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The Journal of Biological Chemistry Sep 1975A bacterial mutagenesis assay and genetic differences in microsomal CO-binding cytochromes were combined in vitro to evaluate the metabolic activation of several known...
A bacterial mutagenesis assay and genetic differences in microsomal CO-binding cytochromes were combined in vitro to evaluate the metabolic activation of several known carcinogens to frameshift mutagens. With the use of liver fractions from C57BL/6N and DBA/2N control mice and mice treated in vivo with 3-methylcholanthrene, beta-naphthoglavone, phenobarbital, or 2,3,7,,-tetrachlorodibenzo-p-dioxin, the in vitro mutagenicity of 3-methylcholanthrene, 6-aminochrysene, and 2-acetylaminofluorene --but not benzo[a]pyrene==is closely associated with the genetically mediated difference in both aromatic hydrocarbon-inducible aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity and new cytochrome P1-450 formation; such an association between 7,12-dimethylbenz[a]anthracene or benz[a]anthracene activation to mutagens in vitro and these genetic differences between C57BL/6N and DBA/2N mouse strains in uncertain. The Salmonella typhimurium histidine mutant TA1538 is more effective than tester strains TA1537 and TA1535 in the determination of 3-methylcholanthrene mutagenesis in vitro. The relationships between the histidine revertant rate as a function of both liver protein concentration per plate and mutagen concentration per plate are illustrated for 3-methylcholanthrene, benzo[a]pyrene, 6-aminochrysene, and 2-acetylaminofluorene. With the use of offspring from the appropriate genetic crosses, the aromatic hydrocarbon-inducible hydroxylase activity appears to be expressed as an autosomal dominant trait, whereas the mutagenesis of 3-methylcholanthrene in vitro appears to be expressed additively; this apparent discrepancy probably reflects different proportional amounts of phenolic benzo[a]pyrene, compared with mutagenic 3-methylcholanthrene metabolites, formed by the monooxygenase(s). 3-Methylcholanthrene, 6-aminochrysene, and 2-acetylaminofluorene--but not benzo[a]pyrene--are each more mutagenic in vitro per molecule of cytochrome P1-450 than per molecule of CO-binding cytochrome other than P1450. Diethylmaleate, a compound which depletes flutathione content in liver, and 1,1,1-trichloropropene-2,3-epoxide, an inhibitor of epoxide hydrase (EC 4.2.1.63), were also studied in vitro. Diethylmaleate, and especially 1,1,1-trichloropropene-2,3-epoxide, increases the mutagenicity of benzo[a]pyrene, whereas no increases occur with 3-methylcholanthrene, 6-aminochrysene, or 2-acetylaminofluorene activation to mutagens in vitro. Both diethylmaleate and 1,1,1-trichloropropene-2,3-epoxide cause decreases in 2-acetylaminofluorene mutagenesis in vitro when liver fractions from phenobarbital-treated mice are used.
Topics: 2-Acetylaminofluorene; 9,10-Dimethyl-1,2-benzanthracene; Animals; Benz(a)Anthracenes; Benzopyrenes; Carcinogens; Crosses, Genetic; Cytochrome P-450 Enzyme System; Female; Genes; Liver; Male; Methylcholanthrene; Mice; Mice, Inbred Strains; Mutagens; Oxygenases; Phenobarbital; Species Specificity
PubMed: 808546
DOI: No ID Found -
The Journal of Biological Chemistry Apr 1994The aromatic hydrocarbon (Ah) receptor is a cytosolic protein that binds halogenated ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and nonhalogenated...
Photoaffinity labeling of the Ah receptor with 3-[3H]methylcholanthrene and formation of a 165-kDa complex between the ligand-binding subunit and a novel cytosolic protein.
