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The Biochemical Journal Jan 19661. A chromatographic investigation of the products of the metabolism of 3-methylcholanthrene by rat-liver homogenates showed the formation of compounds with the...
1. A chromatographic investigation of the products of the metabolism of 3-methylcholanthrene by rat-liver homogenates showed the formation of compounds with the properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene and 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. A glutathione conjugate that is probably S-(11,12-dihydro-12-hydroxy-3-methyl-11-cholanthrenyl)glutathione was also detected. 3-Methylcholanthrene-1- and -2-one and -1,2-quinone were also present, but these products may have arisen by the chemical oxidation of the corresponding hydroxy compounds. 2. Other metabolic products were tentatively identified as 9- and 10-hydroxy-3-methylcholanthrene, 4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene and 3-hydroxymethylcholanthrene. 3. 1- and 2-Hydroxy-3-methylcholanthrene were converted by homogenates into the related ketones and into products with the properties of cis- and trans-1,2-dihydroxy-3-methylcholanthrene: 3-methylcholanthren-1- and -2-one were converted into their related hydroxy compounds and into the isomeric 1,2-dihydroxy compounds. The isomeric 1,2-dihydroxy compounds were each partly converted into the other isomer by these homogenates. All the above substrates also yielded products that appeared to be derivatives of 3-hydroxymethylcholanthrene. 4. 3-Methylcholanthrylene was converted by rat-liver homogenates into products with the properties of trans-1,2-dihydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene and 3-methylcholanthren-2-one. A small amount of the cis-1,2-dihydroxy compound was also formed, together with a glutathione conjugate that is possibly S-(2-hydroxy-3-methyl-1-cholanthrenyl)glutathione or its positional isomer. 5. An unidentified product was detected in the metabolism of 3-methylcholanthrene, the monohydroxy compounds, the ketones and the dihydroxy compounds, the formation of which appeared to involve metabolism at the 1,2-bond. 6. 11,12-Epoxy-11,12-dihydro-3-methylcholanthrene was converted by rat-liver homogenates into products with the properties of 11-hydroxy-3-methylcholanthrene (or, less likely, the 12-isomer), 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and the glutathione conjugate described above. Products with the properties of these compounds were formed when the epoxide was allowed to react with glutathione in an aqueous medium. 7. Mouse-liver homogenate converted 3-methylcholanthrene into products with the chromatographic properties of 1- and 2-hydroxy-3-methylcholanthrene, cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, 3-methylcholanthrene-1- and -2-one and -1,2-quinone and the unidentified hydroxy-3-methylcholanthrenes. 8. The syntheses of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, 3-methylcholanthren-2-one, 2-hydroxy-3-methylcholanthrene, 3-methylcholanthrylene, 11,12-epoxy-11,12-dihydro-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene are described.
Topics: Animals; Chemistry Techniques, Analytical; Chromatography, Thin Layer; In Vitro Techniques; Liver; Methylcholanthrene; Rats; Subcellular Fractions
PubMed: 5938646
DOI: 10.1042/bj0980215 -
Proceedings of the National Academy of... Feb 1981Using partially purified mouse liver 23S mRNA known to be associated with the Ah locus and 3-methylcholanthrene-induced cytochrome P(1)-450, we synthesized...
