ABSTRACT
The development of personalized medicine will require improved knowledge of biological variability, particularly concerning the important impact of each individual's genetic makeup. A five-step strategy can be followed when trying to identify genes and gene products involved in differential responses to cardiovascular drugs: 1) Pharmacokinetic-related genes and phenotypes; (2) Pharmacodynamic targets, genes and products; (3) Cardiovascular diseases and risks depending on specific or large metabolic cycles; (4) Physiological variations of previously identified genes and proteins; (5) Environmental influences on them. After summarizing the most well known genes involved in drug metabolism, we used statins as an example. In addition to their economic impact, statins are generally considered to be of significant importance in terms of public health. Individuals respond differently to these drugs depending on multiple polymorphisms. Applying a pharmacoproteomic strategy, it is important to use available information on peptides, proteins and metabolites, generally gene products, in each of the five steps. A profiling approach dealing with genomics as well as proteomics is useful. In conclusion, the ever growing volume of available data will require an organized interpretation of variations in DNA and mRNA as well as proteins, both on the individual and population level.
Subject(s)
Cardiovascular Agents/pharmacology , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/genetics , Pharmacogenetics , Proteomics , Cardiovascular Agents/pharmacokinetics , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacokinetics , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacologyABSTRACT
Cytochromes P450 (CYPs) are a superfamily of 57 genes coding for drug metabolizing enzymes and endobiotic metabolizing enzymes (steroids, eicosanoids, vitamins...). This is the main metabolizing enzyme system for foreign compounds, including drugs, which has a primary role in organism protection against potential harmful insults from the environment (pollutants, pesticides...). The CYPs regulation is essentially transcriptional: nuclear receptors are recognized as key mediators for the control of drug metabolizing enzymes. Their ligands are exogenous and also endogenous molecules that can up-regulate or down-regulate these transcription factors. Treatment with drugs or xenobiotics, which are nuclear receptor agonists or antagonists, can lead to severe toxicities, loss of therapeutic effect or endobiotic metabolism disorders. Genetic polymorphisms of these enzymes have an important role in their activity and must be taken into account during drug administration. Then, CYP activity depends on genotype and environment; this is recently used as biomarker to determine human exposure to environmental molecules or to predict the susceptibility to certain pathologies.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Xenobiotics/pharmacokinetics , Cytochrome P-450 Enzyme System/chemistry , Gene Expression Regulation, Enzymologic , Homeostasis , Humans , Kinetics , Models, Biological , Models, Molecular , Polymorphism, Genetic , Transcription, GeneticABSTRACT
Psychostimulant drugs abuse is associated with an increased risk of stroke. Cytochromes P450 (CYP), especially the astrocytic members of the CYP2C subfamily may play an important role in the modulation of cerebrovascular functions, by generating vasodilatator metabolites from arachidonic acid (AA). Our study examined the regulation of CYP2C genes in response to cocaine or amphetamine in the human astrocyte-like U373 MG cells, using reverse transcription-polymerase chain reaction (RT-PCR) and western-blot analysis. A treatment for 48h with increasing concentrations of cocaine caused a significant down-regulation of CYP2C8 and CYP2C9 genes and decreased the protein level. These effects were not observed with amphetamine. One mechanism of the CYP2C mRNA regulation implicates various specific receptors including glucocorticoid receptor (GR) and constitutive androstane receptor (CAR). Effects of cocaine on CYP2C were accompanied by a decrease in the GR and CAR gene expression suggesting that these nuclear receptors could be involved in the CYP2C repression by cocaine in the U373 MG cell line. These findings represent a possible molecular mechanism involved in the cerebrovascular risk associated with cocaine abuse.