The aromatic hydrocarbon (Ah) receptor is a cytosolic protein that binds halogenated ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and nonhalogenated ligands such as 3-methylcholanthrene (MC) and benzo[a]pyrene. The best characterized biological response mediated by the Ah receptor is induction of cytochrome P4501A1 (CYP1A1). Photoaffinity labeling of the Ah receptor has been reported only with halogenated ligands such as TCDD and some of its iodinated derivatives. In this study, photolabeling of the Ah receptor was achieved with the nonhalogenated aromatic hydrocarbon [3H]MC. Sources of Ah receptor were the mouse hepatoma cell line Hepa-1c1c9 and the human colon adenocarcinoma line LS180. Cytosolic fractions either were used in a crude form or were enriched by glycerol density gradient centrifugation. These then were incubated with [3H]MC, irradiated with UV light (> 300 nm), precipitated with acetone, and analyzed by SDS-polyacrylamide gel electrophoresis. The yield of photoadduct formation was lower with [3H]MC (approximately 1%) compared with [3H]TCDD (3.5%) in Hepa-1c1c9 cells. The same was true in LS180 cells, i.e. the yield was 0.2% for [3H]MC versus 5.48 +/- 0.26% for [3H]TCDD. The relative molecular mass of the [3H]MC-labeled receptor estimated by SDS-polyacrylamide gel electrophoresis was 94,600 +/- 2,400 (mean +/- S.E.) for Hepa-1c1c9 cells and 113,600 +/- 3,200 for LS180 cells; these are the same molecular masses as determined by photolabeling with [3H]TCDD. In velocity sedimentation assays of mouse cytosol, [3H]MC binds specifically to two cytosolic proteins: the 4 S carcinogen-binding protein and the Ah receptor (9 S). However, no photolabeling of the 4 S protein was detected in our experiments. [3H]MC photolabeling of the human Ah receptor from LS180 cells was detected only in experiments using enriched cytosolic preparations. In addition to the 95-kDa ligand-binding subunit, a specifically radiolabeled protein of 164,900 +/- 5,800 kDa was also detected in Hepa-1c1c9 cytosol photolabeled with [3H]MC, suggesting cross-linking, by MC, of another subunit of the multimeric Ah receptor complex to the ligand-binding subunit. Immunochemical analysis showed that the ligand-binding subunit of the Ah receptor is one component of the 165-kDa complex. The other protein in the complex could not be identified with antibodies to the heat shock proteins hsp90 or hsp70 or with antibodies to the p59 protein or Ah receptor nuclear translocator protein. The identity and function of the protein that becomes cross-linked to the ligand-binding subunit require further investigation.
Topics: Affinity Labels; Animals; Cell Line; Cytochrome P-450 Enzyme System; Cytosol; Enzyme Induction; Kinetics; Liver Neoplasms, Experimental; Macromolecular Substances; Methylcholanthrene; Mice; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Tritium; Tumor Cells, Cultured
PubMed: 8163517
DOI: No ID Found -
The Biochemical Journal Aug 1978The fatty acid compositions of the lipids and the lipid peroxide concentrations and rates of lipid peroxidation were determined in suspensions of liver endoplasmic...
The fatty acid compositions of the lipids and the lipid peroxide concentrations and rates of lipid peroxidation were determined in suspensions of liver endoplasmic reticulum isolated from rats fed on synthetic diets in which the fatty acid composition had been varied but the remaining constituents (protein, carbohydrate, vitamins and minerals) kept constant. Stock diet and synthetic diets containing no fat, 10% corn oil, herring oil, coconut oil or lard were used. The fatty acid composition of the liver endoplasmic reticulum lipid was markedly dependent on the fatty acid composition of the dietary lipid. Feeding a herring-oil diet caused incorporation of 8.7% eicosapentaenoic acid (C(20:5)) and 17% docosahexaenoic acid (C(22:6)), but only 5.1% linoleic acid (C(18:2)) and 6.4% arachidonic acid (C(20:4)), feeding a corn-oil diet caused incorporation of 25.1% C(18:2), 17.8% C(20:4) and 2.5% C(22:6) fatty acids, and feeding a lard diet caused incorporation of 10.3% C(18:2), 13.5% C(20:4) and 4.3% C(22:6) fatty acids into the liver endoplasmic-reticulum lipids. Phenobarbitone injection (100mg/kg) decreased the incorporation of C(20:4) and C(22:6) fatty acids into the liver endoplasmic reticulum of rats fed on a lard, corn-oil or herring-oil diet. Microsomal lipid peroxide concentrations and rates of peroxidation in the presence of ascorbate depended on the nature and quantity of the polyunsaturated fatty acids in the diet. The lipid peroxide content was 1.82+/-0.30nmol of malonaldehyde/mg of protein and the rate of peroxidation was 0.60+/-0.08nmol of malonaldehyde/min per mg of protein after feeding a fat-free diet, and the values were increased to 20.80nmol of malonaldehyde/mg of protein and 3.73nmol of malonaldehyde/min per mg of protein after feeding a 10% herring-oil diet in which polyunsaturated fatty acids formed 24% of the total fatty acids. Addition of alpha-tocopherol to the diets (120mg/kg of diet) caused a very large decrease in the lipid peroxide concentration and rate of lipid peroxidation in the endoplasmic reticulum, but addition of the synthetic anti-oxidant 2,6-di-t-butyl-4-methylphenol to the diet (100mg/kg of diet) was ineffective. Treatment of the animals with phenobarbitone (1mg/ml of drinking water) caused a sharp fall in the rate of lipid peroxidation. It is concluded that the polyunsaturated fatty acid composition of the diet regulates the fatty acid composition of the liver endoplasmic reticulum, and this in turn is an important factor controlling the rate and extent of lipid peroxidation in vitro and possibly in vivo.
