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.

Fas/FasL expression in tumor biopsies: a prognostic response factor to fluoropyrimidines?

Various studies suggested that cytotoxicity induced by 5-fluorouracil (5-FU) is an apoptotic mechanism possibly mediated by the Fas/FasL system. In this preliminary work, we studied retrospectively the role the Fas/FasL expression as a predictive response factor with fluoropyrimidine-based chemotherapies. We developed a real-time PCR method for measuring Fas and FasL expression in various biopsies from patients treated with a FUFOL-like protocol. No correlation was found between Fas or FasL expression and overall survival or partial response. However, the PCR assay was simple and convenient to use for quantitation of Fas/FasL in tumor biopsies.

Involvement of nuclear factor kappaB in c-Myc induction by tubulin polymerization inhibitors.

We showed previously that microtubule disassembly by vinblastine induces the proto-oncogene c-myc in epithelial mammary HBL100 cells. In this study, we demonstrate that vinblastine treatment in these cells, in contrast to what was observed with the colon adenocarcinoma cell line HT29-D4, activated the transcription factor NFkappaB, which has been involved in c-myc regulation. The microtubule disassembly also induced IkappaB degradation. Using transient transfection analysis, we show that the trans-activation of c-myc by vinblastine was decreased when NFkappaB binding sites on c-myc promoter were mutated. Additionally, we demonstrate that microtubule dissolution trans-activated a thymidine kinase-CAT construct containing an NFkappaB binding site at -1180 to -1080 bp relative to the P1 promoter. Thus, vinblastine up-regulates the enhancer activity of the NFkappaB binding site. These results suggest that microtubule disassembly induced by vinblastine can trans-activate the c-myc oncogene through NFkappaB. Taking into consideration the paradoxical roles of both c-myc and NFkappaB in proliferation or apoptosis, this data reveals the complex action mechanism of this microtubule interfering agent.

Serum induces a transcriptional activation of CYP1A1 gene in HepG2 independently of the AhR pathway.

CYP1A1 is largely implicated in carcinogenesis. To date, it is known that this gene is induced by xenobiotics such as polycyclic aromatic hydrocarbons. In this study, we evaluated the effect of serum in the regulation of CYP1A1 gene expression. CYP1A1 mRNA level is induced 1) in HepG2 and HT29-D4 cells by 3-methylcholanthrene 2) only in HepG2 after treatment by serum. The CYP1A1 mRNA induction in HepG2 is the consequence at least in part of a transcriptional activation as was demonstrated by evaluation of the hnRNA level. HepG2 cells were transfected by a plasmid containing the 7.5 Kb of the CYP1A1 promoter and the CAT reporter gene. No CAT stimulation was observed after serum treatment. These results demonstrated that CYP1A1 is induced at a transcriptional level by a physiological compound contained in serum independently of the Ah receptor and the 7.5 Kb promoter region.

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.

Opposite effects of antimicrotubule agents on c-myc oncogene expression depending on the cell lines used.

We investigated the expression of c-myc in HT29-D4, HBL100 and Caco-2 cells treated with microtubule stabilising (paclitaxel) or depolymerising agents (vinblastine, nocodazole). After induction by epidermal growth factor (EGF), c-myc expression decreased in HT29-D4 cells treated with all the antimicrotubule agents. In HBL100 and Caco-2, when microtubules were stabilised with paclitaxel, c-myc expression also decreased. In contrast, its expression increased after treatment with depolymerising agents. In both cell lines, we also observed that depolymerising agents alone induced c-myc expression whilst paclitaxel had no effect. This mRNA induction was confirmed at the protein level. In HT29-D4, no variation of c-myc expression was observed. Then, we showed that the increase of mRNA level was due to activation of gene transcription. These results indicate that modulation of c-myc expression varied depending on the cell lines used and the type of antimicrotubule agents. This work provides a potential link between the microtubular network and c-myc gene expression.

Induction of CYP1A1 by serum independent of AhR pathway.

CYP1A1 is implicated in the bioactivation of procarcinogens such as polycyclic aromatic hydrocarbons. To date, no physiological compounds have been described as inducers of this gene. In this study, we have examined the role of serum in the regulation of CYP1A1 gene expression. After treatment of CaCo-2 cells with fetal bovine serum, CYP1A1 mRNA level increased to the same extent as that observed after 3-methylcholanthrene induction. The same effect was obtained after treatment with adult bovine or human serum. Evaluation of hnRNA level performed on CaCo-2 cells indicates that CYP1A1 induction by serum acts at least in part through transcriptional activation. Promoter region containing the XRE (1.56 kb) was tested in the CAT assay. No stimulation of this reporter gene was detected after serum treatment. These results demonstrate for the first time that physiological compound(s) contained in serum induces CYP1A1 gene expression by transcriptional activation independent of the AhR pathway.

Follicle-like structures formed by intestinal cell lines derived from the HT29-D4 adenocarcinoma cell line: morphological and functional characterization.

