[Pharmacogenetics: from basic research to clinical applications]. / La pharmacogénétique: de la recherche fondamentale aux applications cliniques.

Pharmacogenetics is the study of the genetic factors implicated in the pharmacological or toxicological response to drugs. This response is very variable and depends on three steps: metabolism/transport, target and organism reaction. Each step is very variable as a function of endogenous (patho-physiological, genetics...) or exogenous (environment: drugs, diet, smoking, alcohol...) factors. The scientific bases of pharmacogenetics have been strongly established: genetic polymorphisms have an impact on pharmacological activity or on the toxicity of numerous drugs and examples are given in this review. By contrast, present clinical applications are more limited although, in some instances, medical and economic interest of the pre-treatment determination of genotype or phenotype of the patients has been clearly demonstrated. Certain hospital laboratories now include pharmacogenetic activity. The predictive or explicative value of pharmacogenetics is scientifically demonstrated but prospective studies for validation are now necessary to identify situations where a clinical application will be actually beneficial for patients.

[Molecular pharmacogenetics in hospital laboratories in France: current data and future prospects]. / La pharmacogénétique moléculaire hospitalière en France: données actuelles et perspectives.

Molecular pharmacogenetic units have recently been established in several hospital laboratories in France. The clinical impact of these units is still limited and numerous problems of organizational, ethical, legal, technical, social and economical nature remain to be resolved. However, an increasing number of these units, a rise in their activities and an enlargement of their scope of application are foreseeable in the future. Ultimately, these units would significantly contribute to limit the public health problem caused by interindividual variabilities in drug effects. In view of these prospects, it seems essential that such hospital activity should be quickly recognised by the authorities and the various health sectors in France. It is also essential that the problems that arise from such pharmacogenetic activities should be considered by the authorities and would profit from the organization of a national network and from financial guarantees.

Indirect cytotoxicity of flucloxacillin toward human biliary epithelium via metabolite formation in hepatocytes.

Flucloxacillin, an isoxazolyl-penicillin, causes cholestasis and biliary epithelium injury. The aim of the study was to determine whether flucloxacillin, either directly or through metabolite formation, may induce cytotoxicity in hepatic or biliary cells. Cytotoxicity was assessed by lactate dehydrogenase release in primary cultures of human hepatocytes and of gallbladder-derived biliary epithelial cells (BEC). Metabolite production in microsome and cell preparations was analyzed by chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. While flucloxacillin induced no direct cytotoxicity in any of the hepatocyte (n = 12) and BEC (n = 19) preparations, the conditioned media from cultured hepatocytes preincubated with flucloxacillin (50-500 mg/L) triggered a significant increase in lactate dehydrogenase release over controls in approximately 50% of BEC preparations (7/12), and this effect depended upon flucloxacillin concentration. Remaining BEC preparations exhibited no toxic response. Cytotoxicity in BEC preparations (9/13) was also induced by the supernatants of human liver microsomes and of recombinant human cytochrome P450 (CYP)3A4 preincubated with flucloxacillin (500 mg/L). Supernatants from both liver microsome and CYP3A4 preparations contained one major metabolite which was identified as 5'-hydroxymethylflucloxacillin. The production of this metabolite was inhibited following CYP3A4 inhibition by troleandomycin in human liver microsomes, and markedly enhanced following CYP3A induction by dexamethasone in rat liver microsomes. As opposed to BEC, cultured hepatocytes displayed significant CYP3A activity and produced low amounts of this metabolite. The purified metabolite (0.01-5 mg/L) exerted toxic effects in BEC but not in hepatocytes. In conclusion, hepatocytes mainly via CYP3A4 activity, generate flucloxacillin metabolite(s) including 5'-hydroxymethylflucloxacillin that may induce cytotoxicity in susceptible BEC. These metabolic events may contribute to the pathogenesis of drug-induced cholangiopathies.

Is diclofenac a valuable CYP2C9 probe in humans?

