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Journal of Biochemistry Oct 1991Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA... (Comparative Study)
Comparative Study
Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; Cricetinae; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; DNA; Gene Library; Humans; Liver; Lung; Male; Mesocricetus; Methylcholanthrene; Mice; Molecular Sequence Data; Multigene Family; Oxidoreductases; Rats; Restriction Mapping; Sequence Homology, Nucleic Acid
PubMed: 1778988
DOI: 10.1093/oxfordjournals.jbchem.a123633 -
European Journal of Biochemistry Sep 1997In the present study, we synthesized 14C-labelled 5-nitro-1,2,4-triazol-3-one (NTO) and investigated its hepatic metabolism by dexamethasone-induced murine hepatic...
In the present study, we synthesized 14C-labelled 5-nitro-1,2,4-triazol-3-one (NTO) and investigated its hepatic metabolism by dexamethasone-induced murine hepatic microsomes. Under the nitrogen atmosphere, 5-amino-1,2,4-triazol-3-one was the only detected metabolite of NTO. The microsomal nitroreductase activity was dependent on NADPH, totally inhibited by carbon monoxide and partially inhibited by oxygen. In aerobic conditions, beside a low amount of amine, the major metabolite formed is the 5-hydroxy-triazolone, urazole. This compound resulted from the oxidative denitrification of NTO, which produced equivalent amount of nitrite. This reaction, like the nitroreductase activity, was dependent on NADPH and totally inhibited by carbon monoxide. Both nitroreduction and oxidative denitrification were inhibited by imidazole-related inhibitors: miconazole and methimazole, and to a less extent by N-octylamine. The microsomal denitrification was induced by the treatment of rats with dexamethasone and phenobarbital. The microsomal reductase activity is present in untreated rat microsomes, and recovered with various inducers. The results of this study indicate the role played by cytochrome P-450 in the metabolism of NTO, supported by its transformation with reconstituted cytochrome P-450 systems.
Topics: Animals; Carbon Radioisotopes; Clofibrate; Cytochrome P-450 Enzyme System; Dexamethasone; Fungal Proteins; Glucocorticoids; Male; Methylcholanthrene; Microsomes, Liver; Nitro Compounds; Oxidation-Reduction; Phenobarbital; Rats; Rats, Sprague-Dawley; Triazoles
PubMed: 9346295
DOI: 10.1111/j.1432-1033.1997.00401.x -
The Journal of Biological Chemistry Jul 1978Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is...
Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is proportional to the levels of microsomal cytochrome P-450 from control or phenobarbital-pretreated rats and mice or cytochrome P-448 from 3-methylchol-anthrene-pretreated animals. In the "inducible" C57B/6J strain of mice, 3-methylcholanthrene and phenobarbital pretreatment cause an increase in cytochrome P-448 and P-450 levels, respectively, which is directly proportional to the increase of azoreductase activity. However, in the "noninducible" DBA/2J strain of mice, only phenobarbital treatment causes the increase both in cytochrome P-450 levels and azoreductase activity, while 3-methylcholanthrene has no effect. These experiments suggest that the P-450 type cytochromes are responsible for azoreductase activity in liver microsomes.
Topics: Amaranth Dye; Animals; Azo Compounds; Cytochrome P-450 Enzyme System; Cytochromes; Enzyme Induction; Methylcholanthrene; Mice; Microsomes, Liver; Mixed Function Oxygenases; NADH, NADPH Oxidoreductases; Phenobarbital; Rats; p-Dimethylaminoazobenzene
PubMed: 96118
DOI: No ID Found -
Proceedings of the National Academy of... Jul 2004Using the carcinogen 3-methylcholanthrene (MCA), we demonstrate with Fourier transform-infrared spectroscopy that a cancer DNA phenotype is produced well in advance of...