When cultured in high glucose containing medium, the human colon carcinoma cell line HT29-D4 and a clone derived by transfection with the MDR1 cDNA (MDR31) form multilayers of unorganized cells which are not polarized and are linked by desmosomes. Within these multilayers appear spontaneously large multicellular follicle-like-structures (FLS) where polarized cells linked by tight junctional complexes surround a lumen. Electron microscopy showed that some FLS display well developed brush borders with densely packed microvilli. Others have irregularly oriented microvilli of various lengths or are even completely devoid of apical differentiation. The lumen contains a variable amount of amorphous osmiophilic material. The apical surface of FLS forming cells express dipeptidylpeptidase IV, carcinoembryonic antigen, the mucin MUC1 and for the transfected cells the gp-170 protein. The organic anion fluorescein is transported from the cell to the lumen of FLS. Rhodamine 123, a substrate of the gp-170 ABC transporter is also concentrated in the lumen formed by MDR31 cells. Verapamil and cyclosporine A inhibited this last transport. Cyclic AMP stimulates the formation of these structures since treatment of post-confluent multilayers dramatically increased the number of FLS in HT29-D4 and MDR31 cell cultures within 24 h. The spontaneous formation of these morphologically and functionally polarized structures appeared at random and might respond to the coincidence of fluctuating parameters of the regulatory pathways (cAMP, Ca2+).

Multidrug-resistant phenotype influences the differentiation of a human colon carcinoma cell line.

The human colon carcinoma cell line HT29-D4, which constitutively expresses a very low level of the MDR1 gene product, was made multidrug resistant by transfection with a human MDR1 cDNA from the pHaMDR1/A expression vector and selection by colchicine. Resistant clones were 3- to 15-fold resistant to colchicine and were cross-resistant to doxorubicin (3- to 4-fold). MDR1 gene expression was associated with the expression of functional P-glycoprotein (gp-170); the function was reversed by verapamil and cyclosporin A. HT29-D4 cells are able to differentiate in vitro by replacement of glucose by galactose in the culture medium and also to release the carcinoembryonic antigen (CEA). Under these culture conditions, MDR1 mRNA and gp-170 were always expressed and the protein remained functional. Upon galactose treatment, resistant clones were less differentiated since they showed a heterogeneous monolayer organization accompanied by heterogeneous staining of cell-surface CEA and a high decrease (60-90%) of CEA release.

Metabolism of carbamazepine by CYP3A6: a model for in vitro drug interactions studies.

Carbamazepine (CBZ) is widely used in the treatment of epilepsy. The drug is principally metabolized by CYPs to 10, 11-epoxy carbamazepine (CBZ-E) but this metabolite more toxic than the parent drug, does possess anticonvulsant properties. In humans, CYP3A4, CYP2C8 and CYP1A2 have been shown to be implicated in CBZ biotransformation. Our purpose was to establish an experimental model to determine the interaction of CBZ with other antiepileptic drugs. We first identified the CYP isoforms that metabolized CBZ in rabbit. We used liver microsomes from rabbit treated with various compounds known to induce principally some CYPs subfamilies. Having tested all the compounds we demonstrated that only the animals treated with CYP3A inducers were able to metabolize CBZ strongly. The CBZ biotransformation was inhibited by anti CYP3A antibodies. All the CYP3A subfamily substrates specifically decrease CBZ-E formation. In our experiment we did not observe any inhibition with CYP2C substrate. These data provide evidence that in rabbit the CYP3A subfamily is primarily involved in CBZ metabolism. Using this model we investigated the interaction of CBZ with phenobarbital, phenytoin, ethosuccimide, primidone, progabide, vigabatrin and lamotrigine.

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.

Effect of new thioacridine derivatives on P-gp function and on mdr1 gene expression.

We studied the effect of thioacridine derivatives on the function of P-glycoprotein in MDR mouse T-lymphoma cell line L5178 and in MDR human leukemia cell line K562/ADR by rhodamine 123 uptake assay. The effect of some selected thioacridines was also investigated on the expression of the mdr1 gene. Expression was analysed by RT-PCR. Two compounds: 3-amino-9-thio-(4'-nitrobenzyl)acridinone and 2,7-dimethoxy-9-thio-(2'-diethylaminoethyl) acridinone were able to block the function of the P-gp, and also to decrease significantly mdr1 gene expression. Because these two derivatives exert their positive effects as reversing agents they could be potential candidate anticancer agents for further investigation. The thioacridines, which do not affect P-gp function, do not affect or increase the expression of mdr1 gene. Our results showed the structure-activity relationships of these compounds, providing a direction for the development of new, more active compounds.

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.

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.

Pretreatment by tubulin agents decreases C-MYC induction in human colon carcinoma cell line HT29-D4.