Topics: Animals; Antioxidants; Dietary Fats; Endoplasmic Reticulum; Fatty Acids; Lipid Metabolism; Liver; Male; Methylcholanthrene; Microsomes, Liver; Peroxides; Phenobarbital; Rats
PubMed: 708411
DOI: 10.1042/bj1740585 -
Journal of Biochemistry Dec 1987DT-Diaphorase purified from the liver cytosol of rats treated with a highly toxic PCB congener, 3,4,5,3',4'-pentachlorobiphenyl (PenCB), was compared to those from... (Comparative Study)
Comparative Study
DT-Diaphorase purified from the liver cytosol of rats treated with a highly toxic PCB congener, 3,4,5,3',4'-pentachlorobiphenyl (PenCB), was compared to those from 3-methylcholanthrene (MC)-treated and untreated rats. Treatments with PenCB and MC resulted in about 8- and 7-fold increases of cytosolic DT-diaphorase activity, respectively. Purification of the enzyme preparations from untreated, and PenCB- and MC-treated rats were conducted by using DE-52, DEAE-Sephadex A-50, hydroxylapatite, and Bio-Gel P-150 column chromatographies. Both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that all of the final preparations from the three origins were homogeneous and had the same molecular weight of 59,000, consisting of two subunits with molecular weights of 30,000. Further studies on amino acid composition, Km value, optimum pH, and catalytic activities for various substrates also indicated that both PenCB- and MC-inducible DT-diaphorases were identical with that from the untreated rats. All three DT-diaphorases contained about 2 mol of FAD per mol of enzyme. Partial digestion of the enzymes by trypsin and subsequent analysis by HPLC revealed that the three preparations were indistinguishable. The identity among the three purified DT-diaphorases was finally confirmed by Ouchterlony immunodiffusion employing anti-serum raised against each enzyme preparation.
Topics: Amino Acids; Animals; Chromatography; Chromatography, High Pressure Liquid; Cytosol; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Immunodiffusion; Kinetics; Liver; Male; Methylcholanthrene; Molecular Weight; NAD; NAD(P)H Dehydrogenase (Quinone); Polychlorinated Biphenyls; Quinone Reductases; Rats; Rats, Inbred Strains; Substrate Specificity
PubMed: 3129417
DOI: 10.1093/oxfordjournals.jbchem.a122203 -
The American Journal of Pathology 1956
Topics: Carcinoma; Carcinoma, Squamous Cell; Citrates; Gallium; Methylcholanthrene; Stomach Neoplasms
PubMed: 13275569
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1984Monoclonal antibody (MAb)-based radioimmunoassay (RIA) and immunopurification procedures were used to probe the immunochemical relatedness of cytochromes P-450 in...
