ABSTRACT
It has been previously demonstrated in a human-derived hepatoma cell line (HepG2) that juices from cruciferous vegetables protect against the genotoxicity caused by dietary carcinogens. HepG2 cells possess different enzymes involved in the biotransformation of xenobiotics. Therefore, we investigated the effect of cruciferous juices on the activities of CYP 1A and several phase II enzymes in this cell model. For each experiment, 1 x 10(6) cells were seeded on Petri dishes. After 2 days, the juices (0.5-8 microl/ml of culture medium) were added for 48 h prior to cell harvesting. The addition of juice from water cress (Nasturtium officinalis R. Br) significantly increased the activities of ethoxyresorufin-O-deethylase at high doses only and NAD(P)H-quinone reductase in a dose-dependent manner (1.8- and 5-fold, respectively). The addition of juice from garden cress (Lepidum sativum L.) significantly increased the activities of NAD(P)H-quinone reductase and UDP-glucuronosyl-transferase with a maximal effect around the dose of 2 microl/ml juice (1.4- and 1.2-fold, respectively) while the other enzymes were not altered. Mustard (Sinapis alba L.) juice increased the activities of NAD(P)H-quinone reductase (2.6-fold at the dose of 8 microl/ml), and N-acetyl-transferase (1.4-fold at the dose of 8 microl/ml) in a dose-dependent manner while a maximal induction of UDP-glucuronosyl-transferase was obtained with a dose of 2 microl/ml (1.8-fold). These observations show that the three juices have different induction profiles: only water cress acted as a bifunctional inducer by enhancing both phase I and phase II enzymes. As a consequence, each juice may preferentially inhibit the genotoxicity of specific compounds.
Subject(s)
Brassicaceae , Liver/enzymology , Plant Extracts/pharmacology , Acetyltransferases/biosynthesis , Carcinoma, Hepatocellular , Cell Line, Tumor , Cytochrome P-450 CYP1A1/biosynthesis , Enzyme Induction , Glucuronosyltransferase/biosynthesis , Glutathione Transferase/biosynthesis , Humans , Liver/cytology , Liver/drug effects , NAD(P)H Dehydrogenase (Quinone)/biosynthesis , Sulfotransferases/biosynthesisABSTRACT
Smallanthus sonchifolius (yacon), originating from South America, has become popular in Japan and in New Zealand for its tubers which contain beta-1,2-oligofructans as the main saccharides. The plant is also successfully cultivated in Central Europe in the Czech Republic in particular. Its aerial part is used in Japan and in Brazil as a component in medicinal teas; while aqueous leaf extracts have been studied for their hypoglycemic activity in normal and diabetic rats. We have already demonstrated the high content of phenolic compounds in yacon leaf extracts and their in vitro antioxidant activity. In this paper, we present the effects of two organic fractions and two aqueous extracts from the leaves of S. sonchifolius on rat hepatocyte viability, on oxidative damage induced by tert-butyl hydroperoxide (t-BH) and allyl alcohol (AA), and on glucose metabolism and their insulin-like effect on the expression of cytochrome P450 (CYP) mRNA. All the extracts tested exhibited strong protective effect against oxidative damage to rat hepatocyte primary cultures in concentrations ranging from 1 to 1000 microg/ml, reduced hepatic glucose production via gluconeogenesis and glycogenolysis at 1000 microg/ml. Moreover, the effects of the organic fractions (200 and 250 microg/ml) and to a lesser extent, the tea infusion (500 microg/ml) on rat CYP2B and CYP2E mRNA expression, were comparable to those observed with insulin. The combination of radical scavenging, cytoprotective and anti-hyperglycemic activity predetermine S. sonchifolius leaves for use in prevention and treatment of chronic diseases involving oxidative stress, particularly diabetes.
