ToxSci Advance Access published online on January 25, 2007
Toxicological Sciences, doi:10.1093/toxsci/kfm009
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Underlying Mechanisms of Pharmacology and Toxicity of a Novel PPAR Agonist Revealed Using Rodent and Canine Hepatocytes
Department of Investigative Toxicology, Department of Pathology a Integrative Biology b Statistics and Information Science c Lilly Research Laboratories, Divisions of Eli Lilly and Company, Greenfield, IN 46140
* To whom correspondence should be addressed. Phone: (317) 433-3462. Fax (317) 277-6770. Email: timryan{at}lilly.com
Received December 22, 2006; accepted January 15, 2007
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Abstract |
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Marked species-specific responses to agonists of the peroxisome proliferator activated 
receptor (PPAR) have been observed in rats and dogs, two species typically used to assess the potential human risk of pharmaceuticals in development. In this study, we used primary cultured rat and dog hepatocytes to investigate the underlying mechanisms of a novel PPAR and 
coagonist, LY465608, relative to fenofibrate, a prototypical PPAR agonist. As expected, rat hepatocytes incubated with these two agonists demonstrated an increase in peroxisome number as evaluated by electron microscopy, whereas the peroxisome number remained unchanged in dog hepatocytes. Biochemical analysis showed that rat hepatocytes responded to PPAR agonists with an induction of both peroxisomal and mitochondrial ß-oxidation (PBox and MBox) activities. Dog hepatocytes treated with both PPAR agonists, however, did not show increased PBox activity, but did demonstrate increased MBox activity. Analysis of peroxisomal ß-oxidation gene expression markers by quantitative real time PCR confirmed that PPAR agonists induced the peroxisomal enzymes, acyl CoA oxidase (ACOX), enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (Ehhadh), and 3-ketoacyl-CoA thiolase (Acaa1) at the transcriptional level in rat, but not dog, hepatocytes. Expression of mRNA for the mitochondrial ß-oxidation gene hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase (Hadhb), however, increased in both rat and dog hepatocytes, consistent with biochemical measurements of peroxisomal and mitochondrial ß-oxidation. Repeat-dose nonclinical safety studies of LY465608 revealed abnormities in mitochondrial morphology and evidence of single cell necrosis following 30d of dosing exclusively in dogs, but not rats (Carfagna MA 2006; Reynolds VL 2006). Microarray analysis indicated that dog hepatocytes, but not rat hepatocytes, treated with LY465608 had an expression profile consistent with abnormalities in the regulation of cell renewal and death, oxidative stress, and mitochondrial bioenergetics, which may explain the canine-specific toxicity observed in vivo with this compound. This increased sensitivity to mitochondrial toxicity of canine hepatocytes relative to rat hepatocytes identified using gene expression was confirmed using the fluorescent indicator TMRE and flow cytometry. At doses as low as 0.1 µM LY465608, canine hepatocytes showed a greater shift in fluorescence indicative of mitochondrial damage than observed with rat hepatocytes treated at 10 µM. In summary, using rat and dog primary hepatocytes, we replicated the pharmacologic and toxicologic effects of LY465608 observed in vivo during preclinical development, and propose an underlying mechanism for these species-specific effects.
Key Words: PPAR agonist; hepatocyte; mitochondrial ß-oxidation; peroxisomal ß-oxidation; toxicogenomics; gene expression; LY465608; fenofibrate.