Using the carcinogen 3-methylcholanthrene (MCA), we demonstrate with Fourier transform-infrared spectroscopy that a cancer DNA phenotype is produced well in advance of palpable tumors. We further demonstrate that the administration of cyclophosphamide markedly inhibits the development of the cancer phenotype and concomitantly delays tumor formation. MCA, injected into the hind legs of mice, produced a variety of significant structural changes in the nucleotide bases and phosphodiester-deoxyribose backbone, as reflected in a substantial (34%) difference between the mean DNA spectra of the control and the MCA-injected mice. Strikingly, 57 days before the mean appearance of tumors, we could not distinguish the DNA structure of the histologically normal tissues of the MCA-injected mice from the DNA structure of the tumor tissues. This finding indicates the early development of a cancer phenotype. Confirmatory evidence was obtained when tissues from a group of mice injected with both MCA and cyclophosphamide did not manifest the cancer phenotype, and their mean DNA structure closely resembled that of the control mice. Accordingly, we propose that the cancer DNA phenotype, as evinced by Fourier transform-infrared spectroscopy, is a promising early indicator of tumor formation, and we postulate that agents capable of inhibiting this phenotype may delay or prevent carcinogenesis.
Topics: Animals; Antineoplastic Agents, Alkylating; Cyclophosphamide; DNA; Female; Methylcholanthrene; Mice; Mice, Inbred BALB C; Muscle, Skeletal; Neoplasms; Phenotype; Spectroscopy, Fourier Transform Infrared
PubMed: 15249662
DOI: 10.1073/pnas.0403888101 -
The Journal of Biological Chemistry Nov 1978
Separation and purification of multiple forms of microsomal cytochrome P-450. Partial characterization of three apparently homogeneous cytochromes P-450 prepared from livers of phenobarbital- and 3-methylcholanthrene-treated rats.
Topics: Amino Acids; Animals; Cytochrome P-450 Enzyme System; Immunodiffusion; Isoenzymes; Kinetics; Methylcholanthrene; Microsomes, Liver; Molecular Weight; Phenobarbital; Precipitin Tests; Rats
PubMed: 100498
DOI: No ID Found -
Acta Biochimica Et Biophysica Sinica May 2020
Topics: Animals; Cytokines; Liver; Male; Methylcholanthrene; Mice; Mice, Inbred ICR
PubMed: 32293687
DOI: 10.1093/abbs/gmaa020 -
Journal of Biochemistry Mar 1986The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely...
The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.
Topics: Animals; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP1A2; Cytochrome P-450 Enzyme System; Cytochromes; Enzyme Induction; Female; Male; Methylcholanthrene; Microsomes, Liver; Pharmaceutical Preparations; Phenobarbital; Polychlorinated Biphenyls; Rats; Rats, Inbred Strains; Sex Factors; Steroid 16-alpha-Hydroxylase; Steroid Hydroxylases; Testosterone
PubMed: 3086298
DOI: 10.1093/oxfordjournals.jbchem.a135544 -
Journal of Biochemistry Aug 1980The spectral properties of multiple forms of cytochrome P-450 purified or partially purified from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene...
The spectral properties of multiple forms of cytochrome P-450 purified or partially purified from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rabbits have been studied. Both optical absorption and EPR studies have shown that the oxidized forms of P-450(1), P-450(2) (from PB-treated animals), and P-450(3) (from MC-treated animals) are in the low spin state, having a Soret absorption peak at 417-418 nm. Oxidized P-448(1) (from both PB- and MC-treated animals), on the other hand, shows a Soret peak at 393 nm and a weak band at 646 nm. This and EPR evidence indicate that P-448(1) contains heme which is predominantly in the high spin state, though EPR studies at low temperature indicate the presence of a small amount of low spin ferric heme. The presence of tightly bound MC in P-448(1) purified from MM-treated animals is reflected by characteristic absorption peaks in the ultraviolet region, but this does not affect the absorption spectra in the Soret and visible regions. Emulgen 913, a nonionic detergent, causes the conversion of oxidized P-448(1) from the high to the low spin state, as evidenced by optical absorption and EPR results; bound MC inhibits this conversion in a noncompetitive way. Binding of ethyl isocyanide to reduced P-450(1) and P-448(1) results in the appearance of two Soret peaks in the 430 and 455 nm regions, the relative intensities of which are dependent on pH. At any pH the 455 nm peak of P-448(1) is always higher than that of P-450(1). Benzphetamine and aniline, added to oxidized P-450(1), cause Type I and Type II spectral changes, respectively, but the magnitudes of the changes are small in both cases. The Soret peak of oxidized P-448(1) at 393 nm is completely shifted to 420 nm on addition of aniline, resulting in a reverse Type I spectral change; acetanilide causes the conversion of the Soret peak to the low spin state to only a slight extent. The conversions caused by aniline and acetanilide are both inhibited by the presence of tightly bound MC. On the basis of these and other observations, the spin state of these P-450's are discussed.