Subject(s)
Antioxidants/pharmacology , Asteraceae/chemistry , Liver/drug effects , Oxidative Stress/drug effects , Plant Extracts/pharmacology , Animals , Aryl Hydrocarbon Hydroxylases/biosynthesis , Aryl Hydrocarbon Hydroxylases/genetics , Cell Survival/drug effects , Dose-Response Relationship, Drug , Glucose/biosynthesis , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , Liver/metabolism , Male , Phenols/analysis , Plant Extracts/analysis , Plant Leaves/chemistry , RNA, Messenger/metabolism , Rats , Rats, Wistar , Tumor Cells, CulturedABSTRACT
Glutathione S-transferases (GSTs) are metabolic phase II enzymes that promote reactive metabolite elimination by conjugating them to glutathione (GSH). Because of their important role in xenobiotic metabolism and detoxification, they have been implicated in carcinogenesis processes, especially epithelium transformation. Moreover, their influence on response to chemotherapy in cancer patients has been demonstrated. Genetic polymorphisms for GSTM1, GSTT1 and GSTP1 have been found in human populations and have been shown to have phenotypic consequences. To investigate the role of GST enzymes in carcinogenesis and in response to chemotherapy in patients with head and neck squamous cell carcinoma (HNSCC), GSTP1, GSTM1 and GSTT1 were studied prospectively in a large series of HNSCC patients. Correlations between GST alterations, p53 mutation status and clinical response to chemotherapy were investigated. We showed that the risk of developing laryngeal cancer was increased by 2.6-fold [95% CI 1.6--6.1] in patients with the GSTM1 null genotype and by 2.8-fold [95% CI 0.9--8.1] in patients with the homozygous GSTP1 val105 genotype. Furthermore, individuals with this latter genotype were over-represented in the p53 mutation group (p = 0.05). After storage duration and hemolysis adjustment, a significantly lower plasmatic GSTP1 level was observed in complete responders compared with partial and non-responders (mean: 4.4 +/- 0.06 microg/l, 4.7 +/- 0.06 microg/l and 4.7 +/- 0.07 microg/l; p = 0.05), respectively. The prevalence of p53-mutated tumors was significantly higher in the group of non-responders (81%) compared with partial (60%) and complete responders (64%) (p = 0.05). Two types of multivariate analysis were performed including parameters that have been shown to influence response to chemotherapy significantly in univariate analysis. p53 mutations and high tumor stage are independent factors of non-response to chemotherapy, whereas plasmatic GSTP1 levels and low tumor stage are independent factors of complete response. Our data suggest that GST enzymes are associated with larynx cancer and that their use as predictive factors and treatment targets should be further explored.
Subject(s)
Carcinoma, Squamous Cell/enzymology , Glutathione Transferase/blood , Head and Neck Neoplasms/enzymology , Isoenzymes/blood , Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/blood , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/therapy , Cisplatin/therapeutic use , Female , Gene Frequency , Genotype , Glutathione S-Transferase pi , Head and Neck Neoplasms/blood , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/therapy , Humans , Male , Middle Aged , Multivariate Analysis , Mutation , Neoadjuvant Therapy , Treatment Outcome , Tumor Suppressor Protein p53/geneticsABSTRACT
Smoking is the principal cause of lung cancer. However, not all smokers will develop this disease. Individual susceptibility to chemically induced cancer may be explained in part by genetic differences in the activation and detoxification of procarcinogens. The activation phase of polycyclic aromatic hydrocarbon (PAH) metabolism is governed by the enzyme CYP1A1, induced by PAH when it enters the body. The extent to which PAH induces CYP1A1 activity varies greatly from one subject to another. CYP1A1 inducibility has long been associated, although inconsistently, with an increased risk of lung cancer. In 1982, Kouri corroborated Kellerman's results with a new method for measuring inducibility, but few studies have reported using this method. The glutathione S-transferases (GSTs) are involved in the detoxification phase of PAH, and the allelic deletion of GSTM1 has been also associated with an increased risk of lung cancer. We conducted a case-control study to examine the risk of lung cancer related, separately and together, to CYP1A1 inducibility, GSTM1 polymorphism and cigarette smoking in a French population. The 611 subjects were 310 incident lung cancer cases and 301 hospital control subjects. We were able to constitute a DNA bank for 552 subjects (89.5%) and gather detailed information on smoking history for all of them. Inducibility could be measured for 195 cases and 183 control subjects. Results for GSTM1 polymorphism concern 247 cases and 254 control subjects. GSTM1 polymorphism and inducibility could both be assessed for 179 cases and 166 control subjects. The odds ratio related to inducibility was 1.7 [1.0-3.0] for medium and 3.1 (1.3-7.4) for hyper inducers. The association with GSTM1 was 1.6 (1.0-2.6). With a reference category of subjects who were both low inducers and GSTM1(+), we found an odds ratio for lung cancer of 8.1 (2-31) for the subjects with both risk factors [i.e. GSTM1(-) and hyper inducers]. Our data did not reveal evidence of interaction between smoking and inducibility. On the other hand, we found an interaction of 3.6 (0.6-21) between inducibility and GSTM1.
Subject(s)
Cytochrome P-450 CYP1A1/biosynthesis , Glutathione Transferase/biosynthesis , Lung Neoplasms/enzymology , Base Sequence , Case-Control Studies , Cytochrome P-450 CYP1A1/genetics , DNA Primers , Enzyme Induction , France , Gene Deletion , Glutathione Transferase/genetics , Humans , Inactivation, Metabolic , Polycyclic Compounds/pharmacokinetics , Polymerase Chain Reaction , SmokingABSTRACT
Cytochrome P450 enzymes catalyze the first step of the metabolism and subsequent elimination of hydrophobic xenobiotics. However, the activity of some isoforms, among them CYP1A1 and CYP2E1, may result in cellular insults such as oxidative stress and activation of procarcinogen compounds into reactive metabolites. The regulation of the expression of these enzymes is therefore important. We have previously shown that the CYP1A1 gene promoter was repressed by oxidative stress. We show here that the CYP2E1 gene promoter is down-regulated by exogenous H(2)O(2) addition and glutathione depletion. It is also repressed by the transfection of a CYP2E1 expression vector, which elicits an intracellular H(2)O(2) generation. This autoregulation is limited by catalase (which catalyzes the catabolism of H(2)O(2)), thus implying H(2)O(2) as a mediator of the negative feedback mechanism. Furthermore, we observed that the activity of CYP1A1 resulting either from the stimulation of the endogenous gene by benzo[a]pyrene treatment or from the transfection of an expression vector, repressed the activity of the CYP2E1 gene promoter. Conversely, CYP2E1 overexpression repressed the activity of the CYP1A1 gene promoter. In both cases, catalase and a specific inhibitor of one enzyme prevented the repression of the other. This suggests that the generation of H(2)O(2) during the catalytic cycle of these enzymes is a mediator of the cross-regulatory mechanisms. These novel repressive mechanisms of autoregulation and cross-regulation using H(2)O(2) as a common mediator may limit the potential toxicity resulting from high cytochrome P450 activity within the cell.