Subject(s)
Astrocytes/drug effects , Cocaine/toxicity , Cytochrome P-450 Enzyme System/genetics , Gene Expression/drug effects , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Glucocorticoid/genetics , Transcription Factors/genetics , Astrocytes/enzymology , Astrocytes/metabolism , Astrocytoma , Cell Line, Tumor , Constitutive Androstane Receptor , Down-Regulation , HumansABSTRACT
This study was designed in order to investigate the influence of the genetic polymorphism of CYP2A6 on the amount of smoking. In all, 463 French adults included in the Stanislas cohort were studied and underwent two examinations at 5 years distance (t0 and t(+5) years). Information on their smoking habits was collected. They were genotyped by RFLP for the CYP2A6*1A, CYP2A6*1B and CYP2A6*4 alleles. CYP2A6*1B and CYP2A6*4 allele frequencies were 32 and 4%, respectively. The subjects carrying the CYP2A6*1B allele oxidize nicotine to cotinine faster than subjects with the CYP2A6*1A allele. The number of cigarettes smoked per day was significantly higher in the CYP2A6*1B/*1B group as compared to the CYP2A6*1A/*1A group (P = 0.01 at t0; P = 0.001 t(+5) years), with a larger increase in their daily cigarettes consumption over the 5-year period (P = 0.006). No significant difference in the smoking status was observed according to the CYP2A6 genotype. These data suggest that the CYP2A6*1B is associated with the number of cigarettes smoked per day.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Mixed Function Oxygenases/genetics , Nicotine/metabolism , Polymorphism, Genetic , Smoking/genetics , Adult , Alleles , Cytochrome P-450 CYP2A6 , DNA/analysis , Female , France/epidemiology , Gene Frequency , Genotype , Homozygote , Humans , Longitudinal Studies , Male , Middle Aged , Smoking/epidemiologyABSTRACT
INTRODUCTION: Astrocytes are involved in multiple brain functions in physiological conditions. Cytochromes P450 are expressed in astrocytes and play a role in brain metabolism and in neuroprotection. BACKGROUND: Although the levels of various cytochromes P450 in brain regions are low, these enzymes were reported to be expressed at relatively high level in astroglial cells and may play a critical role in the biotransformation of endogenous or exogenous compounds. Astroglial cytochromes P450 expression suggests a putative capacity to metabolize psychoactive or lipophilic xenobiotics in situ, associated with pharmacological and/or toxicological consequences. Astrocytes appear to be the most active steroidogenic cells in the brain, expressing neurosteroïdogenic cytochromes P450 and producing various neurosteroids. Cytochrome P450 epoxygenase enzymes, which catalyze the formation of vasoactive compounds are also present in astrocytes, contributing to the regulation of the cerebral blood flow. PERSPECTIVES AND CONCLUSION: This review underlines the crucial roles of astroglial cytochromes P450 in brain functions. Identification of the molecular mechanisms involved in the regulation of these enzymes could open therapeutic perspectives and improve our understanding in neuroprotection.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , Cytochrome P-450 Enzyme System/classification , Cytochrome P-450 Enzyme System/metabolism , Neuroprotective Agents/pharmacokinetics , Arachidonic Acid/physiology , Aromatase/physiology , Brain/enzymology , Cholesterol/physiology , Humans , Synaptic Transmission/physiology , Xenobiotics/pharmacology , Xenobiotics/therapeutic useABSTRACT
Chronic venous insufficiency is a complex pathology that is characterised by various symptoms such as venous hypertension, endothelium dysfunction, vascular wall remodelling due to smooth muscle cell hypertrophy and inflammation resulting from the release of pro-inflammatory cytokines from invading leucocytes. Age, hormonal excess, multiparity, sedentariness and prolonged heat exposure represent the main risk factors among many others including hypoxia and shear stress which also influence varicose pathology. Some members of the large cytochrome P450 (CYP) family that are involved in the biotransformation of steroids and arachidonic acid have been shown to be expressed in various cell types (endothelial cells, smooth muscle cells, macrophages) of cardiovascular tissues. The vascular metabolites produced by CYPs are important factors in the regulation of the vascular tone. Most CYPs are markedly expressed in all the cell types of varicose veins in relation to the overall vascular remodelling associated with smooth muscle hypertrophy and periendothelial leucocyte infiltration. Because CYPs produce various vasoactive arachidonic acid metabolites, their increased expression could play a role in the impairement of the vascular tone which is characteristic of varicose veins. Furthermore, polymorphisms, particularly the CYP3A5 polymorphism, may promote changes in the level of expression of CYPs and thus may influence varicose vein formation or functions. This suggests that CYP modulators could be potentially active drugs to treat chronic venous insufficiency symptoms and control its evolution.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Venous Insufficiency/enzymology , Venous Insufficiency/pathology , Animals , Cytochrome P-450 Enzyme System/genetics , Humans , Polymorphism, Genetic/genetics , Venous Insufficiency/geneticsSubject(s)
Ethics, Medical , Genetic Privacy , Genetic Testing , Genetic Predisposition to Disease , Humans , Informed ConsentABSTRACT
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.
