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)

The metabolic activation of the potent carcinogen dibenzo[a,h]anthracene (DB[a,h]A) was investigated with recombinant human cytochrome P450 enzymes 1A2, 2B6, 2C8, 2C9, 2E1, 3A3, 3A4, and 3A5 expressed in hepatoma G2 cells and with 14 different human liver microsomes. Three dihydrodiols, three phenols, and one diphenol were formed and separated by high-performance liquid chromatography and identified by UV absorption and mass spectra. Of all P450s tested, 1A2 and 2C9 were the most active and 2B6 was moderately active in the rate of total DB[a,h]A metabolism (2.5- to 12-fold greater activity than that for other P450s). The trans-3,4-dihydrodiol, generally recognized as a precursor of the ultimate carcinogenic 3,4-diol-1,2-epoxides, was produced most actively by 2C9, then 1A2 and 2B6. The values of enzymatic kinetics (K(m) and V(max)) indicated that 2C9 had the highest catalytic efficiency (V(max)/K(m) = 9.7) in the formation of 3,4-dihydrodiol, in contrast to 1A2 (5.9) and 2B6 (4.4). 1A2 had the highest activity toward production of the 1,2-dihydrodiol, which is considered to be a weakly carcinogenic metabolite. Although specific activities of human liver microsomes in overall metabolism of DB[a,h]A markedly differed between individuals, metabolic patterns were observed similar to that generated from 1A2. Since human 1A1, a predominant enzyme for metabolism of polycyclic aromatic hydrocarbons, is not significantly expressed in the liver, hepatic microsomal 2C9, 1A2, and 2B6 all probably contribute to the metabolic activation of DB[a,h]A.
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PMID:Metabolic activation of the potent carcinogen dibenzo[a,h]anthracene by cDNA-expressed human cytochromes P450. 863 31

A recombinant H4IIE rat hepatoma cell line (H4L1.1c4, H4IIE-luc), containing a luciferase reporter gene under control of dioxin-responsive enhancers, was examined for responsiveness to several polyhalogenated aromatic hydrocarbons (PHAHs). The recombinant cell system was compared with the widely used wild-type cell line (H4IIE-wt), which expresses Ah receptor-mediated cytochrome P450 1A induction. We also report an improved and down-scaled method for the H4IIE-wt bioassay which allows for the rapid screening of environmental samples for Ah-active PhAHs. This method employs 96-well plates, a plate-reading spectrofluorometer, and a fluorescence-based protein assay that enables the simultaneous measurement of resorufin and protein. Both cell lines demonstrated a dose-dependent increase in Ah receptor-mediated response upon exposure to a number of known Ah receptor agonists, including Halowax 1014. H4IIE-luc cells were 3-fold more sensitive than H4IIE-wt cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The detection limit and ED50 for EROD induction by TCDD were 0.6 and 4.9 fmol/well (2,4 and 20 pM), respectively; for luciferase induction they were 0.2 and 1.4 fmol/well (0.8 and 5.6 pM). The detection limit for EROD induction in H4IIE-wt cells was a 50-fold improvement over that reported previously (Tillitt et al., Environ. Sci. Technol. 25, 87-92, 1991) and comparable to that of a chicken embryo primary hepatocyte bioassay (Kennedy et al., Anal. Biochem. 211, 102-112, 1993). The tested PHAHs exhibited a similar structure-activity relationship in H4IIE-luc as in H4IIE-wt cells. Binary mixtures of TCDD, PCB-126, and PCB-77 showed no departure from additivity in their combined responses when tested in H4IIE-wt cells. PCB-153 at the highest tested dose of 14 nmol/well (56 microM) significantly reduced the potency of TCDD and PCB-126 without affecting their efficacy in both H4IIE-wt and H4IIE-luc cells. These findings support the use of H4IIE-luc cells as an alternative bioanalytical tool to the wild-type cells for the detection of Ah agonists in environmental samples.
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PMID:Comparison of Ah receptor-mediated luciferase and ethoxyresorufin-O-deethylase induction in H4IIE cells: implications for their use as bioanalytical tools for the detection of polyhalogenated aromatic hydrocarbons. 866 58

