Activation of the NF κ B Pathway Enhances AhR Expression in Intestinal Caco-2 Cells.

Recent data suggest that apart from its well-known role in the regulation of xenobiotic metabolizing enzymes, AhR is also involved in inflammation. However, the influence of inflammation on AhR expression remains unknown. Here, we demonstrated that proinflammatory conditions induced by either PMA or IL-1 ß enhance AhR expression in Caco-2 cells. This was associated with an increase in AhR promoter activity. By means of directed mutagenesis experiments and the use of proteasome inhibitors, we demonstrated that inflammation-induced AhR expression involved the NF κ B pathway but not AP-1. Moreover, conditioned media from PMA-treated Caco-2 cells were also able to induce AhR expression, and this induction was repressed by anti-IL-1 ß blocking antibodies. Similar results were obtained with conditioned media from PMA-treated THP-1 cells. Taken together, these data suggest that AhR could be involved in vivo in an inflammatory loop. AhR was recently suspected to be implicated in inflammatory bowel disease. Our results support this hypothesis and suggest that AhR could be a new target for inflammatory bowel disease patient management.

PPARalpha transcriptionally induces AhR expression in Caco-2, but represses AhR pro-inflammatory effects.

In this work we demonstrate that Caco-2 cell treatment with WY-14643 (a potent PPARalpha agonist) causes an increase in AhR expression. Luciferase assays and directed mutagenesis experiments showed that induction mainly occurred at transcriptional level and involved a PPRE site located within the AhR promoter. These results were further confirmed by the use of PPARalpha knockout mice in which AhR induction by WY14643 was abrogated. In addition to CYP1 regulation, AhR has been described as being involved in inflammation, so we also studied the effect of AhR regulation by PPARalpha on the expression of some inflammation target genes. 3-Methylcholanthrene (a potent AhR agonist) increased the expression (mRNA) of the major inflammatory targets IL-1beta and MMP9. WY-14643 co-treatment abrogated the 3-methylcholanthrene pro-inflammatory effect. Hence the anti-inflammatory effect of PPARalpha overrides the pro-inflammatory effect of AhR.

Evidence for a new human CYP1A1 regulation pathway involving PPAR-alpha and 2 PPRE sites.

BACKGROUND AND AIMS: Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. METHODS: The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. RESULTS: We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. CONCLUSIONS: We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.

Dietary lipids affect human ethanol-inducible CYP2E1 gene expression in vivo in mononuclear cells.

We tested the hypothesis that dietary cholesterol modulate human ethanol-inducible CYP2E1 expression in vivo in circulating mononuclear cells. Healthy volunteers (n= 10) were submitted to a low fat low cholesterol diet for 4 days (day 0-day 3, LFLC). Cholesterol (595 +/- 56 mg/day) was then reintroduced for 7 days (day 4-day 10, LFHC). In the same time, controls subjects (n=7) did not change their habitual daily diet. CYP2E1 mRNA levels, evaluated in mononuclear cells, decreased in experimental subjects during both LFLC and LFHC from 100% to 53 +/- 5%, (p<0.001) with a main decrease during LFLC period (100% to 71 +/- 16%, p=0.05). Immunoreactive CYP2E1 showed a similar pattern and decreased from 100 to 62 +/- 12% during the trial (p<0.05). No significant change occured in control subjects. Between day 0 and day 11, changes in CYP2E1 mRNA correlated positively with plasma cholesterol (r2=0.67, p<0.001) and HDL cholesterol concentrations (r2=0.61, p<0.001). In contrast, no correlation was found between plasma fatty acids concentrations and CYP2E1 expression. The present results suggest that lipid factors regulate CYP2E1 expression, in vivo, in human mononuclear cells. In particular, plasma cholesterol concentrations may play an important role in this regulation.

Oxidative metabolism of bupivacaine into pipecolylxylidine in humans is mainly catalyzed by CYP3A.