Subject(s)
Acetylcysteine/pharmacology , Aryl Hydrocarbon Hydroxylases/metabolism , Astrocytes/enzymology , Interleukin-1/toxicity , Oxidative Stress/immunology , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/immunology , Astrocytes/drug effects , Astrocytes/immunology , Astrocytoma , Cytochrome P-450 CYP1B1 , Cytochrome P-450 CYP2D6/genetics , Cytochrome P-450 CYP2D6/immunology , Cytochrome P-450 CYP2D6/metabolism , Drug Interactions , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/immunology , Humans , Interleukin-1/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/immunology , Isoenzymes/metabolism , Microsomes/metabolism , Oxidative Stress/drug effects , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Up-Regulation/immunologyABSTRACT
During the last few years the increased presence of proinflammatory cytokines and oxidation products in Alzheimer's disease (AD) has been largely described. Oxysterols, formed by hydroxylation of cholesterol, occur naturally in the brain and are increased in the serum of AD patients. Of these oxysterols, 25-hydroxycholesterol is the most potent regulator of gene transcription. It stimulates the synthesis of apolipoprotein E (apo E) in macrophages. Apo E plays a major role in the brain as a phospholipid and cholesterol carrier molecule in compensatory synaptogenesis. Cytokines might also be able to modulate apo E expression. Accordingly, this study examined the control of apo E secretion by several proinflammatory cytokines and oxysterols in the human astrocytoma cell line CCF-STTG1. A time-dependent stimulation of apo E secretion by 25-hydroxycholesterol was observed. Among several cytokines tested, only tumor necrosis factor (TNF)-alpha inhibited apo E secretion in basal conditions in CCF-STTG1 human astrocytoma cells. In the presence of 25-hydroxycholesterol, TNF-alpha reduced apo E secretion by 80%, while interleukins (IL) IL-1beta, IL-6, and IL-2 had no significant effects. In Alzheimer's disease, the increase in the concentrations of cytokines and the concomitant decrease of cholesterol concentration in the brain could contribute jointly to reduce apo E concentration, and in doing so accelerate neurodegeneration. Surprisingly, oxysterols would be able to limit this phenomenon.
Subject(s)
Apolipoproteins E/metabolism , Astrocytoma , Cytokines/pharmacology , Hydroxycholesterols/pharmacology , Alzheimer Disease/immunology , Alzheimer Disease/metabolism , Apolipoproteins E/biosynthesis , Encephalitis/metabolism , Humans , Interleukin-1/pharmacology , Interleukin-12/pharmacology , Interleukin-6/pharmacology , L-Lactate Dehydrogenase/metabolism , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Oxidative stress has been involved in various neurological disorders and, in the central nervous system, astrocytes represent the cell type that contributes to neuroprotection via glutathione (GSH) metabolism, GSH-metabolizing enzymes like gamma-glutamyltransferase (GGT), and apoE secretion. In this study, using IL-1beta, a proinflammatory and prooxidant cytokine that is increased in numerous pathological situations, cells of astrocytoma cell line U373-MG were exposed to an oxidative stress, leading to c-Jun and c-Fos activation. IL-1beta decreased both GGT activity and intracellular GSH content and increased apoE secretion, initiating astroglial response to injury. We observed that antioxidants inhibit IL-1beta effects on c-Jun and c-Fos proteins, GGT activity and the GSH pool but not on apoE secretion. Our results allow us to conclude that neurological disorders associated with an IL-1beta-induced oxidative stress could be, at least experimentally, reversible in the presence of one antioxidant, N-acetylcysteine.
