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PubMed1978

Drug metabolism in the Novikoff hepatoma: evidence for a mixed function oxidase system and partial purification of cytochrome P-450 reductase.

Novikoff kepatoma microsomes catalyze the hydroxylation of benzphetamine and ethylmorphine at rates less than 1% of those of liver microsomes but catalyze the hydroxylation of p-nitroanisole and p-nitrophenetole at rates about 40% of those of liver microsomes. Benzo[a]pyrene hydroxylation is also catalyzed by Novikoff hepatoma microsomes at about 2% of the rate of liver microsomes. Like the hepatic microsomal system the rates of substrate hydroxylation by Novikoff hepatoma microsomes can be increased by pretreatment with phenobarbital/hydrocortisone or beta-naphthoflavone and inhibited by carbon monoxide, SKF-525A, and 7,8-benzoflavone. In addition, NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) has been partially purified from Novikoff hepatoma ascites cells and some properties are described. The induction and inhibition characteristics of the Novikoff hepatoma microsomal hydroxylation activities and the isolation of a cytochrome P-450 reductase from the hepatoma are consistent with the presence of a functional mixed function oxidase system in the Novikoff hepatoma, analogous to that present in liver endoplasmic reticulum.

Animals↗

PubMed1979

Mutagenicity and irreversible binding of the hepatocarcinogen, 2,4-diaminotoluene.

Mutagenicity of 2,4-diaminotoluene (DAT) in the Salmonella mutagenicity assay was increased with liver fractions from phenobarbital (PB) or beta-naphthoflavone (BNF) treated rats. Substitutions of the hydrogens in the methyl group of 2,4-DAT with deuterium resulted in a decrease in mutagenicity. Incubation of rat liver microsomes with tritiated 2,4-DAT in the presence of NADPH led to the formation of irreversibly bound products to microsomal protein. The rates of binding were not increased using microsomes from PB or BNF-treated rats and was not altered by deuterium substitution in the methyl group. Addition of superoxide dismutase, glutathione (GSH) or rat liver supernatant reduced 2,4-DAT irreversible binding, whereas 2,4-DAT mutagenicity was unaffected by superoxide dismutase addition. Injection of tritiated 2,4-DAT 100 mg/kg to rats lead to its irreversible binding to liver protein and ribosomal RNA and to kidney protein in vivo, again protein binding was not increased after prior treatment with PB or BNF. No irreversible interaction of tritiated 2,4-DAT with DNA either in vitro or in vivo could be demonstrated.

Animals↗

PubMed1979

The development of a "Microtitre" fluctuation test for the detection of indirect mutagens, and its use in the evaluation of mixed enzyme induction of the liver.

The "Microtitre" Fluctuation test recently introduced for the detection of direct mutagens has been adapted for the detection of indirect mutagens through the incorporation of an "S9-mix" metabolic system. It compares favourably with Greens' original method for the detection of a range of chemical mutagens. The technique has been employed in the evaluation of mixed enzyme induction using phenobarbitone and beta-naphthoflavone (benzoflavone), as a safe substitute for Aroclor-1254. The post-mitchondrial preparations from rats induced with the combined inducers had a similar "metabolic competence" to those derived from Aroclor induced animals. Such a combination would therefore provide a useful alternative to Aroclor-1254 for routine screening. It was found that the level of "S9" present in the metabolic system greatly affected the quantitative mutagenic response. This varied considerably from chemical to chemical and underlined the need for such preliminary investigations in routine screening.

Animals↗

PubMed1978

Induction of multiple cytochrome P-450 species in housefly microsomes--SDS-gel electrophoresis studies.

1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains.

Animals↗

PubMed1979

Multiplicity of induction patterns of rat liver microsomal mono-oxygenases and other polypeptides produced by administration of various xenobiotics.

