Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.
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PMID:Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s. 189 86

Tamoxifen (TXF), a triphenylethylene antiestrogen, is the major therapeutic agent for breast cancer. In rare cases, TXF treatment appears to increase incidence of endometrial cancer. Also in rats, TXF was found to induce hepatocellular carcinoma. Previous studies suggested that metabolism of TXF may contribute to its antiestrogenic and anticancer activity. The current study demonstrates a novel route of TXF metabolism. TXF is metabolized by rat and human liver microsomes into a reactive intermediate (txf*) which binds irreversibly to microsomal proteins. The binding requires NADPH and O2 and is inhibited by CO, inhibitors of P-450, and antibodies to rat NADPH-P450 reductase, indicating catalysis by P450. Phenobarbital treatment of rats markedly increases binding, suggesting the involvement of induced P450s. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from incubation of [14C] TXF with phenobarbital-treated microsomes exhibits a major radiolabeled zone which corresponds to a molecular weight of approximately 54,000, suggesting binding to a P-450. Cysteine and glutathione inhibited the binding of TXF without significantly affecting P-450-mediated metabolism of TXF, possibly by reacting with txf* or by competing for the same binding sites. Exposure of phenobarbital-treated microsomes and control-microsomes to 50 degrees C for 90 s, which inactivates the flavin-containing monooxygenase (FMO), diminished binding and pH 8.6 enhanced binding. Also, alternate FMO substrates inhibited binding. These findings indicate that P-450 and possibly FMO catalyze the reactions leading to the formation of txf*. However, incubations with single-labeled and dual-radiolabeled tamoxifen or with [14C]TXF-N-oxide demonstrated that monodesmethyl-TXF and TXF-N-oxide, the principal P-450 and FMO-mediated metabolites, respectively, are not on the major route of txf* formation, indicating that txf* could not be an aldehyde derived from tamoxifen nitrone. Thus, though the structure of txf* was not characterized, certain possibilities were excluded. Speculations on the structure of txf* and on its possible pharmacological and toxicological activity are presented.
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PMID:Cytochrome P-450-mediated activation and irreversible binding of the antiestrogen tamoxifen to proteins in rat and human liver: possible involvement of flavin-containing monooxygenases in tamoxifen activation. 193 68

Phenobarbital is a potent inducer of several liver-specific genes such as those encoding detoxication enzymes, including cytochromes P450. However, the mechanisms of action of the barbiturate are poorly understood. Since both, phenobarbital and glucocorticoids, are capable of inducing the same cytochrome P450 species, we asked whether the glucocorticoid receptor could participate to the phenobarbital induced responses. The results presented here show that phenobarbital was able to induce a two-fold increase in the affinity of the glucocorticoid receptor for the binding of dexamethasone, as well as a 30% increase of the receptor number in Reuber rat hepatoma cells of the Fao line. These effects may have a biological significance since they were paralleled by an enhancement of the dexamethasone-induced tyrosine aminotransferase activity, a glucocorticoid inducible function in rat hepatoma cells and in rat liver. To our knowledge, phenobarbital is the first compound shown to be able to induce, in intact cells, an increase in the affinity of the glucocorticoid receptor for the binding of its ligand.
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PMID:Effect of phenobarbital on the glucocorticoid receptor in rat hepatoma cells. 197 78

We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.
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PMID:Genetic analysis of aflatoxin B1 activation in rat hepatoma cells. 212 92

The cytochrome P450 in the transformable C3H/10T1/2 (10T1/2) cell line has been characterized and compared to the major polycyclic aromatic hydrocarbon (PAH)-inducible hepatic form, cytochrome P450IA1 (P450IA1). The mouse hepatoma cell line, Hepa-1, was used as an in vitro model for P450IA1 expression and regulation by PAH. Microsomes from uninduced and benz[a]anthracene (BA)-induced 10T1/2 cells provided PAH mono-oxygenated product profiles that were totally different from metabolite profiles produced by microsomes from uninduced and BA-induced Hepa-1 cells even though total activities were similar. The proximate carcinogen, 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) was a major product for the 10T1/2 microsomes, while Hepa-1 formed less than 2% of this metabolite. Hepa-1 converted benzo[a]pyrene (BP) to BP-4,5-diol and DMBA to 7-hydroxymethyl-12-methyl-BA, while 10T1/2 did not produce either product. Polyclonal antibody to rat hepatic P450IA1 did not inhibit metabolism of either PAH substrate by 10T1/2 microsomes, but totally inhibited such metabolism by Hepa-1 microsomes. Western immunoblot analysis of BA-induced 10T1/2 microsomes showed that less than 1% of total P450 was P450IA1. The PAH-metabolizing activity of 10T1/2 microsomes was highly inducible (14-fold) by pre-treatment of non-confluent intact cells with BA, but was only half as inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast, the P450IA1 activity of Hepa-1 cells was highly inducible by both compounds. The distinct metabolite profiles, antibody inhibition data and lack of immunoreactivity all indicate that PAH metabolism in 10T1/2 cells is catalyzed by a form of P450 distinct from P450IA1. The anomalous induction patterns suggest that this novel isozyme is predominantly regulated by a mechanism other than the Ah receptor.
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PMID:Characterization of a novel cytochrome P450 from the transformable cell line, C3H/10T1/2. 215 39