Polychlorobiphenyls are potent inducers of hepatic cytochrome P450 in various species. Until now, no model based on cultured cells can be considered as a universal surrogate for in vivo metabolism. In this respect, cultured rat hepatocytes, quail hepatocytes, and human hepatoma (HepG2) cells were used to study the effects of 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB) and Aroclor 1254 on drug-metabolizing enzymes. The presence of dexamethasone in the culture medium allows the expression and the induction of several cytochrome P450 isoenzymes found in adult cells. Induction of ethoxycoumarin-(ECOD) and ethoxyresorufin-O-deethylase (EROD), activities were measured. Induced P450s were identified by immunoblotting and Northern blotting. Aroclor 1254 induced ECOD activity in all three cell types, but the effect was much stronger in fetal rat hepatocytes than in human or quail cells. Aroclor failed to induce EROD activity in quail cells, had a slight inducer effect in HepG2 cells, and a marked effect in rat hepatocytes. 3,3',4,4'-TCB had no effect in HepG2 cells but significantly increased EROD and ECOD activities, especially the latter, in rat and quail cells. On the immunoblots, specific antibodies revealed essentially CYP1A1 in fetal rat hepatocytes, CYP2B1/2 in quail hepatocytes and CYP3A1 in HepG2 cells. Analysis of Northern blots showed an hybridization with CYP1A1, 2B1 and 3A1 mRNA in fetal rat hepatocytes, CYP3A and 1A mRNA in HepG2 cells, and a form of CYP2 mRNA in fetal quail hepatocytes closely related to homolog rat CYP2E or CYP2C. In quail hepatocytes, induction did not increase proportionally with the concentration of inducer in the culture medium. Instead, the dose-response curves (for EROD activity especially) peaked sharply at 1 muM Aroclor 1254, an effect attributed to changes in membrane fluidity or lipid content. Our results highlight the advantage of using several types of cultured hepatocytes to investigate fundamental aspects of drug-metabolism-linked toxicity, the balance between xenobiotic bioactivation and detoxication being differently affected by PCBs in different animal species.
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PMID:P450 induction by Aroclor 1254 and 3,3',4,4'-tetrachlorobiphenyl in cultured hepatocytes from rat, quail and man: interspecies comparison. 866 1

The cytochrome P450-dependent monooxygenases, which represent an extended superfamily, catalyze the biotransformation of many endogenous and exogenous substances. One of these hemoproteins, cytochrome P4501A1, is most closely associated with the bioactivation of polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which may play a role in environmental carcinogenesis. A negative regulatory element (NRE) has been localized in the 5'-upstream region of the cytochrome P4501A1 gene (CYP1A1) at -843 to -746 base pairs from the site of transcription. The purpose of this research was to define any interactions of trans-acting proteins with this cis element. Rat liver nuclei were used as the source of trans-acting proteins and a biotinylated NRE-bearing fragment (-782 to -843 bp) from a plasmid which contained the CYP1A1 was prepared by the polymerase chain reaction technique. Gel mobility shift assays were used to demonstrate interactions between this NRE fragment and nuclear proteins. The specific binding to an octamer-containing motif in the 5'-upstream region of CYP1A1 was demonstrated; this was used as a step in the partial purification from rat liver of the transcription factor, Oct-1. Conventional chromatographic procedures and DNA recognition site affinity chromatography were also used. HepG2 human hepatoma cells were transfected with both pMCoLUC+ which contains the luciferase gene as a reporter gene driven by the CYP1A1 promoter (including the NRE), and an Oct-1 expression vector. Luciferase activity/mg protein in the doubly-transfected cells was significantly lower than in cells containing only pMCoLUC+. A nuclear transcription factor Oct-1 interacts with a portion of the NRE of the rat CYP1A1, suppressing the expression of this gene. These findings may help to explain the low level of basal expression of CYP1A1 in mammalian systems.
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PMID:Nuclear transcription factor Oct-1 binds to the 5'-upstream region of CYP1A1 and negatively regulates its expression. 872 8

