-
The Biochemical Journal Dec 1985Antibodies to four rat liver forms of cytochrome P-450, two phenobarbital-inducible (PB1 and PB2) and two 3-methylcholanthrene-inducible (MC1 and MC2) proteins, have...
Antibodies to four rat liver forms of cytochrome P-450, two phenobarbital-inducible (PB1 and PB2) and two 3-methylcholanthrene-inducible (MC1 and MC2) proteins, have been used to make a structural and functional comparison of rat and human cytochromes P-450. Proteins from both species were identified on Western blots by their reaction with these antibodies. In the human liver preparations, structurally related proteins to PB1 and to PB2 were identified in all the samples tested with apparent Mr values of 51 800 and 54 800 for PB1 and 53 600 and 57 200 for PB2. Considerable variation in the content of the lower-Mr proteins was measured between samples and, as with the rat enzymes, samples which reacted well with anti-PB1 also reacted with anti-PB2, indicating that these proteins are regulated at least to some degree, co-ordinately. The apparent Mr values of the major human proteins identified with anti-MC1 and anti-MC2 were 54 400 and 57 000 respectively. Only six (of 31) human samples contained significant amounts of these proteins. The same six samples which reacted with anti-MC1 also reacted with anti-MC2, again indicating co-ordinate regulation of these two proteins. Antibody inhibition of microsomal 7-ethoxycoumarin and 7-ethoxyresorufin metabolism demonstrated a degree of conservation of substrate specificity related to specific P-450 isoenzymes between the species. However, the contributions of the different P-450 isoenzymes to the human microsomal activity were not always related to the rat enzyme with the highest activity towards these substrates.
Topics: Animals; Antibodies; Coumarins; Cytochrome P-450 Enzyme System; Humans; Isoenzymes; Methylcholanthrene; Microsomes, Liver; Oxazines; Phenobarbital; Rats; Rats, Inbred Strains; Species Specificity
PubMed: 4091826
DOI: 10.1042/bj2320869 -
The Biochemical Journal Oct 19721. The effects of safrole and isosafrole pretreatment on both N- and ring-hydroxylation of 2-acetamidofluorene were studied in male rats and hamsters. 2. Isosafrole...
1. The effects of safrole and isosafrole pretreatment on both N- and ring-hydroxylation of 2-acetamidofluorene were studied in male rats and hamsters. 2. Isosafrole (100mg/day per kg body wt.) pretreatment of rats for 3 days did not have any effect on urinary excretion of hydroxy metabolites of 2-acetamidofluorene. However, similar pretreatment with safrole produced increased urinary excretion of N-, 3- and 5-hydroxy derivatives. 3. Similar treatment with these two chemicals for 3 days increased ring-hydroxylation activity by rat liver microsomal material. Increases in N-hydroxylation were much less than those in ring-hydroxylation. Isosafrole was twice as effective as safrole. 4. Increases in hydroxylating activity due to safrole or isosafrole treatment were inhibited by simultaneous administration of ethionine. Similarly, ethionine inhibition was almost completely reversed by the simultaneous administration of methionine. 5. Safrole or isosafrole (0.1mm and 1mm) inhibited 7-hydroxylation activity by liver microsomal material from control rats. At 1mm these two chemicals inhibited both 5- and 7-hydroxylation activity by liver microsomal material from 3-methylcholanthrene-pretreated rats. 3-Hydroxylation activity was not inhibited by 1mm concentrations of these two chemicals. 6. A single injection of safrole (50100 or 200mg/kg body wt.) 24h before assay had no appreciable effect on either N- or ring-hydroxylation activity by hamster liver microsomal material. However, isosafrole (200mg/kg body wt.) treatment inhibited N-, 3- and 5-hydroxylation activities by hamster liver microsomal material; it had no effect on 7-hydroxylation activity.
Topics: Alkenes; Animals; Carcinogens; Chromatography, Paper; Cricetinae; Dioxoles; Ethionine; Fluorenes; Hydroxylation; In Vitro Techniques; Male; Methionine; Methylcholanthrene; Microsomes, Liver; Rats; Urine
PubMed: 4655827
DOI: 10.1042/bj1290937 -
The Journal of Toxicological Sciences May 1981Pulmonary cytochrome P-448 from 3-methylcholanthrene-pretreated rats was partially purified approximately 20-fold. The purified preparations containing 1.74 nmol per mg...
Pulmonary cytochrome P-448 from 3-methylcholanthrene-pretreated rats was partially purified approximately 20-fold. The purified preparations containing 1.74 nmol per mg protein were essentially free of NADH-cytochrome b5 reductase and NADPH-cytochrome c reductase, and included a small amount of cytochrome b5 spectrophotometrically. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the partially purified cytochrome P-448 gave one major band and several minor bands when stained with Coomassie blue. The major band on which the presence of peroxidase activity could be determined had the apparent molecular weight of 57,000. In the presence of NADPH-cytochrome c reductase, lipid and NADPH, the pulmonary cytochrome P-448 was active in hydroxylation of benzo-[a]pyrene, but catalyzed N-demethylation of benzphetamine in a slow rate.