Twelve forms of human cytochrome P450 were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. The cDNA-expressed enzymes were tested for their ability to oxidize estradiol. Incubation of [14C]estradiol with cell lysates containing P450 IA2 resulted in the production of 2-hydroxy and 4-hydroxy metabolites with substrate turnovers of 2.74 and 0.27 min-1, respectively. P450s IIIA3 and IIIA4 yielded the same metabolites at about one third the rate of P450 IA2. Low levels of estradiol hydroxylation were also catalyzed by P450s IIC9, IIIA5, and IVB1. Six other P450 forms yielded no detectable metabolism. The roles of P450s IA2, IIA3, and IIIA4 were further established by immunoinhibition using antirat P450 antibodies. Antibody that specifically binds to P450 IIIA3 and IIIA4 inhibited 60-70% of estradiol hydroxylation, and antibody against P450 IA2 inhibited 20-40% of the estradiol hydroxylase activity in microsomes from two human liver specimens, suggesting that these enzymes constitute the major forms catalyzing estradiol oxidation in human liver. Immunoinhibition results also suggest that 2-hydroxy- and/or 4-hydroxycatechol estrogens are further metabolized to other yet uncharacterized metabolites by P450s IIIA3 and IIIA4.
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PMID:Estradiol metabolism by complementary deoxyribonucleic acid-expressed human cytochrome P450s. 216 48

Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B1 to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B1 to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B1 to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, IIIA5, and IVB1, did not significantly activate aflatoxin B1 as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B1 activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B1 in human liver involves the contribution of multiple forms of P450.
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PMID:Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1. 216 57

Using the mouse hepatoma Hepa-1c1c7 c37 mutant cell line that exhibits negligible benzo[a]pyrene hydroxylase (Cyp1a1) and acetanilide 4-hydroxylase (Cyp1a2) enzyme activities, we developed stable transfectants of plasmids containing the murine Cyp1a1 (cytochrome P(1)450) and the human CYP1A2 (P(3)450) cDNAs. We show that the assay measuring metabolism of ethoxyfluorescein ethyl ester (EFEE) was invaluable in screening large numbers of individual cell lines for high Cyp1a1 enzyme activity. Nine different plasmid constructs containing various combinations of promoter and enhancer sequences were compared, including: the Drosophila heat shock promoter, the mouse mammary tumor virus long terminal repeat (MMTV LTR) carrying the glucocorticoid-responsive element (GRE), enhancer sequences from simian virus 40 (SV40) and herpes simplex virus type 1 (HSV-1), and the aromatic hydrocarbon-responsive domain (AhRD) of the murine Cyp1a1 gene. Interestingly, only those constructs containing the AhRD produced high levels of Cyp1a1 enzyme activity. In contrast, high levels of CYP1A2 activity were obtained with plasmids carrying the HSV-1 enhancer, as well as the AhRD. These studies suggest that the AhRD, which responds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), provides a post-transcriptional signal necessary for the induction of functional Cyp1a1 enzyme activity. Although untransfected c37 cells exhibit markedly elevated levels of endogenous Cyp1a1 mRNA, the expression of exogenous Cyp1a1 or CYP1A2 enzyme activity in these cells decreases the concentration of this endogenous Cyp1a1 mRNA to negligible levels and restores Cyp1a1 mRNA inducibility by TCDD; these data indicate that the functional product of either the Cyp1a1 gene or the CYP1A2 gene might have a role in an autoregulatory loop controlling the constitutive expression of the Cyp1a1 gene. The cell lines described herein should be valuable in assessing the contribution of these two P450 enzymes to the processes of cytotoxicity, mutagenesis, and carcinogenesis.
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PMID:Stable expression of mouse Cyp1a1 and human CYP1A2 cDNAs transfected into mouse hepatoma cells lacking detectable P450 enzyme activity. 220 99