Subject(s)
Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP2E1/genetics , Gene Expression Regulation, Enzymologic , Hydrogen Peroxide/pharmacology , Promoter Regions, Genetic , Catalysis , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2E1/metabolism , Genetic Vectors , Homeostasis , Humans , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
BACKGROUND: Glutathione conjugation of tacrine reactive metabolites depends in part on the activity of glutathione-S-transferases (GST), of which two isozymes (GST M1 and GST T1) are polymorphically expressed. OBJECTIVE AND METHODS: To determine whether GST M1, GST T1, and the combined GST M1 and GST T1 null genotypes predict individual susceptibility to tacrine hepatotoxicity, 141 patients with mild to moderate Alzheimer's disease treated with tacrine were genotyped. RESULTS: During the treatment period, 52 patients had elevated alanine aminotransferase (ALT) levels at least three times the upper limit of normal, whereas 89 patients had normal ALT values (< or = upper limit of normal). Both groups were comparable in demographic and clinical characteristics. Twenty-eight patients were found to be GST T1-negative (20%; with a 95% confidence interval [95% CI] from 13% to 27%), and 68 patients (48%; 95% CI from 40% to 57%) were GST M1-negative. The combined GST M1-T1 null genotype was observed in 18 patients (13%; 95% CI from 7% to 18%) of whom 13 had an elevated plasma ALT at least three times the upper limit of normal during the study period. Although the cumulative percentage of elevated plasma ALT tended to be higher in the GST M1 null genotype, neither GST M1 nor GST T1 alone could predict individual susceptibility to tacrine hepatotoxicity. Multivariate Cox hazards model showed that the association of the GST M1-T1 null genotype was an independent risk factor of hepatotoxicity. CONCLUSIONS: The presence of combined alleles M1 and T1 deficiencies in glutathione-S-transferase genes increases the susceptibility to tacrine hepatotoxicity.
Subject(s)
Alzheimer Disease/drug therapy , Glutathione Transferase/genetics , Liver/drug effects , Parasympathomimetics/adverse effects , Tacrine/adverse effects , Aged , Alleles , Female , Genetic Predisposition to Disease , Genotype , Glutathione Transferase/drug effects , Humans , Isoenzymes/drug effects , Isoenzymes/genetics , Liver/enzymology , Liver Function Tests , Male , Pharmacogenetics , Polymorphism, Genetic , Proportional Hazards ModelsABSTRACT
PURPOSE: The tumor suppressor gene p53 plays a crucial role in cell cycle control and apoptosis in response to DNA damages. p53 gene mutations and allelic losses at 17p are one of the most common genetic alterations in primary head and neck squamous cell carcinoma (HNSCC). Alterations of the p53 gene have been shown to contribute to carcinogenesis and drug resistance. PATIENTS AND METHODS: In this prospective series, patients with HNSCC were treated with cisplatin-fluorouracil neoadjuvant chemotherapy. p53 status was characterized in 106 patients with HNSCC (p53 mutations, allelic losses at p53 locus, and plasma anti-p53 antibodies) to determine the existence of a relationship between p53 gene status and response to neoadjuvant chemotherapy. RESULTS: Exons 4 to 9 of the p53 gene were analyzed, and mutations were found in 72 of 106 patients with HNSCC. p53 mutations were associated with loss of heterozygosity at chromosome 17p (P <.001). The prevalence of p53-mutated tumors was higher in the group of patients with nonresponse to neoadjuvant chemotherapy than in the group of responders (81% v 61%, respectively; P <.04). When compiling p53 mutations and anti-p53 antibodies in plasma, the correlation between p53 status and response to chemotherapy was significant (87% v 57%, respectively; P =.003). A multivariate analysis showed that p53 status is an independent predictive factor of response to chemotherapy. CONCLUSION: This prospective study suggests that p53 status may be a useful indicator of response to neoadjuvant chemotherapy in HNSCC.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Genes, p53/genetics , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Adult , Carcinoma, Squamous Cell/pathology , Cyclophosphamide/administration & dosage , Female , Fluorouracil/administration & dosage , Head and Neck Neoplasms/pathology , Humans , Loss of Heterozygosity , Male , Neoadjuvant Therapy , Predictive Value of Tests , Prospective Studies , Treatment OutcomeABSTRACT
BACKGROUND: We conducted a case-control study to examine the risk of lung cancer in relation to GSTM1 polymorphism and cigarette smoking (primarily of black tobacco) in a French population. METHODS: The 611 subjects were 301 incident lung cancer cases and 310 hospital controls. We were able to constitute a DNA bank for 547 subjects (89.5%) and gather detailed information on smoking history for all of them. Results presented here concern 247 cases and 254 controls. RESULTS: Taking non- or light smokers as the reference category, we estimated odds ratios (OR) of 4.2 (95% CI: 2.6-6.7) and 5.2 (95% CI: 3.3-8.3) for the medium and heavy smokers respectively. On the other hand we estimated that the crude OR associating GSTM1 with lung cancer was 1.3 (95% CI: 0.9-1.8). Furthermore our data do not depart significantly from a multiplicative model of the combined effects of smoking and GSTM1 deficiency. CONCLUSIONS: We conclude that smoking and the GSTM1 gene are each a risk factor for lung cancer, and that their combined effect does not differ significantly from that of a multiplicative model.