Induction of hepatic microsomal mono-oxygenase species after administration of various xenobiotics is a well-documented phenomenon. To examine the number and specific species of rat liver microsomal membrane polypeptides involved in such responses, we have used sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to analyse microsomal fractions from animals treated with a number of important xenobiotics. The following are the principal points to have emerged from this study. 1. A minimum of twelve electrophoretically distinct patterns of induction of haemopolypeptides and other polypeptides could be distinguished after administration, either singly or in certain combinations, of phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls, 2-acetylaminofluorene, safrole (or isosafrole), pregnenolone-16 alpha-carbonitrile and ethanol. The patterns consisted of various permutations of the amounts of eight polypeptides of 47000-56000 mol.wt., of which at least three were haemopolypeptides. The possible identities of these polypeptides, which included species of cytochrome P-450, cytochrome P-448 and epoxide hydratase, are discussed. 2. Agents (3-methylcholanthrene, benzo[a]-pyrene, polychlorinated biphenyls, 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone) that result in the induction of cytochrome P-448 caused a marked increase in two polypeptides of 54000 and 56000 mol.wt., whereas safrole and isosafrole induced only the former polypeptide. 3. Administration of 2-acetylaminofluorene resulted in the induction of two polypeptides; evidence is presented that suggests that one of these is a species of epoxide hydratase [cf. Levin, Lu, Thomas, Ryan, Kizer & Griffin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3240-3243] ANd that the other may be a novel haemopolypeptide. 4. The overall results emphasize the complexity of the responses exhibited by rat liver microsomal fractions to the administration of xenobiotics.

2-Acetylaminofluorene↗

PubMed1979

Pyrolysis products from amino acids and protein: highest mutagenicity requires cytochrome P1-450.

Pyrolysis products of proteins and amino acids are highly mutagenic, but metabolism of these chemicals by rat liver subcellular fractions is known to be required for production of the mutagenic intermediates. We examined the mutagenesis of seven purified pyrolysis products from tryptophan, lysine, glutamic acid, and soybean globulin with Salmonella typhimurium strain TA98 in the presence of liver fractions from genetically "responsive" C57BL/6N and Ah(b)/Ah(d) or "nonresponsive" DBA/2N and Ah(d)/Ah(d) mice that had been pretreated in vivo with benzo[a]pyrene. For all pyrolysis products tested, mutagenesis is 2-fold to more than 1000-fold greater with C57BL/6N and Ah(b)/Ah(d) than with DBA/2N or Ah(d)/Ah(d) liver fractions. A sucrose density gradient assay for detecting the Ah regulatory gene product, the receptor, was studied with C57BL/6N hepatic cytosol. At levels 100 times in excess of [1,6-(3)H]2,3,7,8-tetrachlorodibenzo-p-dioxin, nonlabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, and beta-naphthoflavone (inducers of cytochrome P(1)-450) are able to displace the radioligand from its hepatic cytosolic receptor; four pyrolysates from tryptophan, glutamic acid, and soybean globulin did not have this capacity. These data indicate that the pyrolysis products tested, although not effective as inducers of cytochrome P(1)-450, are most mutagenic when metabolized by P(1)-450. Potent P(1)-450 inducers-present in pyrolysates during the combustion process-might be present in quantities insufficient to initiate mutagenesis or carcinogenesis but might have a synergistic action, or act as "comutagens" or "cocarcinogens," with the N-containing heterocyclic pyrolysis products. A quantitative relationship between mutagenic and carcinogenic potency of these pyrolysis products remains, however, to be demonstrated.

Amino Acids↗

PubMed1976

Dimethylnitrosamine-demethylase: molecular size-dependence of repression by polynuclear hydrocarbons. Nonhydrocarbon repressors.

