Roque Bort

Down-regulation of human CYP3A4 by the inflammatory signal interleukin-6: molecular mechanism and transcription factors involved

The hepatic drug‐metabolizing cytochrome P‐450 (CYP) enzymes are down‐regulated during inflammation. In vitro studies with hepatocytes have shown that the cytokines released during inflammatory responses are largely responsible for this CYP repression. However, the signaling pathways and the cytokine‐activated factors involved remain to be properly identified. Our research has focused on the negative regulation of CYP3A4 (the major drug‐metabolizing human CYP) by interleukin 6 (IL‐6) (the principal regulator of the hepatic acute‐phase response). CYP3A4 down‐regulation by IL‐6 requires activation of the glycoprotein receptor gp130; however, it does not proceed through the JAK/STAT pathway, as demonstrated by the overexpression of a dominant‐negative STAT3 factor by means of an adenoviral vector. The involvement of IL‐6‐activated kinases such as extracellular signal‐regulated kinase ERK1/2 or p38 is also unlikely, as evidenced by the use of specific chemical inhibitors. It is noteworthy that IL‐6 caused a moderated induction in the mRNA of the transcription factor C/EBPβ (CCAAT‐ enhancer binding protein β) and a marked increase in the translation of C/EBPβ‐LIP, a 20‐kDa C/EBPβ isoform lacking a transactivation domain. Adenovirus‐mediated expression of C/EBPβ‐ LIP caused a dose‐dependent repression of CYP3A4 mRNA, whereas overexpression C/EBPα and C/EBPβ‐LAP (35 kDa) caused a significant induction. Our results support the idea that IL‐6 down‐regulates CYP3A4 through translational induction of C/EBPβ‐LIP, which competes with and antagonizes constitutive C/EBP transactivators. From a clinical point of view, these findings could be relevant in the development of therapeutic cytokines with a less repressive effect on hepatic drug‐metabolizing enzymes.

Long‐term expression of differentiated functions in hepatocytes cultured in three‐dimensional collagen matrix

Hepatocytes entrapped in collagen gel and cultured in serum‐free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c‐fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin‐stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 ± 0.37 and 9 ± 2.7 nmol glucose/h/μg DNA). Collagen‐cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 ± 4 nmol urea/h/μg DNA). The rate of albumin synthesis in collagen‐entrapped cells was maintained above the day‐1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 ± 152 pmol/μg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen‐cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and gluthatione‐transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione‐transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP‐α and ‐β, and HNF‐1 and ‐4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate‐synthetase I). J Cell Physiol 177:553–562, 1998.

Re-expression of C/EBPα induces CYP2B6, CYP2C9 and CYP2D6 genes in HepG2 cells

Cytochrome P450 (CYP) activity is very low or even absent in human hepatomas, a phenomenon that is accompanied by low levels of some liver transcription factors, notably C/EBPα. To investigate a possible link between this transcription factor and hepatic CYP expression, we have stably transfected HepG2 cells with a C/EBPα vector containing a Zn‐inducible metallothionein promoter. Expression of functional C/EBPα up to liver levels concomitantly increased the mRNAs of several members of the CYP2 family (2B6, 2C9 and 2D6), suggesting that this transcription factor may play a relevant role in controlling the hepatic expression of CYP enzymes.

The carcinoGENOMICS project: Critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays. First, a number of solid criteria for the selection of the model compounds are defined. Secondly, the strategy followed, including resources consulted, is described and the selected compounds are briefly illustrated. Finally, limitations and problems encountered during the selection procedure are discussed. Since selecting an appropriate set of chemicals is a frequent impediment in the early stages of similar research projects, the information provided in this paper might be extremely valuable.

Early ERK1/2 activation promotes DRP1-dependent mitochondrial fission necessary for cell reprogramming

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency.

Transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models

As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.

In vitro Investigation of the Molecular Mechanisms of Hepatotoxicity

Iatrogenic Hepatitis: Intrinsic and Idiosyncratic Toxicity. Substances capable of producing liver damage and, more specifically, hepatocyte damage are known as hepatotoxins. They are classified (Zimmerman and Ishak, 1995, Castell et al., 1992) according to whether they exert their effects in all individuals, in a dose-dependent and hence predictable manner (intrinsic hepatotoxins), or in certain individuals, occasionally after several contacts, in a non-dose dependent and therefore unpredictable way (idiosyncratic hepatotoxins). These substances can act directly on cells (active hepatotoxins), or become toxic after biotransformation (latent hepatotoxins). Idiosyncratic hepatotoxicity is the consequence, either of an unusual metabolism of the drug by susceptible individuals which produce too large amounts of toxic metabolites (metabolic idiosincrasy), or is due to an immune-mediated attack to sensitised hepatocytes (drug hypersensitivity).