INTRODUCTION: Besides the low therapeutic index drug tolbutamide, there is no validated in vivo probe to assess the genetically determined CYP2C9 activity in humans. The in vitro CYP2C9-specific substrate diclofenac might be a valuable, well-tolerated probe candidate. In order to validate diclofenac as an in vivo CYP2C9 probe, we planned to show that urinary 4'-hydroxydiclofenac/diclofenac metabolic ratio (MR) would correlate to the apparent partial metabolic clearance of diclofenac into 4'-hydroxydiclofenac (Clmet). PATIENTS AND METHODS: Eighteen healthy volunteers received a single oral dose of 50 mg diclofenac in its enteric-coated form. Blood and urinary pharmacokinetics of diclofenac were studied over 48 h. Identification of the CYP2C9 alleles (CYP2C9*1, CYP2C9*2, and CYP2C9*3) was performed with genomic DNA sequencing. RESULTS: We observed a dramatic inter-individual variability in the delay of diclofenac intestinal absorption since its first detectable blood concentration ranged from 0.5 h to more than 12 h after drug intake. The Clmet of diclofenac could not be determined in two subjects who started to absorb the drug after 12 h. No correlation could be observed between Clmet of diclofenac and the different MRs calculated at 0-4 h, 0-8 h, 0-12 h, 0-24 h and 0-48 h urinary collections. The Clmet of diclofenac in heterozygous subjects tended to be lower than among wild-type homozygous subjects, but this difference did not reach statistical significance due to an insufficient number of subjects studied. CONCLUSION: Diclofenac, in its enteric-coated form, is not a useful in vivo CYP2C9 probe probably because of its highly variable intestinal absorption rate. However, since we found a lower metabolic clearance of diclofenac in heterozygous CYP2C9 subjects, as observed with other CYP2C9 substrates, diclofenac, in another galenic form, might be a potential probe to quantify CYP2C9 activity in humans.

Cytochrome P4502C9 is the principal catalyst of racemic acenocoumarol hydroxylation reactions in human liver microsomes.

The oral anticoagulant acenocoumarol is given as a racemic mixture. The (S)-enantiomer is rapidly cleared and is the reason why only (R)-acenocoumarol contributes to the pharmacological effect. The objective of the study was to establish the cytochrome P450 (CYP) enzymes catalyzing the hydroxylations of the acenocoumarol enantiomers. Of various cDNA-expressed human CYPs, only CYP2C9 hydroxylated (S)-acenocoumarol. Hydroxylation occurred at the 6-, 7-, and 8-position with equal K(m) values and a ratio of 0.9:1:0.1 for V(max). CYP2C9 also mediated the 6-, 7-, and 8-hydroxylations of (R)-acenocoumarol with K(m) values three to four times and V(max) values one-sixth times those of (S)-acenocoumarol. (R)-Acenocoumarol was also metabolized by CYP1A2 (6-hydroxylation) and CYP2C19 (6-, 7-, and 8-hydroxylation). In human liver microsomes one enzyme only catalyzed (S)-acenocoumarol hydroxylations with K(m) values < 1 microM. In most of the samples tested the 7-hydroxylation of (R)-acenocoumarol was also catalyzed by one enzyme only. The 6-hydroxylation was catalyzed by at least two enzymes. Sulfaphenazole could completely inhibit in a competitive way the hydroxylations of (S)-acenocoumarol and the 7-hydroxylation of (R)-acenocoumarol. The 6-hydroxylation of (R)-acenocoumarol could be partially inhibited by sulfaphenazole, 40 to 50%, and by furafylline, 20 to 30%. Significant mutual correlations were obtained between the hydroxylations of (S)-acenocoumarol, the 7-hydroxylation of (R)-acenocoumarol, the 7-hydroxylation of (S)-warfarin, and the methylhydroxylation of tolbutamide. The results demonstrate that (S)-acenocoumarol is hydroxylated by a single enzyme, namely CYP2C9. CYP2C9 is also the main enzyme in the 7-hydroxylation of (R)-acenocoumarol. Other enzymes involved in (R)-acenocoumarol hydroxylation reactions are CYP1A2 and CYP2C19. Drug interactions must be expected, particularly for drugs interfering with CYP2C9. Also, drugs interfering with CYP1A2 and CYP2C19 may potentiate acenocoumarol anticoagulant therapy.

Phase I and phase II drug-metabolizing enzymes are expressed and heterogeneously distributed in the biliary epithelium.

