Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In view of the potential role of the cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) biotransformation enzymes in the metabolism of protoxicants in the circulatory system, we examined CYP and mEH expression in several primary cultures of human umbilical vein endothelial cells (HUVEC), each established from a different individual. Total RNA was isolated from untreated cells and cells 72 hr after exposure to dimethyl sulfoxide (DMSO), Arochlor 1254 (PCB), and beta-naphthoflavone (beta NF). Specific mRNA transcripts were examined by Northern blotting and reverse transcriptase-coupled polymerase chain reaction (RT/PCR) analyses. CYP2E1, CYP3A, and CYP1A2 mRNAs were not detectable in any of the cultures by Northern blot analysis with radiolabeled oligomer probes; however, CYP1A1 mRNA was detected using this procedure in HUVEC cultures exposed to beta NF for 72 hr. Using RT/PCR, constitutive levels of CYP1A1, CYP1A2, CYP2E1, and CYP3A gene expression in HUVEC cultures were evident; however, constitutive CYP2B6 mRNA was not detected. Constitutive CYP1A2 transcript levels were detected in four of six HUVEC cultures, but levels varied between individual cultures. CYP1A2 mRNA levels were also increased in HUVEC cultures exposed to PCB and beta NF. No increases in the levels of CYP2E1 and CYP3A mRNAs were observed in HUVEC cells subsequent to PCB or beta NF exposures. Constitutive CYP2E1 transcript levels were present in all HUVEC cultures examined and varied among individuals. All HUVEC cultures examined for mEH activity exhibited constitutive levels of mEH which varied 40% between individual cultures and produced on average, 1.51 pmol benzo[a]pyrene 4,5-dihydrodiol per milligram protein per minute of reaction. Thus, these results demonstrate that human endothelial cells express CYP and mEH gene products and suggest that these enzymes may play important roles in determining metabolic fates for circulating protoxicants.
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PMID:Expression of cytochrome P450s and microsomal epoxide hydrolase in primary cultures of human umbilical vein endothelial cells. 750 67

The central nervous system is an important potential target for certain environmental protoxins, but relatively little is known regarding brain-specific expression of biotransformation enzyme systems. We undertook the present study to identify regional and cellular expression patterns of individual cytochrome P-450 genes (CYP) and microsomal epoxide hydrolase (mEH) in human brain. Various regions of normal human brain were isolated and examined with respect to mRNA levels of CYP1A1, CYP1A2, CYP2E1, CPY3A, and mEH, using specific oligomer probes and reverse transcriptase-coupled polymerase chain reaction analysis. We also used immunohistochemical techniques, with antipeptide-derived antibodies, to identify specific cells from various regions of the human brain producing CYP1A1 and mEH protein. Relatively equivalent mRNA expression levels of mEH were detected in the cerebellum (C), frontal (F), occipital (O), pons (P), red nucleus (RN), and substantia nigra (SN) regions of brain. The mRNA expression patterns of CYP2E1 and CYP1A2 were similar; although detected in all brain regions examined, the RN and SN exhibited lower levels of CYP2E1 and CYP1A2 mRNA expression compared to other regions. In addition, regional differences in CYP3A and CYP1A1 mRNA expression also were observed, with the highest level of CYP3A mRNA present in the P region compared to the C, F, O, and RN, while no CYP3A mRNA was detected in the SN. CYP1A1 mRNA expression was evident in all brain regions, but the levels of CYP1A1 mRNA in the P and RN were lower than in the C, F, O, and SN. In all cases, the regional mRNA expression levels of these CYP and mEH mRNAs were less than the corresponding levels detected from the same individual's liver. CYP1A1 and mEH immunoreactivity was present in most neurons of the SN, RN, P, median raphae, locus ceruleus, inferior vestibular nucleus, dorsal motor nucleus of the vagus, and thalamus. Some but not all astrocytes within these regions also demonstrated 1A1 and mEH immunoreactivity. These results indicate that many neurons and astrocytes express mEH and CYP1A1 as well as other CYP genes, and suggest that localized biotransformation events within the certain central nervous system may account for toxicities initiated by exposure to certain environmental chemicals.
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PMID:Regiospecific expression of cytochrome P-450s and microsomal epoxide hydrolase in human brain tissue. 769 60

