Meilan Jin

Site-Specific In Vivo Mutagenicity in the Kidney of gpt Delta Rats Given a Carcinogenic Dose of Ochratoxin A

Ochratoxin A (OTA) can induce renal tumors that originate from the S3 segment of the proximal tubules in rodents, but the results of conventional mutagenicity tests have caused controversy regarding the role of genotoxic mechanisms in the carcinogenesis. Human exposure to OTA from various foods is unavoidable. Therefore, an understanding of OTA-induced renal carcinogenesis is necessary for accurate estimates of the human risk hazard. In the present study, a 13-week exposure of gpt delta rats to OTA at a carcinogenic dose induced karyomegaly and apoptosis at the outer stripe of the outer medulla (OM) of the kidney but failed to affect the reporter gene mutations in DNA extracted from whole kidneys. This site specificity resulting from the kinetics of specific transporters might be responsible for the negative outcome of in vivo mutagenicity. The kidney was then macroscopically divided, based on anatomical characteristics, into the cortex, the OM, and the inner medulla, each of which was histopathologically confirmed. Spi⁻ mutant frequencies (MFs) but not gpt MFs in the OM after a 4-week exposure to OTA were significantly higher than in controls despite the absence of cortical changes. There were also no changes in 8-hydroxydeoxyguanosine levels in kidney DNA. These results strongly suggest the involvement of a genotoxic mechanism, with the exception of oxidative DNA damage in OTA-induced renal carcinogenesis. In addition, the reporter gene mutation assay using DNA from target sites could be a more powerful tool to investigate in vivo genotoxicities.

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%.

Gene expression analysis on the dicyclanil-induced hepatocellular tumors in mice.

Our previous studies showed the possibility that oxidative stress, including oxidative DNA damage, is involved in the mechanism of dicyclanil (DC)-induced hepatocarcinogenesis at the preneoplastic stage in mice. In this study, the expression analyses of genes, including oxidative stress-related genes, were performed on the tissues of hepatocellular tumors in a two-stage liver carcinogenesis model in mice. After partial hepatectomy, male ICR mice were injected with N-diethylnitrosamine (DEN) and given a diet containing 0 or 1500 ppm of DC for 20 weeks. Histopathological examinations revealed that the incidence of hepatocellular tumors (adenomas and carcinomas) significantly increased in the DEN + DC group. Gene expression analysis on the microdissected liver tissues of the mice in the DEN + DC group showed the highest expression levels of oxidative stress-related genes, such as Cyp1a1 and Txnrd1, in the tumor areas. However, no remarkable up-regulation of Ogg1—an oxidative DNA damage repair gene—was observed in the tumor areas, but the expression of Trail—an apoptosis-signaling ligand gene—was significantly down-regulated in the tumor tissues. These results suggest the possibility that the inhibition of apoptosis and a failure in the ability to repair oxidative DNA damage occur in the hepatocellular DC-induced tumors in mice.

Effects of p53 knockout on ochratoxin A-induced genotoxicity in p53-deficient gpt delta mice

Ochratoxin A (OTA) is a mycotoxin produced by fungal species and is carcinogenic targeting the S3 segment of the renal proximal tubules in rodents. We previously reported that exposure of gpt delta rats to OTA induced both mutations in the red/gam gene (Spi(-)), suggesting large deletion mutations, and fluctuations in genes transcribed by p53 in the kidneys, which were associated with DNA double-strand break (DSB) repair, particularly homologous recombination (HR) repair. In the present study, to investigate the effects of p53 knockout on OTA-induced mutagenicity, apoptosis, and karyomegaly in renal tubular cells, p53-proficient and p53-deficient gpt delta mice were given 1 and 5mg/kg of OTA for 4 weeks. Significant increases in Spi(-) mutant frequencies (MFs) were observed in the kidneys of p53-deficient gpt delta mice given 5 mg/kg of OTA, but not in the kidneys of p53-proficient gpt delta mice given the same dose. There were no changes in gpt MFs in both genotypes of mice treated with OTA. Western blotting analysis demonstrated that p53 protein levels in the kidneys of p53-proficient mice given OTA were significantly increased compared with the control. Incidences of apoptosis and karyomegaly in not only the outer stripe of outer medulla but also the cortex were significantly higher in p53-deficient at 5mg/kg than in p53-proficient gpt delta mice at same dose, which had no change in the cortex, the inner stripe of outer stripe, and the inner medulla. Given that p53 regulates HR repair in DSBs, these results suggest that OTA may promote large deletion mutations in the process of HR repair for DSBs. Additionally, the lower incidence of karyomegaly and apoptosis found in the p53-proficient gpt delta mice suggests that these phenomena may arise from OTA-induced DNA damage.