Monoclonal antibody (MAb)-based radioimmunoassay (RIA) and immunopurification procedures were used to probe the immunochemical relatedness of cytochromes P-450 in tissues from different species. RIAs based on MAb 1-7-1 and MAb 1-31-2, both to the major 3-methylcholanthrene (MC)-induced rat liver microsomal cytochrome P-450, detected antigenically related forms of cytochrome P-450 in liver microsomes from MC-induced rats, C57BL/6 and DBA/2 mice, hamsters, and guinea pigs, and in lung and kidney microsomes of MC-induced rats. Individual cytochromes P-450 were also isolated from these microsomes by MAb-directed immunopurification. When bound to Sepharose, MAb 1-7-1 adsorbed two species of Mr 56,000 and 57,000 from liver microsomes from rats and C57BL/6 mice, while MAb 1-31-2 adsorbed only the Mr 57,000 polypeptide. These results reveal that livers from both rats and C57BL/6 mice contain a cytochrome P-450 (Mr 56,000) with the epitope for 1-7-1 and a cytochrome P-450 (Mr 57,000) with epitopes for both 1-31-2 and 1-7-1. Additional immunochemical relatedness between the cytochromes P-450 in different tissues was demonstrated by MAb-directed immunopurification of cytochromes P-450 from DBA/2 mouse liver (Mr 56,000), guinea pig liver (Mr 53,000), hamster liver (Mr 57,000), and rat lung (Mr 57,000). These results demonstrate the efficacy of MAb-based RIA and immunopurification procedures for simple and rapid detection and purification of cytochromes P-450 from a variety of tissues.
Topics: Animals; Antibodies, Monoclonal; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Isoenzymes; Kidney; Lung; Male; Methylcholanthrene; Microsomes; Microsomes, Liver; Radioimmunoassay; Rats; Rats, Inbred Strains
PubMed: 6480608
DOI: No ID Found -
The Biochemical Journal Jul 19741. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of...
1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [(32)P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [(32)P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine-phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [(32)P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [(14)C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [(32)P]orthophosphate and [(14)C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [(32)P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [(32)P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.
Topics: Animals; Carbon Radioisotopes; Endoplasmic Reticulum; Glycerol; In Vitro Techniques; Liver; Methionine; Methylcholanthrene; Methyltransferases; Phenobarbital; Phosphates; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Phosphorus Radioisotopes; Rats
PubMed: 4441372
DOI: 10.1042/bj1420019 -
The Journal of Biological Chemistry Apr 1994Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC) cause... (Comparative Study)
Comparative Study
Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC) cause transcriptional activation of the CYP1A1 gene via their interaction with the aromatic hydrocarbon (Ah) receptor. Direct radioligand binding and competitive binding studies demonstrated that the cytosolic Ah receptor from the mouse hepatoma cell line Hepa-1 bound TCDD with an affinity approximately 3-4-fold greater than that for MC. However, TCDD was approximately 1,000-fold more potent than MC as an inducer of CYP1A1-mediated aryl hydrocarbon hydroxylase activity in cultured Hepa-1 cells as assessed at 14 h following exposure to inducer. To understand the basis for this quantitative discrepancy between Ah receptor binding affinity and CYP1A1 induction potency, we systematically compared TCDD and MC for their abilities to activate sequential events in the CYP1A1 induction mechanism that occur subsequent to initial binding to the cytosolic Ah receptor. Using a gel retardation assay, TCDD and MC were shown to be equipotent in causing in vitro transformation of the cytosolic Ah receptor to its DNA-binding form. In addition, the transformed Ah receptor bound to a specific dioxin-responsive enhancer sequence with the same apparent affinity when MC was the ligand as when TCDD was the ligand. At an early time point (i.e. 2 h) in the CYP1A1 induction process, TCDD was only approximately 4-25-fold more potent than MC in stimulating the nuclear uptake of the ligand-Ah receptor complex, and the two ligands displayed a relatively small difference (> or = 10-fold) in CYP1A1 mRNA induction potency. When assessed at 4 h following ligand treatment, TCDD was only approximately 10-fold more potent than MC as an aryl hydrocarbon hydroxylase inducer, suggesting a time-dependent reduction in the potency of MC in intact cells. Exposure of Hepa-1 cells to MC over a 16-h time course resulted in an increased ability of these cells to convert [3H]MC to alkali-extractable metabolites. Our data are consistent with the idea that TCDD and MC display relatively small differences in their intrinsic abilities to activate Ah receptor-mediated events. The reduced biological potency of MC observed in intact cells and whole animals is at least partially due to the more rapid metabolic inactivation of this ligand compared with the poorly metabolized TCDD. By extension, the extraordinary toxicity of TCDD may not be explained solely by its high affinity for the cytosolic Ah receptor.
