Wayne Levin

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels.

Abstract The use of 3,3′,5,5′-tetramethylbenzidine-H2O2 as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.

Purification and characterization of hepatic microsomal cytochrome P-450

Article de synthese sur le cytochrome P450, sa purification et la caracterisation de ses proprietes structurales (electrophorese gel), immunologiques, metaboliques (activite enzymatique). Axe sur le cytochrome P450 de foie de rat, extension a une etude interspecifique

PCBs: Structure-Function Relationships and Mechanism of Action

Numerous reports have illustrated the versatility of polychlorinated biphenyls (PCBs) and related halogenated aromatics as inducers of drug-metabolizing enzymes and the activity of individual compounds are remarkably dependent on structure. The most active PCB congeners, 3,4,4′,5-tetra-, 3,3′,4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexachlorobiphenyl, are substituted at both para and at two or more meta positions. The four coplanar PCBs resembled 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in their mode of induction of the hepatic drug-metabolizing enzymes. These compounds induced rat hepatic microsomal benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) and cytochromes P-450a, P-450c and P-450d. 3,4,4′,5-Tetrachlorobiphenyl, the least active coplanar PCB, also induced dimethylaminoantipyrine N-demethylase and cytochromes P-450b+e and resembled Aroclor 1254 as an inducer of the mixed-function oxidase system. Like Aroclor 1254, all the mono-ortho- and at least eight di-ortho-chloro analogs of the coplanar PCBs exhibited a “mixed-type” induction pattern and induced microsomal AHH, dimethylaminoantipyrine NM-demethylase and cytochromes P-450a–P-450e. Quantative structure–activity relationships (QSARs) within this series of PCBs were determined by comparing their AHH induction potencies (EC50) in rat hepatoma H-4-II-E cells and their binding affinities (ED50) for the 2,3,7,8-TCDD cytosolic receptor protein. The results showed that there was an excellent correlation between AHH induction potencies and receptor binding avidities of these compounds and the order of activity was coplanar PCBs (3,3′,4,4′-tetra-, 3,3′,4,4′,5-penta- and 3,3′,4,4′,5,5′-hexachlorobiphenyls) > 3,4,4′,5-tetrachlorobiphenyl ~ mono-ortho coplanar PCBs > di-ortho coplanar PCBs. It was also apparent that the relative toxicities of this group of PCBs paralleled their biological potencies. The coplanar and mono-ortho coplanar PCBs also exhibit differential effects in the inbred C57BL/6J and DBA/2J mice. These compounds induce AHH and cause thymic atrophy in the former “responsive” mice whereas at comparable or higher doses none of these effects are observed in the nonresponsive DBD/2J mice. Since the responsiveness of these two mice strains is due to the presence of the Ah receptor protein in the C57BL/6J mice and its relatively low concentration in the DBA/2J mice, the results for the PCB cogeners support the proposed receptor-mediated mechanism of action. Although the precise structural requirements for ligand binding to the receptor have not been delineated, the halogenated aromatic hydrocarbons which exhibit the highest binding affinities for the receptor protein are approximate isostereomers of 2,3,7,8-TCDD. 2,3,4,4′,5-Pentachlorobiphenyl elicits effects which are qualitatively similar to that of TCDD and the presence of the lateral 4′-substituent is required for this activity. Thus the 4′-substituted 2,3,4,5-tetrachlorobiphenyls have been used as probes for determining the substituent characteristics which favor binding to the receptor protein. Multiple regression analysis of the competitive binding EC50 values for 13 substituents gave the following equation: log (1/EC50) = 1.53σ + 1.47π + 1.09 HB + 4.08 where σ is electronegativity, π is hydrophobicity, HB is hydrogen bonding and r is the correlation coefficient (r = 0.978). The utility of this equation in describing ligand:receptor interactions and correlations with toxicity are being studied with other halogenated hydrocarbons and PAHs.

