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W Levin

Publications and source records attributed to W Levin.

At least 91 records · Page 5Linked to original sources
PubMed1987

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes.

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.

Animals↗
PubMed1987

A regulatory polymorphism for rat hepatic cytochrome P-450g.

Rat hepatic cytochrome P-450g is a male-specific hemoprotein found at significant levels only in adult animals. In the present study, two-dimensional gel electrophoretic and immunochemical methods were used to study a polymorphism of this isozyme and its ontogenetic regulation. Inbred ACI/Hsd and WF/Hsd rats were found to express high and low levels of cytochrome P-450g, respectively. F1 hybrids of these strains showed additive inheritance for this trait and the responsible gene was found to be autosomal. Cytochrome P-450g and another male-specific form of the enzyme, cytochrome P-450h, were characterized by a similar time-course for their ontogenetic expressions. However, unlike cytochrome P-450g, the level of cytochrome P-450h was indistinguishable in hepatic microsomes from mature ACI/Hsd and WF/Hsd rats. Considering these results, we tentatively conclude that the gene regulating the level of cytochrome P-450g is Cis-acting.

Aging↗
PubMed1987

Purification and characterization of ethanol-inducible human hepatic cytochrome P-450HLj.

Human hepatic cytochrome P-450HLj was purified in a catalytically active state from microsomes obtained from an ethanol-intoxicated man. The electrophoretically homogeneous preparation of HLj was compared to rat P-450j and found to have a slightly different apparent molecular mass (54 vs 51.5 kDa) but highly similar immunochemical, spectral, and catalytic properties. Purified HLj exhibited high activity toward N-nitro-sodimethylamine (NDMA) and aniline metabolism, low but measurable activity toward benzphetamine and 7-ethoxycoumarin, and no detectable activity toward benzo[a]pyrene, testosterone, and progesterone. Antibody against rat P-450j reacted with HLj in immunoblot analyses and, when added directly to HLj before reconstitution with NADPH-cytochrome P-450 reductase and lipid, the antibody inhibited (96%) NDMA metabolism by HLj almost completely. However, if HLj was reconstituted with the other components before the addition of the anti-P-450j IgG, the ability of the antibody to inhibit the metabolism of NDMA was greatly diminished. This suggests that the interactions between reductase and HLj are similar to those previously observed between rat P-450j and reductase, and appear to prevent the complete access of anti-P-450j. The addition of cytochrome b5 to reconstitution systems containing HLj resulted in a small increase in the Vmax from NDMA demethylation accompanied by a decrease in Km,app (1.3 to 0.3 mM) as has been observed in reconstitution systems with rat P-450j. Therefore, in reconstituted systems, cytochrome b5 appears to play an important role in the biotransformations mediated by HLj and P-450j. In conclusion, this study demonstrates that HLj is functionally related to ethanol-inducible rat P-450j and rabbit LM3a.

Amino Acid Sequence↗
PubMed1987

Role of diaxial versus diequatorial hydroxyl groups in the tumorigenic activity of a benzo[a]pyrene bay-region diol epoxide.

Tumorigenic activities of the (7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro derivatives of benzo[a]pyrene [(+)-B[a]P diol epoxide-2] and 6-fluorobenzo[a]pyrene (6-FB[a]P diol epoxide-2) were evaluated in newborn CD-1 mice. A total dose of 14 nmol of either diol epoxide was administered to preweanling mice, and tumorigenic activity was determined when the mice were 32 to 36 weeks old. At the termination of the study, 13% of solvent-treated control mice had developed lung tumors with an average of 0.19 tumor per mouse. No other tumors were observed in control animals. (+)-B[a]P diol epoxide-2 induced pulmonary tumors in 60% of the mice with an average of 1.9 tumors per mouse, and 14% of the male mice developed hepatic tumors with an average of 0.18 tumor per mouse. In contrast, 6-FB[a]P diol epoxide-2 had no significant tumorigenic activity at the 14-nmol dose. Although both bay-region diol epoxides have the same absolute configuration, (7R,8S,9S,10R), the hydroxyl groups of (+)-B[a]P diol epoxide-2 prefer the pseudoequatorial conformation whereas the hydroxyl groups of 6-FB[a]P diol epoxide-2 prefer the pseudoaxial conformation. The tumorigenicity results reported here are the first direct demonstration that conformation of the hydroxyl groups in a bay-region diol epoxide, in addition to the documented effect of absolute configuration, is an important determinant in the tumorigenic activity of these ultimate carcinogens.

Animals↗
PubMed1987

The P450 gene superfamily: recommended nomenclature.

A nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1-2 years. Assignment of orthologous genes is presently uncertain in some cases--between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowledge.

Alleles↗
PubMed1987

Purification of a human liver cytochrome P-450 immunochemically related to several cytochromes P-450 purified from untreated rats.

