Marie-José Vilarem

Interleukin 1β inhibits CAR‐induced expression of hepatic genes involved in drug and bilirubin clearance

During the inflammatory response, intrahepatic cholestasis and decreased drug metabolism are frequently observed. At the hepatic level, the orphan nuclear constitutive androstane receptor (CAR) (NR1I3) controls phase I (cytochrome P450 [CYP] 2B and CYP3A), phase II (UGT1A1), and transporter (SLC21A6, MRP2) genes involved in drug metabolism and bilirubin clearance in response to xenobiotics such as phenobarbital or endobiotics such as bilirubin. We investigated the negative regulation of CAR, a glucocorticoid‐responsive gene, via proinflammatory cytokine interleukin 1β (IL‐1β) and lipopolysaccharides (LPSs) in human hepatocytes. We show that IL‐1β decreases CAR expression and decreases phenobarbital‐ or bilirubin‐mediated induction of CYP2B6, CYP2C9, CYP3A4, UGT1A1, GSTA1, GSTA2, and SLC21A6 messenger RNA. This occurs via nuclear factor κB (NF‐κB) p65 activation, which interferes with the enhancer function of the distal glucocorticoid response element that we have identified recently in the CAR promoter. We demonstrate that: (1) LPSs, IL‐1β, or overexpression of p65RelA inhibit glucocorticoid receptor (GR)‐mediated CAR transactivation; (2) these suppressive effects can be blocked both by pyrrolidine dithiocarbamate, an inhibitor of NF‐κB activation, or by overexpression of SRIkBα, a NF‐κB repressor; and (3) the GR agonist dexamethasone induces histone H4 acetylation at the proximal CAR promoter region, whereas LPSs and IL‐1β inhibit this acetylation as assessed via chromatin immunoprecipitation assay. In conclusion, GR/NF‐κB interaction affects CAR gene transcription through chromatin remodeling and provide a mechanistic explanation for the long‐standing observation that inflammation and sepsis inhibit drug metabolism while inducing intrahepatic cholestasis or hyperbilirubinemia. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004.)

Ketoconazole and Miconazole Are Antagonists of the Human Glucocorticoid Receptor: Consequences on the Expression and Function of the Constitutive Androstane Receptor and the Pregnane X Receptor

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) play a major part in the control of drug metabolism and transport. We have previously shown that PXR and CAR expression is controlled by the glucocorticoid receptor (GR) and proposed the existence of a signal transmission cascade GR-(PXR/CAR)-drug metabolizing and transporter systems. In the current study, we investigated the effect of ketoconazole and other azole-derived drugs, miconazole and fluconazole, on the transcriptional activity of the human GR (hGR) in HeLa and HepG2 cells, and in primary human hepatocytes. The data show that ketoconazole inhibits GR transcriptional activity and competes with dexamethasone for hGR binding. In primary human hepatocytes, ketoconazole inhibits the expression of 1) GR-responsive genes tyrosine aminotransferase and both PXR and CAR; 2) CAR and PXR target genes, including cytochromes P450 (P450) CYP2B6, CYP2C9, and CYP3A4; UDP-glucuronosyltransferase 1A1, glutathione S-transferases A1 and A2; and transporter proteins (phase III) solute carrier family 21 form A6 and multidrug resistance protein 2. In parallel experiments, ketoconazole affected neither the expression of GR, the expression of glyceraldehyde-3-phosphate dehydrogenase, nor the inducible expression of CYP1A1 and 1A2. Miconazole behaved like ketoconazole, whereas fluconazole had no effect. We conclude that, in addition to their well known inhibitory effect on P450 enzyme activities, ketoconazole and miconazole are antagonists of hGR. These results provide a novel molecular mechanism by which these compounds may exert adverse and toxic effects on drug metabolism and other functions in human.

CHARACTERIZATION OF THE TOPOISOMERASE II-INDUCED CLEAVAGE SITES IN THE c-myc PROTO-ONCOGENE

In an attempt to get an insight into the activity of mAMSA (a DNA topoisomerase II-mediated drug) on the human proto-oncogene c-myc, an in vitro system consisting of purified calf thymus DNA topoisomerase II and a c-myc DNA inserted in lambda phage was utilized. The occurrence of discrete bands, detected by hybridization of Southern blots with appropriate c-myc probes, indicated the presence of cleavage sites in the sole presence of DNA topoisomerase II. The band intensity increased in the presence of mAMSA, while no significant difference occurred in the cleavage pattern. The location of the cleavage sites along the c-myc locus revealed a striking correspondence with that of some DNase hypersensitive sites. These results indicate that DNA topoisomerase II is most certainly implicated in the mAMSA activity and that the drug stimulates the topoisomerase II cleaving activity at specific sites, which may be involved in the biological activity of the drug.