Topics: Animals; Benzopyrene Hydroxylase; Catalysis; Cytochrome P-450 CYP1A2; Cytochromes; Electrophoresis, Polyacrylamide Gel; Liver; Lung; Male; Methylcholanthrene; NADPH-Ferrihemoprotein Reductase; Rats
PubMed: 6792366
DOI: 10.2131/jts.6.71 -
The Journal of Biological Chemistry Jan 1980The synthesis of cytochrome P-450 (phenobarbital inducible) and cytochrome P-448 (3-methylcholanthrene inducible) have been studied in rat liver in vivo and in the wheat...
The synthesis of cytochrome P-450 (phenobarbital inducible) and cytochrome P-448 (3-methylcholanthrene inducible) have been studied in rat liver in vivo and in the wheat germ cell-free system using anti-cytochrome P-450 and anti-cytochrome P-448 antibodies. The major mature forms synthesized in vivo correspond to a molecular weight of 47,000 for cytochrome P-450 and 53,000 for cytochrome P-448. Translation of poly(A)-containing RNA from phenobarbital-treated rats in the wheat germ cell-free system reveals that the cell-free product immunoprecipitated with anti-cytochrome P-450 antibody has a molecular weight close to 47,000. In the case of 3-methylcholanthrene, the cell-free product immunoprecipitated with anti-cytochrome P-448 antibody shows a molecular weight around 59,000. Significant conversion of the 59,000 species to the 53,000 species can be demonstrated when the translation is carried out in the presence of microsomal membranes isolated from rat liver. Phenobarbital and 3-methylcholanthrene enhance the translatable messenger RNA contents for cytochrome P-450 and cytochrome P-448, respectively.
Topics: Animals; Cytochrome P-450 Enzyme System; Liver; Methylcholanthrene; Molecular Weight; Phenobarbital; Plants; Protein Biosynthesis; Rats; Triticum
PubMed: 7356631
DOI: No ID Found -
The Biochemical Journal Aug 1986Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of...
Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.
Topics: Animals; Arachidonic Acid; Arachidonic Acids; Benzoflavones; Cobalt; Cytochrome P-450 Enzyme System; Heme Oxygenase (Decyclizing); Kidney; Male; Methylcholanthrene; Microsomes; Microsomes, Liver; Oxygenases; Rabbits; beta-Naphthoflavone
PubMed: 3099765
DOI: 10.1042/bj2380283 -
Environmental Health Perspectives Apr 1979The influence of vitamin A on the development of chemically induced lung carcinomas in rats was investigated. Rats were maintained on low, "normal" and excess levels of...
The influence of vitamin A on the development of chemically induced lung carcinomas in rats was investigated. Rats were maintained on low, "normal" and excess levels of retinyl acetate (RA). Respiratory tract-squamous carcinomas were induced by intratracheal injections of 3-methylcholanthrene (3-MCA). The carcinogen doses used ranged from 1.25 to 10.0 mg of 3-MCA. Serial sacrifices conducted during the first 20 weeks following carcinogen exposure showed that metaplastic lung nodules, presumed to be precursors of later appearing carcinomas, occurred earlier and at higher incidence in rats maintained on low levels of RA than in rats maintained on moderate or high levels of RA. The development of invasive pulmonary carcinomas was enhanced at all four carcinogen doses in rats receiving low levels of RA as compared to rats receiving moderate or high levels of RA. No consistent difference in lung cancer incidence existed between the groups receiving normal and high levels of RA. The data clearly show an increased susceptibility of vitamin A-deficient rats to develop chemically induced lung cancers. Possible mechanisms underlying this effect are discussed.
Topics: Animals; Carcinogens; Carcinoma, Squamous Cell; Drug Interactions; Female; Lung Neoplasms; Male; Methylcholanthrene; Rats; Respiratory Tract Neoplasms; Vitamin A
PubMed: 510247
DOI: 10.1289/ehp.792989 -
The Journal of Biological Chemistry Aug 1988Primary cultures of adult rat hepatocytes grown in serum-free hormonally defined medium were shown, for the first time, to be capable of supporting the...
Primary cultures of adult rat hepatocytes grown in serum-free hormonally defined medium were shown, for the first time, to be capable of supporting the 3-methylcholanthrene-inducible expression of cytochrome P-450d. Such cultures were used to investigate the mechanism of the induction of cytochrome P-450c and P-450d mRNAs. After 1 day of growth in culture, P-450c and P-450d mRNAs were induced 33- and 28-fold, respectively, by 3-methylcholanthrene treatment. A similar magnitude of induction was achieved after 2-5 days growth in culture. However, the relative abundance of the two mRNAs before and after treatment decreased linearly over the 5-day time course. Kinetic analysis revealed that induction of both genes was rapid and could be observed less than 2 h following treatment. Accumulation of both mRNAs was linear for 8 h, reaching a plateau by 12 h. Expression then remained constant for at least 12 additional hours. In vitro nuclear run-on experiments revealed a 3.9- and 2.0-fold transcriptional induction of the P-450c and P-450d genes, respectively. This is in contrast to the large induction of accumulation of these mRNAs observed at steady state. Thus, the 3-methylcholanthrene induction of P-450c and P-450d mRNAs in the hepatocyte cultures appeared to be mediated primarily at the post-transcriptional level. Experiments on rat liver showed that, in vivo, P-450d is also regulated primarily at the post-transcriptional level. However, P-450c was found to be regulated primarily transcriptionally.