Studies with 58 polynuclear aromatic hydrocarbons have shown that to repress demethylation of dimethylnitrosamine (DMN) in rat liver, the hydrocarbons must satisfy specific requirements of molecular geometry regarding size, shape, and coplanarity. Expressing the molecular size of these planar compounds by the two-dimensional area occupied, the size for maximal repressor activity ranges between about 85 and 150 A2. In addition to being within the correct molecular size range the hydrocarbons must have an elongated-rather than compact-molecular shape; circularly shaped and/or highly symmetrical hydrocarbons, such as coronene, triphenylene, ovalene, and tetrabenzonaphthalene, have very low activity or are inactive, in spite of being in the optimum size range. Coplanarity of the molecule is a critical requirement; thus, the potent carcinogen, 9,10-dimethyl-1,2-benzanthracene, is inactive as repressor of DMN-demethylase synthesis. Two exceptions, fluoranthene and benzol[ghi] fluoranthene, showed significant induction of DMN-demethylase. The molecular size distribution of hydrocarbons that repress the DMN-demethylase shows a mirror-image relationship with respect to the earlier reported molecular size requirement for indcution of azo dye N-demethylase. Compounds other than hydrocarbons also show the mirror-image relationship in the sense that pregnenolene-16alpha-carbonitrile, alpha- and beta-naphthoflavone, and Aroclor 1254 (known to be inducers of various mixed-function oxidases) are strong repressors of DMN-demethylase. Aminoacetonitrile, a strong inhibitor of carcinogenesis by DMN, is also a potent repressor of DMN-demethylase. The enzyme is inhibited by pretreatment of the animals with cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450. Pregnenolone-16alpha-carbonitrile and 3-methylcholanthrene, despite their similarity of action on DMN-demethylase, have different effects on azo reductase, which is repressed by the former and induced by the latter compound.

Animals↗

PubMed1978

Quinoline: conversion to a mutagen by human and rodent liver.

Quinoline, a hepatocarcinogen in rats, and 23 quinoline derivatives were tested for mutagenic activity with the Ames Salmonella typhimurium assay. Quinoline, 5-hydroxyquinoline, and 8-hydroxyquinoline were mutagenic in strain TA 100 when Aroclor 1254-induced rat (male outbred Sprague-Dawley) liver homogenate was present in the incubation mixture. Enzyme preparations from rats pretreated with P-448-dependent aryl hydrocarbon hydroxylase inducers [3-methylcholanthrene (MCA) and beta-naphthoflavone] and MCA-treated "responsive" C57BL mice also metabolized quinoline to a mutagen, but phenobarbital and pregnenolone-16alpha-carbonitrile pretreatment did not yield active preparations. The mutagenicity of quinoline was blocked by the in vitro addition of menadione, butylated hydroxytoluene, alpha-naphthoflavone, vitamin A acetate, and glutathione to the test system. Depletion of glutathione by diethyl maleate pretreatment in vivo enhanced the mutagenic potential of the liver enzyme preparation. Mutagenic activity was correlated to the formation of water-soluble quinoline metabolites, and we suggested that the reactive quinoline intermediate is quinoline-2,3-epoxide. Microsomal enzymes isolated from human liver tissue, but not lung tissue, also converted quinoline to a mutagen.

Animals↗

PubMed1978

Characterization of electron transport enzymes in the envelope of rat liver nuclei.

Nuclei and microsomes were prepared from the livers of normal, phenobarbital (PB)-treated and beta-naphthoflavone (beta-NF)-treated rats, and the contents of several enzymes in both subcellular fractions were examined. In normal rats, the enzyme activities in the nuclear fraction were about one-third of those of microsomes on a phospholipid basis. The induction of some particular enzymes by the drugs was observed with nuclei as well as with microsomes. Cytochrome P-450 and NADPH-cytochrome c reductase were increased by PB treatment and cytochrome P-448 was induced by beta-NF treatment both in nuclei and in microsomes. The extents of inhibition of nuclear enzyme activities by the antibodies against corresponding microsomal enzymes were almost the same as those of the microsomal activities. It was concluded that a microsomal type electron transport system exists in rat liver nuclei, and that nuclear drug-oxidizing activities are inducible by PB or beta-NF as their microsomal counterparts are.

Animals↗

PubMed1978

Effect of polycyclic aromatic hydrocarbons on hepatic microsomal enzymes and disposition of methylnaphthalene in rainbow trout in vivo.