Bupivacaine is used to provide prolonged anesthesia and postoperative analgesia. The human cytochrome P450 (CYP) involved in bupivacaine degradation into pipecolylxylidine (PPX), its major metabolite, has, to our knowledge, never been described. Microsome samples were prepared from six human livers and incubated in the presence of bupivacaine. The concentrations of PPX in the microsomal suspensions were assessed, and K(m) and V(max) values were calculated. Bupivacaine incubations were then performed with specific CYP substrates and inhibitors. For each sample of hepatic microsomes, the correlation between the rate of PPX formation and the corresponding erythromycin N-demethylase activity was analyzed. Finally, an immunoinhibition study using an anti-rabbit CYP3A6 antibody and assays with cDNA-expressed human CYP were conducted. The apparent K(m) and V(max) values of bupivacaine were, respectively, 125 microM and 4.78 nmol/min/mg of microsomal protein. The strongest inhibition of bupivacaine metabolism was obtained for troleandomycin (-95% at 50 microM), a specific CYP3A inhibitor. The correlation between PPX formation and erythromycin N-demethylase activity showed an R value of 0.99 whereas anti-rabbit CYP3A6 antibody inhibited the degradation of bupivacaine into PPX by 99%. Finally, CYP1A2 and CYP2E1 cDNA-expressed forms of human CYP did not allow PPX formation, CYP2C19 and CYP2D6 produced only small amounts whereas CYP3A4 most efficiently metabolized bupivacaine into PPX. These results demonstrated that bupivacaine degradation into PPX was mediated in humans by CYP3A.

Panax vietnamensis protects mice against carbon tetrachloride-induced hepatotoxicity without any modification of CYP2E1 gene expression.

In order to explore the effect of Panax vietnamensis on carbon tetrachloride-induced hepatotoxicity, mice were pretreated for 7 days with either crude extract or total saponins. Crude extract and total saponins dramatically decreased carbon tetrachloride-induced increase of serum GST alpha level (-50.0%, -49.5% respectively). Serum AST level was significantly decreased only with total saponins (-52.2%) and ALT level was slightly modified. In vitro experiments shown that both preparations at high concentrations (> 2000 micrograms/ml) are able to inhibit CYP2E1 enzymatic activity in mouse and human microsomes. However, we did not observe any modification of Cyp2e1 gene expression (enzymatic activity, protein and mRNA levels) in mice treated with either crude extract or total saponins. Taken together, these data demonstrated that Panax vietnamensis could be used as an hepatoprotectant. However, the mechanism of action is not associated with CYP2E1 expression, as previously suggested in vitro in rat for total saponins from Panax ginseng.

CYP2E1 and CYP3A1/2 gene expression is not associated with the ursodeoxycholate effect on ethanol-induced lipoperoxidation.

Ethanol is a well-known hepatotoxicant inducing steatosis and membrane lipoperoxidation. The aim of the present study was to investigate in rats, whether the protective effect of UDC on ethanol-induced lipid peroxidation may be related with CYP2E1 and CYP3A1/2 gene expression. We showed that UDC treatment in ethanol-fed rats induced a significant decrease in liver triglyceride concentration which was closely correlated with a reduction in malondialdehyde and hydroxyalkenal levels. In chronically ethanol-fed rats, CYP2E1 and CYP3A1/2 gene expressions were increased by a post-transcriptional mechanism. These inductions, mainly of CYP2E1, could take part in alcohol-induced hepatic lipoperoxidation. UDC modified neither the specific activity, nor the protein level, nor the mRNA level of CYP2E1 when compared with control. UDC supplementation to alcohol diet did not prevent the increase in CYP2E1 expression of ethanol-fed rats. Furthermore, CYP3A1/2 protein levels were similarly increased by ethanol and ethanol plus UDC treatment. Therefore, UDC protective effect against ethanol-induced lipoperoxidation was not associated with a modification of CYP2E1 and CYP3A1/2 expression.

Modulation of MDR1 and CYP3A expression by dexamethasone: evidence for an inverse regulation in adrenals.

