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)

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

Delavirdine, a non-nucleoside inhibitor of HIV-1 reverse transcriptase, is metabolized primarily through desalkylation catalyzed by CYP3A4 and CYP2D6 and by pyridine hydroxylation catalyzed by CYP3A4. It is also an irreversible inhibitor of CYP3A4. The interaction of delavirdine with CYP2C9 was examined with pooled human liver microsomes using diclofenac 4'-hydroxylation as a reporter of CYP2C9 catalytic activity. As delavirdine concentration was increased from 0 to 100 microM, the K(M) for diclofenac metabolism rose from 4.5+/-0.5 to 21+/-6 microM, and V(max) declined from 4.2+/-0.1 to 0.54+/-0.08 nmol/min/mg of protein, characteristic of mixed-type inhibition. Nonlinear regression analysis revealed an apparent K(i) of 2.6+/-0.4 microM. There was no evidence for bioactivation as prerequisite to inhibition of CYP2C9. Desalkyl delavirdine, the major circulating metabolite of delavirdine, had no apparent effect on microsomal CYP2C9 activity at concentrations up to 20 microM. Several analogs of delavirdine showed similar inhibition of CYP2C9. Delavirdine significantly inhibited cDNA-expressed CYP2C19-catalyzed (S)-mephenytoin 4'-hydroxylation in a noncompetitive manner, with an apparent K(i) of 24+/-3 microM. Delavirdine at concentrations up to 100 microM did not inhibit the activity of CYP1A2 or -2E1. Delavirdine competitively inhibited recombinant CYP2D6 activity with a K(i) of 12.8+/-1.8 microM, similar to the observed K(M) for delavirdine desalkylation. These results, along with previously reported experiments, indicate that delavirdine can partially inhibit CYP2C9, -2C19, -2D6, and -3A4, although the degree of inhibition in vivo would be subject to a variety of additional factors.
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PMID:Interaction of delavirdine with human liver microsomal cytochrome P450: inhibition of CYP2C9, CYP2C19, and CYP2D6. 1112 28

The capacity of three clinically available nonnucleoside reverse transcriptase inhibitors (NNRTIs) to inhibit the activity of human cytochromes P450 (CYPs) was studied in vitro using human liver microsomes. Delavirdine, nevirapine, and efavirenz produced negligible inhibition of phenacetin O-deethylation (CYP1A2) or dextromethorphan O-demethylation (CYP2D6). Nevirapine did not inhibit hydroxylation of tolbutamide (CYP2C9) or S-mephenytoin (CYP2C19), but these CYP isoforms were importantly inhibited by delavirdine and efavirenz. This indicates the likelihood of significantly impaired clearance of CYP2C substrate drugs (such as phenytoin, tolbutamide, and warfarin) upon initial exposure to these two NNRTIs. Delavirdine and efavirenz (but not nevirapine) also were strong inhibitors of CYP3A, consistent with clinical hazards of initial cotreatment with either of these drugs and substrates of CYP3A. The in vitro microsomal model provides relevant predictive data on probable drug interactions with NNRTIs when the mechanism is inhibition of CYP-mediated drug biotransformation. However, the model does not incorporate interactions attributable to enzyme induction.
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PMID:Inhibition of human cytochrome P450 isoforms by nonnucleoside reverse transcriptase inhibitors. 1122 65