Purification, characterization and regulation of five rat hepatic microsomal cytochrome P-450 isozymes

1. Five hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, P-450e) have been purified to apparent homogeneity from immature male rats treated with various xenobiotics. 2. The unique electrophoretic properties, substrate specificities and spectral characteristics of these haemoproteins have been compared and contrasted. 3. Structural studies of these cytochrome P-450 isozymes have included NH2-terminal amino acid sequence analyses, as well as electrophoretic profiles of limited proteolytic digests and cyanogen bromide fragments of the haemoproteins. 4. Specific antibodies have been prepared against four of the isozymes and used to evaluate immunochemical relationships among these cytochrome P-450s by Ouchterlony double-diffusion analyses. 5. The levels of some of these cytochrome P-450 isozymes have been quantified immunologically in hepatic microsomal preparations from untreated rats and following induction by phenobarbital, 3-methylcholanthrene or Aroclor 1254. 6. Antibodies directed against cytochromes P-450a and P-450b have been used to establish the presence of more than one 7 alpha- and 16 alpha-testosterone hydroxylase in rat hepatic microsomes. The relative contributions of cytochromes P-450c and P-450d to the overall microsomal metabolism of benzo[a]pyrene have been evaluated using antibodies to these haemoproteins.

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

Summary Cytochromes P-450 (from rats treated with phenobarbital) and P-448 (from rats treated with 3-methylcholanthrene) were solubilized and partially purified from rat liver microsomes. The final preparation had a specific activity (nmoles per mg protein) of 5.5 for cytochrome P-450 and 5.3 for cytochrome P-448. The partially purified cytochromes P-450 and P-448 exhibited different substrate specificities for benzphetamine and 3,4-benzpyrene.

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (−)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (−)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (−)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (−)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ∼30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ∼5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.

Lipid peroxidation and the degradation of cytochrome P-450 heme.

Abstract In an in vitro system consisting of rat liver microsomes and NADPH, significant lipid peroxidation was observed along with a concomitant loss of cytochrome P-450. This spectral loss of cytochrome P-450 was shown to be the result of a breakdown of cytochrome P-450 heme. Inhibitors of lipid peroxidation also prevented the loss of cytochrome P-450, demonstrating a direct relationship between lipid peroxidation and breakdown of cytochrome P-450 heme. This breakdown of cytochrome P-450 heme during lipid peroxidation is unrelated to the degradation of cytochrome P-450 heme by an active metabolite of certain allyl-containing barbiturates and barbiturate related compounds. In addition, it appears that different breakdown products of heme are produced by these two mechanisms.

Purification and characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls☆

Abstract A minor form of hepatic microsomal cytochrome P -450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P -450 e , is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P -450 b and P -450 c , have also been highly purified during the isolation of cytochrome P -450 e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P -450 b , highly purified cytochrome P -450 e is immunochemically identical to cytochrome P -450 b but does not cross-react with antibodies prepared against other rat liver cytochromes P -450 ( P -450 a , P -450 c , P -450 d ) or epoxide hydrolase. Purified cytochrome P -450 e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P -450 b or P -450 d (52,000) but clearly distinct from cytochromes P -450 a (48,000) and P -450 c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P -450 e is at 450.6 nm, whereas the peak of cytochrome P -450 b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P -450 e and P -450 b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P -450 b and P -450 e , respectively. Metyrapone binds to reduced cytochromes P -450 e and P -450 b (absorption maximum at 445–446 nm) but not cytochromes P -450 a , P -450 c , or P -450 d . Metabolism of several substrates catalyzed by cytochrome P -450 e or P -450 b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P -450 e usually paralleled that of cytochrome P -450 b except that the rate of metabolism of benzphetamine, benzo[ a ]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P -450 e was only 15–25% that of cytochrome P -450 b . In contrast, cytochrome P -450 e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P -450 b . Cytochrome P -450 d , however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P -450 a , P -450 b , P -450 c , or P -450 e . The peptide fragments of cytochromes P -450 e and P -450 b , generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of Salmonella typhimurium and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of S. typhimurium and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of S. typhimurium . The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17-18, 37-46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (greater than 90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.