A strong overlap exists between gp170 and CYP3A substrates and inducers. In order to investigate a putative coregulation of MDR and CYPA gene expression, we measured their transcripts in human liver and after dexamethasone treatment in HepG2 cells or in different mouse tissues. In human liver, we observed no correlation between MDR1 and CYP3A4 expression, whereas these genes were coinduced by dexamethasone in HepG2 cells. In mouse liver treated with dexamethasone, mdr1b and Cyp3a were induced (5- and 2-fold, respectively). In adrenals, the main expressing gp170 tissue, Cyp3a, was increased while mdr1b was repressed (-51%). The expression of mdr1b increased in heart, brain, and colon and decreased in lung and kidney but Cyp3a was not detectable. In conclusion, human hepatic CYP3A4 and MDR1 are not corregulated but are coinducible. In vivo murine mdr1b and Cyp3a are coregulated by dexamethasone in liver and inversely regulated in adrenals.

Effects of tobacco smoke on the gene expression of the Cyp1a, Cyp2b, Cyp2e, and Cyp3a subfamilies in mouse liver and lung: relation to single strand breaks of DNA.

Cigarette smoking is a worldwide health problem and is the greatest risk factor for lung cancer. By activating procarcinogens, hepatic and extrahepatic cytochromes P450 can participate in lung carcinogenesis. Tobacco smoke contains numerous cytochrome P450 inducers, substrates, and inhibitors. In the present study we investigated, in male NMRI mice, the effects of cigarette smoke on hepatic and pulmonary cytochrome P450 expression and their possible role in the induction of DNA lesions such as DNA single strand breaks (SSB). Hepatic and pulmonary mouse cytochrome P450 isozymes involved in carcinogenesis (Cyp1a, 2b, 2e, 3a) were differently induced by cigarette smoke. Cyp2e1 mRNA was dramatically enhanced (12.7-fold increase) while Cyp2b10 mRNA remained unchanged and Cyp1a1 was decreased or not detected. Cyp3a protein and mRNA were not detected in lung, suggesting that this isozyme is not expressed in mouse pulmonary tissue. The SSB of DNA increased in lung and liver treated mice. In contrast no modification was observed in lymphocytes that barely expressed cytochromes P450. Cimetidine and propylene glycol reduced SSB of DNA induced by smoking in liver and lung cells. The inhibition (-70%) observed in lung following treatment by propylene glycol, a CYP2E1 inhibitor, suggested that this isozyme is at least in part involved in pulmonary DNA damage induced by tobacco smoke. The high concentration of CYP2E1 function and regulation in mammals suggests that this protein could be involved in pulmonary carcinogenesis in human smokers.

Effect of cigarette smoke on UDP-glucuronosyltransferase activity and cytochrome P450 content in liver, lung and kidney microsomes in mice.

The effect of cigarette smoke on the expression of several cytochromes P450 (CYP) and UDP-glucuronosyl-transferases (UGT) was studied in mice. The animals were exposed to cigarette smoke for 4 to 30 days. Enzymatic activities supported by CYP1A1, 1A2, 2B, 2E1 and the glucuronidation activity toward phenols were measured in lung, liver and kidney microsomes. Cigarette smoke induced several CYPs, especially in lung. CYP2E1 was more induced than CYP1A1 in this organ. The expression of CYP2E1 was also increased in kidney (5.6 times after 30 days). The glucuronidation in kidney was non-sensitive to the treatment whatever substrate used. In contrast, this activity was enhanced in liver and particularly in lung, in which the glucuronidation of 1-naphthol and 2-hydroxybiphenyl was increased by 122 and 180%, respectively. Interestingly, the times of induction differed according to the substrate used, thus suggesting the presence of different UGTs active toward phenols that were differentially affected by cigarette smoke. The UGT activities toward phenols were low in lung, when compared with those measured in liver or kidney. In conclusion, cigarette smoke greatly affected both glucuronidation activity and the hydroxylation reactions supported by CYPs in mouse liver and lung.