Synthetic estrogens act as tumor promoters in rat liver. Because estrogen treatment markedly increases the secretion of pituitary prolactin, also shown to be a tumor promoter in rat liver, the possibility of a pituitary influence in estrogen promotion was investigated in Wistar rats. In diethylnitrosamine (DEN)-initiated hypophysectomized (hx) female rats, 24 weeks of ethinyl estradiol (EE) administration (500 microg/kg/d, intraperitoneally) did not increase the number of hepatocyte nodules and did not induce hepatocellular carcinoma (HCC) in a 2-year study. Very few placental forms of glutathione-S-transferase (GST-P)-positive foci were observed at the end of EE administration. Estrogen receptor (ER) messenger RNA (mRNA) levels in hx females were 20% of the levels in intact females. EE administration (range, 160-210 microg/kg/d, subcutaneous release pellets) to DEN-initiated intact males and females increased the number and size of hepatocyte foci. A significant increase in HCC frequency was observed in EE-treated females compared with females receiving sham-release pellets, and the latency period for HCC induction was decreased by EE in both males and females. Inhibition of prolactin (PRL) secretion by bromocriptine (Brc) (ParlodelLAR, slow intramuscular release vehicles) during EE treatment decreased the number of foci without affecting their size and markedly prolonged the latency period in both sexes. EE treatment also significantly increased the expression of c-myc, and c-jun, enhanced the levels of growth hormone receptor (GHr) mRNA in females and the levels of ER mRNA in males and "feminized" the expression of the GH-regulated genes cytochrome P450 (CYP), 2C11, CYP 2C12, and GHr in male liver. Brc administration decreased the mRNA levels of the female-predominant CYP 2C12 in EE-treated males but otherwise had no effects. In conclusion, a decreased promotive effect of EE was obtained by decreasing the PRL levels, indicating that estrogens exert at least part of their promotion effects indirectly, by increasing the levels of pituitary PRL.
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PMID:Role of the pituitary in tumor promotion with ethinyl estradiol in rat liver. 885 87

The adrenal steroid dehydroepiandrosterone (DHEA) stimulates a dramatic increase in both the size and the number of peroxisomes present in liver when given at pharmacological doses to rodents. Structurally diverse chemicals including many fatty acids, hypolipidemic drugs and other foreign chemicals, can also induce such a peroxisome proliferative response. This response is associated with a dramatic induction of perosisomal fatty acid beta-oxidation enzymes and microsomal cytochrome P450 4A fatty acid hydroxylases and, long-term, can lead to induction of hepatocellular carcinoma. This review examines the underlying mechanisms by which DHEA induces peroxisome proliferation and evaluates the possible role of peroxisome proliferator-activated receptor (PPAR) in this process. Like DHEA, the 17 beta-reduced metabolite 5-androstene-3 beta. 17 beta-diol (ADIOL) is an active peroxisome proliferator when administered in vivo, whereas androgenic and estrogenic metabolites of DHEA are inactive. In primary rat hepatocytes, however, DHEA and ADIOI are inactive as inducers of P450 4A and peroxisomal enzymes unless first metabolized by steroid sulfotransferase to the 3 beta-sulfates, DHEA-S and ADIOL-S. Investigations as to whether DHEA utilizes the same induction mechanism employed by classic, foreign chemical peroxisome proliferators, namely, activation of the intracellular receptor molecule PPAR, have shown that DHEA-S and ADIOL-S are ineffective with respect to PPAR activation in transient transfection/trans-activation assays. This inactivity of DHEA-S in vitro suggests a requirement for specific cellular transport or for further metabolism of the steroid which is only met in liver cells. Alternatively, the action of DHEA-S may require accessory proteins or other nuclear factors that modulate the activity of PPAR, such as retinoid X receptor (RXR), hepatocyte nuclear factor-4 (HNF-4) or chick ovalbumin upstream promoter transcription factor (COUP-TF). Investigations using Ca(2+)-channel blockers such as nicardipine suggest that there are important mechanistic similarities between the foreign chemical- and DHEA-S-stimulated induction responses, and support the hypothesis that these two classes of peroxisome proliferators both activate Ca(2+)-dependent signaling pathways. Further studies are required to ascertain whether this potential of DHEA and its sulfated metabolites to serve as physiological modulators of fatty acid metabolism and peroxisome enzyme expression contributes to the striking anti-carcinogenic and other useful chemoprotective properties that DHEA is known to possess.
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PMID:Role of metabolism in the activation of dehydroepiandrosterone as a peroxisome proliferator. 894 97