Using partially purified mouse liver 23S mRNA known to be associated with the Ah locus and 3-methylcholanthrene-induced cytochrome P(1)-450, we synthesized double-stranded cDNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) and the Klenow A fragment of Escherichia coli DNA polymerase I. The double-stranded cDNA was inserted into pBR322 plasmid DNA by Pst I cleavage and homopolymeric "tailing" and cloned in E. coli LE392. Clone 46 hybridized with [(32)P]cDNA made from 23S mRNA from "Ah-responsive" C57BL/6N mice but did not hybridize with similarly prepared [(32)P]cDNA from "Ah-nonresponsive" DBA/2N mice. Clone 30 was positive, and clone 7 was negative, with both C57BL/6N and DBA/2N [(32)P]cDNA probes; these two clones were therefore used as "positive" and "negative" control clones, respectively. By translation-arrest experiments, clone 46 DNA and clone 30 DNA were shown to be associated with anti-P(1)-450- and anti-albumin-precipitable material, respectively. By agarose gel electrophoresis of Pst I digests, the clone 46 DNA insert was shown to be 1100 base pairs in total length and to contain one internal Pst I site. The cDNA made from total mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mice hybridized to the two fragments of Pst I-digested DNA from clone 46, whereas similarly prepared cDNA from 3-methylcholanthrene-treated DBA/2N and control C57BL/6N and DBA/2N mice did not. Of 11 restriction endonucleases used, two (Pst I and Xba I) had sites within the clone 46 DNA insert. After hybridization of clone 46 (32)P-labeled nick-translated DNA to EcoRI fragments from A/HeJ mouse genomic DNA and fractionation by RPC-5 chromatography and gel electrophoresis, only one positive band (3-4 kilobase pairs appeared. These data demonstrate conclusively that pBR322 clone 46 DNA is associated with mRNA controlled by the murine Ah locus, presumably the structural gene encoding 3-methylcholanthrene-induced P(1)-450.
Topics: Animals; Cloning, Molecular; Cytochrome P-450 Enzyme System; DNA Restriction Enzymes; DNA, Recombinant; Liver; Methylcholanthrene; Mice; Nucleic Acid Hybridization; Plasmids; Protein Biosynthesis; RNA, Messenger
PubMed: 6262772
DOI: 10.1073/pnas.78.2.800 -
Journal of Zhejiang University.... Aug 2005A comparative study was made on the tissue specific expression of glutathione transferases (GST) in brain and testis after exposure of rat to phenobarbitol (PB) and...
A comparative study was made on the tissue specific expression of glutathione transferases (GST) in brain and testis after exposure of rat to phenobarbitol (PB) and b-methylcholanthrene (MC). Glutathione transferases, a family of multifunctional proteins are involved in intracellular transport processes and in detoxication of electrophilic xenobiotics by catalyzing reactions such as conjugation, isomerization, reduction and thiolysis. On purification, the yield of GST proteins by affinity chromatography was 39% in testis and 32% in brain. The affinity purified testis GSTs were resolved by chromatofocusing into six anionic and four cationic isozymes, and in brain glutathione transferases were resolved into four anionic and three cationic isozymes, suggesting the presence of multiple isozymes with Yc, Yb, Ybeta and Ydelta in both of them. In testis and brain, these isozymes at identical pI values showed variable functions with a battery of substrates and the cationic isozymes of brain and testis showed identical properties in CHP (cumene hydroperoxide) at pH values of above 7.0. Substrate specificity studies and immunoblot analysis of testis and brain proteins revealed that they play a predominant role in the detoxication of phenobarbitol or beta-methylcholanthrene. Expression of the isozymes in testis and brain on exposure to PB and MC indicated elevated subunit variation. In both testis and brain, Ydelta of pi class was expressed on PB treatment and Yc of alpha class and Ybeta of mu class was expressed in MC treated testis and only Yc was predominantly expressed in MC treated brain. Thus these subunits expression is considered as markers for carcinogenesis and specific to chemical toxicity under phenobarbitol and beta-methylcholanthrene stress.
Topics: Animals; Brain; Exercise Test; Gene Expression Regulation, Enzymologic; Glutathione Transferase; Male; Methylcholanthrene; Phenobarbital; Rats; Rats, Wistar; Testis
PubMed: 16052709
DOI: 10.1631/jzus.2005.B0759 -
Journal of Biochemistry Mar 1984Four forms of cytochrome P-450, tentatively designated PB-1, PB-2, MC-1, and MC-2, were purified from liver microsomes of rats treated with phenobarbital (PB-1 and PB-2)...