Transformants with stable expression of a series of human cytochrome P450 (CYP) subtypes in the human hepatic cell line, HepG2, were established. These transformants are designated Hepc/1A1.4, Hepc/1A2.9, Hepc/2A6L.14, Hepc/2B6.68, Hepc/2C8.46, Hepc/2C9.1, Hepc/2C19.12, Hepc/2D6.39, Hepc/2E1.3-8 and Hepc/3A4.2-30, which stably expressed human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively. The expression of the CYP subtypes in the transformants was confirmed by both determination of enzyme activities and the reverse transcriptase polymerase chain reaction (RT-PCR) procedure. The apparent K(m) values of the expressed CYP subtypes for their specific substrates were close to those of human liver microsomes. In addition to their CYP activities, these transformants retained glucuronide- and sulfate-conjugating activities. Furthermore, the activities of CYP2C9, CYP2D6 and CYP3A4 were inhibited by their specific inhibitors. The cytotoxicity of acetaminophen (APAP), cyclophosphamide (CPA) and benz[a]anthracene (BA) were analyzed by CYP-expressing transformants. The cytotoxicity depended on the expression of CYP subtypes and increased in a dose-dependent manner. These results show the metabolic activation of APAP, CPA and BA by the specific CYP subtypes expressed in the transformants and demonstrate the usefulness of these transformants for in vitro metabolic and toxicological studies in human liver.
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PMID:Establishment of the transformants expressing human cytochrome P450 subtypes in HepG2, and their applications on drug metabolism and toxicology. 1137 97

Environmental chemicals are one of the risk factors in breast cancer genesis. Cytochrome P450 (CYP) enzymes play a major role in the activation of these chemicals. Using highly specific and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. the expression profile of all major xenobiotic metabolizing CYP forms was screened in breast tumour and surrounding tumour free (control) breast tissue in a series of 20 sample pairs obtained from females with infiltrating ductal carcinoma. The levels of CYPIAI mRNA were very low in both tumour and normal tissue. CYP1B1, CYP2B6, CYP2C, CYP2D6, CYP2E1, CYP4B1, and CYP11A1 expressions were positive in both tumours and control tissue. CYP2A6, CYP2A7, CYP2A13, CYP2F1, CYP3A4, CYP3A5. and CYP3A7 mRNAs were expressed neither in tumours nor in control tissue. These results show that several CYPs. responsible for the activation of a quite large number of procarcinogens and genotoxic estrogen metabolites. are expressed in breast tissue with a lack of qualitative differences in CYP expression at mRNA level between breast tumours and surrounding normal breast.
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PMID:The expression of cytochrome P450 enzymes in human breast tumours and normal breast tissue. 1176 4

The human drug oxidizing cytochrome P450, CYP2D6, is expressed at highly variable levels mainly due to a common genetic polymorphism which leads to the poor metabolizer phenotype in carriers of two nonfunctional alleles and to the extensive metabolizer phenotype in carriers of one or more functional alleles. Investigation of the role of CYP2D6 mRNA for expression and the possibility of using mRNA expression as a surrogate marker has been hampered by the presence of two pseudogenes, CYP2D7P and CYP2D8P. We therefore developed highly specific TaqMan real-time reverse transcriptase-PCR assays for the discriminative quantification of CYP2D6 and CYP2D7/8P transcripts. By in vitro transcription of plasmids containing the CYP2D6 cDNA or a hybrid CYP2D6/7 cDNA constructed by in vitro mutagenesis, authentic cRNAs were synthesized to be used for specificity testing and for absolute quantification. The method was used to determine CYP2D transcripts in a large number of human livers samples. CYP2D6 was not normally distributed with a median mRNA content of 3.2 transcripts per picogram of total RNA in all livers (range 0.32-14.8, N = 74). Expression in genetic poor metabolizers (1.81, N = 6) was significantly lower compared to extensive metabolizers (3.33, N = 68, P = 0.022). Similar expression levels were found for CYP2D7/8P (median 3.38 transcripts/pg, range 0.46-14.3), which were correlated to CYP2D6 mRNA (r(S) = 0.46, P < 0.0001) but did not depend on CYP2D6 genotype. These data demonstrate genotype-dependent mRNA expression for CYP2D6 and they emphasize the necessity of differentiating between the functional CYP2D6 and the CYP2D pseudogenes.
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PMID:Discriminative quantification of cytochrome P4502D6 and 2D7/8 pseudogene expression by TaqMan real-time reverse transcriptase polymerase chain reaction. 1177 2