Evidence for a tissue-specific induction of cutaneous CYP2E1 by dexamethasone.

We studied in mouse the effect of topical application of dexamethasone or salicylic acid, on CYP2E1 and CYP3A expression (proteins and/or mRNA) in liver and skin. Dexamethasone was also administered by intraperitoneal injection. Topical application or intraperitoneal injection of dexamethasone increased cutaneous CYP2E1 (8 and 4-fold respectively) whereas the hepatic level of this isoform showed a slight decrease and hepatic CYP3A expression was increased (3-fold). Cutaneous CYP2E1 was increased (3-fold) after topical treatment by salicylic acid. This compound had no effect on hepatic CYP3A and CYP2E1 expression. Cutaneous CYP3A (protein and mRNA) was not detectable in all groups (control or treated animals). Dexamethasone and salicylic acid increased cutaneous CYP2E1 mRNA level (2.5 and 1.4-fold respectively). In conclusion, dexamethasone and salicylic acid induced cutaneous CYP2E1 protein and mRNA level. Cutaneous CYP2E1 induction by dexamethasone is a tissue-specific process.

Effects of different exposure times to tobacco smoke intoxication on carboxyhemoglobin and hepatic enzymate activities in mice.

The aim of this work was to determine the effects of different exposure times to smoke on carboxyhemoglobin (HbCO) and hepatic enzymate activities in order to adapt a tobacco smoke intoxication model in mice. Mice were exposed to tobacco smoke for various durations of either 2 (group S2), 4 (group S4), 8 (group S8), or 31 days (group S31) using the Hamburg II machine. Controls (nonexposed animals) were used under the same experimental conditions. On the 2nd, 4th, 8th, and 31st day, mice were sacrificed by decapitation, and blood carboxyhemoglobin level and hepatic enzymate activities catalysed by CYP 450 families were measured. Our data with regard to the exposed group indicated first that HbCO was significantly increased after 4 or 8 days of exposure and decreased after 31 days compared to controls (where HbCO was constant for the duration of the 31 days) and second, the enzymate activities were significantly higher during the period of exposure. In conclusion, a 4- and 8-day exposure period with eight cigarettes per day seems to be the model of tobacco smoke intoxication in mice to be chosen.

High inducibility of mouse renal CYP2E1 gene by tobacco smoke and its possible effect on DNA single strand breaks.

Several studies have shown in humans an association between renal carcinoma and cigarette smoking. Cigarette smoke contains numerous cytochrome P450 inducers and substrates. In the present study we investigated the effect of cigarette smoke on the regulation of murine cytochrome P450 expression in kidney and its possible role in the induction of single strand breaks in DNA. Results demonstrated that CYP2E1 (activity, protein, and MRNA) was induced by tobacco smoke (2.1, 5.6 and 20.8, respectively). We did not detect any CYP1A, CYP2B, and CYP3A using Western blot and RT-PCR experiments. We have analyzed the renal single strand breaks of DNA in control and treated mice. The results indicated an increase of single strand breaks of DNA in kidney from treated mice which paralleled the high inducibility of the CYP2E1. No significant difference was observed between lymphocytes (which expressed very low or undetectable cytochrome P450 levels) of control and treated mice.

Glucuronidation of propofol in microsomal fractions from various tissues and species including humans: effect of different drugs.