Subject(s)
Astrocytoma , Interleukin-1/pharmacology , Oxidative Stress/physiology , Acetylcysteine/pharmacology , Apolipoproteins E/metabolism , Ascorbic Acid/pharmacology , Blotting, Western , Cell Survival , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Free Radical Scavengers/pharmacology , Glutathione/metabolism , Humans , Oxidative Stress/drug effects , Proto-Oncogene Proteins c-fos/analysis , Proto-Oncogene Proteins c-jun/analysis , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymology , Vitamin E/pharmacology , gamma-Glutamyltransferase/metabolismABSTRACT
A tamoxifen-resistant cell line (MCF7TAM) was established from tamoxifen-sensitive MCF-7 human breast cancer cells expressing estrogen receptors. Though the resistant cell line grows in the presence of tamoxifen, estrogen receptors continue to be expressed at similar levels as in the parental cell line. However, estrogen receptors appeared to be altered in the resistant cell line since important discrepancies are observed between results obtained with ligand binding assays and immunoenzymatic assays, tending to show modifications of estrogen receptor ligand binding capacity. The intracellular distribution of tamoxifen in sensitive and resistant cell lines was investigated using fluorescence of eosin-tamoxifen ionic association. Fluorescence emission spectra of eosin, tamoxifen and eosin-tamoxifen complex (lambda(ex)=480 nm) were analyzed and the maximal fluorescence intensity found for the complex (lambda(em)=540 nm) was four times higher than that of eosin alone, while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be mainly located surrounding the nucleus, although nuclear fluorescence intensity was significantly lower. No highly fluorescent granules were observed in the resistant cell lines as opposed to sensitive cells. Improvement of this fluorescence microscopy methodology could appear of interest, taking into account the complexity of tamoxifen resistance molecular pathways.
Subject(s)
Adenocarcinoma/metabolism , Antineoplastic Agents, Phytogenic/metabolism , Breast Neoplasms/metabolism , Eosine Yellowish-(YS)/chemistry , Tamoxifen/metabolism , Adenocarcinoma/pathology , Antineoplastic Agents, Phytogenic/chemistry , Breast Neoplasms/pathology , Drug Resistance, Neoplasm , Humans , Microscopy, Fluorescence , Spectrometry, Fluorescence , Tamoxifen/chemistry , Tumor Cells, CulturedABSTRACT
The CYP4A1 isoenzyme induced in rodents by peroxisome proliferators is known to be repressed at a pretranslational level by interferon. Interleukin-1beta (IL-1beta) also reduces CYP4A1-related 12-laurate hydroxylase activity in cultured fetal rat hepatocytes after induction by clofibric acid. In this fetal hepatocyte model, IL-1beta and interleukin-6 (IL-6) were tested for their ability to reduce 12-laurate hydroxylase activity, CYP4A1 apoprotein content, and the CYP4A1 mRNA level. IL-1beta and IL-6 strongly diminished CYP4A1 activity and apoprotein and mRNA levels in a dose- and time-dependent manner. CYP4A1 expression is thus down-regulated at a pretranslational level by these cytokines. As it has been shown that the peroxisome proliferator-activated receptor alpha (PPAR alpha) mediates the induction of the CYP4A1 gene by a peroxisome proliferator, the capacity of IL-1beta or IL-6 to modulate the PPAR alpha mRNA level was tested. It was found that IL-1beta and IL-6 both repress the induction of PPAR alpha expression exerted by the combined action of clofibric acid and dexamethasone. However, even at the highest concentration (10 ng/mL) tested for both cytokines, IL-1beta as well as IL-6 failed to abolish the induction of CYP4A1 by dexamethasone. The mechanism of the protective effect of the synthetic glucocorticoid on CYP4A1 repression by interleukins is discussed.