No abstract

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative among the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the endocrine organs (pituitary gland, pineal gland, thyroid gland, parathyroid glands, adrenal glands and pancreatic islets) of laboratory rats and mice, with color photomicrographs illustrating examples of the lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for endocrine lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Drug-induced hepatotoxicity is a major issue for drug development, and toxicogenomics has the potential to predict toxicity during early toxicity screening. A bead-based Illumina oligonucleotide microarray containing 550 liver specific genes has been developed. We have established a predictive screening system for acute hepatotoxicity by analyzing differential gene expression profiles of well-known hepatotoxic and nonhepatotoxic compounds. Low and high doses of tetracycline, carbon tetrachloride (CCL4), 1-naphthylisothiocyanate (ANIT), erythromycin estolate, acetaminophen (AAP), or chloroform as hepatotoxicants, clofibrate, theophylline, naloxone, estradiol, quinidine, or dexamethasone as nonhepatotoxic compounds, were administered as a single dose to male Sprague-Dawley rats. After 6, 24, and 72 h, livers were taken for histopathological evaluation and for analysis of gene expression alterations. All hepatotoxic compounds tested generated individual gene expression profiles. Based on leave-one-out cross-validation analysis, gene expression profiling allowed the accurate discrimination of all model compounds, 24 h after high dose treatment. Even during the regeneration phase, 72 h after treatment, CCL4, ANIT, and AAP were predicted to be hepatotoxic, and only these three compounds showed histopathological changes at this time. Furthermore, we identified 64 potential marker genes responsible for class prediction, which reflected typical hepatotoxicity responses. These genes and pathways, commonly deregulated by hepatotoxicants, may be indicative of the early characterization of hepatotoxicity and possibly predictive of later hepatotoxicity onset. Two unknown test compounds were used for prevalidating the screening test system, with both being correctly predicted. We conclude that focused gene microarrays are sufficient to classify compounds with respect to toxicity prediction.

Our current approach focused on the identification of potential early protein biomarker signatures which are indicative of the carcinogenic processes in rats exposed to 20 mg/kg of the liver carcinogen N-nitrosomorpholine (NNM). Treated liver was investigated at different timepoints. Therefore, proteins were separated by two-dimensional gel electrophoresis as a first step prior to identification of differentially expressed proteins by mass spectrometry. Proteomic analysis of liver samples after one day of exposure revealed significant upregulation of proteins involved in response to cellular stress induced by NNM (superoxide dismutase, heat shock protein 60, peroxiredoxin). Eighteen weeks after withdrawal of NNM, we were able to identify cancer-related proteins in rat liver bearing malignant, transformed cells (caspase-8 precursor, vimentin, Rho GDP dissociation inhibitor). Some of these proteins were already deregulated after three weeks of exposure indicating their potential usefulness as early predictive biomarkers for liver carcinogenicity (annexin A5, fructose-1,6-bisphosphatase). As regulatory toxicology approaches usually include the investigation of carcinogenicity in two-years studies in rodents, especially the detection of early protein biomarker signatures which precede the appearance of neoplasia, demonstrates the high potential of proteomics approaches to substantially reduce the time and costs of carcinogenicity testing.

Today, toxicoproteomics still relies mainly on 2-DE followed by MS for detection and identification of proteins, which might characterize a certain state of disease, indicate toxicity or even predict carcinogenicity. We utilized the classical 2-DE/MS approach for the evaluation of early protein biomarkers which are predictive for chemically induced hepatocarcinogenesis in rats. We were able to identify statistically significantly deregulated proteins in N-nitrosomorpholine exposed rat liver tissue. Based on literature data, biological relevance in the early molecular process of hepatocarcinogenicity could be suggested for most of these potential biomarkers. However, in order to ensure reliable results and to create the prerequisites necessary for integration in routine toxicology studies in the future, these protein expression patterns need to be prevalidated using independent technology platforms. In the current study, we evaluated the usefulness of iTRAQ reagent technology (Applied Biosystems, Framingham, USA), a recently introduced MS-based protein quantitation method, for verification of the 2-DE/MS biomarkers. In summary, the regulation of 26 2-DE/MS derived protein biomarkers could be verified. Proteins like HSP 90-beta, annexin A5, ketohexokinase, N-hydroxyarylamine sulfotransferase, ornithine aminotransferase, and adenosine kinase showed highly comparable fold changes using both proteomic quantitation strategies. In addition, iTRAQ analysis delivered further potential biomarkers with biological relevance to the processes of hepatocarcinogenicity: e.g. placental form of glutathione S-transferase (GST-P), carbonic anhydrase, and aflatoxin B1 aldehyde reductase. Our results show both the usefulness of iTRAQ reagent technology for biomarker prevalidation as well as for identification of further potential marker proteins, which are indicative for liver hepatocarcinogenicity.