Topics: Acetanilides; Aniline Compounds; Animals; Binding Sites; Cytochrome P-450 Enzyme System; Electron Spin Resonance Spectroscopy; Isoenzymes; Male; Methylcholanthrene; Microsomes, Liver; Oxidation-Reduction; Phenobarbital; Protein Binding; Rabbits; Spectrophotometry
PubMed: 6252201
DOI: 10.1093/oxfordjournals.jbchem.a132997 -
The Journal of Biological Chemistry Feb 1979
Separation and characterization of highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, and 3-methylcholanthrene.
Topics: Animals; Aroclors; Carbon Monoxide; Cytochrome P-450 Enzyme System; Immunodiffusion; Male; Methylcholanthrene; Microsomes, Liver; Molecular Weight; Oxidation-Reduction; Peptide Fragments; Phenobarbital; Polychlorinated Biphenyls; Rats; Spectrophotometry
PubMed: 105007
DOI: No ID Found -
The Journal of Biological Chemistry Nov 1984Metabolism of benzo(a)pyrene (BP) and 7,8-dihydrodiol by 3-methylcholanthrene (MC)-induced rat liver microsomes are both subject to severe inhibition by primary...
Metabolism of benzo(a)pyrene (BP) and 7,8-dihydrodiol by 3-methylcholanthrene (MC)-induced rat liver microsomes are both subject to severe inhibition by primary metabolites of BP, which was analyzed by determining individual inhibition constants for all primary BP metabolites for both BP and 7,8-dihydrodiol metabolism. Monooxygenation of 7,8-dihydrodiol was, surprisingly, 5 to 10 times more sensitive than monooxygenation of BP to inhibition by all primary metabolites, even though both reactions require the same enzyme, cytochrome P-450c. Two representative products, 1,6-quinone and 9-phenol, were both strong, competitive inhibitors of BP metabolism with Ki values of 0.12 and 0.74 microM, respectively. The total effect of product inhibition on the overall reactions was determined by fitting progress curves of BP, 7,8-dihydrodiol, and anti-7,8-dihydrodiol 9,10-oxide (determined as 7,10/8,9-tetrol) over a range of BP concentrations to integrated steady-state equations using experimental Vmax and Km values. The effective product inhibition factors for BP and 7,8-dihydrodiol metabolism, determined from progress curve fits, were only 2-fold higher than the corresponding calculated theoretical values. The effective product inhibition factors, obtained from progress curve analysis, confirmed that 7,8-dihydrodiol metabolism was substantially more sensitive to inhibition by primary BP metabolites than BP metabolism itself. This difference probably reflects the much higher affinity of cytochrome P-450c for BP (Kd = 6 nM), as compared to 7,8-dihydrodiol (Kd = 175 nM) that was established spectrophotometrically both for the purified cytochrome and for MC microsomes. The Km for BP metabolism is 50 to 100 times higher than the Kd, while the Km is similar to the Kd for 7,8-dihydrodiol metabolism. The discrepancy for BP between Km and Kd suggests that standard Michaelis-Menten kinetics may be perturbed by either slow substrate or product dissociation.
Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Amines; Animals; Benzo(a)pyrene; Benzopyrenes; Cytochrome P-450 Enzyme System; Kinetics; Male; Mathematics; Methylcholanthrene; Microsomes, Liver; Rats; Rats, Inbred Strains
PubMed: 6438082
DOI: No ID Found