This in vitro study was conducted to evaluate propofol glucuronidation and the effect of concomitantly administered drugs in various species. Propofol glucuronidation was studied in microsomal fractions from rat, rabbit, and human livers. Extrahepatic metabolism was investigated using lung and kidney microsomes. The propofol-uridine diphosphate-glucuronosyltransferase (UGT) activity measured in liver microsomes was higher in rabbit than in rat. Among the three tested species, human livers exhibited the highest activity, with only small variability in the three samples studied. Animal kidney, but not lung (animal or human), microsomes were able to glucuronidate propofol, meaning that extrahepatic metabolism of propofol exists, at least in the kidney, in the tested species (rat and rabbit). Since metabolic interactions are potential sources of prolonged drug effect or overdose, we screened the effect of 21 compounds (known substrates of various UGT or potentially coadministered drugs) on the glucuronidation of propofol by human liver microsomes. Inhibitions obtained with chemicals or drugs glucuronidated by either UGT1 or UGT2 families (1-naphtol, 4-hydroxybiphenyl, carvacrol, n-propylgallate, ketoprofen, chloramphenicol, acetylsalicylic acid) indicated that at least two UGT isoforms are involved in propofol glucuronidation. Inhibition was observed with several drugs potentially coadministered during pre-, per, or postoperative periods (e.g., acetylsalicyclic acid, ketoprofen, oxazepam, fentanyl). Although not directly transposable to the in vivo situation, these results indicate that such interactions are theoretically possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cigarette smoke on hepatic and pulmonary cytochromes P450 in mouse: evidence for CYP2E1 induction in lung.

Pulmonary and liver microsomes of male NMRI mice were used to study the inductive effect of cigarette smoke on various cytochrome P450 isoforms implicated in precarcinogen and premutagen bioactivation. The enzymatic activities catalyzed by CYP1A1, CYP2B, CYP2C, CYP2D, CYP2E1 and CYP3A were induced in liver microsomes. Immunoquantification of lung and liver CYP1A1, 2E1 and 3A demonstrated that 1) CYP1A1 was induced in lung and liver, 2) CYP3A subfamily was induced in liver and not detected in lung, 3) CYP2E1 was slightly induced in liver whereas its pulmonary expression was more largely increased (6.8 fold) than CYP1A1 (2.0 fold). This latter data suggests that CYP2E1, which is known to be expressed in human lung, could actively participate in pulmonary carcinogenesis induced by cigarette smoke.

Abbott PKS system: a new version for applied pharmacokinetics including Bayesian estimation.

Abbott Laboratories has developed a new software package (Abbottbase pharmacokinetic system or PKS package) that employs the principles of pharmacokinetics to assist clinical pharmacologists and clinicians in designing dosage regimens. This software, which runs on IBM PC compatibles, allows Bayesian estimation of individual pharmacokinetic parameters. The aim of the present study was to validate this new system in routine clinical practice for amikacin (40 intensive care unit patients) and theophylline (20 patients). By using the program one or more times during the treatment (50 cases for amikacin and 46 cases for theophylline), dosing recommendations were obtained in real time for all patients. The predictive performance (bias and precision) was assessed by comparing predicted drug concentrations with those measured 24-48 h after dosage recommendation. In the case of amikacin, precision and bias were computed separately for peak and trough levels. In all cases, no statistically significant bias was observed. Finally, our results demonstrate the accuracy of the program in predicting drug levels for amikacin and theophylline. Consequently, the PKS package is reliable for the choice of optimal dosage regimen for amikacin and theophylline.

Predictive performance of two software packages (USC*PACK PC and Abbott PKS system) for the individualization of amikacin dosage in intensive care unit patients.

Many dosing methods (nomogram, pharmacokinetic methods, Bayesian methods) can be used for the individualization of amikacin dosing. Among these methods, it is now well known that the Bayesian method provides a rapid and accurate means for individualizing dosage requirements for patients with diverse pharmacokinetic profiles. However, one problem has not been fully resolved. Should we use population-based parameters reflecting the patient population being monitored or should we used general population parameters? The aim of this study was to answer this question using two widely used software programs (USC*PACK PC and Abbott PKS system) and two different population parameters sets. Predictive performance of these methods was assessed with respect to the prediction of amikacin serum concentrations in intensive care unit (ICU) patients. Our results show that the differences between predicted and measured concentrations were unbiased when the population parameters used were adequate. Precision values were comparable with previously reported values. The predictive performance of the two tested software programs are very comparable in ICU patients. In addition, we demonstrated that performance can be enhanced when using population-based parameters which reflect the patient population being monitored. It is therefore advisable for each user to properly characterize each particular patient population.