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Dexamethasone/pharmacology , Interleukin-1/pharmacology , Interleukin-6/pharmacology , Mixed Function Oxygenases/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , Animals , Cells, Cultured , Clofibric Acid/pharmacology , Cytochrome P-450 CYP4A , Down-Regulation/drug effects , Female , Gene Expression Regulation, Enzymologic/drug effects , Liver/cytology , Liver/embryology , Male , RNA, Messenger/genetics , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVES: Previous uncontrolled studies have suggested an interaction between ticlopidine, a major antiplatelet agent, and cyclosporin in heart- and kidney-transplant recipients. The aims of this study were to examine in a randomised, double-blind fashion, the possible interaction between cyclosporin A and ticlopidine (250 mg per day) and the tolerability of this combination in heart-transplant recipients. METHODS: Twenty heart-transplant recipients were randomised into either a treated or a placebo group. Blood samples were drawn for time-course evaluation of cyclosporin blood levels over a period of 12 h, following the morning intake of cyclosporin and, for platelet aggregation studies, before and after 14 days of ticlopidine administration. Twenty four-hour urine samples were collected for 6-beta-hydroxycortisol measurements, before and after 14 days of ticlopidine. RESULTS: Although given at half the recommended daily dosage, ticlopidine significantly reduced platelet aggregation. Pharmacokinetic parameters indicate that the bioavailability of cyclosporin A was not significantly modified by ticlopidine. However, one patient in the ticlopidine group was withdrawn because of a major fall in cyclosporin blood level within 3 days of treatment. Urinary excretion of 6-beta-hydroxycortisol was augmented after treatment in the ticlopidine group compared with the placebo group, suggesting that induction of drug metabolism might have occurred. Data also show quite a large intra-individual variability in cyclosporin bioavailability in the placebo group, suggesting that poor absorption of the drug formulation and/or poor compliance might have contributed to the decreased cyclosporin blood levels in the patient withdrawn from this study and in previous uncontrolled studies. CONCLUSION: Cyclosporin bioavailability was not clearly modified by a half dosage of ticlopidine in this study. We, however, recommend closely monitoring cyclosporin blood levels when prescribing ticlopidine. Further studies will be needed with new formulations of cyclosporin or when using the full dosage of ticlopidine.
Subject(s)
Cyclosporine/pharmacokinetics , Cytochrome P-450 Enzyme System/metabolism , Heart Transplantation , Immunosuppressive Agents/pharmacokinetics , Mixed Function Oxygenases/metabolism , Platelet Aggregation Inhibitors/pharmacology , Ticlopidine/pharmacology , Adolescent , Adult , Aged , Analysis of Variance , Cyclosporine/blood , Cytochrome P-450 CYP3A , Double-Blind Method , Drug Interactions , Heart Transplantation/physiology , Humans , Hydrocortisone/analogs & derivatives , Hydrocortisone/urine , Immunosuppressive Agents/blood , Male , Middle Aged , Platelet Aggregation Inhibitors/blood , Prospective Studies , Ticlopidine/bloodABSTRACT
1. Expression of various P450 subfamilies (1A, 2A, 2B, 2C, 3A) have been studied in cultured foetal rat hepatocytes after treatment with clofibric acid, a peroxisome proliferator and prototypic CYP4A inducer in vitro. Ethoxyresorufin O-deethylase activity (EROD, a CYP1A-related activity) as well as 7 alpha-, 16 alpha-, 2 alpha- and 6 beta-testosterone hydroxylase activities (CYP2A, 2B, 2C11 and 3A respectively) were determined during culture. Levels of the corresponding P450 apoproteins were measured by Western blotting. 2. Clofibric acid was able to induce all the P450-dependent activities studied. In most cases this induction required the additional presence of dexamethasone, an agent which promotes differentiation and favours long-term maintenance of the hepatocytes. 3. The major pro-inflammatory cytokines, IL-1 beta and IL-6, decrease the levels of the clofibric acid-induced P450 isoforms, except CYP1A, which was insensitive to IL-6, previous studies having shown that IL-1 beta represses lauric acid 12-hydroxylase activity after induction by clofibric acid. The effects of these cytokines were clearly dose- and time-dependent. The decrease in enzyme activity correlated with a decrease in apoprotein content. 4. The ability of clofibric acid to induce P450 isoforms highlights the complexity of P450 regulation by peroxisome proliferators. Our results confirm, moreover, that different P450 subfamilies are differentially affected by IL-1 beta and IL-6.