Topics: Animals; Cell Nucleus; Cells, Cultured; Cytochrome P-450 Enzyme System; Gene Expression Regulation; Kinetics; Liver; Methylcholanthrene; Nucleic Acid Hybridization; RNA, Messenger; Rats; Rats, Inbred Strains; Transcription, Genetic; Uridine Triphosphate
PubMed: 3403555
DOI: No ID Found -
Carcinogenesis Jul 2018The development of alternative methods to animal testing is a priority in the context of regulatory toxicology. Carcinogenesis is a field where the demand for...
The development of alternative methods to animal testing is a priority in the context of regulatory toxicology. Carcinogenesis is a field where the demand for alternative methods is particularly high. The standard rodent carcinogenicity bioassay requires a large use of animals, high costs, prolonged duration and shows several limitations, which can affect the comprehension of the human relevance of animal carcinogenesis. The cell transformation assay (CTA) has long been debated as a possible in vitro test to study carcinogenesis. This assay provides an easily detectable endpoint of oncotransformation, which can be used to anchor the exposure to the acquisition of the malignant phenotype. However, the current protocols do not provide information on either molecular key events supporting the carcinogenesis process, nor the mechanism of action of the test chemicals. In order to improve the use of this assay in the integrated testing strategy for carcinogenesis, we developed the transformics method, which combines the CTA and transcriptomics, to highlight the molecular steps leading to in vitro malignant transformation. We studied 3-methylcholanthrene (3-MCA), a genotoxic chemical able to induce in vitro cell transformation, at both transforming and subtransforming concentrations in BALB/c 3T3 cells and evaluated the gene modulation at critical steps of the experimental protocol. The results gave evidence for the potential key role of the immune system and the possible involvement of the aryl hydrocarbon receptor (AhR) pathway as the initial steps of the in vitro transformation process induced by 3-MCA, suggesting that the initiating events are related to non-genotoxic mechanisms.
Topics: 3T3 Cells; Animals; Biological Assay; Carcinogenesis; Carcinogenicity Tests; Carcinogens; Cell Transformation, Neoplastic; Methylcholanthrene; Mice; Mice, Inbred BALB C; Receptors, Aryl Hydrocarbon
PubMed: 29554273
DOI: 10.1093/carcin/bgy037 -
The Journal of Biological Chemistry Mar 1972
Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds, and endogenous substrates. II. Role of the cytochrome P-450 and P-448 fractions in drug and steroid hydroxylations.
Topics: Animals; Benzopyrenes; Carbon Isotopes; Catalysis; Cytochromes; Dealkylation; Enzyme Induction; Hydroxylation; Lipid Metabolism; Male; Methylcholanthrene; Microsomes, Liver; NADP; Oxidoreductases; Phenobarbital; Piperazines; Rats; Testosterone
PubMed: 4401153
DOI: No ID Found -
The Biochemical Journal Jan 1980At 2-3 h after phenobaribtal administration, the drug has no effect on nucleoplasmic RNA synthesis and decreases nucleolar RNA synthesis. However, at this time there is... (Comparative Study)
Comparative Study
At 2-3 h after phenobaribtal administration, the drug has no effect on nucleoplasmic RNA synthesis and decreases nucleolar RNA synthesis. However, at this time there is an increase in the labelling of cytoplasmic poly(A)-containing RNA, even though there is decreased labelling of total polyribosomal RNA. The decrease in labelling of nucleolar and total polyribosomal RNA owing to phenobarbital is a transient phenomenon. Under similar conditions, 3-methylcholanthrene has no effect on nucleolar RNA synthesis, but leads to an increase in synthesis of nucleoplasmic and cytoplasmic poly(A)-containing RNA. Cytosol isolated from phenobarbital-treated, but not from 3-methyl-cholanthrene-treated, animals facilitates an enhanced transport of RNA from nuclei. At the time points investigated, 3-methylcholanthrene or its metabolite shows a 10-15-fold higher concentration in the chromatin than that of phenobarbital or its metabolite. It is suggested that the primary effect of phenobarbital is at the cytoplasmic level, promoting the transport of RNA from the nuclei, which can act as a trigger for enhanced transcription at later periods. 3-Methylcholanthrene or its metabolite directly binds to the chromatin and evokes a selective transcriptional response.
Topics: Animals; Biological Transport; Cell Nucleus; Cytoplasm; Kinetics; Liver; Male; Methylcholanthrene; Phenobarbital; Polyribosomes; RNA; Rats
PubMed: 6154460
DOI: 10.1042/bj1860081