Possible involvement of sulfotransferase 1A1 in estragole-induced DNA modification and carcinogenesis in the livers of female mice.

Estragole (ES), a natural organic compound, is frequently used as a flavoring in food even though it is a hepatocarcinogen in mice. Although formation of ES-specific DNA adducts following conversion from ES to the nucleophilic metabolite by sulfotransferase 1A1 (SULT1A1) has been reported, the modes of action underlying ES-induced hepatocarcinogenesis remain uncertain because conventional genotoxicity tests for ES yield negative results. In the present study, taking notice of the fact that there is a sex difference in SULT1A1 activity in the mouse liver, we assessed the frequency of micronuclei in polychromatic erythrocytes and the mutant frequency (MF) of reporter genes in female gpt delta mice treated with ES at doses of 0 (corn oil), 37.5, 75, 150 or 300mg/kg body weight (bw) by gavage for 13 weeks. Results were compared with those obtained in males. Since one female was found dead at week one, the highest dose was reduced to 250mg/kg bw in females from week two. As reported previously in C57BL/6 mice, the mRNA levels of Sult1a1 in female gpt delta mice were significantly higher than those in the males. The levels of ES-specific DNA adducts in the females were higher than those in the males at all doses except the highest dose. In addition, MFs of the gpt gene were significantly increased from doses of 75mg/kg bw of females, but the increment was observed only at the highest dose in males. There were no changes in the micronucleus test among the groups. Thus, the overall data suggest that specific DNA modifications by the SULT1A1-mediated carbocation formation and the resultant genotoxicity are key events in the early stage of ES-induced hepatocarcinogenesis of mice.

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.

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.

Comprehensive toxicity study of safrole using a medium-term animal model with gpt delta rats.

In order to investigate a medium-term animal model using reporter gene transgenic rodents in which general toxicity, genotoxicity and carcinogenicity are evaluated, F344 gpt delta rats were given a diet containing 0.1% and 0.5% (a carcinogenic dose) safrole for 13 weeks. Serum biochemistry and histopathological examinations revealed overt hepatotoxicity of safrole, in line with previous reports. In the current study, safrole treatment possibly resulted in renal toxicity in male rats. In the in vivo mutation assays, an increase or a tendency to increase of the gpt mutant frequencies (MFs) was observed in both sexes at the carcinogenic dose. The number and area of foci of glutathione S-transferase placental form (GST-P) positive hepatocytes, ratio of proliferating cell nuclear antigen (PCNA)-positive hepatocytes and 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA were significantly increased in both sexes of the 0.5% group. The overall data suggested that the present model might be a promising candidate for investigating comprehensive toxicities of the agents. In addition, data demonstrating the base modification and cell proliferation due to exposure to safrole could contribute to understanding safrole-induced hepatocarcinogenesis, which imply expanding in application of this model.

In vivo genotoxicity of methyleugenol in gpt delta transgenic rats following medium-term exposure

Methyleugenol (MEG), which is commonly used as a fragrance and flavoring agent, has been shown to induce hepatocellular tumors in rodents. However, the role of genotoxicity as a possible mechanism of action is not fully understood even though the DNA-reactive metabolite of MEG has been identified. In this study, a gpt delta transgenic rat model was used to clarify whether genotoxic mechanisms are involved in MEG-induced hepatocarcinogenesis following medium-term exposure. F344 gpt delta rats were subjected to repeated oral administration of MEG at dosages of 0, 10, 30, or 100mg/kg (a carcinogenic dose) for 13 weeks. The relative weight of the liver of the male and female rats that were administered 100mg/kg MEG and the absolute weight of the liver of the male rats that were administered 100mg/kg MEG were significantly increased. In addition, the number and area of glutathione S-transferase placental form (GST-P) positive foci and proliferating cell nuclear antigen (PCNA) positive cell ratios in the hepatocytes were significantly increased in the male and female rats that were administered 100mg/kg MEG compared with the control animals. In the in vivo mutation assays, a significant increase in the gpt and Spi(-) mutant frequencies was observed in both sexes at the carcinogenic dose. These results suggest the possible participation of genotoxic mechanisms in MEG-induced hepatocarcinogenesis.

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.