Tissue expression of drug-metabolizing enzymes influences susceptibility to drugs and carcinogens. Because the biliary epithelium, exposed to bile-borne chemicals, may give rise to drug-induced cholangiopathies and to cholangiocarcinomas, we determined the pattern of expression of drug-metabolizing enzymes in this epithelium. We first demonstrated by blot analyses that biliary epithelial cells (BEC) isolated from human gallbladders display cytochrome P450 (CYP) 1A, 2E1, and 3A, microsomal epoxide hydrolase (mEH), alpha, mu, and pi glutathione S-transferase (GST), transcripts and proteins. We also identified CYP-associated steroid 6beta-hydroxylase activity in BEC. CYP and mEH expression was 5- to 20-fold lower in BEC than in autologous hepatocytes, and further differed by a higher ratio of CYP3A5/CYP3A4, and by CYP1A1 predominance over CYP1A2. alphaGST was highly expressed in both hepatocytes and BEC, while piGST was restricted to BEC. In approximately 50% of individuals, muGST was expressed in hepatocytes and at lower levels in BEC. By using the same antibodies as those used in immunoblots, we could show by immunohistochemistry that CYP2E1, CYP3A, mEH, alpha, mu, and piGST immunoreactivities are expressed and display a heterogeneous distribution in the epithelium lining the entire biliary tract except for small intrahepatic bile ducts that were devoid of CYP3A and alphaGST immunoreactivities. In conclusion, BEC contribute to phase II, and although to a lesser extent than hepatocytes, to phase I biotransformation. The distribution of drug-metabolizing enzymes in BEC suggest that they are heterogeneous in their ability to generate and detoxicate reactive metabolites, which may contribute to specific distributions of cholangiopathies.

High yield purification and characterization of engineered human P450 1A2 and generation of immuno-inhibitor antibodies.

P450 S12, an engineered human P450 1A2 containing the 88-first amino-acids of the P450 1A1, demonstrates particularly high expression level in yeast while exhibiting catalytic properties very similar to the moderately expressed natural human P450 1A2. To facilitate P450 purification by nickel chelate chromatography, C-terminal extensions including histidine tags were tested. The -G(H)4 extension was found to be particularly efficient for permitting high expression levels without any catalytic alteration. This engineered P450 was purified to electrophoretic homogeneity (18 nmol/mg of protein) at a very high yield (87%) without any detectable formation of P420. P450 S12 activities were reconstituted in the presence of yeast and Arabidopsis thaliana (ATR1) NADPH-P450 reductases. The plant reductase supported better ethoxyresorufin-, methoxyresorufin- and phenacetin-O-dealkylase activities than the yeast reductase in reconstituted systems. Interestingly, polyclonal antibodies raised against purified P450 S12 selectively recognized in Western blot and fully immuno-inhibited the natural or recombinant P450 1A2 with very limited or no cross-reaction with P450 1A1 and other isoenzymes.

Drug-induced immunotoxicity.

Immune-related drug responses are one of the most common sources of idiosyncratic toxicity. A number of organs may be the target of such reactions; however, this review concentrates mostly on the liver. Drug-induced hepatitis is generally divided into two categories: acute hepatitis in which the drug or a metabolite destroys a vital target in the cell; immunoallergic hepatitis in which the drug triggers an adverse immune response directed against the liver. Their clinical features are: a) low frequency; b) dose independence; c) typical immune system manifestations such as fever, eosinophilia; d) delay between the initiation of treatment and onset of the disease; e) a shortened delay upon rechallenge; and f) occasional presence of autoantibodies in the serum of patients. Such signs have been found in cases of hepatitis triggered by drugs such as halothane, tienilic acid, dihydralazine and anticonvulsants. They will be taken as examples to demonstrate the recent progress made in determining the mechanisms responsible for the disease. The following mechanisms have been postulated: 1) the drug is first metabolized into a reactive metabolite which binds to the enzyme that generated it; 2) this produces a neoantigen which, once presented to the immune system, might trigger an immune response characterized by 3) the production of antibodies recognizing both the native and/or the modified protein; 4) rechallenge leads to increased neoantigen production, a situation in which the presence of antibodies may induce cytolysis. Toxicity is related to the nature and amount of neoantigen and also to other factors such as the individual immune system. An effort should be made to better understand the precise mechanisms underlying this kind of disease and thereby identify the drugs at risk; and also the neoantigen processes necessary for their introduction into the immune system. An animal model would be useful in this regard.

