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2006 ACVP Annual Meeting

2006 Society of Toxicologic Pathology Student Speaker Award

CHRONIC SUBLETHAL MICROCYSTIN EXPOSURE RESULTS IN PROLIFERATION AND CHANGES IN MITOTIC GENE EXPRESSION IN HOMOZYGOUS p53 KNOCKOUT MICE.S. Clark1, S. Hooser2, T. Ryan3, and M. Davis3.1Biomedical Sciences and Pathobiology, Virginia Tech University, 2Department of Veterinary Pathobiology, Purdue University, 3Investigative Toxicology, Eli Lilly and Company, Greenfield, IN.

The homozygous p53 knockout mouse model was used to assess the mechanisms of hepatotoxicity and tumor promotion by the protein phosphatase inhibitor, microcystin-LR (MCLR). Microcystin-LR is a cyclic heptapeptide hepatotoxin produced primarily by the cyanobacteria (blue-green algae) Microcystis aeruginosa. In addition to its acute hepatotoxicity, MCLR is also a tumor promoter and chronic exposure may be associated with the increased risk of liver cancer in some areas of the world. Over 50% of all human cancers bear mutations in the p53 gene that inactivate its function. The p53 protein functions as a transcription factor that regulates downstream genes involved in cell cycle arrest, DNA repair and apoptosis. Mutations in the p53 tumor suppressor gene mutations have been shown to play a major role in hepatocarcinogenesis. Trp53 homozygous mice (inactivated p53) and age-matched wild type control mice were randomly assigned to various dosing groups. Forty micrograms of MCLR per kilogram of body weight or saline control were given daily by intraperitoneal injection for up to 28 days. At 4 hours, 24 hours, 4 days, 14 days and 28 days after the first MCLR dose, the mice were necropsied and liver tissue was collected for histopathology, immunohistochemistry and RNA analysis. Blood was also collected for plasma biochemistry evaluation. The RNA from the 28 day study was processed and hybridized onto Mouse Genome 430A 2.0 GeneChip arrays. The RNA from the other time points was processed for quantitative-PCR. In the 28-day treatment group, there were greater hyperplastic/dysplastic changes morphologically, and an increase in Ki-67 and phospho-Histone H3 (mitotic marker) immunoreactivity in the livers of Trp53 mutant mice. Gene expression analysis revealed differences in cell cycle regulation and cellular proliferation between the mutant and wild-type mice. We conclude that p53 plays a role in microcystin-induced toxicity and tumor promotion. These data suggest that the regulation of the cell cycle by p53 is important in preventing the proliferative response associated with chronic, sublethal microcystin exposure.

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