Cooking of protein-rich food generates procarcinogenic heterocyclic aromatic amines (HCA) in amounts that range in the part per billion levels. HCA have been reported to induce xenobiotic metabolizing enzymes of the cytochrome P450 1A (CYP1A) subfamily, most notably the CYP1A2 isoform. The regulator mechanism of the CYP1A induction by HCA is, however, unclear. Studies in vivo in rats and in primary hepatocyte cultures revealed "that MelQx induced both CYP1A1/1A2 proteins and the corresponding catalytic activities. In contrast to previous studies, no preferential induction of CYP1A2 was observed in the present study. CYP1A1 and CYP1A2 are target genes of the intracellular dioxin receptor. This receptor interacts with xenobiotic response elements (XREs) of target promoters upon binding the environmental pollutant dioxin or related compounds. HCA exhibited capacity to activate the dioxin receptor to a form which interacts with XRE in vitro. Taken together, these results suggest that MelQx regulates both CYP1A isozymes by the same mechanism involving the dioxin receptor. Another group of putative dioxin receptor ligands of dietary orgin are the indolocarbazoles, which are produced in vivo from precursor molecules in cruciferous plants. Indolocarbazoles potently regulated gene expression of a reporter gene driven by a minimal XRE in both mouse and human hepatoma cells. The indolocarbazole-induced human receptor appeared to form more stable complexes with XRE in vitro relative to those generated by the dioxin-activated receptor. This study indicates that the HCA and the indolocarbazoles both represent distinct classes of dietary dioxin receptor agonists.
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PMID:Interactions of procarcinogenic heterocyclic amines and indolocarbazoles with the dioxin receptor. 896 Mar 75

Pollutants of River Lambro, a tributary River Po, were monitored by their potential to induce 1A1 isoform of cytochrome P450 in the FaO hepatoma cell line. Extracts of water samples taken during different months over about one year were fractionated by reverse phase HPLC technique. Six fractions of decreasing polarity were collected, concentrated, freeze-dried and suspended in DMSO for the treatment of the cells. Aliquots of such suspensions were dissolved in the growth medium and left for 48 h in contact with FaO cells, that were maintained in 24-well plates. Cellular monolayers were also exposed to a mixture of the six fractions, to evaluate the effects of all the pollutants mixed together in the original water sample. The CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity was fluorimetrically detected as a measure of inducing potential, and total protein content was evaluated as cytotoxicity end-point. The results showed significant increases of EROD activity over the controls in nearly all the fractions with marked reduction of viability only in two of the mixed samples. The effects of the mixtures were not simply additive, thus suggesting both synergistic and antagonistic interactions. The potential utility of this simple and fast bioassay in environmental risk/hazard assessment is clearly apparent.
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PMID:An "in vitro" approach to water pollution monitoring. 900 49

1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1'(E),3' (E)-dien-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (EB1089) is a novel synthetic analog of 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D3] with potential for use in the treatment of hyperproliferative disorders. It has an altered side-chain structure compared to 1,25-(OH)2D3, featuring 26,27 dimethyl groups, insertion of an extra carbon atom (24a) at C-24, and two double bonds at C-22,23 and C-24,24a. In vitro metabolism of EB1089 was studied in a human keratinocyte cell model, HPK1A-ras, previously shown to metabolize 1,25-(OH)2D3. Four metabolites were formed, all of which possessed the same UV chromophore as EB1089, indicating the retention of the side-chain conjugated double bond system. Two metabolites were present in sufficient quantities to identify them as 26-hydroxy EB1089 (major product) and 26a-hydroxy EB1089 (minor product), based on mass spectral analysis and cochromatography with synthetic standards. Similar metabolites were generated in vivo and using a liver postmitochondrial fraction in vitro (Kissmeyer et al., companion paper). Studies with the human hepatoma Hep G2 gave rise to 2 isomers of 26-hydroxy EB1089. Studies using ketoconazole, a general cytochrome P450 inhibitor, implicated cytochrome P450s in the formation of the EB1089 metabolites. COS-1 transfection cell experiments using vectors containing CYP27 and CYP24 suggest that these cytochrome P450s are probably not involved in 26- or 26a-hydroxylation of EB1089. Other experiments that examined the HPK1A-ras metabolism of related analogs containing only a single side-chain double bond: 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-1' (E)-en-1'-yl)-9,10-secopregna-5(Z),7(E),10(19)-triene (MC1473; double bond at C-22,23) and 1(S),3(R)-dihydroxy-20(R)-(5'-ethyl-5'-hydroxy-hepta-3'(E)-en-1'-yl)-9, 10-secopregna-5(Z),7(E),10(19)-triene (MC1611; double bond at C-24,24a) revealed that the former compound was subject to 24-hydroxylation and the latter compound was mainly 23-hydroxylated. Metabolism experiments involving EB1089, MC1473, and MC1611 in competition with [1 beta-3H]1,25-(OH)2D3 in HPK1A-ras confirmed that CYP24 is probably not involved in the metabolism of EB1089 whereas, in the case of MC1473 and MC1611, it does appear to carry out side-chain hydroxylation. Our interpretation is that the conjugated double bond system in the side-chain of EB1089 is responsible for directing the target cell hydroxylation to the distal positions, C-26 and C-26a. We conclude that EB1089 is slowly metabolized via unique in vitro metabolic pathways, and that these features may explain the relative stability of EB1089 compared to other analogs in vivo.
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PMID:Metabolism of the vitamin D analog EB1089 by cultured human cells: redirection of hydroxylation site to distal carbons of the side-chain. 911 99