Identification of proteomic changes during human liver development by 2D-DIGE and mass spectrometry

BACKGROUND/AIMS The aim of this study was to identify human liver proteins that are associated with different stages of liver development. METHODS We collected liver samples from 14 fetuses between 14 and 41 weeks of development, one child and four adults. Proteins which exhibited consistent and significant variations during development by two-dimensional differential in gel electrophoresis (2D-DIGE) were subjected to peptide mass fingerprint analysis by MALDI-TOF mass spectrometry. Real-time PCR analysis confirmed, at the transcriptional level, the data obtained by the proteomic approach. RESULTS Among a total of 80 protein spots showing differential expression, we identified 42 different proteins or polypeptide chains, of which 26 were upregulated and 16 downregulated in developing in comparison to adult liver. These proteins could be classified in specific groups according to their function. By comparing their temporal expression profiles, we identified protein groups that were associated with different developmental stages of human fetal liver and suggest that the changes in protein expression observed during the 20- to 36-week time window play a pivotal role in liver development. CONCLUSIONS The identification of these proteins may represent good markers of human liver and stem cells differentiation.

Possible relationship between pyrido-pyrrolo-isoquinoline derivative (BD-40) cell uptake and cell viability

BD-40 is a pyrido-pyrrolo-isoquinoline derivative which possesses an heterocyclic nucleus very closely related to ellipticine and a diethylaminopropyl amino lateral chain (Rivalle et al., 1979). Cellular effects of the drug have been studied. Viability and morphology of the cells were irreversibly impaired by 24 hours drug treatments at concentrations exceeding 0.10 micro M. Spectrofluorimetric determinations of the drug repartition between cytoplasm and nucleus showed that 10 per cent of BD-40 was found in the nucleus for external drug concentrations lower than 0.10 micro M. On the other hand, for cytotoxic doses exceeding 0.10 micro M, 40 to 50 per cent of the total intracellular BD-40 were found in the nuclear fraction. Although possible exchange phenomena during the course of the cell fractionation cannot be excluded, these results suggest that a direct relationship exists between BD-40 nuclear content and cell lethality. Comparisons of intracellular drug contents in the absence and the presence of a metabolic inhibitor, sodium azide, indicated that drug content of azide-treated cells was approximately twice that of untreated cells. Besides, sodium azide blocked the release of the drug when previously loaded cells were placed in BD-40-free medium. These results are in agreement with the existence of an active outward transport (efflux), which is sodium azide-sensitive.

Expression and transcriptional activities of nuclear receptors involved in regulation of drug-metabolizing enzymes are not altered by colchicine: Focus on PXR, CAR, and GR in primary human hepatocytes

Recent findings show that colchicine (COL) in submicromolar concentrations downregulates the expression of major drug-metabolizing P450 enzymes in human hepatocytes. Concomitantly, the expression of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) was diminished by COL, whereas expression of glucocorticoid receptor (GR) remained unaltered. A tentative mechanism is perturbation of the GR-PXR/CAR-CYP2/3 signaling cascade, resulting in restricted transcriptional activity of GR receptor by colchicine. In this work we focused on 10-demethylcolchicine (colchiceine; EIN), a structural analogue and a putative metabolite of COL that lacks tubulin-binding activity. We investigated the effects of EIN on the expression of PXR, CAR, and GR receptors in primary cultures of human hepatocytes. In contrast with the effects of COL, EIN does not alter the expression of PXR, CAR, and/or GR receptors mRNAs. In addition, EIN had no effects on transcriptional activities of PXR, CAR, and GR receptors in reporter gene assays using transfected cell lines. Considering that COL and EIN are structurally very close and differ only in their tubulin-binding activity, the data presented imply that the deleterious effects of COL on the GR-PXR/CAR-CYP2/3 cascade are primarily due to perturbation of the microtubule network. Our data support the idea of replacing COL by EIN, which is less toxic and does not interact with xenoreceptors.

Response to Zhou et al.