Subject(s)
Genetic Predisposition to Disease/epidemiology , Glutathione Transferase/genetics , Lung Neoplasms/epidemiology , Lung Neoplasms/genetics , Polymorphism, Genetic , Smoking/adverse effects , Adult , Age Distribution , Aged , Analysis of Variance , Case-Control Studies , Confidence Intervals , Female , France/epidemiology , Glutathione Transferase/metabolism , Health Surveys , Humans , Incidence , Male , Middle Aged , Odds Ratio , Reference Values , Risk Assessment , Risk Factors , Sampling Studies , Smoking/epidemiologyABSTRACT
Human cytochrome (CYP)2B6 cDNA was cloned and expressed in bacteria and in yeast. Its expression in Saccharomyces cerevisiae enabled us to obtain, at a high level, an active yeast-expressed CYP2B6 protein, so as to assess its role in the metabolism of ethoxyresorufin, pentoxyresorufin, benzyloxyresorufin, ethoxycoumarin, testosterone and cyclophosphamide. Kinetic analysis showed that human CYP2B6 preferentially metabolized benzyloxyresorufin and pentoxyresorufin, although other CYPs also metabolized these substrates in human liver microsomes. CYP2B6 also manifested a strong 4-hydroxycyclophosphamide activity. Its expression in Escherichia coli enabled us to produce a very specific anti-human CYP2B6 antibody. No cross reactivity of this antibody was observed with CYPs1A1, 1A2, 3A4, 3A5, 2C8, 2C9, 2C18, 2C19, 2D6 or 2E1. This antibody enabled us to study the hepatic and extrahepatic expression of CYP2B6 in man, as well as its expression and inducibility in primary cultured human hepatocytes and in different human cell lines. Immunoblot analysis revealed that the CYP2B6 protein was expressed in 43 of the 48 human liver samples tested, with levels ranging from 0.4 to 8 pmol/mg of microsomal protein with a mean of 1.7 pmol/mg protein. CYP2B was also expressed in human brain, intestine and kidney, and at a lower level in the lung. CYP2B mRNA was detected in human liver, kidney, lung, trachea and intestine. We also found that CYP2B6 is induced at protein and mRNA levels by phenobarbital (2 mM) and cyclophosphamide (1 mM), an anticancer drug known to be metabolized by CYP2B6. No expression or inducibility of CYP2B6 was observed in any of the human cell lines tested.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Oxidoreductases, N-Demethylating/metabolism , Base Sequence , Catalysis , Cell Line , Cloning, Molecular , Cyclophosphamide/pharmacology , Cytochrome P-450 CYP2B6 , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/genetics , DNA Primers , Enzyme Induction , Humans , Liver/drug effects , Liver/enzymology , Oxidoreductases, N-Demethylating/biosynthesis , Oxidoreductases, N-Demethylating/genetics , Phenobarbital/pharmacology , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
Molecular studies have revealed that microsatellite instability and loss of heterozygosity occurred in head-and-neck cancer, suggesting the involvement both of suppressor and of mutator pathways in head-and-neck carcinogenesis. There is evidence for relations between tumor phenotype and clinical parameters. Indeed, replication-error phenotype, characterized by microsatellite instability, was associated with decreased sensitivity to chemotherapeutic agents in cell lines. Loss of heterozygosity is a frequent mechanism of inactivation of tumor-suppressor genes, which might be implicated in resistance to chemotherapy. In head-and-neck cancer, chemosensitivity is inconstant, and no marker is available to predict response to treatment. In order to evaluate the role of tumor phenotype on resistance to chemotherapy, we analyzed 56 primary head-and-neck squamous-cell carcinomas collected at time of diagnosis and a sub-group of 23 resistant tumors collected after chemotherapy at 22 microsatellite loci. At time of diagnosis, only one tumor showed MSI-H phenotype. Loss of heterozygosity (LOH) was observed in 75% of tumors, indicating the dominant role of the suppressor in comparison with the mutator pathway in HNSCC carcinogenesis. No change in microsatellite patterns was observed after treatment, suggesting that chemotherapy did not select mismatch-repair-deficient clones. Univariate analyses showed that LOH at 9p or 17p was significantly associated with drug resistance. In a multivariate analysis, only LOH at 17p remains predictive of low response to chemotherapy, with a relative risk of 3.7 and 95% CI of 1.1-13, indicating that p53 alterations could play a role in chemotherapy resistance in HNSCC. Int. J. Cancer (Pred. Oncol.) 84:410-415, 1999.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Loss of Heterozygosity , Microsatellite Repeats , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/therapy , Chi-Square Distribution , Chromosomes, Human, Pair 17 , Chromosomes, Human, Pair 18 , Chromosomes, Human, Pair 3 , Chromosomes, Human, Pair 8 , Chromosomes, Human, Pair 9 , Cisplatin/administration & dosage , Combined Modality Therapy , Female , Fluorouracil/administration & dosage , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/therapy , Humans , Male , Middle Aged , Neoplasm Staging , Predictive Value of Tests , Prospective Studies , Regression AnalysisABSTRACT