1. The effects of 3-methylcholanthrene, 2,3-benzanthracene and beta-naphthoflavone on xenobiotic metabolism in rainbow trout were studied. 2. These three polycyclic aromatic hydrocarbons increase hepatic arylhydrocarbon (benzo[alpha]pyrene) hydroxylase activity without altering glucuronyl transferase activity. 3. All three polycyclic aromatic hydrocarbons increased hepatic microsomal cytochrome P-450 levels by approximately 50%. 4. Pretreatment of trout with 2,3-benzanthracene resulted in an increase in the metabolism and biliary excretion of 2-methylnaphthalene in vivo. 5. These studies demonstrate that the induction of mono-oxygenation by polycyclic aromatic hydrocarbons can result in significant effects upon the metabolism and excretion of xenobiotics by fish in vivo.

Animals↗

PubMed1979

Enhancing effect of inducers of liver microsomal enzymes on induction of hyperplastic liver nodules by N-2-fluorenylacetamide in rats.

The effects of inducers of liver microsomal enzymes on the induction of hyperplastic liver nodules by N-2-fluorenylacetamide (2-FAA) were studied in male F344 rats. The rats were fed a diet containing 200 ppm 2-FAA for 2 weeks and then given test chemicals for the following 8 weeks. Partial hepatectomies were performed at the end of the third week of the experiment. The compounds were tested, and their concentrations (in ppm) in the diet were as follows: 1,000 polychlorinated biphenyls (PCB), 500 PCB, 500 PCB plus 70 3-methylcholanthrene (MCA); 500 PCB plus 500 phenobarbital, 70 MCA, 500 phenobarbital, 500 phenobarbital plus 500 beta-naphthoflavone, and 2,500 3-(3,5-dichlorophenyl)-5,5-dimethyloxazoline-dione-2,4 (DDOD). All experimental groups developed significantly greater numbers and total areas of hyperplastic nodules than did the controls. In groups treated similarly with the test chemicals but without partial hepatectomy, the numbers and total areas of hyperplastic nodules were significantly less than those in the experimental groups with partial hepatectomy. Administration of test chemicals except 2-FAA to partially hepatectomized rats did not induce hyperplastic nodules. The present results showed the early detection of the enhancing effect on induction of hyperplastic liver nodules by a system consisting of the following three procedures: 1) 2-FAA feeding; 2) administration of test chemicals, and 3) partial hepatectomy during administration of test chemicals. In this system DDOD enhanced the induction of hyperplastic nodules of the liver.

2-Acetylaminofluorene↗

PubMed1976

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.

Amino Acid Sequence↗

PubMed1979

Induction, inhibition, and biological properties of aryl hydrocarbon hydroxylase in a stable human B-lymphocyte cell line, RPMI-1788.

Aryl hydrocarbon hydroxylase was induced in the absence of mitogens by several compounds in a stable, human B-lymphocyte cell line (RPMI-1788). Over the dose ranges tested and on molar basis the inducers, in decreasing order of potency, were 2,3,7,8-tetrachlorodibenzo-p-dioxin, dibenz(a,h)-anthracene, 3-methylcholanthrene, benzo(a)pyrene, and 1,2-benzanthracene. Potential inducers which, paradoxically, diminished basal aryl hydrocarbon hydroxylase, included 7,12-dimethylbenzanthracene, 2,5-diphenyloxazole, and chyrsene. Induction under optimal culture conditions ensured maximal activities 3- to 4 fold above basal aryl hydrocarbon hydroxylase. The characteristics of the induced [dibenz(a,h)anthracene] and basal enzymes were found virtually identical. Both had similar pH curves (optima at 8.25) and inhibitor specificity (alpha- and beta-naphthoflavones, metyrapone, and 2-diethylaminoethyl-2,2-diphenylvalerate in decreasing potency). Induced and basal enzymes exhibited similar half-lives (41, 46 hr), apparent activation energies (16.7, 16.6 kcal/mol), temperature optima (37-38, 38-39 degrees), temperature-dependence of denaturation (range, 42-50 degrees), and apparent Km's with benzo(a)pyrene (1.8, 0.8 microM). The small difference in the apparent Km was related to enzyme concentration in the incubation rather than to the quality of the enzyme.

Aryl Hydrocarbon Hydroxylases↗

PubMed1978

Activation of carcinogens and mutagens by rat colon mucosa.