Osteomalacia is a frequent complication resulting from long-term therapy with drugs such as phenytoin, carbamazepine, and phenobarbital. We have investigated the crosstalk between pregnane X receptor (PXR) and vitamin D receptor (VDR) and have reported that (a) PXR binds to and transactivates vitamin D–responsive elements in the 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter, and (b) PXR agonists increase CYP24 mRNA expression in human hepatocytes and mouse kidney (1). Since the CYP24 enzyme converts 1,25-dihydroxyvitamin D3 to inactive metabolites, these observations provide an objective explanation for the induction of osteomalacia by PXR agonists. In their report on this crosstalk in the June issue of the JCI, Zhou et al. (2) concluded instead that CYP24 is not induced but rather downregulated by PXR agonists. These authors observed neither binding to nor transactivation of the CYP24 promoter by PXR, nor did they find induction of CYP24 mRNA in human hepatocytes. As soon as we became aware of this work, we repeated key experiments using extracts from new hepatocyte cultures and new inducers (SR128123 and CITCO). Moreover, we evaluated the constitutive androstane receptor–VDR (CAR-VDR) crosstalk. Our new results fully confirm our previous findings. In human hepatocyte cultures, the CYP24 mRNA level was increased by rifampicin and 2 other PXR agonists, phenobarbital and SR128123. EMSA not only confirmed the binding of PXR to vitamin D–responsive elements–I and –II of CYP24 but also revealed that CAR binds these responsive elements. This last point is consistent with the finding that CITCO, a specific agonist of CAR, was a weak inducer of CYP24 in hepatocytes and that androstanol, an inverse agonist of CAR, inhibited CAR-mediated transactivation of the CYP24 promoter. We believe that these discrepancies between our work and that published by Zhou et al. (2) could be due to methodological differences in hepatocyte cultures (culture medium), transfection assays (cell lines), and EMSA. More work is currently being done to identify the origin of these discrepancies.

Regulation of Xenobiotic Detoxification by PXR, CAR, GR, VDR and SHP Receptors: Consequences in Physiology

Cytochromes P450 (CYP) from families CYP1/2/3 are involved in xenobiotic detoxification and are regulated by numerous agents including xenobiotics, plant and fungal toxins, bile salts, etc. Nuclear receptors PXR (pregnane X receptor, NR1I3) and CAR (constitutive androstane receptor, NR1I2) control the expression of distinct but overlapping arrays of genes including the CYP2/3 families. These receptors are involved in a tangle of regulatory networks including those pathways controlling vitamin D and cholesterol/bile salt homeostasis. They are expressed under the control of the glucocorticoid receptor and are inhibited by the short heterodimer partner (SHP). The crosstalk between both PXR and CAR and other nuclear receptors allows for the establishing and prediction of functional interferences between the detoxification and other signaling and metabolic pathways. These interferences provide new clues for the interpretation of xenobiotic adverse effects, such as bone demineralization, and for understanding how physiopathological factors, such as biliary salts or inflammation and infections, affect an organism’s ability to detoxify xenobiotics.

Effects of BD-40, an ellipticine analogue (aza-ellipticine) on cell cycle traverse and DNA synthesis in cultures of synchronized mouse fibroblasts

BD-40 is a pyrido-pyrrolo-isoquinoline analogue of ellipticine, which possesses oncostatic in vivo activity on experimental tumors, and dose-dependent cytostatic and cytotoxic activities on mammalian cells in culture. In order to appreciate the effects of the drug on the replication of DNA, cultures of murine fibroblasts were synchronized by thymidine double block, and BD-40 was added at the time of the block release. The drug did not interfere with the entry of cells in S phase, but a delay in S-phase transit was observed, regardless of the dose employed. In agreement with these data, DNA synthesis started at the same time in control cells and in BD-40 treated cells, but a significant reduction of 3H thymidine incorporation was found in drug-treated cells. This inhibition was not likely to result from a diminution of the specific activity of 3H-dTTP, since nuclei, isolated from cells previously incubated with the drug, also presented a strong diminution of synthetic activity in the presence of the four nucleoside triphosphate precursors (dNTPs). Analysis by alkaline sucrose gradient centrifugation of DNA synthesized in the presence of BD-40 showed that primary fragments, probably corresponding to the duplication of initial replicons, were normally formed but were not further elongated in cells treated with cytotoxic doses, while they were normally processed (although at a slower rate than in control) with a cytostatic drug concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of c-Ki-ras coexists with c-myc amplification in cells from a nude mouse tumor induced by the human breast carcinoma cell line SW 613-S.

In vitro transfection experiments have shown that cooperation between two different oncogenes can confer a fully malignant phenotype to primary rodent cells. We have previously reported that SW 613‐Tul cells, derived from a tumor induced in a nude mouse by the human breast carcinoma cell line SW 613‐S, showed a 30‐fold amplification of the c‐ myc gene. In the present work, we show that these cells also harbor an activated c‐Ki‐ras gene capable of inducing the formation of foci upon transfection of NIH 3T3 cells with SW 613‐Tul genomic DNA. Our results suggest that both the c‐myc and c‐Ki‐ras oncogenes, activated by two different mechanisms, may cooperate in the full expression of the tumorigenic phenotype of SW 613‐Tul cells.