Inter-laboratory study of human in vitro toxicogenomics-based tests as alternative methods for evaluating chemical carcinogenicity: a bioinformatics perspective.

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.

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.