Colon mucosal cells can catalyze the activation of precarcinogens to mutagenic metabolites without the intermediacy of intestinal bacteria as shown in a mutagenesis assay system composed of Salmonella typhimurium strain TA100 and the 9000 X g supernatant fraction of rat colon mucosal cells. Pretreatment of rats with beta-naphtoflavone increased the activation of 2-aminoanthracene 10- to 20-fold and the activation of benzo(a)pyrene 4-fold. Pretreatment of rats with Aroclor 1254 doubled the activation of 2-aminoanthracene over control but had no effect on the activation of benzo(a)pyrene. The activation of 2-aminoanthracene and benzo(a)pyrene by liver was induced significantly by pretreatment with beta-naphthoflavone and Aroclor 1254. Phenobarbital/hydrocortisone pretreatment did not increase the activation by the colon system of any precarcinogen tested but did increase the activation of 2-aminoanthracene, cyclophosphamide, and isophosphamide by the liver system. The activation of precarcinogens in the bacterial test system is directly correlated with the activities of the pretreated colon and liver preparations toward several drug and polycyclic hydrocarbon substrates assayed in vitro.

Animals↗

PubMed1979

Drug-mediated induction of cytochrome(s) P-450 and drug metabolism in cultured hepatocytes maintained in chemically defined medium.

Cytochrome P-450 was induced in monolayer cultures of chick embryo hepatocytes incubated in a chemically defined medium. Phenobarbital induced the de novo synthesis of a microsomal protein of Mr = 51,000, whereas beta-naphthoflavone induced the de novo synthesis of a microsomal protein of Mr = 55,000. The increases in these proteins were associated with differential increases in aminopyrine-N-demethylation and benzo-(a)pyrene hydroxylation.

Animals↗

PubMed1977

Genetic regulation of UDP-glucuronosyltransferase induction by polycyclic aromatic compounds in mice. Co-segregation with aryl hydrocarbon (benzo(alpha)pyrene) hydroxylase induction.

Induction of hepatic 4-methylumbelliferone UDP-glucuronosyltransferase (EC 2.4.1.17) by polycyclic aromatic compounds, such as 3-methylcholanthrene or beta-naphthoflavone, occurs in C57BL/6N, A/J, PL/J, C3HeB/FeJ, and BALB/cJ but not in DBA/2N, AU/SsJ, AKR/J, or RF/J inbred strains of mice. This pattern of five responsive and five nonresponsive mouse strains parallels that of the Ah locus, which controls the induction of aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (EC 1.14.14.2). Induction of the transferase is maximal in C57BL/6N mice with 200 mg of 3-methylcholanthrene/kg body weight; no induction occurs in nonresponsive DBA/2N mice even at a dose of 400 mg/kg. The rise of inducible transferase activity lags 1 or more days behind the rise of inducible hydroxylase activity and peaks 5 days after a single dose of 3-methylcholanthrene. In offspring from the appropriate backcrosses and intercross between C57BL/6N and DBA/2N parent strains, the genetic expression of 3-methylcholanthrene-inducible transferase activity is inherited as an additive (co-dominant) trait. This expression differs distinctly from that of the inducible hydroxylase activity, which is inherited almost exclusively as a single autosomal dominant trait in these same animals. The more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the transferase more than 3-fold in C57BL/6N mice and less than 2-fold in DBA/2N mice, whereas the hydroxylase is induced equally (about 8-fold) in both strains. A dose of 3-methylcholanthrene given 3 days after 2,3,7,8-tetrachlorodibenzo-p-dioxin, at a time when hydroxylase induction in both strains is very high, does not enhance the rise in inducible transferase activity seen in C57BL/6N or DBA/2N mice which have received 2,3,7,8-tetrachlorodibenzo-p-dioxin alone. These data indicate that (a) the inducibility of two metabolically coordinated membrane-bound enzyme activities may be regulated by a single genetic locus, and (b) although the hydroxylase can be fully induced in the nonresponsive DBA/2N strain by 2,3,7,8-tetrachlorodibenzo-p-dioxin prior to 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene, presumably present in the liver, are incapable of inducing further the transferase activity. The difference in sensitivity between 3-methylcholanthrene and the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin for both the hydroxylase and the transferase activities suggests the possibility of a common receptor in regulating both enzyme induction processes.