Epidemiological evidence suggests that the presence of human papillomaviruses (HPV), when combined with smoking behaviors, considerably enhances the risk of developing oral, cervical, vulvar, and/or anal carcinomas. It is well established that the cytochrome P450 (CYP), microsomal epoxide hydrolase (mEH), and other biotransformation enzymes are important modulators of the bioactivation and detoxification of many environmental chemicals, including constituents of tobacco smoke such as certain nitrosamines and polycyclic aromatic hydrocarbons (PAH). Since there is little information regarding oral and cervical epithelial-specific expression of these genes, established primary and HPV-immortalized oral and cervical epithelial cell lines were analyzed for morphology, mRNA and protein expression patterns of specific CYPs and mEH. Primary human oral and cervical epithelial cells were immortalized using retroviral infection with HPV-16 E6/E7 genes. Primary human keratinocyte cells were immortalized by transfection of HPV-18 and made tumorigenic with nitrosomethylurea treatment. Expression profiles for mEH, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP3A, and CYP2E1 were evaluated in these cultures in the presence or absence of a PAH inducer, using reverse transcriptase-coupled polymerase chain reaction analysis. mEH gene expression was evident in all cultures, while CYP2A6 mRNA was not detected in any of the cell lines, regardless of culture conditions. CYP2E1 mRNA expression was greatest in the oral epithelial cultures and detectable in all other epithelial cultures except for the HPV-18 immortalized keratinocyte cell line. Elevated levels of CYP2D6 mRNA existed in both oral epithelial cell lines and the HPV-16 immortalized cervical epithelial cells when compared to the other cell lines examined. CYP1A1 and CYP1A2 mRNAs were detected in all the cells and several cultures were inducible by PAH exposure. To corroborate the RT/PCR data, Western immunoblotting experiments were conducted on selected samples. Using these methods, CYP1A1 and CYP2E1 proteins were detected in primary and HPV-immortalized oral and cervical epithelial cultures. These data indicate that both primary and HPV immortalized cells appear to express certain biotransformation enzymes necessary for the activation of tobacco-specific nitrosamines and PAHs. Although the overall impact of HPV gene infection on expression of these systems remains to be fully elucidated, as in vitro system is characterized which should prove useful in examining interactive mechanisms of HPV with xenobiotic activation in the etiology of squamous cell carcinomas.
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PMID:Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial cells immortalized by human papillomavirus type 16 E6/E7 genes. 761 6

In the present study, we developed a very sensitive, semiquantitative assay based on the reverse transcriptase-coupled polymerase chain reaction to measure, in a region-selective manner, mRNA expression patterns within the brain for microsomal epoxide hydrolase and several cytochrome P-450s (P-450s) known to be induced by prototypic agents in other tissues. The P-450s assessed included the polyaromatic hydrocarbon-inducible CYP1A1 and CYP1A2 systems, together with the phenobarbital-inducible P-450s, CYP2B1, CYP2B2, CYP3A1, which were examined 18 hr after a single intraperitoneal dose of the respective inducing agents. Highly region-specific patterns of expression were evident for P-450 mRNAs within the rat brain. In the control, uninduced brain, CYP1A1 mRNAs were readily detected in the striatum and in the hypothalamus, and to a lesser extent in the other regions examined. The regional pattern of expression was similar for CYP1A2; however, a major difference was noted in the olfactory bulbs, characterized by a relatively high level of CYP1A2 mRNA but correspondingly low levels of CYP1A1. Within the brain regions examined, the highest content of CYP2B1 and CYP2B2 mRNAs were present in the striatum and in the cerebellum, whereas CYP3A1 levels varied only slightly across the respective regions. In contrast to the P-450s, microsomal epoxide hydrolase mRNAs were expressed at relative homogeneous amounts throughout the brain. beta-Naphthoflavone markedly increased the CYP1A1 and CYP1A2 mRNA contents of each brain region investigated, although this agent did not affect levels of epoxide hydrolase. At 18 hr post-treatment with phenobarbital, an optimal time period for hepatic induction, brain expression was characterized by a complex pattern of effects, with increased levels noted for CYP2B1 mRNA content in the medulla oblongata, midbrain, and cortex, but decreased contents measured in the cerebellum, the hypothalamus, and the striatum. In each of these respective regions, CYP2B2 content was profoundly decreased whereas epoxide hydrolase expression was slightly increased by the same treatment. These results establish that the central nervous system actively expresses a number of different biotransformation gene products in a regional specific and inducer-dependent manner, and suggest that for tissues exhibiting low regenerative capacity, like the brain, such reactions are likely to be of critical toxicological significance.
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PMID:Regional distribution and expression modulation of cytochrome P-450 and epoxide hydrolase mRNAs in the rat brain. 824 22

