Jihei Nishimura

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%.

Molecular Expression Analysis of β-Naphthoflavone-induced Hepatocellular Tumors in Rats

The present study was performed to characterize molecular expression levels of preneoplastic and neoplastic lesions induced by β-naphthoflavone (BNF), an aryl hydrocarbon receptor (AhR) agonist in rat hepatocarcinogenesis. Male F344 rats were initiated with an intraperitoneal injection of 200 mg/kg N-diethylnitrosamine, and two weeks later, they were fed a diet containing 0% or 1% BNF for twenty-eight weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and sacrificed at week 30. Histopathologically, BNF increased the incidence and multiplicity of altered foci (1.7-fold and 3.3-fold) and hepatocellular adenomas (HCAs) (4.0-fold and 4.7-fold). Immunohistochemically, BNF increased the number of proliferating cell nuclear antigen (PCNA)-positive cells in altered foci (2.3-fold) and HCAs (6.7-fold) compared with the surrounding tissue and decreased the staining of cell cycle regulators (P21, C/EBPα). In addition, loss of reactivity for AhR-regulated (CYP1A1, CYP1B1) molecules and increased reactivity of Nrf-2-regulated (AKR7, GPX2) molecules were also observed in proliferative lesions. Furthermore, increased staining of histone deacetylase (HDAC1) in the nucleus was prominent in HCAs. The differential expression patterns were confirmed at mRNA levels by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. These results suggest that enhanced cell proliferation and protection against oxidative stress play an important role in BNF-induced hepatocarcinogenesis in rats.

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.

Role of Nrf2 and Oxidative stress on Fenofibrate-Induced Hepatocarcinogenesis in Rats

Regional specific relationships between oxidative stress and the development of glutathione S-transferase placental form (GST-P)-positive or GST-P-negative lesions in rats, induced by fenofibrate (FF), a peroxisome proliferator, were examined using a two-stage hepatocarcinogenesis model in F344 rats. Animals were initiated with a single ip injection of 200 mg/kg N-diethylnitrosamine (DEN) and from 2 weeks later were fed a diet containing 3000 or 0 ppm FF for 28 weeks. Animals were subjected to a two-third partial hepatectomy at week 3 and sacrificed at week 28. The development of hepatocellular proliferative lesions, which were mainly attributed to GST-P-negative lesions, was significantly increased in the FF-treated groups. Immunohistochemically, GST-P-positive lesions were devoid of intracytoplasmic nuclear factor-erythroid 2-related factor 2 (Nrf2) expression, whereas GST-P-negative lesions expressed higher levels of cytoplasmic Nrf2. On the other hand, nuclear accumulation of Nrf2 was observed in some cells of GST-P-positive lesions that were negative for Nrf2 in the cytoplasm and in GST-P-negative lesions of the DEN-FF group that were positive for Nrf2 in the cytoplasm. The mRNA expression levels of Gpx2 or Gsta2, Nrf2-inducible enzymes, were increased in GST-P-positive tumors or GST-P-positive lesions, respectively. These results suggest that the activation of Nrf2, due to nuclear translocation, occurs in the GST-P-positive lesions. In addition, the development of continuous oxidative stress was identified by mRNA expression analyses as well as by measurements of GST activity and 8-hydroxydeoxyguanosine. These results suggest that the relative inhibition of nuclear translocation of Nrf2 in GST-P-negative lesions aggravated the condition of oxidative stress in the liver of rats given FF, resulting in enhanced tumor promotion in FF-induced hepatocarcinogenesis.

Involvement of glycogen synthase kinase-3β signaling and aberrant nucleocytoplasmic localization of retinoblastoma protein in tumor promotion in a rat two-stage thyroid carcinogenesis model

To investigate the cell cycle kinetics during the tumor promotion process induced by hypothyroidism in a rat model of thyroid follicular cell carcinogenesis, immunohistochemical analysis of cell cycle molecules and related signaling molecules was performed in conjunction with analysis of cell proliferation activity in an initiation-promotion model. Male F344 rats were injected with N-bis(2-hydroxypropyl)nitrosamine, and one week later treated with 6-propyl-2-thiouracil (PTU) at 12ppm in the drinking water for 4, 10 or 15 weeks. At each time point, proliferative lesions increased the expression of cyclin A, cyclin D, cyclin E and cyclin-dependent kinase (Cdk)-2, in association with the development of lesion stages from the early focal hyperplasia to the late carcinoma, while a subpopulation of proliferative lesions showed decreased numbers of both cell division cycle-2- and Ki-67-positive cells at week 15 compared with that at week 10, suggesting a reduced promoting effect of serum thyroid-stimulating hormone in the sensitive cellular population after long-term exposure to PTU. On the other hand, increased immunolocalization of phosphorylated and inactive glycogen synthase kinase (GSK)-3beta was observed in a subpopulation of proliferative lesions, in parallel with the cyclins and Cdk2. Nuclear immunoreactivity of phosphorylated and inactive retinoblastoma (Rb) protein was also increased in association with lesion development, with carcinomas showing increased cytoplasmic localization. The results suggest that proliferative lesions activate the cell cycle machinery following tumor promotion via a regulatory mechanism involving inactivation of GSK3beta and Rb protein, the latter signaling mechanism involving its aberrant nucleocytoplasmic transport for the acquisition of a malignant phenotype.