Topics: Animals; Aryl Hydrocarbon Hydroxylases; Base Sequence; Binding Sites; Binding, Competitive; Cell Line; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cytochrome P-450 Enzyme System; Cytosol; Enzyme Induction; Kinetics; Liver Neoplasms, Experimental; Methylcholanthrene; Mice; Molecular Sequence Data; Oligodeoxyribonucleotides; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Radioligand Assay; Receptors, Aryl Hydrocarbon; Transfection; Tritium; Tumor Cells, Cultured
PubMed: 8163516
DOI: No ID Found -
European Journal of Biochemistry Feb 1980Rat liver microsomes were shown to catalyze the conjugation of 1-chloro-2,4-dinitrobenzene with glutathione and this activity has been characterized. It cannot be...
Rat liver microsomes were shown to catalyze the conjugation of 1-chloro-2,4-dinitrobenzene with glutathione and this activity has been characterized. It cannot be removed from the microsomes by washing or other procedures which release loosely bound material from membranes. The microsomal glutathione S-transferase can be activated up to eight fold by treatment with N-ethylmaleimide. This activation also affects the apparent Km of the enzyme(s) for both glutathione and 1-chloro-2,4-dinitrobenzene. Upon subcellular fractionation of the liver the N-ethylmaleimide-activateable glutathione S-transferase distributes in the same manner as a marker for the endoplasmic reticulum and unlike markers for the other organelles and for the cytoplasm. Treatment of microsomes with proteases revealed that the enzyme is at least partially exposed on the cytoplasmic surface of the endoplasmic reticulum. Finally, three inducers of drug-metabolizing systems-i.e. phenobarbital, methylcholanthrene, and trans-stilbene oxide-all increase the activity of the cytoplasmic glutathione S-transferases, but they do not affect the microsomal activity. These and other considerations indicate that the microsomal glutathione S-transferase(s) is distinct from the cytoplasmic enzymes catalyzing similar reactions. The microsomal enzyme is likely to be involved in drug metabolism and the possibility of activating it through attack on a sulfhydryl group may represent an important physiological response to certain xenobiotics.
Topics: Animals; Cytosol; Ethylmaleimide; Glutathione Transferase; Kinetics; Liver; Male; Methylcholanthrene; Microsomes, Liver; Peptide Hydrolases; Phenobarbital; Rats; Subcellular Fractions; Substrate Specificity
PubMed: 6989596
DOI: 10.1111/j.1432-1033.1980.tb04412.x -
Journal of Biochemistry Jun 1987Two cDNA clones, pHPah1 and pHPah2, encoding polycyclic hydrocarbon-inducible forms of rabbit liver microsomal cytochrome P-450 were isolated and their nucleotide...
Two cDNA clones, pHPah1 and pHPah2, encoding polycyclic hydrocarbon-inducible forms of rabbit liver microsomal cytochrome P-450 were isolated and their nucleotide sequences were determined. The inserts of pHPah1 and pHPah2 contained open reading frames specifying the entire primary structures of cytochrome P-450s, consisting of 518 and 516 amino acid residues, respectively. The deduced amino acid sequences for pHPah1 and pHPah2 are 76 and 73% homologous with rat P-450c and P-450d, respectively, and 96% homologous with rabbit P-450 forms 6 and 4, respectively. We conclude that pHPah1 and pHPah2 encode the rabbit counterparts of rat P-450c and P-450d, respectively. A region highly conserved in all species of cytochrome P-450 so far examined, called the HR2 region, can be detected in the pHPah1 and pHPah2 primary structures, but another conserved region, HR1, cannot be observed. Northern hybridization analysis of total RNAs from livers of untreated and drug-treated rabbits demonstrated that the pHPah1 and pHPah2 genes are expressed in untreated animals, induced considerably by administration of 3-methylcholanthrene or beta-naphthoflavone, and suppressed by phenobarbital and isosafrole.
Topics: Amino Acid Sequence; Animals; Base Sequence; Benzoflavones; Cytochrome P-450 Enzyme System; DNA; Enzyme Induction; Gene Expression Regulation; In Vitro Techniques; Male; Methylcholanthrene; Microsomes, Liver; Molecular Sequence Data; Polycyclic Compounds; RNA, Messenger; Rabbits; Sequence Homology, Nucleic Acid; beta-Naphthoflavone
PubMed: 3667560
DOI: 10.1093/oxfordjournals.jbchem.a122017