For HT29-D4 cell line, we confirmed the interaction between c-Myc protein and microtubules by immunoprecipitation. We then studied the effect of antimitotic agents, nocodazole and the taxoids [paclitaxel (taxol) and docetaxel (taxotere)] on c-myc oncogene expression. The expression was analyzed by RT-PCR and Western blot. Taxol (1 microM), taxotere (1 microM) and nocodazole (3 microM) inhibited by 30-50% the c-myc induction produced by growth factors in culture medium. According to the flow cytometry analysis, the inhibition is not linked to the mitotic block. These results observed for both stabilizing and depolymerizing agents suggest that microtubular system is involved in c-myc expression more through its dynamic properties which influence signal transduction and intracellular transports than through its direct interaction with c-Myc protein.

The Salmonella sulA-test: a new in vitro system to detect genotoxins.

The Salmonella sulA-test is a newly developed colorimetric assay to detect genotoxins. This technique is based on the ability of DNA-damaging agents to induce the sulA gene, one of the SOS response genes. A constructed plasmid, pEM1968, carrying a fused sulA'::'lacZ was introduced into Salmonella typhimurium TA1538. Monitoring sulA gene expression was performed by assaying the beta-galactosidase activity in the transformed strain S. typhimurium TA1538/pEM1968. A simple, fast and sensitive liquid incubation procedure has been developed after optimization of the S9 mix composition and beta-galactosidase assay. The SOS-inducing potency (SOSIP, microM-1) was defined as the slopes of the non-linear dose-response relationships. Twenty-one chemicals with different modes of action were examined for a preliminary evaluation of the test. Nineteen chemicals were genotoxic in the Salmonella sulA-test. The SOSIP ranged from 1.2 x 10(-4) microM-1 (ethyl methanesulfonate) to 419.9 microM-1 (bleomycin). Sodium azide and 5-fluorouracil were not genotoxic. Frameshift, base-pair and oxidative genotoxins were detected by the tester strain. The calculated SOSIP and the minimum concentrations detected (MCD) in the Salmonella sulA-test were compared to the reported values obtained with two similar assays: the SOS Chromotest and umu-test. The SOSIP values of 12 compounds were the highest in this new assay. Five chemicals tested in the Salmonella sulA-test gave similar SOSIP values with those of one of the two other tests. ICR-191 had the highest SOSIP with the SOS Chromotest and 3-methylchloranthrene showed the highest SOSIP with the umu-test. Similarly, the lowest MCD values were found for 12 compounds in the Salmonella sulA-test. Four compounds had close MCD values in this assay and one of the two other techniques. The SOS Chromotest remained the most sensitive assay for cisplatin and ICR 191. The umu-test was the technique of choice for 3-methylchloranthrene.

Effect of isoprinosine on 3'-Azido-3'-deoxythymidine uptake and phosphorylation in human peripheral blood mononuclear cells.

The effect of the immunomodulating agent Isoprinosine on AZT uptake and phosphorylation in human blood monocytes/macrophages was examined. AZT (1-15 microM) uptake in monocytes/macrophages was rapid and dose- and time-dependent. The main metabolite formed after 1 h was AZT-5'-monophosphate. Isoprinosine cotreatment (5-50 microM) had no significant effect on AZT (5 microM) uptake. On the other hand, Isoprinosine increased the formation of AZT-diphosphate metabolites and, more intensively, of AZT-triphosphate (AZT-TP) metabolites after 1 h incubation. Extended exposure (4 h) resulted in complete absence of AZT-TP due to dephosphorylation of these metabolites or blocking of AZT phosphorylation.

Steady state pharmacokinetics of carbamazepine-phenobarbital interaction in patients with epilepsy.

Two carbamazepine (CBZ) tablet formulations (conventional, CBZ-CO, or controlled release, CBZ-CR) are commonly prescribed in monotherapy or in comedication with phenobarbital (PB) in the treatment of epilepsies. This study compares the pharmacokinetics of CBZ-CO against CBZ-CR in patients with epilepsies chronically treated with CBZ in monotherapy or CBZ-PB in bitherapy, the effect of PB on CBZ-CO and CBZ-CR pharmacokinetic parameters, and the effect of the two formulations of CBZ on PB pharmacokinetic parameters. The absorption rate constant (Ka), apparent steady state volume of distribution (Vdss/F), and apparent total clearance (CL/F) were computed with the APIS software using blood level profiles from 34 patients divided into four groups: patients receiving either CBZ-CO or CBZ-CR in monotherapy, or CBZ-CO or CBZ-CR in comedication with PB. The results show that the lowest dispersion of pharmacokinetic parameters was in patients receiving CBZ-CR in monotherapy. The CBZ formulation alters CBZ Ka, (Vdss/F) and (CL/F) values. CBZ (CL/F) also depends on the treatment (presence or absence of comedication by PB). In patients receiving PB in comedication with CBZ, the formulation of CBZ has no effect on PB pharmacokinetic parameters. These changes may be clinically significant and should be taken into account.