Age Factors↗

PubMed1977

Genetic differences in induction of cytosol reduced-NAD(P):menadione oxidoreductase and microsomal aryl hydrocarbon hydroxylase in the mouse.

The stimulation of reduced-NAD(P):menadione oxidoreductase (EC 1.6.99.2) activity in liver cytosol is highly correlated with the stimulation of hepatic microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) activity in 3-methylcholanthrene-, beta-naphthoflavone-, phenobarbital-, or pregnenolone-16alpha-carbonitrile-treated inbred C57BL/6N and DBA/2N mice and in eight other inbred strains treated with 3-methylcholanthrene. No oxidoreductase activity is detectable in mouse liver microsomes. Cytochrome c and 2,6-dichlorophenolindophenol are equally good electron acceptors for the oxidoreductase. There is no preferential in vitro inhibition of induced versus control oxidoreductase activities by either alpha-naphthoflavone or metyrapone. In 3-methylcholanthrene-treated F1 and F2 progeny and offspring from backcrosses between the F1 and either C57BL/6N or DBA/2N parent, however, there is not a strict correlation between induced or noninducible aryl hydrocarbon hydroxylase and oxidoreductase activities. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, at doses (80 mug kg-1) sufficiently high to induce the hydroxylase almost as well in DBA/2N as in C57BL/6N mice, induces the oxidoreductase about 3-fold in C57BL/6N and less than 50% in DBA/2N mice. All the data are consistent with an hypothesis that two loci (Ox-1 and Ox-2) regulate oxidoreductase induction by 3-methylcholanthrene, that one of the genes is linked to the Ah locus (with an estimated recombination frequency between 2% and 23%), and that the other gene is not linked to the Ah locus. These experimental data might be useful in the protein activator hypothesis of the Britten-Davidson model for gene regulation.

Animals↗

PubMed1992

Rat pulmonary microsomal cytochrome P-450 enzymes involved in the activation of procarcinogens.

Rat lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic specificities towards activation of several procarcinogens to genotoxic metabolites in Salmonella typhimurium TA1535/pSK1002. We first examined the roles of rat liver microsomal P-450 enzymes in the activation of benzo[a]pyrene and its 7,8-diol enantiomers to genotoxic products, and found that P-450 1A1 is a major catalyst for the activation of these potential procarcinogens in rat livers. Using lung microsomes isolated from rats treated with various P-450 inducers we obtained evidence that at least three P-450 enzymes are involved in the activation of several procarcinogens. Immunoinhibition studies support the view that benzo[a]pyrene and its 7,8-diol derivatives, other dihydrodiol derivatives of polycyclic aromatic hydrocarbons, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole are activated to genotoxins mainly by rat P-450 1A1, which is inducible in rat lungs by 5,6-benzoflavone and the polychlorinated biphenyl mixture Aroclor 1254. Activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline and 2-amino-3-methylimidazo[4,5-f]quinoline may be catalyzed by another P-450 enzyme because the activities were not induced by treatment with 5,6-benzoflavone or Aroclor 1254. The observation that both activities were inhibited by antibodies raised against P-450 1A2 and by 7,8-benzoflavone suggests a role for an enzyme of P-450 1A family, probably P-450 1A2, in rat lung microsomes. The activation of aflatoxin B1 and sterigmatocystin appears to be catalyzed by other P-450 enzyme(s) rather than the P-450 1A family as judged by the different responses of activities to the P-450 inducers and the specific antibodies in rat lung microsomes. Interestingly, lung microsomal activation of several procarcinogens was found to be suppressed in rats treated with isosafrole and pregnenolone 16 alpha-carbonitrile. Thus, the results support the roles of different P-450 enzymes in the activation of procarcinogens in rat lung microsomes.

Aflatoxins↗