Sayaka Matsumoto

Threshold dose of piperonyl butoxide that induces reactive oxygen species-mediated hepatocarcinogenesis in rats

To determine the threshold dose of piperonyl butoxide (PBO) that induces hepatocellular tumor-promoting effects, reactive oxygen species (ROS) generation, and drug-metabolizing enzymes that protect against ROS generation, partial hepatectomized rats were fed diets containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.

Crosstalk between PTEN/Akt2 and TGFβ signaling involving EGF receptor down-regulation during the tumor promotion process from the early stage in a rat two-stage hepatocarcinogenesis model

The present study investigated the involvement of signaling of phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/protein kinase B (Akt) and transforming growth factor‐β (TGFβ) as well as receptor tyrosine kinases in the tumor promotion processes in a two‐stage hepatocarcinogenesis model using male F344 rats. The cellular localization of related molecules was examined in liver cell foci expressing glutathione S‐transferase placental form (GST‐P) at the early stage of tumor promotion by fenbendazole (FB), piperonyl butoxide, or thioacetamide. Distribution in the liver cell foci and neoplastic lesions positive for GST‐P was also examined at the later stage of FB promotion. In contrast to the initiation‐alone cases, subpopulations of GST‐P‐positive foci induced by promotion for 6 weeks, regardless of the promoting chemicals used, enhanced down‐regulation of PTEN and up‐regulation of phosphorylated (active) Akt2 and phosphorylated substrate(s) of Akt‐kinase activity. Also, up‐regulation of TGFβ receptor I and down‐regulation of epidermal growth factor receptor (EGFR) were enhanced in the subpopulation of GST‐P‐positive foci in all promoted cases. A similar pattern of cellular distribution of these molecules was also observed in the neoplastic lesions at the late stage. These results suggest a crosstalk between Akt2 and TGFβ signaling that involves a mechanism requiring EGFR down‐regulation during the entire tumor promotion process starting from the early stage. In particular, a shift in subcellular localization of phosphorylated substrate(s) of Akt from the cell membrane in liver cell foci to the cytoplasm in carcinomas was observed, suggesting an alteration of the function or activity of the corresponding molecule(s). (Cancer Sci 2009; 100: 813–820)

Hepatocarcinogenic Susceptibility of Fenofibrate and Its Possible Mechanism of Carcinogenicity in a Two-Stage Hepatocarcinogenesis Model of rasH2 Mice

Fenofibrate (FF) has previously been shown to induce hepatocellular neoplasia in a conventional mouse bioassay (NDA 1993), but there has been no report to examine the carcinogenic susceptibility of rasH2 mice to this chemical. In the present study, male rasH2 mice were subjected to a two-thirds partial hepatectomy (PH), followed by an N-diethylnitrosamine (DEN) initiation twenty-four hours after PH, and given a diet containing 0, 1200, or 2400 ppm FF for seven weeks. The incidences of preneoplastic foci were significantly increased in mice from the FF-treated groups. Immunohistochemistry revealed that significant increases in proliferating cell nuclear antigen (PCNA)-positive cells and cytokeratin 8/18 positive foci were observed in FF-treated groups. In addition, the transgene and several downstream molecules such as c-myc, c-jun, activating transcription factor 3 (ATF3), and cyclin D1 were overexpressed in these groups. These results suggest that the hepatocarcinogenic activity of rasH2 mice to FF can be detected in this hepatocarcinogenesis model and that up-regulation of genes for the ras/MAPK pathway and cell cycle was probably involved in the hepatocarcinogenic mechanism of rasH2 mice.

Modification of dietary copper levels on the early stage of tumor-promotion with propylthiouracil in a rat two-stage thyroid carcinogenesis model

To investigate the role of copper (Cu)-related cellular responses on thyroid carcinogenesis, the expression of ceruloplasmin (Cp) and metallothionein (MT)-1/2 were examined in relation to the activities of cell proliferation/apoptosis in the thyroid of rats at an early stage of tumor promotion under different dietary Cu levels. Male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine by single subcutaneous injection at 2800 mg/kg body weight, and 1 week later promoted with 6-propyl-2-thiouracil at 12 ppm in the drinking water for 4 weeks. Animals were fed a diet containing Cu at 0.6, 6 or 60 ppm from the time point of initiator-treatment to create marginally deficient, normal, or non-toxic supplementary levels of Cu. At both 0.6 and 60 pm, the multiplicity of preneoplastic focal follicular cell hyperplasias (FFCHs) was decreased as compared with 6 ppm Cu, while adenomas also decreased at 0. 6 ppm Cu. Both 0.6 and 60 ppm Cu levels revealed decreased Ki-67-immunoreactive proliferating cells in both FFCHs and surrounding follicles accompanied by mRNA downregulation of Cdc2a and Ccnb1, while TUNEL-positive apoptotic cells were unaltered with change of dietary Cu. Both Cp and MT-1/2 were immunolocalized in FFCHs and adenomas, with higher distribution in the latter. At both 0.6 and 60 ppm, the immunoreactivities and/or thyroidal mRNA levels of Cp and MT-1/2 were also decreased. Transcript levels of several antioxidant enzymes were up- or downregulated in the same direction at both Cu levels. Serum levels of thyroid-related hormones were unaltered at both Cu levels, except for non-significant reduction of thyroid-stimulating hormone at 0.6 ppm. These results suggest an involvement of Cp and MT-1/2 on the thyroid tumor promotion that can be suppressed by dietary Cu level through inhibition of cell proliferation associated with altered redox balance.