During the first year of the project, the research conducted by the different partners has yielded the following achievements:
• The suitability of different hepatocyte cultures for predictomics analysis has been investigated. Collagen cultures have also been examined for their ability to maintain long term cultures, and at the same time to meet the requirements for further genomic analysis
• Studies on the role of gene expression and regulation of hepatocyte adult phenotype have been initiated, using trichostatin analogues and vectors encoding for key transcription factors
• The suitability of hepatic progenitor cells to derive hepatocytes suitable for the purpose of the project has been examined. Based on the results obtained, it is concluded that other sources of stem cells should be investigated
• Methodology to examine multiple hepatocyte metabolism indicators has been set up and adapted for rapid FACS analysis.
• An in vitro experimental procedure to reproduce the esteatosis induced by drugs has been developed and cytomic analysis has been developed and evaluated.
• Methodology for optimise the extraction and evaluation of mRNA from cultures with a limited number of hepatic cells has been developed to meet project requirements.
• Standard operating procedures for renal proximal tubular cell cultures have been produced and disseminated . A perfusion based culture model has been developed and optimizedl
• A characterization of rat primary proximal tubular cells and the cell line NRK-52E as compared to rat kidney in vivo has been conducted by using gene expression profiling .
• Cytomic data has been generated from HK-2 cells intoxicated with ochratoxin A, cyclosporine A, FK506, rapamycin and cadmium chloride .
• Close proximity indirect filter based renal co-culture systems with epithelial/endothelial and epithelial/fibroblast partner cells have been developed .
• Gene array experiments have been conducted comparing plastic grown HK-2 cells, filter grown HK-2 cells in monoculture and HK-2 cells cultured in co-culture with microvascular endothelial cells .
• Identification of altered gene expression following exposure to CsA, FK506 and rapamycin and ochratoxin A.
Second Year Achievements
1. A decision was adopted concerning the suitability of different hepatocyte cultures for predictomics analysis. Primary rat hepatocytes treated with HDAC inhibitors better retain the adult phenotype in culture, and may constitute a suitable models (P3).
2. The role of transcription factors (HNF4), and co-activators and co-repressors (SRC1, SRC2, PGC1á, PCAF) have been investigated in detail to understand the lack of expression of CYP´s in the human hepatoma HepG2, with the view of restoring the metabolic competence in this cell line (P1). Adenoviral expression vectors have been constructed which have allowed a detailed analysis of these factors. Among them, PGC1á, has shown the most relevant effects: sobreexpresión of this co-activator resulted in the upregulation of key hepatic genes, including CYPs (P1).
3. Hepatic progenitor cells have been successfully isolated from human liver and hepatocytes have been derived of. In an appropriate differentiation culture media, cells acquire many of the biochemical features displayed by adult hepatocytes. Nevertheless, they show a gradual process of senescence after several passages (P4).
4. An in vitro experimental procedure that reproduces the esteatosis induced by drugs has been developed and applied to genomic, proteomic and cytomic analysis. The genomic analysis has allowed identifying a number of genes (ca. 30), which are consistently upregulated as consequence of the lipid accumulation in hepatocytes (P1, P9). Several proteins could be identified as well, displaying a characteristic behaviour in steatosic hepatocytes. The proteins could be properly identified on the basis of tryptic digestion and MS analysis (P9). A cytomic analysis was also established that enables an accurate measurement of lipid content in hepatocytes treated with steatosic drugs (P1, P1-S, P3). This integrated strategy appears to be suitable to identify this class of hepatotoxicants.
5. Studies to assess the cholestasic potential of drugs were conducted thanks to the preparation and use of new bile acid derivatives (P1) which have been successfully adapted to cytometric analysis (P1-Sc). Initial studies on the effect of cholestasic drugs on gene expression in hepatocytes have been conducted, although no conclusive results have been obtained yet (P9).
6. Optimised, stable, and well characterised renal mono- and co-cultures for in vitro nephrotoxicity studies have been established. All SOPs have been optimized and are in place (P2, P5). Adoption of SOPs for culture techniques used for simple monocultures and renal co-culture systems and perfusion culture conditions has been achieved (P2, P5). Exploration of on-line cell function monitoring in renal epithelial culture systems under perfusion conditions has been completed (P2).