Four forms of cytochrome P-450, tentatively designated PB-1, PB-2, MC-1, and MC-2, were purified from liver microsomes of rats treated with phenobarbital (PB-1 and PB-2) or 3-methylcholanthrene (MC-1 and MC-2). Each purified form showed a single protein-staining band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56,000 (MC-1), 53,000 (PB-1), 53,000 (MC-2), or 49,000 (PB-2). PB-1 and MC-1 were the major cytochrome P-450 components inducible by phenobarbital (PB) and 3-methylcholanthrene (MC), respectively. Antibodies prepared against each form of purified cytochrome P-450 did not cross-react with heterologous antigens in Ouchterlony double diffusion tests, confirming the immunological distinctness of the four forms. The CO-compounds of reduced PB-1 and PB-2 had an absorption maximum at 450 nm, whereas those of MC-1 and MC-2 had a maximum at 447 nm. Judging from the oxidized absolute spectra, MC-2 was of high spin type and the others were of low spin type. Amino acid analysis revealed considerable differences among the purified four forms of cytochrome P-450, and the amino acid sequences of their NH2-terminal portions confirmed that the four forms were different proteins. In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, PB-1 and PB-2 oxidized benzphetamine at high rates, but their oxidation of benzo(a)pyrene was much slower than that by MC-1, which catalyzed rapid hydroxylation of benzo(a)pyrene but had low activity with benzphetamine. The quantity of each form of cytochrome P-450 in microsomes was determined by quantitative immunoprecipitation, and selective induction of PB-1 and MC-1 by PB and MC, respectively, was confirmed. Some induction of PB-2 and MC-2 by the corresponding inducers was also noticed. PB group P-450's were not increased by MC treatment, nor were MC group P-450's by PB.
Topics: Amino Acids; Animals; Cytochrome P-450 Enzyme System; Electrophoresis, Polyacrylamide Gel; Immunodiffusion; Male; Methylcholanthrene; Microsomes, Liver; Phenobarbital; Rats; Rats, Inbred Strains; Substrate Specificity
PubMed: 6427199
DOI: 10.1093/oxfordjournals.jbchem.a134660 -
The Journal of Biological Chemistry Apr 1984With the use of cDNA probes reverse transcribed from purified glutathione S-transferase mRNA templates, four cDNA clones complementary to transferase mRNAs have been...
Rat liver glutathione S-transferases. Complete nucleotide sequence of a glutathione S-transferase mRNA and the regulation of the Ya, Yb, and Yc mRNAs by 3-methylcholanthrene and phenobarbital.
With the use of cDNA probes reverse transcribed from purified glutathione S-transferase mRNA templates, four cDNA clones complementary to transferase mRNAs have been identified and characterized. Two clones, pGTB38 and pGTB34, have cDNA inserts of approximately 950 and 900 base pairs, respectively, and hybridize to a mRNA(s) whose size is approximately 980 nucleotides. In hybrid-select translation experiments, pGTB38 and pGTB34 select mRNAs specific for the Ya and Yc subunits of rat liver glutathione S-transferases. Clone pGTB33, which harbors a truncated cDNA insert, hybrid-selects only the Ya mRNA. All of the clones, pGTB38, pGTB34, and pGTB33, hybrid-select another mRNA which is specific for a polypeptide with an electrophoretic mobility slightly greater than the Ya subunit. The entire nucleotide sequence of the full length clone, pGTB38, has been determined and the complete amino acid sequence of the corresponding polypeptide has been deduced. The mRNA codes for a protein comprising 222 amino acids with Mr = 25,547. We have also identified a cDNA clone complementary to a Yb mRNA of the rat liver glutathione S-transferases. This clone, pGTA/C36, hybrid-selects only Yb mRNA(s) and hybridizes to a mRNA(s) whose size is approximately 1200 nucleotides. Although the Ya, Yb, and Yc mRNAs are elevated coordinately by phenobarbital and 3-methylcholanthrene, the Ya-Yc mRNAs are induced to a much greater extent compared to the Yb mRNA(s). These data suggest that the mRNAs for each transferase isozyme are regulated independently.
Topics: Amino Acid Sequence; Animals; Cloning, Molecular; DNA; DNA Restriction Enzymes; Glutathione Transferase; Liver; Methylcholanthrene; Molecular Weight; Nucleic Acid Hybridization; Phenobarbital; Plasmids; Polyribosomes; RNA, Messenger; Rats; Transcription, Genetic
PubMed: 6325423
DOI: No ID Found -
Aquatic Toxicology (Amsterdam,... Jan 2008The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE, respectively) that...
