Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat.

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

Iconix Biosciences, Inc.

Iconix Biosciences has developed leading products and services that apply novel and proprietary genomic technologies to profile candidate drug compounds in early discovery through to preclinical development, leading to a better understanding of candidate drugs in a faster, more cost-effective manner. The toxicology community is embracing this approach to increase the accuracy, sensitivity and speed of toxicity testing. Changing this paradigm will significantly impact the failure rate of late-stage preclinical compounds and provide a compelling return on investment. Through strategic growth and research, the company has identified the factors and is creating the environment that will lead to a 'tipping point' in the pharmaceutical and biotechnology industries, such that toxicogenomics becomes a standard practice in the drug discovery and development process.

Toxicogenomics in drug discovery and development: mechanistic analysis of compound/class-dependent effects using the DrugMatrix database.

A range of genomics technologies are increasingly becoming integrated with existing scientific disciplines to broaden and strengthen existing capabilities and open new avenues of research in drug discovery and development. Examples of these new research fields are proteomics, pharmacogenomics, metabolomics and toxicogenomics. Here we review the application of toxicogenomics to improve the evaluation of drug safety, mechanism of action and toxicity in the drug discovery and development process.

Specificity of short interfering RNA determined through gene expression signatures.

Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.

T cell activation induces a noncoding RNA transcript sensitive to inhibition by immunosuppressant drugs and encoded by the proto-oncogene, BIC.

In a search for novel early T cell activation transcripts, we identified expressed sequence tags (ESTs) more abundantly expressed in normal human CD4(+) T lymphocytes fully activated by a 5 h exposure to CD3 plus CD28 mAbs, compared to the same cells stimulated with either CD3 mAb or CD28 mAb alone. An EST was identified that hybridized with a 1.7 kb transcript expressed in activated T cells but was undetectable by Northern blot analysis in resting T cells or other normal tissues. The T cell transcript was maximally induced within 6 h and remained elevated for at least 47 h. Induction of the transcript was blocked by cyclosporin A, FK506, and dexamethasone but not by rapamycin. The transcript was polyadenylated but lacked an open reading. A BLAST search of the NCBI database revealed that the transcript shared identity with the recently reported human BIC proto-oncogene that encodes a noncoding mRNA (W. Tam, Gene 274 (2001) 157). Our data demonstrate that transcriptional activation of the BIC proto-oncogene is an early and sustained T cell activation event and suggest an important role for noncoding mRNA in T cell function.

The promise of toxicogenomics.

As human genetics and genomics have progressed, culminating in the completion of the rough draft of the human genome in February 2001, new tools and technologies have been developed to identify and quantify global gene expression changes occurring in the cell. These new technologies are allowing researchers to gain an increased understanding of the function and regulation of genes at the systems level, and are transforming virtually all areas of biological research. In the field of toxicology, a new subdiscipline termed toxicogenomics has emerged which promises to identify and characterize the molecular mechanisms that lead to toxicity. Gene expression profiling, through the use of microarray technology, is rapidly becoming a standard analysis in toxicology studies, and has the potential to play a pivotal role in all stages of drug safety evaluation. This review focuses on recent studies in toxicogenomics, and discusses the promises and future challenges in this field.

A Colorimetric DNA Hybridization Method for the Detection of Escherichia coli in Foods.

A colorimetric DNA hybridization assay has been developed for the rapid detection of Escherichia coli in foods. The method employs two oligonucleotide probes which are specific for the 16S ribosomal RNA of E. coli . Probes are added to lysates of test cultures and allowed to hybridize to target rRNA if present. The probe-target complex is captured via hybridization to a polystyrene dipstick. The immobilized target is detected using an antibody-horseradish peroxidase conjugate which binds to the immobilized probe-target complex. The probe-target-antibody complex generates a colorimetric signal when exposed to a substrate/chromogen mixture. A total of 233 E. coli isolates representing typical, toxigenic, invasive, hemorrhagic serotype 0157:H7, and other pathogenic strains all resulted in a positive assay signal. Dose-response experiments indicate the sensitivity of the assay is approximately 1 × 106 CFU/ml. Specificity of the assay was determined by testing 207 strains of non- E. coli species at 109 CFU/ml. All of the non- E. coli organisms tested were negative with the exception of Escherichia fergusonii and Shigella species. A total of 345 enriched samples including inoculated, uninoculated, and naturally-contaminated foods was tested for the presence of E. coli by the hybridization assay and a conventional cultural method. The false-negative rate for the hybridization assay was 1.2%. By comparison, the false-negative rate for the culture method in these studies was 23.4%. Based on these data, the DNA hybridization method is significantly more accurate than conventional methods for the detection of E. coli in foods.