Human bronchial epithelial cells (BEC), a primary defense against inhaled materials, are the progenitor cells for bronchogenic carcinomas and have important metabolic capabilities. We used reverse transcriptase-polymerase chain reaction (RT-PCR) to identify xenobiotic metabolism enzymes expressed in primary BEC and alveolar macrophages (AM) of non-smoking volunteers. Cytochromes P450 (CYP) 1A1, 1B1, 2B7, 2E1, and 4B1 and microsomal epoxide hydrolase (mEH) were expressed in BEC but not AM. CYP2F1 was expressed in BEC, but it was expressed at barely detectable levels or not at all in AM. NADPH oxidoreductase (NADPH OR), microsomal glutathione transferase (GST 12), glutathione transferase mu, phenol sulfotransferase (PST), thermolabile phenol sulfotransferase (TL PST), and the clara cell-specific gene, CC10 were expressed in both BEC and AM. CYP3A4 and glucuronosyl transferases-1 and 2 were not expressed in either BEC or AM. In contrast to primary BEC, of the genes evaluated, the immortalized human bronchial epithelial cell line BEP2D constitutively expressed only CYP1A1, CYP2E1, NADPH OR, glucuronosyl transferase 1, GST 12, GST mu, PST, TL PST, and CC10. The loss of xenobiotic metabolism enzyme gene expression in the BEP2D cell line may result from either reduced exposure to inducing agents, or loss of differentiative characteristics in culture. It is clear from the data comparing BEC and AM that there are important intertissue differences in expression of xenobiotic metabolism enzymes.
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PMID:Xenobiotic metabolism enzyme gene expression in human bronchial epithelial and alveolar macrophage cells. 884 77

Bronchial epithelial cells (BEC) are the progenitors of bronchogenic carcinomas and are exposed to polycyclic aromatic hydrocarbon (PAH) procarcinogens through inhalation of combustion products. PAH are converted to carcinogenic molecules through a combination of monoxygenation by cytochrome p450 (CYP) enzymes in the presence of NADPH oxidoreductase (OR) and hydrolysis by microsomal epoxide hydrolase (mEH). In artificial systems, the relative expression of these genes determines whether carcinogenic or noncarcinogenic species are generated during metabolism. This relationship was explored in humans by using quantitative competitive reverse transcriptase polymerase chain reaction amplification to determine the range of expression of CYP1A1, CYP1B1, mEH, and NADPH OR in BEC recovered from 10 nonsmokers and 9 smokers. CYP2B7 expression was evaluated because, although little is known of its substrate specificity, it is expressed at high levels in human lung tissue. CYP1A1 and CYP1B1 were expressed in BEC at significantly different levels (P < 0.05) in the 9 smokers at 1.4 +/- 2.3 x 10(4) and 2.4 +/- 3.2 x 10(3) molecules/10(6) beta-actin molecules (mean +/- STD), respectively, but each was measurable in only one of the 10 nonsmokers. There was significant inter-individual variation (P < 0.05) in both CYP1A1 and CYP1B1 expression among the subjects for whom sufficient data were obtained. The inducibility of human BEC CYP1A1 gene by PAH exposure was confirmed in vitro by incubating cultured immortalized human BEC with beta-naphthoflavone and observing a > 6-fold induction of CYP1A1 after 24 h. In contrast to BEC, alveolar macrophages expressed CYP1A1 at low (30-70 molecules/10(6) beta-actin molecules) to unmeasurable levels in both smokers and nonsmokers. There was no significant difference in expression of mEH, CYP2B7, or NADPH OR in smokers compared with nonsmokers. The inter-individual variation in absolute and relative expression of PAH metabolism enzymes in BEC reported here supports the hypothesis that inter-individual variation in ability to activate/inactivate inhaled PAH carcinogens accounts for at least some of the inter-individual variation in risk for bronchogenic carcinoma.
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PMID:Quantitative RT-PCR measurement of cytochromes p450 1A1, 1B1, and 2B7, microsomal epoxide hydrolase, and NADPH oxidoreductase expression in lung cells of smokers and nonsmokers. 922 17