Among characterized forms of liver microsomal cytochromes P-450 in rats are four related isozymes (P-450f-i) notable for their lack of inducibility. Immunoblot analyses demonstrated that human livers microsomes contained several proteins related to these rat P-450s. A human liver P-450, termed HLx, was purified and found by immunochemical assays to resemble rat P-450g. Analysis of the NH2-terminal amino acid sequence of HLx indicates that it is related to rat P-450s f-i and human liver P-450MP. A monoclonal antibody was used to measure the amounts of HLx in 21 human liver specimens. No correlation between the levels of HLx protein in these specimens and the patients' environmental histories was observed. However, statistical analysis of the data suggests that the distribution of HLx is at least bimodal. We conclude that HLx is a member of a family of human liver P-450s that resembles in its structure, and possibly in its distribution, several liver P-450s found in other animals.

Adult↗
PubMed1987

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.

Animals↗
PubMed1987

Co-induction of cytochrome P-450 isozymes in rat liver by 2,4,5,2',4',5'-hexachlorobiphenyl or 3-methoxy-4-aminoazobenzene.

A multitude of xenobiotics have been demonstrated to co-induce either cytochromes P-450c and P-450d or cytochromes P-450b and P-450e in rat hepatic microsomes. Recently, the compounds 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) and 3-methoxy-4-aminoazobenzene (3-MeO-AAB) have been suggested as selective inducers of cytochrome P-450b (Eur. J. Biochem. 151:67 (1985)) and P-450d (Biochem. Biophys. Res. Commun. 133:1072 (1985)), respectively. Since the identification of inducers with such unique characteristics would have implications with regard to the mechanism of induction of all four isozymes, we have examined the induction of cytochromes P-450b and P-450e by HCB and cytochromes P-450c and P-450d by 3-MeO-AAB in liver microsomes from adult male rats. Immunoblot analysis with monoclonal antibodies directed against cytochromes P-450b and P-450e indicate that HCB induces both isozymic species at the three dosage levels examined (10, 90, and 180 mg/kg). Similarly, 3-MeO-AAB does not appear to represent a unique inducer. Immunoblots of hepatic microsomes from animals treated with three different dosage regimens of 3-MeO-AAB demonstrate that, even at the lowest dosage level (50 mg/kg), both cytochromes P-450c and P-450d are induced. Moreover, immunoinhibition of 7-ethoxyresorufin O-deethylase (EROD) activity by monospecific antibody against either cytochrome P-450c or P-450d confirms this result. 3-MeO-AAB increases this enzyme activity 10-fold; approximately one-third of this induced activity is inhibited with monospecific anti-P-450c, while two-thirds is inhibited with monospecific anti-P-450d. This study also demonstrates that hepatic EROD activity is not an accurate estimate of cytochrome P-450c content since the majority of this enzyme activity in control and 3-MeO-AAB-treated rats is inhibited with monospecific anti-P-450d but not with monospecific anti-P-450c.

Animals↗
PubMed1987

Computers in the cognitive rehabilitation of brain-injured persons.

Currently there is a rapid expansion of work among rehabilitation professionals in applying computer-assisted instructional technology to remediate cognitive deficits resulting from brain injury. The present article presents a framework for relating the various theoretical, empirical, and clinical challenges raised by the coalescence of two such recently emerging disciplines as computer-assisted instruction and cognitive rehabilitation. These challenges are presented from the perspectives of diverse disciplines, including cognitive science, rehabilitation professions, and computer science. A set of guiding principles are derived for evaluating the potential efficacy of currently existing programs and for directing future developmental work in software design, evaluation research, and service delivery.

Animals↗
PubMed1986

Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase.

Through the use of monospecific antibodies directed against hepatic cytochrome P-450j, an enzyme induced in rats treated with ethanol or isoniazid, we have purified from human liver the related cytochrome P-450 termed HLj. HLj resembles rat P-450j and P-450 LM3a, the homologous cytochrome in rabbit liver, in its NH2-terminal amino acid sequence, in being in highest concentration in liver microsome samples prepared from two patients intoxicated by ethanol and one patient given isoniazid, and in catalyzing the metabolic activation of the procarcinogen N-nitrosodimethylamine. Furthermore, each of nine human liver RNA samples contained a species of mRNA hybridizable to a cloned HLj cDNA. We conclude that HLj is related by structure, function, and some regulatory characteristics to rat P-450j and rabbit P-450 LM3a, cytochromes critical for metabolism of several clinically relevant cytotoxic and carcinogenic agents.

Adult↗
PubMed1986

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone.

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.

Acetophenones↗
PubMed1986

Chemical modification and inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone.