The environmental contaminant benzo[c]phenanthrene (B[c]Ph) has weak carcinogenic activity in rodent bioassays; however, the fjord region diol epoxides of B[c]Ph, B[c]Ph-3,4-diol 1,2-epoxides (B[c]PhDE), are potent carcinogens. To determine the role of cytochrome P450 isozymes in the activation of B[c]Ph in MCF-7 cells and the low activation of B[c]Ph in mouse skin, cells of the MCF-7 and the human hepatoma HepG2 cell lines were treated with the potent Ah receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prior to exposure to B[c]Ph for 24 h. Mice were treated topically with 1 microg of TCDD or vehicle (control) for 73 h and then with 2 micromol of B[c]Ph for 24 h. In MCF-7 cells, TCDD exposure increased B[c]PhDE-DNA adduct levels more than 3-fold with a 10-fold increase in the (-)-B[c]PhDE-2-dA(t) adduct. Treatment of HepG2 cells with TCDD prior to B[c]Ph application did not increase B[c]PhDE-DNA binding. Total B[c]PhDE-DNA adducts increased 3-fold in TCDD-treated mouse epidermis: the majority of the increase resulted from (+)-B[c]PhDE-1-dA adducts. Analysis of P450 enzymes by Western blotting detected a large increase of P4501B1 but almost no increase in P4501A1 in MCF-7 cells exposed to 10 microM B[c]Ph for 24 or 48 h. In HepG2 cells, there were no detectable levels of P4501A1 or P4501B1 after treatment with 10 microM B[c]Ph for 24 h. In contrast, topical application of 2 micromol of B[c]Ph to mouse skin for 48 or 72 h increased P4501A1, but no P4501B1 was detected. As a measure of P450 activity, the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) was analyzed in microsomes prepared from MCF-7 and HepG2 cells exposed to 0.1% DMSO, 10 microM B[c]Ph, or 10 nM TCDD for 24 or 48 h and from mouse epidermis treated with 1 microg of TCDD, or vehicle control for 72 h, or 2 micromol of B[c]Ph for 48 h. The levels of DMBA metabolites were low or undetectable in microsomes from B[c]Ph-treated MCF-7 and HepG2 cells, but a metabolite pattern consistent with P4501A1 metabolism of DMBA was present in B[c]Ph-exposed mouse epidermal microsomes. TCDD-treated MCF-7 cells, HepG2 cells, and mouse epidermis had DMBA metabolism patterns characteristic of P4501A1 activity. Microsomes from TCDD-treated human cells formed a higher proportion of the proximate carcinogenic metabolite DMBA-3,4-dihydrodiol (16% of total identified metabolites) than TCDD-treated mouse epidermis (2%). In mouse epidermis, the weak ability of B[c]Ph to increase hydrocarbon-metabolizing activity and the increase in mainly P4501A1, leading to formation of the less carcinogenic stereoisomer B[c]PhDE-1, may explain the low carcinogenic activity of B[c]Ph. In a human mammary carcinoma cell line, treatment with B[c]Ph increases mainly P4501B1 and results in formation of a higher proportion of the more carcinogenic B[c]PhDE-2. This indicates that cells in which B[c]Ph treatment increases P4501B1 levels effectively activate B[c]Ph to potent carcinogenic metabolites.
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PMID:Role of cytochrome P450 enzyme induction in the metabolic activation of benzo[c]phenanthrene in human cell lines and mouse epidermis. 916 60


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