7. A wide number of cytomic assays (including matrix metalloproteinase secretion, tissue inhibitor of matrix metalloproteinase secretion (P6), IL-6 secretion, RANTES secretion (P5), ATP production, inner mitochondrial membrane potential (JC-1), ROS production, LDH release, lactate production, DNA synthesis and resazurin reduction) (P2) have been examined for their usefulness for in vitro toxicity testing. Three assays have been selected based on their ease of application, global toxicological relevance and sensitivity, which will be utilised to identify sublethal concentrations of the test compounds. These assays are LDH release (cytotoxicity), BrdU incorporation (DNA synthesis) and resazurin reduction (relative viable cell number).
8. Gene expression profile experiments for OTA have been conducted using HK-2 cells, human primary proximal tubular cells and rat primary proximal tubular cells. Experimental data has been analysed in detail and compared to in vivo data in the rat (P2, P7). Comparison of different renal culture models have been completed and a consensus reached as to the model of choice for further testing and toxicogenomic profiling (P2, P5, P6, P7). This model will be HK-2 cells cultured on plastic supports in monoculture. Plastic cultured confluent HK-2 monolayers will be treated in the standard serum free defined medium with sub-lethal concentrations of test compounds (defined as minimum LDH release and no decrease in resazurin conversion) for 12 and 48 hr.
9. Gene expression profile experiments for CsA (P2, P5, P6, P7), CdCl2, diquat dibromide (P2), FK506, and rapamycin (P6) have been conducted using the HK-2 cell model. A number of genes have been identified which are considered to be potentially related to cell injury process triggered by nephrotoxin exposure. This list will be expanded once the data for all nephrotoxins has been completed and statistically analysed.
10. Preliminary proteomic analysis using CsA as a test compound has indicated a number of differentially expressed proteins
Third Year Achievements (Final)
Renal group: A human proximal tubule model has been established for the purposes of conducting gene expression microarrays with the Affymetrix HGU-133 plus 2 platform. An interlaboratory comparison across four laboratories, using the model nephrotoxin CsA, has confirmed that the model is standardised, robust, reproducible and transferable. The predictively of the model with an additional 11 nephrotoxic compounds has been investigated and a preliminary prediction model developed. A number of limitations of the model have been identified including poor performance where phase I metabolism is necessary for toxic action and an intrinsic aminoglycoside resistance. A number of marker genes have been identified which are potential markers of early response to toxicity.
Liver group: Elucidation of the transcription factors needed for hepatocytes to express their adult phenotype, poorly expressed in dedifferentiated human hepatoma cells, has been achieved. Seven adenoviral vectors for expression of hepatic transcription factors, coactivators and nuclear receptors have been developed. HepG2, as prototype, have been transiently transfected with these vectors, and the expression of CYPs, and typical hepatic genes was partially recovered.. The reexpression of CYPs was further increased with the combination of recombinant adenovirus for transcription factors and HDAC inhibitors. Other major achievement has been the isolation, characterisation and differentiation to the hepatocyte lineage of adult intrahepatic progenitor cells to be used as new tools for metabolism and toxicity testings of xenobiotics and drugs. We have designed and prevalidated a new fast method to predict drug induced liver cholestasis by means of flow cytometry. Three types of novel fluorescent analogs have been synthesized as probes for this purpose. The method allows evaluation of the real-time kinetics of bile acid derivatives uptake in fresh suspensions of hepatocytes and the uptake impairment by drugs. It has also developed a new multiparametric method to predict drug induced liver steatosis by flow cytometry allowing to discriminate between early and late effects of steatosic compounds. Cultured hepatocytes treated with model cholestatic and steatotic hepatotoxins were analysed on genomics platforms on a genome-wide scale to identify gene products whose expression levels or post-translational features change as a consequence of exposure to the drugs. A steatosis fingerprint has been defined and will be exploited in the form of a DNA microarray. Finally, an integrated strategy of cytomic and genomic platforms has been defined which will be useful for screening steatotic and cholestatic drugs at pre-clinical stages