Glucuronidation and sulfonation, in vitro, of the major endocrine-active metabolites of methoxychlor in the channel catfish, Ictalurus punctatus, and induction following treatment with 3-methylcholanthrene.
The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE, respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2mg MXC/kg daily for 6 days and sacrificed 24h after the last dose. The 3-MC treatment was a single 10mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the V(max) value (mean+/-S.D., n=4) for glucuronidation of OH-MXC was 270+/-50pmol/min/mg protein, higher than found for HPTE (110+/-20pmol/min/mg protein). For each substrate, the V(max) values observed in intestinal microsomes were approximately twice those found in the liver. The K(m) values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32+/-0.04mM for OH-MXC and 0.26+/-0.06mM for HPTE. The K(m) for the co-substrate, UDPGA, was higher in liver (0.28+/-0.09mM) than intestine (0.04+/-0.02mM). Treatment with 3-MC but not MXC increased the V(max) for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The V(max) values for sulfonation of OH-MXC and HPTE, respectively, in liver cytosol were 7+/-3 and 17+/-4pmol/min/mg protein and in intestinal cytosol were 13+/-3 and 30+/-5pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics of the conjugation pathways with those published for the demethylation of MXC showed that formation of the endocrine-active metabolites was more efficient than either conjugation pathway. Residues of OH-MXC and HPTE were detected in extracts of liver microsomes from MXC-treated fish. This work showed that although OH-MXC and HPTE could be eliminated by glucuronidation and sulfonation, the phase II pathways were less efficient than the phase I pathway leading to formation of these endocrine-active metabolites.
Topics: Acetates; Animals; Benzo(a)pyrene; Biotransformation; Female; Glucuronidase; Glucuronides; Hydrocarbons, Chlorinated; Ictaluridae; Intestinal Mucosa; Intestines; Male; Methylcholanthrene; Microsomes; Phenols; Reproducibility of Results; Sulfur Radioisotopes; Water Pollutants, Chemical
PubMed: 18078677
DOI: 10.1016/j.aquatox.2007.11.003 -
Biological & Pharmaceutical Bulletin 2022The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies...
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.
Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Female; Gene Expression; Humans; MCF-7 Cells; Methylcholanthrene; Receptors, Aryl Hydrocarbon; V-Set Domain-Containing T-Cell Activation Inhibitor 1
PubMed: 35650105
DOI: 10.1248/bpb.b21-01068 -
Scientific Reports Feb 2018Cancer incidence appears to be higher amongst firefighters compared to the general population. Given that many cancers have an environmental component, their...
Cancer incidence appears to be higher amongst firefighters compared to the general population. Given that many cancers have an environmental component, their occupational exposure to products of carbon combustion such as polycyclic aromatic hydrocarbons (PAHs) is of concern. This is the first UK study identifying firefighters exposure to PAH carcinogens. Wipe samples were collected from skin (jaw, neck, hands), personal protective equipment of firefighters, and work environment (offices, fire stations and engines) in two UK Fire and Rescue Service Stations. Levels of 16 US Environmental Protection Agency (EPA) PAHs were quantified together with more potent carcinogens: 7,12-dimethylbenzo[a]anthracene, and 3-methylcholanthrene (3-MCA) (12 months post-initial testing). Cancer slope factors, used to estimate cancer risk, indicate a markedly elevated risk. PAH carcinogens including benzo[a]pyrene (B[a]P), 3-MCA, and 7,12-dimethylbenz[a]anthracene PAHs were determined on body surfaces (e.g., hands, throat), on PPE including helmets and clothing, and on work surfaces. The main exposure route would appear to be via skin absorption. These results suggest an urgent need to monitor exposures to firefighters in their occupational setting and conduct long-term follow-up regarding their health status.