Subject(s)
Clofibric Acid/pharmacology , Cytochrome P-450 Enzyme System/biosynthesis , Interleukin-1/pharmacology , Interleukin-6/pharmacology , Isoenzymes/biosynthesis , Liver/enzymology , Animals , Cells, Cultured , Clofibric Acid/antagonists & inhibitors , Enzyme Induction , Fetus , Microbodies/drug effects , Microsomes, Liver/enzymology , RatsABSTRACT
The antiestrogen tamoxifen (TAM) has been successfully used to treat breast cancer expressing estrogen and progesterone receptors (ER+ and PR+). However, the development of antiestrogen resistance is frequently observed in patients following long-term treatment. To better understand the mechanism of action of TAM and its main metabolites: N-desmethyltamoxifen (N-des-TAM) and 4-hydroxytamoxifen (4-OH-TAM), their growth inhibitory effect was studied in 5 breast cancer cell lines characterized by different estrogen receptor levels: MDA-MB 231 (ER-), MCF-7 R (ER-), T47D (ER+), ZR-75/1 (ER+) and MCF-7 (ER+) trying to reproduce a cellular heterogeneity encountered in human breast tumors. In this study, the effects of TAM, N-des-TAM and 40-H-TAM on the cell growth were tested at concentrations ranging from 10(-8) to 10(-6)M with or without estradiol (10(-8)M). Only 4-OH-TAM showed a clear antiestrogen dose-dependent effect. Moreover, the finding of an antiproliferative activity at the highest dose (10(-6)M) for TAM, 4-OH-TAM and N-des-TAM in the ER- and PR- cell line MDA-MB 231 supports the hypothesis that TAM could be effective on ER+ as well as ER- tumors by an ER-independent mechanism. Despite ER+ and PR+ status after 2, 4 and 6 days of treatment, the T47D cell line displayed an increased growth rate with N-des-TAM at 10(-6)M. It should be noted that such concentration is within the range of the plasma level of N-des-TAM (10(-6)M) in patients receiving TAM per os (40 mg/day). These results and the well-known cell heterogeneity of human breast tumors may significantly account for some failure of antiestrogen treatment.
Subject(s)
Adenocarcinoma/metabolism , Antineoplastic Agents, Hormonal/toxicity , Breast Neoplasms/metabolism , Tamoxifen/toxicity , Adenocarcinoma/pathology , Antineoplastic Agents, Hormonal/metabolism , Antineoplastic Agents, Hormonal/pharmacology , Biotransformation , Breast Neoplasms/pathology , Cell Division/drug effects , Cell Line , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Female , Humans , In Vitro Techniques , Receptors, Estrogen/drug effects , Receptors, Estrogen/metabolism , Receptors, Progesterone/drug effects , Receptors, Progesterone/metabolism , Tamoxifen/metabolism , Tamoxifen/pharmacologyABSTRACT
Possible liver damage induced by chemicals or drugs must be detected early during drug development or industrial exposure, although damage is still difficult to predict, especially when immunotoxicity is involved. Liver toxicity may result from cytolysis, steatosis, cholestasis, phospholipidosis, or vascular lesions, most the outcome of a disadvantageous balance between chemicals or metabolites vs protective mechanisms, resulting from chemical dosage, genetic factors, or the immunoallergic status of the patient. Drug metabolism, lipid peroxidation, and thiol oxidation are frequently involved in liver toxicities. Classical guidelines in toxicology propose many methods for liver toxicity assessment: histology; chemical changes in hepatic tissue (lipids, glutathione, enzymes); physiological changes in biosynthesis (proteins, glycoproteins); excretion function (fructose); drug metabolism; and concentrations of related enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase) in blood. In vitro studies in human or animal hepatocytes or tumor-derived cell lines are useful in detecting hepatocellular lesions by cell viability, glutathione concentration, amount of lactate dehydrogenase released, cellular ATP, morphology (blebs), and drug metabolism.
Subject(s)
Chemical and Drug Induced Liver Injury , Liver/drug effects , Xenobiotics/adverse effects , Apoptosis/drug effects , Cell Line , Cells, Cultured , Drug Evaluation , Energy Metabolism , France , Humans , Liver/pathology , Liver/ultrastructure , Microscopy, Electron , Occupational Exposure , Risk Assessment , Xenobiotics/metabolism , Xenobiotics/urineABSTRACT
Various types of cells lines are used in pharmacotoxicology. Established cell lines are easily available, with few ethical restrictions. Some specific properties are preserved, although they have kept the phenotype of the original tissue, which is frequently a tumor phenotype. They are usually more resistant to toxic compounds than freshly isolated cells. Some drug-metabolizing enzymes are expressed and regulated in these cells. Immortalized cell lines are also of interest in toxicology. They are mainly examined for their potential in mutagenicity testing. These cells and numerous others of animal or human origin can be transfected with cDNA coding for human enzymes. They are used for determination of the individual enzyme involved in a particular metabolic pathway, or, when multiple transfections are successfully achieved, for mutagenicity testing. Regulation studies are also possible in such cells after transfection of DNA elements regulating gene transcription.
