Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity

Direct communication between hepatocytes, mediated by gap junctions, constitutes a major regulatory platform in the control of liver homeostasis, ranging from hepatocellular proliferation to hepatocyte cell death. Inherent to this pivotal task, gap junction functionality is frequently disrupted upon impairment of the homeostatic balance, as occurs during liver toxicity and carcinogenicity. In the present paper, the deleterious effects of a number of chemical and biological toxic compounds on hepatic gap junctions are discussed, including environmental pollutants, biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. Particular attention is paid to the molecular mechanisms that underlie the abrogation of gap junction functionality. Since hepatic gap junctions are specifically targeted by tumor promoters and epigenetic carcinogens, both in vivo and in vitro, inhibition of gap junction functionality is considered as a suitable indicator for the detection of nongenotoxic hepatocarcinogenicity

Characterization of spontaneous cell death in monolayer cultures of primary hepatocytes

Monolayer cultures of primary hepatocytes, isolated from freshly removed livers, represent widely used in vitro tools in the area of liver physiology and pathology, pharmacology and toxicology. However, a major shortcoming of these systems is that they cope with dedifferentiation, which is accompanied by spontaneous cell death. The goal of the present study was to elucidate the mechanisms that drive the process of self-generated cell demise in primary hepatocyte cultures. For this purpose, isolated rat hepatocytes were cultivated under conventional conditions, and the occurrence of apoptosis and necrosis was monitored during 4 days by performing a set of acknowledged cell death assays. These included examination of cell morphology by light microscopy, quantification of apoptotic and necrotic cell populations by Hoechst 33342 and propidium iodide in situ staining, assessment of apoptotic and necrotic activities by measuring caspase 3-like activity and extracellular leakage of lactate dehydrogenase, and studying the expression of apoptosis regulators through immunoblot analysis. In essence, two cell death peaks were observed, namely shortly after cell seeding and in the final stages of the cultivation period, both involving apoptotic and necrotic actions. The outcome of this study not only sheds new light onto the molecular processes that underlie spontaneous cell death in primary hepatocyte cultures, but also opens perspectives for the establishment of strategies to increase cell survival in these popular in vitro systems

Comparison of genotoxicant-modified transcriptomic responses in conventional and epigenetically stabilized primary rat hepatocytes with in vivo rat liver data

The concept of mechanistic toxicogenomics implies that compound-induced changes in gene expression profiles provide valuable information about their mode of action. A growing number of research groups have presented evidence that whole-genome gene expression profiling techniques might be used as tools for in vivo and in vitro generation of gene signatures and elucidation of molecular mechanisms after exposure to toxic compounds. An important issue to be investigated is the in vivo relevance of in vitro-obtained data. In the current study, we compare the gene expression profiles generated in vitro, after exposing conventional and epigenetically stabilized primary rat hepatocytes to well-known genotoxic hepatocarcinogens (aflatoxin B1, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 2-nitrofluorene) with those derived in vivo after oral exposure of rats to these compounds. Similar statistical tools were applied on both sets of data. The major molecular pathways affected in the in vivo setting were DNA damage, detoxification and cell survival response, as previously described. In the conventional hepatocyte cultures, two of the three genotoxicants showed quite similar responses as in vivo with respect to these pathways. The third compound (2-nitrofluorene) revealed in vitro response which was not observed in vivo. In the epigenetically stabilized hepatocytes, in contrast to what was expected, the responses were less relevant for the in vivo situation. This study highlights the importance of in vitro/in vivo comparison of data that are generated using in vitro models and shows that conventional primary rat hepatocyte cultures represent an appropriate in vitro model to retrieve mechanistic information on the exposure to genotoxicants

TESTING CHEMICAL CARCINOGENICITY BY USING A TRANSCRIPTOMICS HEPARG-BASED MODEL?

The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances

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.JRC.I.2-In-vitro Toxicolog