Halothane hepatitis occurs because susceptible patients mount immune responses to trifluoroacetylated protein antigens, formed following cytochrome P450-mediated bioactivation of halothane to trifluoroacetyl chloride. In the present study, an in vitro approach has been used to investigate the cytochrome P450 isozyme(s) which catalyze neoantigen formation and to explore the protective role of non-protein thiols (cysteine and reduced glutathione). Significant levels of trifluoroacetyl protein antigens were generated when human liver microsomes, and also microsomes from livers of rats pre-treated with isoniazid, phenobarbital or beta-naphtoflavone, were incubated with halothane plus a nicotinamide adenine dinucleotidephosphate (NADPH) generating system. Immunoblotting studies revealed that the major trifluoroacetyl antigens expressed in vitro exhibited molecular masses of 50-55 kDa and included 60 and 80 kDa neoantigens recognized by antibodies from patients with halothane hepatitis. Much lower concentrations of halothane were required to produce maximal antigen generation in isoniazid-induced rat microsomes, as compared with phenobarbital or isosafrole-induced microsomes (0.5 vs 12.5 microl/ml). In isoniazid-induced microsomes, antigen generation was inhibited > 90% by the nucleophiles cysteine and glutathione and by the CYP2E1-selective inhibitors diallylsulfide and p-nitrophenol, but was unaffected by inhibitors of other P450 isozymes (furafylline, sulfaphenazole or triacetyloleandomycin). Neoantigen formation in six human liver microsomal preparations was inhibited in the presence of diallylsulfide, but not by furafylline, sulfaphenazole or triacetyloleandomycin, and exhibited marked variability which correlated with CYP2E1 levels. These results suggest that the balance between metabolic bioactivation by CYP2E1 and detoxication of reactive metabolites by cellular nucleophiles could be an important metabolic risk factor in halothane hepatitis.
Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Cytochrome P-450 Enzyme System/metabolism , Halothane/toxicity , Hepatitis Antigens/biosynthesis , Isoenzymes/metabolism , Animals , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/immunology , Chloroacetates , Cysteine/metabolism , Enzyme Activation/drug effects , Glutathione/metabolism , Halothane/pharmacokinetics , Inactivation, Metabolic , Individuality , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Peptides/immunology , Peptides/metabolism , Rabbits , Rats , Rats, Inbred F344 , Rats, Sprague-Dawley , Trichloroacetic Acid/pharmacokineticsABSTRACT
To investigate whether interindividual variation in CYP2E1 levels can be explained by genetic polymorphism, we analysed DNA samples from 40 healthy individuals by single-strand conformational polymorphism analysis for polymorphisms in the CYP2E1 coding sequence and promoter region. DNA sequencing of samples showing mobility shifts on single-strand conformational polymorphism detected polymorphisms at positions -316 (A to G), -297 (T to A), -35 (G to T), 1107 (G to C; intron 1), 4804 (G to A Val179Ile; exon 4) and 10157 (C to T; exon 8). All individuals positive for either A(-316)G, G(-35)T, G(4804)A or the previously described RsaI polymorphism at -1019 were also positive for T(-297)A, which had the highest allele frequency of the observed polymorphisms (0.20). A(-316)G, G(-35)T and G(4804)A were detected at allele frequencies of 0.022, 0.052 and 0.013, respectively. The functional significance of the upstream polymorphisms was examined by preparing constructs of positions -549 to +3 of CYP2E1 containing the observed combinations of the polymorphisms fused to luciferase reporter genes and transfecting HepG2 cells. For the G(-35)T/T(-297)A construct, a 1.8-fold increase in luciferase activity compared with the wild-type sequence (P = 0.06) and 2.5-fold compared with T(-297)A only (P = 0.025) was observed. No significant difference in activity was observed between the other constructs. The significance of the predicted Val179Ile base change from G(4804)A was determined by expression of the wild-type and mutated full length cDNAs in lymphoblastoid cells. No significant difference in kinetic constants for chlorzoxazone hydroxylation between mutant and wild-type was observed. In summary, this study demonstrated six novel CYP2E1 polymorphisms, including three upstream of the promoter, but with the possible exception of G(-35)T, none appeared to be of functional significance.