Mechanistic Study on Hepatocarcinogenesis of Piperonyl Butoxide in Mice

To clarify the mechanism of piperonyl butoxide (PBO)-induced hepatocarcinogenesis in mice, male mice were subjected to a two-thirds partial hepatectomy, N-diethylnitrosamine (DEN) initiation, and a diet containing 0.6% PBO for eight weeks. The incidence of γ-glutamyl transpeptidase (GGT)-positive foci and PCNA-positive cells was significantly increased in the DEN + PBO group compared with the DEN-alone group. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis showed up-regulation of genes related to metabolism, such as cytochrome P450 1A1 and 2B10, and metabolic stress, such as Por, Nqo1, Nrf2, abcc3, and abcc4. Early responsive genes downstream of mitogen-activated protein kinase (MAPK), such as c-fos, c-jun, c-myc, and activating transcription factor 3 (ATF3), were also up-regulated in this group. Positive immunohistochemical staining for ATF3 was diffusely observed in nonproliferating hepatocytes of the DEN + PBO group, but altered foci were negative or weakly positive for ATF3. The nuclei of hepatocytes within ATF3-negative foci were positive for cyclin D. Thus PBO can induce oxidative stress, activate the MAPK pathway, and increase ATF3 transcript levels in hepatocytes outside the altered foci during the early stage of PBO-induced hepatocarcinogenesis in mice.

Induction of liver preneoplastic foci in F344 rats subjected to 28-day oral administration of diheptyl phthalate and its in vivo genotoxic potential

To investigate possible potential inducing preneoplastic lesions in liver and in vivo genotoxic potential of diheptyl phthalate (DHP), male F344 rats were subjected to repeated oral administration of DHP at 0, 2.5 or 5 g/kg/day for 28 days. In addition, F344 rats were subjected to once or 14 repeated oral administrations of 5 g/kg/day of DHP, and their livers were subjected to analysis in an alkaline single-cell gel electrophoresis (comet) assay. Furthermore, based on the results of these studies, partial hepatectomized male F344 rats given once, three times, and 14 repeated oral administration of 0, 2.5 or 5 g/kg body weight of DHP were examined by an in vivo liver initiation assay. In a 28-day repeated dose toxicity study, the number and area of glutathione-S-transferase placental form (GST-P) positive foci, a marker of hepatocellular preneoplastic lesions in rats, were significantly increased in DHP-treated groups compared with controls. At 24h after the 14 repeated administrations of DHP, DNA migration, a marker of DNA damage in the comet assay, was significantly induced in DHP-treated rat livers, whereas single treatment did not show such an alteration. In an in vivo liver initiation assay, a significant increase in the number and area of GST-P positive foci was observed in DHP-treated groups subjected to 14 repeated oral administrations of DHP as compared with the control group. These results indicate that DHP may induce altered hepatocellular foci in liver of rats which suggests that DHP is a genotoxic carcinogen in the liver of rats.

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.

Effects of compound X, a novel potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, on the adrenal gland of rats.

To investigate the adrenal toxicity of a novel inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase, compound X (CX), histopathological examinations, fat staining, adrenal cholesterol measurement, blood biochemistry, plasma corticosterone and ACTH measurement, ACTH-stimulation assay, and adrenal gene-expression analyses were done in rats in repeated-dose studies (experiment 1: 0, 3, 10, 30 and 150mg/kg for 4, 8, 15 and 28 days; experiment 2: 0, 3, 10,30 and 150mg/kg for 28 days; experiment 3: 0, 10, 30, 100 and 300mg/kg for 28 days). CX induced morphologic changes such as vacuolation and hypertrophy in the zona fasciculata (ZF) at ≥10mg/kg, and eosinophilic changes in the ZF at 150mg/kg. Vacuolation decreased in a dose-dependent manner and was replaced by eosinophilic changes. Inflammatory and fibrous changes were observed at ≥30mg/kg. These changes were expressed at early stages of dosing and were not exacerbated by extension of the administration period. Oil-red-O/Filipin staining showed depletion of cholesterol ester in dose-dependent manner and enabled adrenal cholesterol measurement. Filipin staining also revealed vacuoles to be composed of cholesterol esters. No significant changes were observed during the dosing period of CX for plasma corticosterone and ACTH levels. Gene-expression analyses showed up-regulation of Star and Abca1 mRNA levels at 300mg/kg. In conclusion, CX induced adrenal toxicity, but CX did not influence adrenocortical functions, and exacerbation of adrenal toxicities by extension of the administration period was not observed. Up-regulation of genes related to the transport of FC, such as Star and Abca1, were observed in CX groups, and these genes may be involved in the maintenance of adrenal structure and function in rats given CX.