Polycyclic aromatic hydrocarbon (PAH)-induced C3H/10-T1/2/CL8 mouse embryo fibroblasts (10T1/2) and mouse hepatoma-derived Hepa 1c1c7 cells (Hepa-1), exhibit comparable total cytochrome P450 levels and total PAH-metabolizing activities but very different distributions of PAH metabolites. Based on anti-P450IA1-IgG inhibition data, P450IA1 contributes essentially all PAH metabolism in Hepa-1 microsomes but is not involved in PAH metabolism by 10T1/2 cells. In addition, the microsomal epoxide hydratase (EHm) in Hepa-1 cells is far less effective in dihydrodiol (diol) formation compared to that in 10T1/2 microsomes [Pottenger, L.H. and Jefcoate, C.R. Carcinogenesis, 11, 321-327 (1990)]. In the present study, the levels of expression of P450IA1 and EHm proteins and the corresponding mRNAs, both prior to and following exposure to benz[a]anthracene (BA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been correlated with microsomal PAH metabolism by each cell type. In 10T1/2 cells, P450IA1 protein (56 kd) and mRNA (2.6 kb) were detectable at extremely low levels in only two of five cell preparations and then only after maximum induction by TCDD and BA. Thus although 10T1/2 cells contain functional Ah receptors, their capacity to induce P450IA1 is highly suppressed, representing at most 2% of the total P450. TCDD (10 nM) was 4-fold more effective than BA (10 microM) in inducing P450IA1 mRNA, while the levels of immunodetectable protein were comparable. An even greater discrepancy between P450IA1 mRNA and protein levels was seen in BA-induced Hepa-1 cells, where a 4-fold increase in mRNA was paralleled by a 20-fold increase in protein. This difference is probably due to the greater effect of BA depletion on mRNA compared to protein levels. In 10T1/2 cells, BA and TCDD were equally effective at increasing expression of an unidentified 1.9 kb mRNA sequence that blotted very weakly with the P450IA1 cDNA probe. The expression of this mRNA was independent from that of P450IA1. A similar band was visible in Hepa-1 cells less than 1% of the P450IA1 mRNA. EHm mRNA was almost 3-fold higher in 10T1/2 compared to Hepa-1 cells and was unaffected by cell treatments. In Hepa-1 cells, BA and TCDD elevated EHm protein and hydrating activity to levels comparable to those expressed in 10T1/2 cells. It is, therefore, suggested that the relative ineffectiveness of Hepa-1, compared to 10T1/2 EHm, to hydrate low levels of PAH-epoxides is due to differences between the two proteins or their disposition in the microsomal membrane.
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PMID:Differences in the modulation of P450IA1 and epoxide hydratase expression by benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse embryo versus mouse hepatoma-derived cell lines. 220 84

The mouse hepatoma cell line Hepa-1 was studied for aryl hydrocarbon hydroxylase (AHH) inducibility by sixteen compounds known to be inducers of cytochrome P450 of different "classes". Both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and sodium phenobarbital induced AHH activity. A cytochrome P450IA1-specific (P1-450) mouse cDNA probe was used to quantitate mRNA induction. There was a good correlation between the amount of cytochrome P450IA1 mRNA induced and AHH activity. Immunoblots with monoclonal antibody 1-7-1, which recognizes rat liver P450IA1 and P450IA2 (P450c and P450d, respectively), showed that both phenobarbital and TCDD increase the amount of a P450 isozyme immunorelated to P450IA1 in this cell line. Hepa-1 mutants with no AHH inducibility (no functional P450IA1 structural gene; no Ah receptor; no nuclear translocation of the inducer-receptor complex; and presence of dominant repressor) did not respond to phenobarbital. The cytosolic receptor for TCDD (Ah receptor) was characterized to see if phenobarbital induced cytochrome P450IA1 mRNA and the hydroxylase enzyme through the same mechanism as TCDD. 20 mM Phenobarbital almost completely abolished the binding of 3H-TCDD to the cytosolic receptor. These data indicate that phenobarbital can be a weak ligand for the Ah receptor and thus induce cytochrome P450IA1 and AHH activity. The observation increases the list of different P450 forms inducible by phenobarbital.
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PMID:Induction of cytochrome P450IA1 in mouse hepatoma cells by several chemicals. Phenobarbital and TCDD induce the same form of cytochrome P450. 254 28


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