Subject(s)
Cytochrome P-450 CYP2E1/genetics , Polymorphism, Single-Stranded Conformational , Base Sequence , Cell Line , Chlorzoxazone/metabolism , DNA Primers , Gene Frequency , Genetic Linkage , Humans , Microsomes, Liver/enzymology , Polymerase Chain Reaction , Transcription, GeneticABSTRACT
Cytochrome P450 mediated metabolism of methyleugenol to the proximate carcinogen 1'-hydroxymethyleugenol has been investigated in vitro. Kinetic studies undertaken in liver microsomes from control male Fischer 344 rats revealed that this reaction is catalyzed by high affinity (Km of 74.9 +/- 9.0 microM, Vmax of 1.42 +/- 0.17 nmol/min/nmol P450) and low affinity (apparent Km several mM) enzymic components. Studies undertaken at low substrate concentration (20 microM) with microsomes from livers of rats treated with the enzyme inducers phenobarbital, dexamethasone, isosafrole and isoniazid indicated that a number of cytochrome P450 isozymes can catalyze the high affinity component. In control rat liver microsomes, 1'-hydroxylation of methyleugenol (assayed at 20 microM substrate) was inhibited significantly (P < 0.05) by diallylsulfide (40%), p-nitrophenol (55%), tolbutamide (30%) and alpha-naphthoflavone (25%) but not by troleandomycin, furafylline, quinine or cimetidine. These results suggested that the reaction is catalyzed by CYP 2E1 and by another as yet unidentified isozyme(s) (most probably CYP 2C6), but not by CYP 3A, CYP 1A2, CYP 2D1 or CYP 2C11. Administration of methyleugenol (0-300 mg/kg/day for 5 days) to rats in vivo caused dose-dependent auto-induction of 1'-hydroxylation of methyleugenol in vitro which could be attributed to induction of various cytochrome P450 isozymes, including CYP 2B and CYP 1A2. Consequently, high dose rodent carcinogenicity studies are likely to over-estimate the risk to human health posed by methyleugenol. The rate of 1'-hydroxylation of methyleugenol in vitro in 13 human liver samples varied markedly (by 37-fold), with the highest activities being similar to the activity evident in control rat liver microsomes. This suggests that the risk posed by dietary ingestion of methyleugenol could vary markedly in the human population.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Eugenol/analogs & derivatives , Isoenzymes/metabolism , Microsomes, Liver/enzymology , Animals , Biotransformation , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Eugenol/metabolism , Eugenol/pharmacokinetics , Humans , Hydroxylation , Isoenzymes/antagonists & inhibitors , Male , Rats , Rats, Inbred F344ABSTRACT
Ethylene dibromide (1,2-dibromoethane, EDB) is metabolized by two routes: a conjugative route catalyzed by glutathione S-transferases (GST) and an oxidative route catalyzed by cytochrome P450 (P450). The GST route is associated with carcinogenicity. An approach is presented to use human purified GST and P450 enzymes to explore the importance of these metabolic pathways for man in vivo. This strategy basically consists of four steps: (i) identification of the most important isoenzymes in vitro, (ii) scaling to rate per milligram cytosolic and microsomal protein, (iii) scaling to rate per gram liver, and (iv) incorporation of data in a physiologically based pharmacokinetic (PBPK) model. In the first step, several GST isoenzymes were shown to be active toward EDB and displayed pseudo-first-order kinetics, while the EDB oxidation was catalyzed by CYP2E1, 2A6, and 2B6, which all displayed saturable kinetics. In the second step, the predictions were in agreement with the measured activity in a batch of 21 human liver samples. In the third step, rat liver P450 and GST metabolism of EDB was predicted to be in the same range as human metabolism (expressed per gram). Interindividual differences in GST activity were modeled to determine "extreme cases." For the most active person, an approximately 1.5-fold increase of the amount of conjugative metabolites was predicted. Lastly, it was shown that the GST route, even at low concentrations, will always contribute significantly to total metabolism. In the fourth step, a PBPK model describing liver metabolism after inhalatory exposure to EDB was used. The saturation of the P450 route was predicted to occur faster in the rat than in man. The rat was predicted to have a higher turnover of EDB from both routes. Nevertheless, when all data are combined, it is crucial to recognize that the GST remains significantly active even at low EDB concentrations. The limitations and advantages of the presented strategy are discussed.