Autoantibodies against a kidney--liver protein associated with quinolone-induced acute interstitial nephritis or hepatitis.

In the present study we report on four cases of acute interstitial nephritis (AIN) and two cases of hepatitis induced by quinolone. We show by immunoblotting analysis that all sera from these patients contained autoantibodies that recognize a 65-kDa protein expressed in normal human kidney and liver microsomes. Only 6% of sera from healthy individuals who did not ingest quinolone recognized the same protein. These findings suggest that the presence of autoantibodies could be used as a sensitive marker and that a modification of microsomal proteins by quinolone itself or by a metabolite could generated an autoimmune response.

Epitope mapping of human CYP1A2 in dihydralazine-induced autoimmune hepatitis.

Dihydralazine-induced hepatitis is characterized by the presence of anti-liver microsomal (anti-LM) autoantibodies in the sera of patients. Cytochrome P450 1A2 (CYP1A2), involved in the metabolism of dihydralazine, was shown to be a target for autoantibodies. In order to investigate further the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, and since the specificity of anti-LM autoantibodies towards CYP1A2 has been determined, the antigenic site was further localized. By constructing fragments derived from CYP1A2 cDNA and probing the corresponding proteins with several anti-LM sera, we were able to define a region (amino acid 335-471) which was immunoreactive with 100% of sera. An internal deletion in this region led to the loss of recognition by anti-LM autoantibodies, confirming that the epitope was conformational. Epitope mapping studies had previously been performed for CYP2D6, CYP17, CYP21A2, and recently for CYP3A1 and CYP2C9. Those data were compared with results obtained in the present study for CYP1A2.

The use of human in vitro metabolic parameters to explore the risk assessment of hazardous compounds: the case of ethylene dibromide.

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.

Immunotoxicology of the liver: adverse reactions to drugs.

Liver is a frequent target for drug-induced hepatitis. They can be classified in two categories: the hepatitis in which the drug or a metabolite reach a vital target in the cell and the hepatitis in which the drug triggers an adverse immune response directed against the liver. We will discuss essentially this second kind of disease. They have key clinical features such as the low frequency, the dose independence, the delay between the beginning of drug intake and the triggering of the disease, the shortening of the delay upon rechallenge and very often the presence of autoantibodies in the serum of the patients. Such signs were found in hepatitis triggered by drugs such as halothane, tienilic acid, dihydralazine, anticonvulsants. They will be taken as examples to show the recent progress in the understanding of the mechanisms leading to the disease. It has been postulated that the drug is metabolised into a reactive metabolite binding to the enzyme which generated it; therefore the neoantigen might trigger an immune response characterised by the production of antibodies recognising the native and or the modified protein. Most of these steps were proven in the cases of halothane, tienilic acid and dihydralazine. Several points seem important in the development of the disease; the equilibrium between toxication and detoxication pathways, the nature and amount of neoantigen, the individual immune response. However, many points remain unclear: for instance, the reason for the very low frequency of this kind of disease; the precise mechanism of the adverse immune response; the risk factors for developing such adverse reactions. Efforts should be made to better understand the mechanisms of this kind of disease: for instance, an animal model, tests to identify drugs at risk for such reactions, the role of these drugs in the processing of P450s and the processing of the neoantigens for their presentation to the immune system.

Inter-individual variability in the oxidation of 1,2-dibromoethane: use of heterologously expressed human cytochrome P450 and human liver microsomes.

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.

Tienilic acid-induced autoimmune hepatitis: anti-liver and-kidney microsomal type 2 autoantibodies recognize a three-site conformational epitope on cytochrome P4502C9.