A cDNA was cloned from Japanese monkey liver mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers based on the marmoset cytochrome P450 2D19 (CYP2D19) nucleotide sequence. The full-length cDNA encoded a 497 amino acid protein (designated CYP2D29) that is 96, 91, and 88% homologous to human CYP2D6, cynomolgus monkey CYP2D17, and marmoset monkey CYP2D19, respectively. Yeast cells (Saccharomyces cerevisiae AH-22 strain) transfected with pGYR1 vectors containing the CYP2D29 cDNA were cultured, and microsomal fractions were obtained. Reduced carbon monoxide-difference spectra and western blot analysis using polyclonal antibodies raised against rat CYP2D2 demonstrated that in yeast cell microsomal fractions, the level of CYP2D29 holoenzyme was similar to that of CYP2D6 holoenzyme. However, western blot analysis indicated that the level of CYP2D29 in Japanese monkey liver microsomes might be much higher than that of CYP2D6 in human liver microsomes. Japanese monkey liver microsomes exhibited much higher activities than did human liver microsomes, expressed as nmol/min/mg protein, for debrisoquine (DB) 4-hydroxylation and bufuralol (BF) 1"-hydroxylation (typical reactions catalyzed by CYP2D6), whereas recombinant CYP2D29 activity, expressed as nmol/min/nmol CYP, was similar to that of CYP2D6 for DB and BF hydroxylation. In kinetic analyses, the K(m) value of CYP2D29 for DB 4-hydroxylation was much lower than that of Japanese monkey liver microsomes, whereas the K(m) value of CYP2D6 for DB 4-hydroxylation was similar to that of human liver microsomes. In contrast, K(m) values for BF 1"-hydroxylation were similar for Japanese monkey and human liver microsomes and yeast cell microsomal fractions expressing recombinant CYP2D29 or CYP2D6. These results suggest that the properties of Japanese monkey CYP2D29 are similar to those of human CYP2D6, but their populations and/or some other factors in liver microsomes may cause the difference in microsomal DB 4-hydroxylase activities between Japanese monkeys and humans.
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PMID:Complementary DNA cloning and characterization of cytochrome P450 2D29 from Japanese monkey liver. 1223 13

The present work aims to determine the relevance of an astrocytoma cell line U373 MG, for assessing the role of some astroglial cytochrome P450 in neurotoxicity and neuroprotection. CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP2J2, CYP2E1 and CYP4A11 mRNA were detected by reverse transcriptase-polymerase chain reaction in control U373 MG cell cultures. Among them we focused on CYP1B1 expression. After 48 h treatment with a range of concentrations of interleukin-1beta (1, 5, 10 ng/ml) used to simulate stress conditions, CYP1B1 mRNA expression was enhanced in a dose-dependent way. This increased expression was followed 24 h later by an increase in protein level, determined by Western-blot. N-acetylcysteine (NAC) partially inhibited this effect both on the mRNA and protein levels. As CYP1B1 activates procarcinogenic compounds to reactive metabolites, an increase in this P450 isoform will participate to toxic consequences of an inflammatory/oxidative stress. NAC will prevent this deleterious effect.
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PMID:Astroglial CYP1B1 up-regulation in inflammatory/oxidative toxic conditions: IL-1beta effect and protection by N-acetylcysteine. 1256 1

