Dene E. Ryan

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.

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.

Highly purified cytochrome P-448 and P-450 from rat liver microsomes

Abstract Cytochrome P-448 from 3-methylcholanthrene-treated rats has been purified to a specific content of greater than 20 nmoles/mg protein, and cytochrome P-450 from phenobarbital-treated rats to greater than 17 nmoles/mg protein. Both cytochromes are catalytically active when reconstituted with lipid and NADPH-cytochrome c reductase and exhibit differential substrate specificities for benzphetamine and benzo[a]pyrene. Cytochrome P-448 has a minimum molecular weight of approximately 53,000, and cytochrome P-450, 48,000 by SDS polyacrylamide gel electrophoresis.

Preparation of monospecific antibodies against two forms of rat liver cytochrome P-450 and quantitation of these antigens in microsomes.

Abstract Antibodies prepared against purified cytochrome P -450 and P -448 from phenobarbital- and 3-methylcholanthrene-pretreated rats have been shown to recognize several forms of hepatic cytochrome P -450 ( P. E. Thomas, A. Y. H. Lu, D. Ryan, S. B. West, J. Kawalek, and W. Levin, 1976, Mol. Pharmacol. 12 , 746–758 ). These antibodies have been made monospecific for a single form of cytochrome P -450 by immunoadsorption with partially purified solid-phase cytochrome P -450 from rats treated with a different inducer than that used for isolation of the antigen. Each monospecific antibody did not react with different forms of cytochrome P -450 present in the heterologous antigen preparation. These monospecific antibodies, covalently bound to Sepharose, were used to purify the antigens (catalytically inactive) from microsomes in a single step. The high specificity of these antibodies for a single form of cytochrome P -450 was used to quantitate two forms of cytochrome P -450 in rat liver microsomes by radial immunodiffusion. The percentage of the total cytochrome P -450 in microsomes that is represented by each of these two forms of cytochrome P -450 varied from 3 to 89% depending on the xenobiotic pretreatment of the rats.

Mutagenicity of metabolically activated benzo[a]anthracene 3,4-dihydrodiol: evidence for bay region activation of carcinogenic polycyclic hydrocarbons.

Summary Benzo[a]anthracena and the 5 metabolically possible trans dihydrodiols of benzo[a]anthracene were metabolized, in the presence of S. thyphimurium strain TA100, by a highly purified hepatic microsomal monooxygenase system. The metabolic product(s) of benzo[a]anthracene 3,4-dihydrodiol was nearly 10 times as mutagenic to the bacteria as were the metabolites of benzo[a]anthracene and the other four dihydrodiols. The marked activation of benzo[a]anthracene 3,4-dihydrodiol, presumably to the 3,4-diol-1,2-epoxide is consistent with and supports the hypothesis that bay region epoxides of unsubstituted polycyclic hydrocarbons are ultimately reactive forms of these carcinogenic compounds.

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-4501

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).

High mutagenicity of metabolically activated chrysene 1,2 dihydrodiol: Evidence for bay region activation of chrysene

Abstract Chrysene and the 3 metabolically possible vicinal trans dihydrodiols of chrysene were tested for mutagenicity towards S. typhimurium strain TA100 in the presence of hepatic microsomes or a highly purified hepatic microsomal monooxygenase system. The products formed during the metabolic activation of chrysene 1,2-dihydrodiol were more than 20 times as mutagenic to the bacteria than the metabolites formed from chrysene, chrysene 3,4-dihydrodiol or chrysene 5,6-dihydrodiol. When the double bond in the 3,4-position of chrysene 1,2-dihydrodiol was saturated, the resulting tetrahydrodiol could not be metabolically activated. These results, which strongly suggest that chrysene 1,2-dihydrodiol is activated by metabolism to either or both of the diastereomeric chrysene 1,2-diol-3,4-epoxides, provide additional support for the bay region theory of polycyclic hydrocarbon carcinogenicity.