We developed a quantitative competitive reverse transcriptase-polymerase chain reaction (QC RT-PCR) assay to measure mRNA levels of seven human cytochrome P450 (P450, CYP) genes and microsomal epoxide hydrolase (EH) simultaneously. This assay employs an exogenous recombinant RNA (rcRNA) molecule as an internal standard that shares PCR primer and hybridization probe sequences with CYP1A1, CYP1A2, CYP2A6/7, CYP2D6, CYP2E1, CYP2F1, CYP3A4/5/7, and EH mRNA. Because each rcRNA molecule contains several primer sequences, an entire battery of genes that exhibit differential responsiveness to various classes of xenobiotics may be measured simultaneously from one population of cDNA molecules. In this study, we demonstrated the precision and power of the assay using small amounts of human liver total RNA. We also report for the first time quantitative profiles of P450 and EH mRNA abundance in eight human livers. Cytochrome P450 2E1 mRNA maintained the highest abundance (average 6.67 x 10(7) molecules/microg of total RNA) and least variation (13 fold) in all livers examined. Cytochrome P450 1A2, CYP2A6/7, CYP2D6, CYP3A4/5, and EH mRNAs were approximately one order of magnitude less abundant than CYP2E1 transcripts, with CYP2D6 levels exhibiting the greatest variation (220 fold) between individuals. This QC RT-PCR assay should prove valuable for measuring basal and induced mRNAs in different cell types in vitro, as well as in biomonitoring applications where individuals are exposed or hypersusceptible to certain xenobiotic-initiated toxicities.
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PMID:Quantification of multiple human cytochrome P450 mRNA molecules using competitive reverse transcriptase-PCR. 953 3

To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase-coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 10(5) molecules/microg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 10(5) and 1.8 x 10(5) molecules/microg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 10(4) molecules/microg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., beta-naphthoflavone (beta-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.
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PMID:Fingerprinting of cytochrome P450 and microsomal epoxide hydrolase gene expression in human blood cells. 1082 67

Studies suggest that resveratrol (trans-3,4',5-trihydroxystilbene), which is a diphenolic antioxidant found in plants and foods, has cancer chemopreventive and chemotherapeutic potential. A lower risk of lung cancer among consumers of wine compared with consumers of other beverages has been observed, which may be partly attributed to the high content of resveratrol particularly in red wine. We have studied the effect of resveratrol on the expression of genes involved in the metabolism of polycyclic aromatic hydrocarbons in the human bronchial epithelial cell line BEP2D. Expression of the cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase P1 (GSTP1) genes was measured by quantitative reverse transcriptase-polymerase chain reaction. The cells were treated either with benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin in the presence or absence of resveratrol. Resveratrol inhibited both the constitutive and the induced expression of CYP1A1 and CYP1B1 in a dose-dependent manner. In contrast, the expression of the mEH gene was increased in response to resveratrol and no change in the expression of GSTP1 was found. The altered gene expression in response to resveratrol was reflected in a reduced overall level of benzo[a]pyrene metabolism. These data indicate that resveratrol may exert lung cancer chemopreventive activity through altering the expression of genes involved in the metabolism of polycyclic aromatic hydrocarbons, resulting in altered formation of carcinogenic benzo[a]pyrene metabolites in human bronchial epithelial cells.
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PMID:Lung carcinogenesis: resveratrol modulates the expression of genes involved in the metabolism of PAH in human bronchial epithelial cells. 1127 1

It is controversial whether women have a higher lung cancer susceptibility compared to men. We previously reported higher levels of smoking-related bulky DNA adducts in female lungs. In a pilot study (27 cases), we also found a higher level of female lung cytochrome P4501A1 (CYP1A1) gene expression. In the present extended study we report on the pulmonary expression of several genes involved in polycyclic aromatic hydrocarbon bioactivation in relation to sex, smoking and DNA adducts. CYP1A1, CYP1B1, aryl hydrocarbon receptor and microsomal epoxide hydrolase gene expression was measured by quantitative real-time reverse transcriptase-PCR in 121 normal lung tissue samples. The expression of CYP1A1 and CYP1B1 was significantly higher among current smokers compared to ex-smokers and never-smokers. Among current smokers, females had a 3.9-fold higher median level of CYP1A1 compared to males (p = 0.011). CYP1B1 expression was not related to sex. Lung DNA adducts (measured by 32P-postlabeling) were highly significantly related to CYP1A1 (p < 0.0001) irrespective of smoking-status. Our results are consistent with the hypothesis that CYP1A1 plays a significant role in lung DNA adduct formation and support a higher susceptibility to lung cancer among females.
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PMID:Sex differences in risk of lung cancer: Expression of genes in the PAH bioactivation pathway in relation to smoking and bulky DNA adducts. 1655 73


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