Subject(s)
Cell Line/drug effects , Drug Evaluation, Preclinical/methods , Drug Screening Assays, Antitumor/methods , Humans , Tumor Cells, CulturedABSTRACT
Many studies on drug metabolism have been carried out during the last decades using protein purification, molecular cloning techniques and analysis of polymorphisms at phenotype and genotype levels. These researchers led to a better understanding of the role of drug metabolizing enzymes in the biotransformation of drugs, pollutants or foreign compounds and of their use in laboratory medicine. The metabolic processes commonly involved in the biotransformation of xenobiotics have been classified into functionalization reaction (phase I reactions), which implicate lipophilic compounds. These molecules are modified via monooxygenation, dealkylation, reduction, aromatization, hydrolysis and can be substrates for the phase II reactions, often called conjugation reactions as they conjugate a functional group with a polar, endogenous compound. This review, devoted to cytochromes P-450 (CYP) and UDP-glucuronosyltransferases (UGT), describes essentially the genetic polymorphisms found in humans, their clinical consequences and the methods to assess the phenotypes or genotypes, with a view to studying the interindividual differences in drug monooxygenation and drug glucuronidation. Variations in drug glucuronidation reported here focused essentially on variations due to physiological factors, induction, drug interactions and genetic factors in disorders such as Gilbert's Syndrome and Crigler-Najjar type I and II diseases.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Pharmaceutical Preparations/metabolism , Genotype , Humans , Isoenzymes , Polymorphism, GeneticABSTRACT
Induction of drug metabolism enzymes is defined as a de novo synthesis of an enzyme protein. Not all, but a certain number of sub-families of cytochromes P450 are inducible among 27 families. Each group of inducers is relatively specific of one corresponding P450 subfamily; Polycyclic hydrocarbons and P4501A; Phenobarbital and P4502B; glucocorticoids and P4503A; Ethanol and P4502E; Peroxisome proliferations and P4504A. P450 induction has pharmacological implications specially concerning drug interactions, and inducers are themselves drugs of environmental compounds. Last 10 years have offered progresses in the knowledge of molecular mechanisms of induction such as mediation by receptors (Ah or PPAR), transcriptional regulation; Stabilisation of RNAm or of enzyme proteins. Repression of P450s synthesis is for less understood, as an example cytokines repress more than one P450-subfamily. At least transcriptional and post-translational mechanisms are involved.
Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Animals , Cytochrome P-450 Enzyme System/classification , Enzyme Induction , Enzyme Repression , Humans , In Vitro Techniques , RatsABSTRACT
The role of individual rat liver cytochrome P-450 isozymes in the metabolism of the skeletal muscle relaxant, dantrolene, was studied. Following incubation of dantrolene with hepatic microsomes from 3-methylcholanthrene-treated rats, two major hydroxylated metabolites were identified. Using inhibitory antibodies specific for individual cytochrome P-450 isozymes, cytochromes P-450 1A1, 1A2, and 3A were identified to be involved in dantrolene hydroxylations. In liver microsomes from 3-methylcholanthrene-treated rats, antibodies specific for cytochrome P-450 1A1 and 1A2 inhibited hydroxylation of dantrolene by 60% and 20%, respectively. Kinetics studies using these microsomes showed that dantrolene hydroxylation was biphasic with a low KM (0.06-0.08 microM) and high KM (5-7 microM). Cytochrome P-450 1A1 was responsible for the low KM hydroxylation of dantrolene, whereas cytochrome P-450 1A2 was responsible for the high KM. In hepatic microsomes from pregnenolone-16 alpha-carbonitrile-treated rats, an antibody specific for cytochrome P-450 3A completely inhibited the formation of 5-hydroxydantrolene, the major metabolite formed by these microsomes.