Prediction of carcinogenic potential by a toxicogenomic approach using rat hepatoma cells

Abstract

The long-term rodent bioassay is the standard method to predict the carcinogenic hazard of chemicals for humans. However, this assay is costly, and the results take at least two years to produce. In the present study, we conducted gene expression profiling of cultured cells exposed to carcinogenic chemicals with the aim of providing a basis for rapid and reliable prediction of carcinogenicity using microarray technology. We selected 39 chemicals, including 17 rat hepatocarcinogens and eight compounds demonstrating carcinogenicity in organs other than the liver. The remaining 14 were non-carcinogens. When rat hepatoma cells (MH1C1) were treated with the chemicals for 3 days at a non-toxic dose, analysis of gene expression changes with our in-house microarray allowed a set of genes to be identified differentiating hepatocarcinogens from non-carcinogens, and all carcinogens from non-carcinogens, by statistical methods. N umerous studies have investigated relationships among chemical exposure, toxicity, and disease states. The long-term rodent bioassay, the standard method to predict carcinogenic hazard of chemicals to humans, is costly in terms of time and material resources, therefore interest has concentrated on shortor medium-term bioassays.(1-5) One approach is to study change in gene expression in biological models in response to chemical exposure. Signatures of genetic alteration cannot be defined using classical methods for investigation of individual genes but with the advent of toxicogenomics, and the use of cDNA microarrays, the potential roles of multiple genes in concert can now be readily investigated in high throughput assays that are more sensitive and predictive than, for example, cell death assays. Recently, use of this approach for predictive toxicology using gene expression profiles has been reported by several groups. (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) In the present study, we aimed to provide a basis for a rapid and easy method to predict carcinogenicity of chemicals based on microarray technology with cultured MH1C1 rat hepatoma cells, selected as a model system to minimize complicating factors such as cell type heterogeneity and interindividual differences of animals. For this purpose, 39 chemicals that have been well characterized for carcinogenicity were first tested for cytotoxicity in cells using reductase activity at 3 days and non-toxic doses were determined for measurement of gene expression. Genes for prediction for carcinogen and non-carcinogen groups were selected with statistical methods and analyzed by clustering analysis and an SVM. Additionally, cross-validation using individual external data was carried out for confirmation. For some selected genes, semiquantitative RT-PCR analyses were used to confirm the microarray results. Materials and Methods

A Gene Expression Biomarker Provides Early Prediction and Mechanistic Assessment of Hepatic Tumor Induction by Nongenotoxic Chemicals

“…(B) Test chemicals that were correctly predicted positive by the signature (beta-napthoflavone, DES, chloroform, DEHP) clustered most closely to the representative reference compounds with similar MOA. Kramer et al, 2004;Tsujimura et al, 2006), more extensive independent validation is needed to increase the confidence in their general utility. Likewise, other attempts using larger data sets (Nie et al, 2006) show promise and await further independent validation.…”

Genetic toxicology in the 21st century: Reflections and future directions

“…Toxicogenomics (mRNA transcript profiling) to identify genes that respond to groups or categories of chemicals and statistical classification methods together with machine learning [Auerbach et al, 2010] clearly have progressed in their ability to predict carcinogenicity. Recent studies reviewed in Waters et al [in press] by Van Delft et al [2004, 2005], Ellinger-Ziegelbauer et al [2005, 2008, 2009a], Nakayama et al [2006], Nie et al [2006], Tsujimura et al [2006], Fielden et al [2007, 2008], Thomas et al [2007] suggest that it is possible to screen for carcinogenicity and to discern the potential mode of action (MOA) of a chemical based on analysis of gene expression using toxicogenomics methods, with the potential for screening of putative MOA by quantitative RT-PCR [Ellinger-Ziegelbauer et al, 2009b]. The strategy used involves selecting a training set of known carcinogenic (genotoxic or nongenotoxic) and non-carcinogenic compounds to which to expose mice or rats in vivo or cultured cells in vitro for periods of from 1 to 90 days.…”

A novel toxicogenomics-based approach to categorize (non-)genotoxic carcinogens

“…Alternative tests are therefore required. The application of transcriptomics approaches to detect carcinogenic features of chemicals has been extensively investigated in vivo as well as in vitro (EllingerZiegelbauer et al 2009a;Fielden et al 2011;Thomas et al 2009;Tsujimura et al 2006;Yamada et al 2012). Overall, these studies have yielded biologically relevant gene signatures, which can be employed to distinguish different chemical classes, e.g, (non-) genotoxic carcinogens versus noncarcinogens (Ellinger-Ziegelbauer et al 2008;Melis et al 2014; Thomas et al 2009;Watanabe et al 2012;Yamada et al 2012).…”

“…In vitro systems in which toxicogenomics has been explored for assessment of the carcinogenic features of substances are, just like in vivo, mainly of hepatic origin: (primary) hepatocytes or hepatic (cancer-) cell lines (e.g., HepG2, MH1C1) (Ellinger-Ziegelbauer et al 2009b;Magkoufopoulou et al 2012;Mathijs et al 2009;Tsujimura et al 2006). The results of these studies indicated that toxicogenomicsbased in vitro assays are promising tools to detect mainly genotoxic carcinogens.…”