Topics: 9,10-Dimethyl-1,2-benzanthracene; Benzopyrenes; Carcinogens; Environmental Monitoring; Firefighters; Humans; Incidence; Methylcholanthrene; Neoplasms; Occupational Diseases; Occupational Exposure; Polycyclic Aromatic Hydrocarbons; Protective Clothing; Skin; Skin Absorption; United Kingdom
PubMed: 29410452
DOI: 10.1038/s41598-018-20616-6 -
The Journal of Biological Chemistry Dec 1986The cDNAs encoding ethanol-inducible forms of rat and human cytochrome P-450s have been isolated, sequenced, and used to study the expression of this cytochrome P-450... (Comparative Study)
Comparative Study
The cDNAs encoding ethanol-inducible forms of rat and human cytochrome P-450s have been isolated, sequenced, and used to study the expression of this cytochrome P-450 during development and by various inducing agents. Polyclonal antibody against ethanol-inducible cytochrome P-450 was used to screen rat and human lambda gt11 cDNA expression libraries. The longest cDNAs obtained from each library were completely sequenced, and the deduced amino acid sequence of the rat cDNA was found to correspond to P450j based on the published amino-terminal sequence. The rat and human cytochrome P-450s both contained 493 amino acids and calculated molecular masses of 56,634 and 56,916 daltons, respectively. Human P450j shared 75% nucleotide and 78% amino acid similarities to the respective orthologous rat cDNA and deduced amino acid sequences. Amino acid alignment also revealed that P450j was 48% similar to P450b and P450e, the major phenobarbital-inducible forms, and 54% similar to P450PB1 and P450f, two developmentally regulated forms. Southern blot analyses of rat and human genomic DNAs verified that only a single gene shared extensive homology with P450j. The expression of P450j was found to be developmentally regulated. No immunodetectable protein or P450j mRNA was present in newborn rats; however, rapid increases in P450j mRNA and protein occurred within 1 week after birth in both male and female rats. The levels of P450j and its mRNA thereafter remained elevated up to 12 weeks of age in both sexes. The increases in both P450j and its mRNA paralleled the change in aniline hydroxylase activity during development. Run-on transcriptional analysis confirmed that these increases were due to transcriptional activation of the P450j gene. In contrast to transcriptional activation during development, induction of P450j by various agents such as pyrazole, 4-methylpyrazole, and acetone might be due to post-transcriptional events. A 4-fold elevation in both enzymatic activity and immunodetectable P450j was not accompanied by an increase in P450j mRNA level.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cytochrome P-450 Enzyme System; DNA; Enzyme Induction; Ethanol; Female; Humans; Imidazoles; Kinetics; Liver; Male; Methylcholanthrene; Nucleic Acid Hybridization; Phenobarbital; Pyrazoles; Rats; Rats, Inbred Strains; Species Specificity
PubMed: 3782137
DOI: No ID Found -
British Journal of Cancer Aug 1975The uptake of 3-methycholanthrene and its metabolism to water-soluble derivatives were both determined in organ cultures of mouse and rat tissues, including prostate,...
The uptake of 3-methycholanthrene and its metabolism to water-soluble derivatives were both determined in organ cultures of mouse and rat tissues, including prostate, skin, lung and skeletal muscle. All the tissues concentrated the carcinogen from the medium and metabolized part of it to water-soluble compounds. The uptake of tritiated 3-methylcholanthrene was highest in the absence of serum and declined with rising serum concentration. Except for skeletal muscle, it was consistently higher in the murine tissues. The uptake of the hydrocarbon by rat and mouse prostates rose rapidly with time, reaching a maximum after 18 h incubation; the amounts of carcinogen in the tissue then declined and remained at a lower level for the rest of the observation period. The major part of the radioactivity was released within 5 h of transferring the explants to medium without the tracer; 25-40% of the peak concentration of carcinogen, however, still remained in the tissue and further medium changes could not remove any more. Addition of unlabelled 3-methylcholanthrene to the initial incubation increased the radioactivity taken up and caused substantially larger quantities of the carcinogen to be retained after the medium had been changed. The explants converted between 15% and 30% of the 3-methylcholanthrene which they had incorporated to water-soluble derivatives within 48 h but there was no obvious relationship between the amounts of hydrocarbon taken up by the different tissues and the proportions metabolized. A considerable part of the 3-methylcholanthrene in the explants remained unconverted 24 h after its removal from the medium.
Topics: Animals; Lung; Male; Methylcholanthrene; Mice; Mice, Inbred C3H; Muscles; Organ Culture Techniques; Prostate; Rats; Skin; Solubility; Time Factors; Water
PubMed: 1240005
DOI: 10.1038/bjc.1975.152