Subject(s)
Ethylene Dibromide/pharmacokinetics , Ethylene Dibromide/toxicity , Glutathione Transferase/drug effects , Hazardous Substances/metabolism , Hazardous Substances/toxicity , Liver/drug effects , Animals , Cytochrome P-450 Enzyme System/drug effects , Cytochrome P-450 Enzyme System/metabolism , Cytosol/metabolism , Glutathione Transferase/metabolism , Humans , In Vitro Techniques , Kidney/enzymology , Liver/enzymology , Microsomes, Liver/metabolism , Models, Biological , Placenta/enzymology , Rats , Risk Assessment , Species SpecificityABSTRACT
1,2-Dibromoethane (1,2-DBE) is mainly used as an additive in leaded gasoline and as a soil fumigant and it is a suspected carcinogen in humans. In this study, the oxidative bioactivation of 1,2-DBE to 2-bromoacetaldehyde (2-BA) was studied using heterologously expressed human cytochrome P450 (P450) isoenzymes and human liver microsomes. Out of ten heterologously expressed human P450 isoenzymes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2C9, CYP2C18, CYP3A4 and CYP3A5), only human CYP2A6, CYP2B6 and CYP2E1 metabolized 1,2-DBE, albeit with strongly differing catalytic efficiencies. The apparent Km and Vmax values were 3.3 mM and 0.17 pmol/min per pmol P450 for CYP2A6, 9.7 mM and 3.18 pmol/min per pmol P450 for CYP2B6 and 42 microM and 1.3 pmol/min per pmol P450 for CYP2E1, respectively. In all of 21 human liver samples studied, 1,2-DBE was oxidized with activities ranging from 22.2 to 1027.6 pmol/min per mg protein, thus showing a 46-fold inter-individual variability. The kinetics of the oxidative metabolism of 1,2-DBE to 2-BA in human liver microsomes were linear, indicating the involvement of primarily one single P450 isoenzyme. There was a tendency towards a positive correlation between the oxidative metabolism of 1,2-DBE in the human liver microsomes and the 6-hydroxylation of chlorzoxazone, a selective substrate for CYP2E1. Furthermore, the oxidative metabolism of 1,2-DBE was inhibited by the specific CYP2E1 inhibitors disulfiram (DS) and diethyldithiocarbamate (DDC). In contrast, a poor correlation was found between the immunochemically quantified amount of CYP2E1 and the microsomal chlorzoxazone 6-hydroxylation or the 1,2-DBE oxidation. The results indicate that CYP2E1 is probably the major P450 isoenzyme involved in the oxidative hepatic metabolism of 1,2-DBE in humans. The inter-individual variability in the oxidative bioactivation of 1,2-DBE in humans, largely due to inter-individual variability in the catalytic activity of hepatic CYP2E1, may have important consequences for the risk assessment for human exposure to 1,2-DBE.
Subject(s)
Carcinogens/metabolism , Cytochrome P-450 Enzyme System/metabolism , Ethylene Dibromide/metabolism , Isoenzymes/metabolism , Microsomes, Liver/enzymology , Acetaldehyde/analogs & derivatives , Acetaldehyde/metabolism , Humans , Hydroxylation , Kinetics , Oxidation-Reduction , Structure-Activity RelationshipABSTRACT
CYP2E1 is involved in the activation of various carcinogens, including N-nitrosamines, which are believed to be important in human carcinogenesis. Humans exhibit wide interindividual variability in levels of CYP2E1 mRNA and protein, which might explain interindividual differences in susceptibility to carcinogens activated by CYP2E1. Such variability could be due either to genetic polymorphisms observed in the CYP2E1 gene (Rsa I in the 5'-flanking region, Dra I in intron 6 and Taq I in intron 7) or to varying inducibility by xenobiotics. The aim of the present study was to establish whether, in a Caucasian population (n = 93), there existed a relationship between allelic forms of the CYP2E1 gene and the phenotype determined in vitro by hepatic ability to 6-hydroxylate chlorzoxazone. Rates of chlorzoxazone-6-hydroxylation were significantly correlated with levels of immunochemically measured CYP2E1 (p < 0.001). CYP1A2, 2C8, 2C9, 2C18, 2D6, 3A4 and 3A5 did not appear to be significantly involved in chlorzoxazone metabolism, whereas the participation of CYP1A1 could not be excluded. Frequencies of the rare alleles for the three polymorphism sites were 2.2% for RsaI, 7.5% for DraI and 8.5% for TaqI. Despite substantial interindividual variations in chlorzoxazone hydroxylase activity, no relationship between any of the three polymorphisms and CYP2E1 activity was established. Therefore, in humans, interindividual variability in CYP2E1 levels is probably due to differing induction levels as a result of environmental factors, or to genetic factors other than those studied in this work.
