Microarrays are a new technology used to study global gene expression and to decipher biological pathways. In the current study, microarrays were used to examine gene expression patterns associated with cisplatin-mediated nephrotoxicity. Sprague-Dawley rats received either single or seven daily ip doses of cisplatin (0.5 or 1 mg/kg/day) or the inactive isomer transplatin (1 or 3 mg/kg/day). Histopathological evaluation revealed renal proximal tubular necrosis in animals that received cisplatin for 7 days, but no hepatotoxic findings. Microarray analyses were performed using rat specific arrays containing 250 toxicity-related genes. Prominent gene expression changes were observed only in the kidneys of rats that received cisplatin for 7 days. Mechanistically, the gene expression pattern elicited by cisplatin (e.g., Bax upward arrow and SMP-30 downward arrow) suggested the occurrence of apoptosis and the perturbation of intracellular calcium homeostasis. The induction of multidrug resistance genes (MDR1 upward arrow, P-gp upward arrow) and tissue remodeling proteins (clusterin upward arrow, IGFBP-1 upward arrow, and TIMP-1 upward arrow) indicated the development of cisplatin resistance and tissue regeneration. Select gene expression changes were further confirmed by TaqMan analyses. Gene expression changes were not observed in the liver following cisplatin administration. In contrast to these in vivo findings, studies using NRK-52E kidney epithelial cells and clone-9 liver cells suggested that liver cells were more sensitive to cisplatin treatment. The discrepancies between the in vivo and in vitro results suggest that caution should be taken when extrapolating data from in vivo to in vitro systems. Nonetheless, the current study elucidates the biochemical pathways involved in cisplatin toxicity and demonstrates the utility of microarrays in toxicological studies.

DNA microarrays, used to measure the gene expression of thousands of genes simultaneously, hold promise for future application in efficient screening of therapeutic drugs. This will be aided by the development and population of a database with gene expression profiles corresponding to biological responses to exposures to known compounds whose toxicological and pathological endpoints are well characterized. Such databases could then be interrogated, using profiles corresponding to biological responses to drugs after developmental or environmental exposures. A positive correlation with an archived profile could lead to some knowledge regarding the potential effects of the tested compound or exposure. We have previously shown that cDNA microarrays can be used to generate chemical-specific gene expression profiles that can be distinguished across and within compound classes, using clustering, simple correlation, or principal component analyses. In this report, we test the hypothesis that knowledge can be gained regarding the nature of blinded samples, using an initial training set comprised of gene expression profiles derived from rat liver exposed to clofibrate, Wyeth 14,643, gemfibrozil, or phenobarbital for 24 h or 2 weeks of exposure. Highly discriminant genes were derived from our database training set using approaches including linear discriminant analysis (LDA) and genetic algorithm/K-nearest neighbors (GA/KNN). Using these genes in the analysis of coded liver RNA samples derived from 24-h, 3-day, or 2-week exposures to phenytoin, diethylhexylpthalate, or hexobarbital led to successful prediction of whether these samples were derived from livers of rats exposed to enzyme inducers or to peroxisome proliferators. This validates our initial hypothesis and lends credibility to the concept that the further development of a gene expression database for chemical effects will greatly enhance the hazard identification processes.

Methapyrilene (MP) exposure of animals can result in an array of adverse pathological responses including hepatotoxicity. This study investigates gene expression and histopathological alterations in response to MP treatment in order to 1) utilize computational approaches to classify samples derived from livers of MP treated rats based on severity of toxicity incurred in the corresponding tissue, 2) to phenotypically anchor gene expression patterns, and 3) to gain insight into mechanism(s) of methapyrilene hepatotoxicity. Large-scale differential gene expression levels associated with the exposure of male Sprague-Dawley rats to the rodent hepatic carcinogen MP for 1, 3, or 7 days after daily dosage with 10 or 100 mg/kg/day were monitored. Hierarchical clustering and principal component analysis were successful in classifying samples in agreement with microscopic observations and revealed low-dose effects that were not observed histopathologically. Data from cDNA microarray analysis corroborated observed histopathological alterations such as hepatocellular necrosis, bile duct hyperplasia, microvesicular vacuolization, and portal inflammation observed in the livers of MP exposed rats and provided insight into the role of specific genes in the studied toxicological processes.Keywords. Toxicogenomics; gene expression; methapyrilene; toxicity classification; rat liver; histopathology; phenotypic anchorage; hepatotoxicity.INTRODUCTION Methapyrilene (MP) is an antihistaminic compound once used as a popular over-the-counter sleep-aid component and also used in cold and allergy medications. It was found to induce hepatocellular carcinomas and cholangiocarcinomas in rats (20, 31, 33) and was subsequently withdrawn from the market. However, its carcinogenicity appears to be species-specific because no evidence has been found of MPassociated carcinogenesis in mice (3), guinea pigs, hamsters (32), or humans (36).MP was negative in the DNA adduct formation assay (8, 9, 34) and has not been found to be mutagenic with the Ames assay or other mutation assays (7, 38). Furthermore, MP did not induce unscheduled DNA synthesis (4) and did not cause sister-chromatid exchange (24). These data are consistent with the hypothesis that MP is carcinogenic in rats via nonmutagenic mechanisms (36,54). MP is extensively metabolized by the liver (29, 53), and phase I metabolism plays a major role in its toxicity because cytochrome P450 inhibitors afford protection from the toxicity of MP (43). The oxidative potential of methapyrilene and/or metabolites and increased cellular proliferation have been proposed to play a central role in the observed toxicity (9,45,49). Bile duct cannulation of MP treated rats affords protection from MP hepatic toxicity suggesting that enterohepatic recirculation of glucuronidated metabolites plays a role in MP toxicity (45). In humans, methapyrilene has a very short half-life, a relatively high apparent volume of distribution, and total

Heterozygous p53 +/-transgenic mice are being studied for utility as a short-term alternative model to the 2-yr rodent carcinogenicity bioassay. During a 26-wk study to assess the potential carcinogenicity of oxymetholone using p-cresidine as a positive control, glass/ polypropylene microchips (

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