Tatyana Y. Doktorova

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.

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.

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.

Opportunities for an alternative integrating testing strategy for carcinogen hazard assessment

The 2-year rodent carcinogenicity bioassay evolved more than 40 years ago, and although it is complex, long lasting, expensive, and animal consuming, it is still the only generally accepted test for assessing the carcinogenicity of chemicals. Over time, different alternative approaches have been developed with the final goal to replace the bioassay. Unfortunately, at present, none of these strategies alone provides sufficient assurance of accurate prediction. In this review paper, we discuss the major advantages and pitfalls of the existing alternative methodologies to the carcinogenicity bioassay. Finally, based on the available scientific data in the public domain, we propose what we would like to call a “feasible integrated testing strategy” which incorporates some promising alternatives, providing at the same time information on the mechanism of action and the toxic nature of the compounds tested. It is, however, clear that the adoption of whatever “new” testing scheme should be considered with caution and its effectiveness should be experimentally demonstrated in advance by addressing a reasonable number of chemical carcinogens and non-carcinogens from a variety of structural and functional classes.

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.

Effect of the Histone Deacetylase Inhibitor Trichostatin A on the Metabolome of Cultured Primary Hepatocytes

Trichostatin A (TSA) is a histone deacetylase inhibitor that has antiproliferative and differentiation-inducing effects on cancer cells, and in cultures of primary hepatocytes has been shown to maintain xenobiotic metabolic capacity. Using an NMR-based metabolic profiling approach, we evaluated if the endogenous metabolome was stabilized and the normal metabolic phenotype retained in this model. Aqueous soluble metabolites were extracted from isolated rat hepatocytes after 44 and 92 h exposure to TSA (25 muM) together with time-matched controls and measured by (1)H NMR spectroscopy. Multivariate analysis showed a clear difference in the global metabolic profile over time in control samples, while the TSA treated group was more closely clustered at both time points, suggesting that treatment reduced the time related effect on metabolism that was observed in the control. TSA treatment was associated with decreases in glycerophosphocholine, 3-hydroxybutyric acid, glycine and adenosine, an increase in glycogen, and a reduction in the decrease of inosine, hypoxanthine, and glutathione over time. Collectively, our data suggest that TSA treatment reduces the loss of a normal metabolic phenotype in cultured primary hepatocytes, improving the model as a tool to study endogenous liver metabolism, xenobiotic metabolism, and potentially affecting the accuracy of all biological assays in this system.

Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors

SummaryThe vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

Way forward in case of a false positive in vitro genotoxicity result for a cosmetic substance

The currently used regulatory in vitro mutagenicity/genotoxicity test battery has a high sensitivity for detecting genotoxicants, but it suffers from a large number of irrelevant positive results (i.e. low specificity) thereby imposing the need for additional follow-up by in vitro and/or in vivo genotoxicity tests. This could have a major impact on the cosmetic industry in Europe, seen the imposed animal testing and marketing bans on cosmetics and their ingredients. Afflicted, but safe substances could therefore be lost. Using the example of triclosan, a cosmetic preservative, we describe here the potential applicability of a human toxicogenomics-based in vitro assay as a potential mechanistically based follow-up test for positive in vitro genotoxicity results. Triclosan shows a positive in vitro chromosomal aberration test, but is negative during in vivo follow-up tests. Toxicogenomics analysis unequivocally shows that triclosan is identified as a compound acting through non-DNA reactive mechanisms. This proof-of-principle study illustrates the potential of genome-wide transcriptomics data in combination with in vitro experimentation as a possible weight-of-evidence follow-up approach for de-risking a positive outcome in a standard mutagenicity/genotoxicity battery. As such a substantial number of cosmetic compounds wrongly identified as genotoxicants could be saved for the future.

In silico tools and transcriptomics analyses in the mutagenicity assessment of cosmetic ingredients: a proof-of-principle on how to add weight to the evidence

Prior to the downstream development of chemical substances, including pharmaceuticals and cosmetics, their influence on the genetic apparatus has to be tested. Several in vitro and in vivo assays have been developed to test for genotoxicity. In a first tier, a battery of two to three in vitro tests is recommended to cover mutagenicity, clastogenicity and aneugenicity as main endpoints. This regulatory in vitro test battery is known to have a high sensitivity, which is at the expense of the specificity. The high number of false positive in vitro results leads to excessive in vivo follow-up studies. In the case of cosmetics it may even induce the ban of the particular compound since in Europe the use of experimental animals is no longer allowed for cosmetics. In this article, an alternative approach to derisk a misleading positive Ames test is explored. Hereto we first tested the performance of five existing computational tools to predict the potential mutagenicity of a data set of 132 cosmetic compounds with a known genotoxicity profile. Furthermore, we present, as a proof-of-principle, a strategy in which a combination of computational tools and mechanistic information derived from in vitro transcriptomics analyses is used to derisk a misleading positive Ames test result. Our data shows that this strategy may represent a valuable tool in a weight-of-evidence approach to further evaluate a positive outcome in an Ames test.

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.