Increased toxicity of cocaine on human hepatocytes induced by ethanol : Role of GSH

Increased toxicity of cocaine to human hepatocytes is observed when cells are simultaneously incubated with ethanol. Ethanol might exacerbate cocaine hepatocyte toxicity by three different pathways: a) by increasing the oxidative metabolism of cocaine and hence the oxidative damage; b) by the formation of a more toxic metabolite, namely cocaethylene; or c) by decreasing the defence mechanisms of the cell (i.e. GSH). In the present study, experiments were conducted to investigate the feasibility of these hypotheses. In hepatocytes preincubated for 48 hr with ethanol, neither significant changes in cocaine metabolism nor cytotoxicity were found despite differences in hepatocyte p-nitrophenol hydroxylase (largely CYP2E1 activity). Cocaethylene, the transesterification product of cocaine and ethanol, was found to be more toxic than cocaine for human hepatocytes (3x). However, the small amount formed when human hepatocytes were incubated with cocaine and ethanol would hardly explain the increased toxicity observed. On the other hand, the simultaneous presence of cocaine and ethanol caused a sustained decline in the intracellular GSH content that was larger than that observed in cocaine- or ethanol-treated cultures. Parallel to this phenomenon, a significant increase in lipid peroxidation was observed, as compared to cells treated with equimolar amounts of cocaine, ethanol, or cocaethylene. Finally, depletion of hepatocyte GSH with diethylmaleate down to levels similar to those found in ethanol-treated cells made hepatocytes more susceptible to cocaine. Taken together, the results of this research suggest that by decreasing GSH levels, ethanol makes human hepatocytes more sensitive to cocaine-induced oxidative damage.

Molecular mechanism of diclofenac hepatotoxicity: Association of cell injury with oxidative metabolism and decrease in ATP levels.

A certain number of case reports of adverse hepatic reactions to diclofenac are known, suggesting that diclofenac-associated hepatitis may be more common than previously recognized. In order to discriminate among possible molecular mechanisms of toxicity, the following were investigated: (a) cytotoxicity of diclofenac on metabolizing (rat hepatocytes) and non-metabolizing hepatic cells (HepG2, FaO); (b) changes in calcium homoeostasis, glutathione (GSH), lipid peroxidation and ATP levels, and (c) diclofenac metabolism in relation to cytotoxicity. The results indicate that toxicity is associated with the oxidative metabolism of the drug, and correlated with the formation of a minor oxidation metabolite. Inhibitors of diclofenac metabolism concomitantly reduced the toxicity of the drug. Hepatocyte injury was preceded by a decrease in ATP levels. No oxidative stress (no changes in GSH, no lipid peroxidation) could be demonstrated at this early stage. Cytotoxicity was prevented when cells were incubated with fructose, suggesting that the inability of mitochondria to produce ATP is the probable cause of diclofenac hepatotoxicity.

Interaction between Hhex and SOX13 Modulates Wnt/TCF Activity

Fine-tuning of the Wnt/TCF pathway is crucial for multiple embryological processes, including liver development. Here we describe how the interaction between Hhex (hematopoietically expressed homeobox) and SOX13 (SRY-related high mobility group box transcription factor 13), modulates Wnt/TCF pathway activity. Hhex is a homeodomain factor expressed in multiple endoderm-derived tissues, like the liver, where it is essential for proper development. The pleiotropic expression of Hhex during embryonic development and its dual role as a transcriptional repressor and activator suggest the presence of different tissue-specific partners capable of modulating its activity and function. While searching for developmentally regulated Hhex partners, we set up a yeast two-hybrid screening using an E9.5–10.5 mouse embryo library and the N-terminal domain of Hhex as bait. Among the putative protein interactors, we selected SOX13 for further characterization. We found that SOX13 interacts directly with full-length Hhex, and we delineated the interaction domains within the two proteins. SOX13 is known to repress Wnt/TCF signaling by interacting with TCF1. We show that Hhex is able to block the SOX13-dependent repression of Wnt/TCF activity by displacing SOX13 from the SOX13·TCF1 complex. Moreover, Hhex de-repressed the Wnt/TCF pathway in the ventral foregut endoderm of cultured mouse embryos electroporated with a SOX13-expressing plasmid. We conclude that the interaction between Hhex and SOX13 may contribute to control Wnt/TCF signaling in the early embryo.