Tienilic acid-induced hepatitis is characterized by the presence of anti-liver and -kidney microsomal (anti-LKM2) autoantibodies in patient sera. Cytochrome P4502C9(CYP2C9), involved in the metabolism of tienilic acid, was shown to be a target for tienilic acid-reactive metabolites and for autoantibodies. To further investigate the relationship between drug metabolism and the pathogenesis of this drug-induced autoimmune disease, the specificity of anti-LKM2 autoantibodies toward CYP2C9 was first determined, and the antigenic sites on CYP2C9 were localized. By constructing several deletion mutants derived from CYP2C9 cDNA and by probing the corresponding proteins with different anti-LKM2 sera, we defined three regions (amino acids 314-322, 345-356, and 439-455); they interacted to form a major conformational autoantibody binding site. This binding site was immunoreactive with 100% of sera and allowed removal of the entire reactivity of the sera tested by immunoblotting. Epitope mapping studies have been performed for CYP2D6, CYP17, CYP21A2, and, recently, CYP3A. Those data were compared with the results obtained in the current study with CYP2C9 in an attempt to elucidate one of the mechanisms by which CYP becomes immunogenic.

Anti-cytochrome P450 autoantibodies in drug-induced disease.

Drugs may induce hepatitis through immune mechanisms. In this review we have used the examples of 2 drugs to elucidate the first steps leading to the triggering of such disease, namely tienilic acid (TA) and dihydralazine (DH). These drugs are transformed into reactive metabolite(s) by cytochrome P450 (2C9 for TA and 1A2 for DH) (step 1). The reactive metabolites produced are very short-lived and bind directly to the enzymes which generated them (step 2). A neoantigen is thus formed which triggers an immune response (step 3), characterized by the presence of autoantibodies in the patient's serum (step 4). The autoantibodies are directed against the cytochrome P450 which generated the metabolite(s). Although the process by which TA and DH induce-hepatitis has been elucidated, further studies are necessary to generalize this mechanism. In addition, an animal model will also be useful to fully understand the immune mechanism of this type of disease.

Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies.

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.

CYP3A5 is the major cytochrome P450 3A expressed in human colon and colonic cell lines.

CYP3A is known to be expressed in liver, small intestine and colon. However, its isoform distribution (CYP3A4, 3A5 and 3A7) and inducibility have not been clearly elucidated in the colon. Therefore, we analyzed CYP3A in human colon and compared its expression and inducibility to the human colonic cell lines HT29 and Caco2, which were used as models. Patients suffered either from sigmoiditis or colonic adenocarcinoma. Patients as well as HT29 and Caco2 cells were treated with rifampicin. CYP3A protein expression was analyzed in the colon of patients and in the cells by immunoblot and by isoelectric focusing enabling separation of CYP3A isoforms, while mRNA expression was determined using specific reverse transcription-polymerase chain reaction. In both human colon and cells, CYP3A5 was the main isoform expressed at the protein and mRNA levels. Rifampicin treatment had no effect on CYP3A expression. HT29 and Caco2 cells exhibiting the same CYP3A expression and inducibility might therefore be useful in vitro models for studying xenobiotic metabolism in human colon.

Insulin down-regulates cytochrome P450 2B and 2E expression at the post-transcriptional level in the rat hepatoma cell line.

Cytochromes P450 (P450s) are inducible drug-metabolizing enzymes involved in the metabolism of numerous endogenous and exogenous substrates. The regulation of some of these enzymes during experimental diabetes has been reported, but the direct involvement of insulin and the mechanism of its action remain unclear. The aim of our work was to study the effects of insulin on P450 2B and 2E expression in differentiated Fao hepatoma cells. Exposure of the cells to 0.1 microM insulin caused 60% and 80% decreases in the steady state levels of P450 2B and 2E proteins, respectively, within 24 hr. Before this, a rapid decrease in the corresponding messages was observed. Indeed, 5-6 hr of insulin treatment produced 80 and 50% decreases in P450 2B and 2E mRNA levels, respectively. Nuclear run-on transcription and mRNA turnover studies were performed to determine the mechanism (transcriptional and/or post-transcriptional) by which insulin modulated these mRNA levels. From our results, it can be concluded that insulin down-regulates the expression of P450 2B by shortening the half-life of its mRNA (half-lives of 6.9 hr without insulin and 3.6 hr with insulin), whereas it down-regulates the expression of P450 2E both by weak repression of the transcription rate (-30%) and, in particular, by acceleration of its mRNA turnover (half-lives of 8.5 hr without insulin and 3.3 hr with insulin).