Subject(s)
Cytochrome P-450 CYP2E1/genetics , Alleles , Chlorzoxazone/analogs & derivatives , Chlorzoxazone/metabolism , Cytochrome P-450 CYP2E1/metabolism , Genotype , Humans , Hydroxylation , PhenotypeABSTRACT
Involvement of cytochrome P-4501A1 (CYP1A1) in the regulation of sucrase-isomaltase and hexose transporters was analyzed in low (TC7)- and high (PF11)-glucose-consuming Caco-2 clones. CYP1A1 mRNA is elevated in exponentially growing cells concomitantly with high rates of glucose consumption and high levels of GLUT-1 and GLUT-3 mRNA. After confluency, CYP1A1 is not detectable in TC7 cells; this is associated with a decreased glucose consumption, a downregulation of GLUT-1 and GLUT-3, and an upregulation of sucrase-isomaltase, SGLT-1, GLUT-2, and GLUT-5. In PF11 cells CYP1A1 mRNA remains elevated, along with high glucose consumption, high levels of GLUT-1 and GLUT-3, and minimal expression of sucrase-isomaltase, SGLT-1, GLUT-2, and GLUT-5. Exposure of TC7 cells to inducers of CYP1A1 results in high levels of CYP1A1 mRNA, a 10-fold increase of glucose consumption after confluency, an upregulation of GLUT-1 and GLUT-3, and a downregulation of sucrase-isomaltase, GLUT-2, and, to a lesser extent, SGLT-1 and GLUT-5. These results suggest that activation of CYP1A1, whether spontaneous or drug induced, is involved in the variations of glucose utilization and in the associated modifications of expression of sucrase-isomaltase and hexose transporters.
Subject(s)
Caco-2 Cells/metabolism , Cytochrome P-450 Enzyme System/physiology , Glucose/metabolism , Monosaccharide Transport Proteins/metabolism , Sucrase-Isomaltase Complex/metabolism , Caco-2 Cells/pathology , Cell Cycle , Clone Cells , Glucose/pharmacology , Humans , Lactates/biosynthesis , Lactic AcidABSTRACT
Rat liver epithelial cells (RLECs) isolated by trypsinization of the livers of normal 10 day old rats are largely used in co-culture with primary hepatocytes. The aim of the present study was to investigate the expression of biotransformation enzyme-encoding genes in three preparations of RLEC lines. Although no expression of cytochrome P450 1A1/2 (CYP1A1/2), CYP2B1/2, CYP2C6 or CYP3A mRNAs could be detected, we found that all of the different preparations of RLECs expressed a high level of CYP2E1 mRNA. We demonstrated the presence of the CYP2E1 apoprotein in microsomes of RLECs by immunoblot analyses, together with chlorzoxazone 6-hydroxylation, an activity known to be mainly catalyzed by CYP2E1. In addition, acetone treatment of these cells resulted in an increase in both CYP2E1 apoprotein and chlorzoxazone 6-hydroxylation activity levels. Finally, we showed the susceptibility of RLECs to N-methyl formamide- and diethylnitrosamine-induced toxicity, suggesting metabolic activation by CYP2E1. Thus, RLECs may cooperate with hepatocytes to CYP2E1-mediated metabolism in the co-culture model. In addition, transfection experiments with a CYP2E1 promoter construct, in which the proximal 539 bp containing the binding site for HNF1alpha were inserted upstream of the chloramphenicol acetyl transferase gene, demonstrated a strong induction upon co-transfection with an HNF1alpha expression plasmid. Thus, RLECs provide a useful tool for studying metabolism and cytotoxicity of CYP2E1 substrates in the absence of other expressed CYPs, and for analyzing CYP2E1 promoter function.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Liver/enzymology , Oxidoreductases, N-Demethylating/metabolism , Animals , Blotting, Western , Cytochrome P-450 CYP2E1 , Cytochrome P-450 Enzyme System/genetics , Epithelium/enzymology , Male , Oxidoreductases, N-Demethylating/genetics , Promoter Regions, Genetic , RNA, Messenger/analysis , Rats , Rats, Inbred F344 , Rats, Sprague-DawleyABSTRACT
Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics. Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate. Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies. These antibodies were characterized for their specificity and sensitivity by immunoblots; anti-CYP3A4 was immunoinhibitor. These antibodies could be used in association with other means to identify the CYPs responsible for production of a given metabolite. The use of our human recombinant CYP1A2 as antigen and the corresponding specific antibody enabled us to quantify the CYP1A2 content in 43 human livers. The average level was 69 pmol of CYP1A2/mg of microsomal proteins. Finally, these antibodies were also used to evaluate the level of heme incorporation in human microsomal CYP expressed in yeasts.
Subject(s)
Antibodies/immunology , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/immunology , Antibody Formation , Antibody Specificity , Blotting, Western , Cloning, Molecular , Cytochrome P-450 CYP1A2 , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression , Heme/analysis , Humans , Microsomes, Liver/enzymology , Oxidoreductases/metabolism , Oxidoreductases, N-Demethylating/antagonists & inhibitors , Recombinant Proteins/immunologyABSTRACT
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.