Tetsuya Sakairi

Untargeted Metabolomic Profiling as an Evaluative Tool of Fenofibrate-Induced Toxicology in Fischer 344 Male Rats

Peroxisome proliferator-activated receptor-α (PPARα) agonists such as fenofibrate are used to treat dyslipidemia. Although fenofibrate is considered safe in humans, it is known to cause hepatocarcinogenesis in rodents. To evaluate untargeted metabolic profiling as a tool for gaining insight into the underlying pharmacology and hepatotoxicology, Fischer 344 male rats were dosed with 300 mg/kg/day of fenofibrate for 14 days and the urine and plasma were analyzed on days 2 and 14. A combination of liquid and gas chromatography mass spectrometry returned the profiles of 486 plasma and 932 urinary metabolites. Aside from known pharmacological effects, such as accelerated fatty acid β-oxidation and reduced plasma cholesterol, new observations on the drug’s impact on cellular metabolism were generated. Reductions in TCA cycle intermediates and biochemical evidence of lactic acidosis demonstrated that energy metabolism homeostasis was altered. Perturbation of the glutathione biosynthesis and elevation of oxidative stress markers were observed. Furthermore, tryptophan metabolism was up-regulated, resulting in accumulation of tryptophan metabolites associated with reactive oxygen species generation, suggesting the possibility of oxidative stress as a mechanism of nongenotoxic carcinogenesis. Finally, several metabolites related to liver function, kidney function, cell damage, and cell proliferation were altered by fenofibrate-induced toxicity at this dose.

Gene expression in livers of BALB/C and C57BL/6J mice fed a high-fat diet.

We previously demonstrated that high-fat diet (HFD)–induced hepatic lipid accumulation is more severe in BALB/c mice than in C57BL/6J (B6) mice. To understand the changes in liver metabolism, we studied blood chemistry, gene expression, and histopathological changes of the liver in nine-week HFD-fed BALB/c and B6 mice and one- or four-week HFD-fed BALB/c mice. Serum total cholesterol and triglyceride levels were significantly increased in all HFD-fed groups, and one- and four-week HFD-fed BALB/c groups, respectively. Histopathology revealed that vacuolation of hepatocytes was severe in nine-week HFD-fed BALB/c mice, although it was less severe in the other groups. Microarray analysis of mRNA expression of nine-week HFD-fed BALB/c mice showed up-regulation of genes involved in fatty acid uptake and biosynthesis, such as Cd36, Acaca, Acly, and Fasn. Some changes were observed in the one- and four-week HFD-fed BALB/c groups and the nine-week HFD-fed B6 group, however these changes in mRNA expression were not so marked. In conclusion, the fatty accumulation observed in BALB/c mice may be caused, at least in part, by up-regulation of fatty acid uptake and biosynthesis. Cd36, Acaca, Acly and Fasn may be involved in these metabolic processes.

Greater Expression of Transforming Growth Factor α and Proliferating Cell Nuclear Antigen Staining in Mouse Hepatoblastomas Than Hepatocellular Carcinomas Induced by a Diethylnitrosamine-Sodium Phenobarbital Regimen

Transforming growth factor α (TGF-α) is a potent stimulator of normal hepatocyte proliferation, considered to have relationship to the liver regeneration or carcinogenesis. In this study, we investigated immunohistochemically the association between expression of TGF-α and cell proliferation activity in mouse hepatoblastoma s (HBs) and hepatocellular carcinomas (HCCs) induced in B6C3F1 mice by diethylnitrosamine and sodium phenobarbital. The TGF-α-positive rate in HBs (29.2%) was significantly higher than that in HCCs (12.7%). Likewise, the proliferating cell nuclear antigen-positive rate (22.2%) was higher than the HCC value (14.5%). On the individual data for both TGF-α and PCNA, most of the HBs showed higher positive rates than HCCs. In HBs, TGF-α was localized only in the nuclei, whereas some HCC cells stained positive both in their nuclei and cytoplasm (0.6%). These results suggest expression of TGF-α and its localization might be linked to cell proliferation and play a role in malignant progression of mouse HBs.

Skeletal Myopathy in Transgenic Mice Carrying Human Prototype c-Ha-ras Gene

Skeletal myopathy was found in almost all-transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mouse). Microscopically, variation of the muscle fiber size, centrally placed nuclei, regenerating fibers, and interstitial fibrosis were evident; hyalinization and necrosis were sometimes observed in the skeletal muscle (femoralis and pectoralis) of the rasH2 mice. Inflammatory changes in the skeletal muscle or abnormality of adjacent peripheral nerve were not observed. The features were essentially similar to those of muscular dystrophy. Although the severity was relatively mild compared to 34-week-old rasH2 mice, the skeletal myopathy was also observed in younger male (10 weeks of age) rasH2 mice. In nontransgenic littermates, skeletal myopathy was not observed. The mRNA of human c-Ha-ras product was detected in femoral muscle from the rasH2 mice by RT-PCR. In conclusion, these data suggest that skeletal myopathy is occurring in almost all rasH2 mice. Integration of c-Ha- ras gene is thought to be crucial to pathogenesis of skeletal myopathy in the rasH2 mice. Further characterization of the muscular lesion and its pathogenesis are needed to explore the possibility of rasH2 mouse becoming a new model for muscular dystrophy.