Lung is a target organ for the toxicity of inhalated compounds. The respiratory tract is frequently exposed to elevated concentrations of these compounds and become the primary target site for toxicity. Occupational, accidental or prolonged exposure to a great variety of chemicals may result in acute or delayed injury to cells of the respiratory tract. Nevertheless, lung has a significant capability of biotransforming such compounds with the aim of reducing its potential toxicity. In some instances, the biotransformation of a given compound can result in the generation of more reactive, and frequently more toxic, metabolites. Indeed, lung tissue is known to activate pro-carcinogens (i.e. polycyclic aromatic hydrocarbons or N-nitrosamines) into more reactive intermediates that easily form DNA adducts. Lungs express several enzymes involved in the metabolising of xenobiotics. Among them, cytochrome P450 enzymes are major players in the oxidative metabolism as well metabolic bioactivation of many organic toxicants, including pro-carcinogens. Xenobiotic-metabolising P450 enzymes are expressed in bronchial and bronchiolar epithelium, Clara cells, type II pneumocytes, and alveolar macrophages Individual CYP isoforms have different patterns of localisation within pulmonary tissue. With the aid of sensitive techniques (i.e. reverse transcriptase-polymerase chain reaction, RT-PCR) it has become possible to detect CYP1A1, CYP1B1, CYP2A6, CYP2B6, CYP2E1 and CYP3A5 mRNAs in lung cells. Less conclusive results have been obtained concerning CYP2Cs, CYP2D6 and CYP3A4. CYP3A5 protein appears to be widely present in all lung samples and is localised in the ciliated and mucous cells of the bronchial wall, bronchial glands, bronchiolar ciliated epithelium and in type I and type II alveolar epithelium. Lung cells also express Phase II enzymes such as epoxide hydrolase, UGT1A (glucuronyl transferase) and GST-P1 (glutathione S-transferase), which largely act as detoxifying enzymes. A key question concerning organ-specific chemical toxicity is whether the actual target has the capacity to activate (or efficiently inactivate) chemicals. Results of several studies indicate that the different xenobiotic-metabolising CYPs, present in the human lung and lung-derived cell lines, likely contribute to in situ activation of pulmonary toxins, among them, pro-carcinogens. Some CYPs, in particular CYP1A, are polymorphic and inducible. Interindividual differences in the expression of these CYPs may explain the different risk of developing lung toxicity (possibly cancer), by agents that require metabolic activation. Few cell lines, principally A549, have been used with variable success as an experimental model for investigating the mechanisms of toxicity. Although RT-PCR analysis has evidenced the presence of the major human pulmonary CYP mRNAs, the measurable P450 specific activities are, however, far below those present in human lungs. Detection of the toxicity elicited by reactive metabolites requires the use of metabolically competent cells; consequently, better performing cells are needed to ensure realistic in vitro prediction of toxicity. Genetic manipulation of lung-derived cells allowing them to re-express key biotransformation enzymes appear to be a promising strategy to improve their functionality and metabolic performance.
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PMID:Metabolism and bioactivation of toxicants in the lung. The in vitro cellular approach. 1609 27

We determined changes in mRNA expression in specific enzymes involved in the biosynthesis of morphine in human white blood cells via microarray. Leukocyte exposure to morphine down-regulated catechol-O-methyl transferase (COMT) and CYP2D6 by approximately 50% compared with control values. The treatment did not alter DOPA decarboxylase and dopamine beta-hydroxylase expression, demonstrating the specificity of morphine actions. The verification of the microarray data was accomplished via real-time Taqman reverse transcriptase polymerase chain reaction (RT-PCR) focused on CYP2D6 and COMT expression in different blood samples treated with morphine. The analysis showed similar changes in the expression of CYP2D6 and COMT mRNA. The expression was reduced by 47 +/- 7% for CYP2D6, substantiating the microarray finding of a 54% reduction. Furthermore, exposure of white blood cells to 10(-6) M S-nitroso-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, reduced the expression of CYP2D6 and COMT. Prior naloxone (10(-6) M) or N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) addition abrogated morphine's down-regulating activity, demonstrating morphine was initiating its actions via stimulating constitutive NO synthase derived NO release via the mu3 opiate receptor splice variant. In the past we demonstrated that UDP-glucurosyltransferase is involved in metabolizing morphine to morphine 6-glucuronide in adrenal chromaffin cells. In the present study its expression was not found in controls and morphine-treated cells, suggesting that morphine 6-glucuronide may not be synthesized in white blood cells. Taken together, it appears that morphine has the ability to modulate its own synthesis via autocrine and paracrine signaling.
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PMID:Endogenous morphine signaling via nitric oxide regulates the expression of CYP2D6 and COMT: autocrine/paracrine feedback inhibition. 1757 83


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