN metabolism decreases the functional coupling between thereductase and the P-450 complex, leading to an increased electronflow from the reductase towards molecular oxygen to form reducedoxygen species (HO⁺) at the expense of the monooxygenase functions.