The alkylating agent 2-bromo-4'-nitroacetophenone (BrNAP) binds covalently to each of 10 isozymes of purified rat liver microsomal cytochrome P-450 (P-450a-P-450j) but substantially inhibits the catalytic activity of only cytochrome P-450c. Regardless of pH, incubation time, presence of detergents, or concentration of BrNAP, treatment of cytochrome P-450c with BrNAP resulted in no more than 90% inhibition of catalytic activity. Alkylation with BrNAP did not cause the release of heme from the holoenzyme or alter the spectral properties of cytochrome P-450c, data that exclude the putative heme-binding cysteine, Cys-460, as the major site of alkylation. Two residues in cytochrome P-450c reacted rapidly with BrNAP, for which reason maximal loss of catalytic activity was invariably associated with the incorporation of approximately 1.5 mol of BrNAP/mol of cytochrome P-450c. Two major radio-labeled peptides were isolated from a tryptic digest of [14C]BrNAP-treated cytochrome P-450c by reverse-phase high performance liquid chromatography. The amino acid sequence of each peptide was determined by microsequence analysis, but the identification of the residues alkylated by BrNAP was complicated by the tendency of the adducts to decompose when subjected to automated Edman degradation. However, results of competitive binding experiments with the sulfhydryl reagent 4,4'-dithiodipyridine identified Cys-292 as the major site of alkylation and Cys-160 as the minor site of alkylation by BrNAP in cytochrome P-450c.

Acetophenones↗
PubMed1986

Age- and sex-related expression of cytochromes p450f and P450g in rat liver.

We have previously shown that rat hepatic cytochromes P450f, P450g, P450h, and P450i possess a high degree of immunochemical and, presumably, structural relatedness. Polyclonal antibodies directed against cytochromes P450f and P450g were made monospecific by immunoabsorption against the cross-reactive proteins. The specificity of the immunoabsorbed antibodies was established by using Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays (ELISA), and immunoblots. Since factors regulating the expression of cytochromes P450f and P450g are unknown, a competitive ELISA employing the monospecific antibodies was developed to quantitate each of these isozymes in hepatic microsomes from control and treated rats. The results obtained showed that expression of cytochrome P450f is developmentally regulated in both male and female rat liver. Cytochrome P450f levels rise from less than 1% in young animals to approximately 7 and 14% of total cytochrome P450 in adult male and female rats, respectively. Cytochrome P450g is sex-specific since it is expressed only in male rat liver where it also is developmentally regulated. Levels of cytochrome P450g rise from less than 1% in 3-week-old male rats to an average value of 17% of total cytochrome P450 in 6-week-old adult animals. However, there appear to be at least two subpopulations of adult male Long Evans rats, one of which expresses low levels (less than 1%) of cytochrome P450g and the other high levels (greater than or equal to 10%). This expression appears to be independent of serum testosterone levels. Treatment of immature and adult male rats with 20 xenobiotics that are known inducers of certain cytochrome P450 isozymes revealed that cytochromes P450f and P450g are relatively refractory to induction, although Kepone appears to be a weak inducer of cytochrome P450f.

Age Factors↗
PubMed1986

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.

1-Butanol↗
PubMed1986

Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system.

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.

Animals↗
PubMed1986

7-Dehydrocholesterol 5,6 beta-oxide as a mechanism-based inhibitor of microsomal cholesterol oxide hydrolase.

7-Dehydrocholesterol 5,6 beta-oxide covalently modifies and inactivates the rat liver microsomal enzyme cholesterol oxide hydrolase. The covalent modification is presumed to occur at the active site of the enzyme since 5,6 alpha-iminocholestanol, a potent competitive inhibitor of the enzyme, blocks incorporation of 3-[3H]-7-dehydrocholesterol 5,6 beta-oxide into the protein. Kinetics of the inactivation were measured both by following the loss of catalytic activity and by monitoring incorporation of 3-[3H]-7-dehydrocholesterol 5,6 beta-oxide into microsomal protein. Both the loss of catalytic activity and the incorporation of label followed first order kinetics. Linear plots of the reciprocal of the pseudo-first order rate constants for the loss of catalytic activity and for the incorporation of radioactivity versus reciprocal of inhibitor concentrations indicated saturation kinetics. The kinetic parameter kinac is found to be (2.83 +/- 0.43)10(-3) s-1 measured either by incorporation of tritium (300 mM potassium phosphate buffer, pH 8.0, 2.4 mg of microsomal protein/ml at 37 degrees C) or by the loss of catalytic activity (300 mM potassium phosphate buffer, pH 7.5, 0.99 mg of microsomal protein/ml at 37 degrees C). Unlike xenobiotic microsomal epoxide hydrolase (EC 3.3.2.3) which is not inactivated or inhibited by 7-dehydrocholesterol 5,6 beta-oxide, cholesterol oxide hydrolase appears to hydrolyze cholesterol oxides via a positively charged transition state.

Animals↗