Gene expression profiling in rat liver treated with compounds inducing elevation of bilirubin.

We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for the diagnosis of elevated total bilirubin (TBIL) and direct bilirubin (DBIL), we extracted 59 probe sets of rat hepatic genes from the data for seven typical drugs, gemfibrozil, phalloidin, colchicine, bendazac, rifampicin, cyclosporine A, and chlorpromazine, which induced this phenotype from 3 to 28 days of repeated administration in the present study. Principal component analysis (PCA) using these probes clearly separated dose- and time-dependent clusters in the treated groups from their controls. Eighteen more drugs in the database, reported to elevate TBIL and DBIL, were estimated by PCA using these probe sets. Of these, 12 drugs, that is methapyrilene, thioacetamide, ticlopidine, ethinyl estradiol, alpha-naphthylisothiocyanate, indomethacin, methyltestosterone, penicillamine, allyl alcohol, aspirin, iproniazid, and isoniazid were also separated from the control clusters, as were the seven typical drugs causing elevation of TBIL and DBIL. The principal component 1 (PC1) value showed high correlation with TBIL and DBIL. In the cases of colchicine, bendazac, chlorpromazine, gemfibrozil, and phalloidin, the possible elevation of TBIL and DBIL could be predicted by expression of these genes 24 h after single administration. We conclude that these identified 59 probe sets could be useful to diagnose the cause of elevation of TBIL and DBIL, and that toxicogenomics would be a promising approach for prediction of this type of toxicity.

Erythropoietin mRNA in hepatocellular carcinomas and kidney in male B6C3F1 mice with secondary polycythemia.

The purpose of this study was to investigate the cause of polycythemia occurring in hepatocellular carcinoma-bearing control male B6C3F1 mice from 2-yr carcinogenicity studies. Erythrocyte counts and plasma levels of erythropoietin in mice with hepatocellular carcinomas were significantly increased compared with the values in non-tumor-bearing mice. Erythropoietin mRNA in 4 of 5 non-tumor-bearing mice was detected in the kidney, but no visible signals for hepatic erythropoietin mRNA in 5 of 5 non-tumor-bearing mice were detected by the reverse transcriptase competitive polymerase chain reaction method. Erythropoietin mRNA was expressed in neoplastic hepatocytes from 8 of 9 hepatocellular carcinoma-bearing mice, and this expression was accompanied by decreased expression of erythropoietin mRNA in the kidneys from these mice. The present findings show that polycythemia in hepatocellular carcinoma-bearing mice occurs secondary to excess synthesis of erythropoietin mRNA by neoplastic hepatocytes.

Secondary polycythemia in male B6C3F1 mice with spontaneously occurring hepatocellular carcinomas.

The purpose of this study was to investigate the cause of polycythemia occurring in control male B6C3F1 mice with hepatocellular carcinomas from 2-yr carcinogenicity studies. Erythrocyte counts and plasma erythropoietin levels in these mice were significantly increased compared to those in nontumor-bearing mice. Hepatocellular carcinomas in the mice were well differentiated, and the neoplastic hepatocytes contained either or both of 2 types of intracytoplasmic inclusion bodies; one was relatively large and weakly eosinophilic (pale inclusion body), while the other was relatively small and strongly eosinophilic (globular inclusion body). The pale eosinophilic inclusions but not the globular ones were immunohistochemically positive for erythropoietin. Ultrastructurally, the erythropoietin-positive inclusions were characterized by granular materials in dilated cisternae of rough endoplasmic reticulum, suggesting increased protein synthesis. Erythropoietin-negative inclusions were dense bodies that were not surrounded by a delimiting membrane. These findings indicate that polycythemia in hepatocellular carcinoma-bearing mice occurs secondary to excess synthesis and secretion of erythropoietin by neoplastic hepatocytes.

Auditory brainstem response (ABR) and effects of furosemide on ABR in conscious F344 rats.

The present study was conducted to evaluate the variations in the normal auditory brainstem response (ABR) between the F344 and Wistar rats. It was also investigated whether strain differences exist in the ABR profiles of each strain of rat given furosemide, which induced ototoxicity. The typical waveform of ABRs in the F344 rats were similar to those observed in the Wistar rats. There were no significant differences between the strains in almost all parameters, but peak latencies from P1 to P3 in the F344 rats tended to be longer than those in the Wistar rats. After a single intravenous injection of furosemide, the minimum effective dosage levels, at which each peak of the ABR disappeared, were 53.3 +/- 8.2 and 70.0 +/- 8.9 mg/kg in the F344 and Wistar rats, respectively. A single intravenous injection of 100 mg/kg furosemide delayed the reversal of decrease in amplitude, prolongation of peak latency and the reappearance time in F344 rats. Thus, the normal waveform of ABR was comparable in the F344 and Wistar rats, but a tendency towards a prolongation in each latency was noted in the F344 rat. The results also suggested that the ABR of F344 rats were more vulnerable to furosemide ototoxicity than that of Wistar rats.