ß-catenin Mutations Are Not Involved in Early-stage Hepatocarcinogenesis Induced by Protoporphyrinogen Oxidase Inhibitors in Mice.

We previously reported the contribution of constitutive androstane receptor (CAR) in cytotoxicity-related hepatocarcinogenesis induced by oxadiazon (OX) or acifluorfen (ACI), two pesticides categorized as protoporphyrinogen oxidase (PROTOX) inhibitors. The molecular characteristics of preneoplastic and neoplastic lesions induced by OX and ACI were immunohistochemically compared to those by phenobarbital (PB), a typical CAR activator, in wild-type (WT) and CAR knockout (CARKO) mice after diethylnitrosamine initiation. We focused on changes in ß-catenin and its transcriptional product glutamine synthetase (GS). In PB-promoted foci and adenomas, nuclear accumulation of mutated ß-catenin was increased with high frequency. PB treatment also increased the multiplicity and area of GS-positive foci and adenomas in WT mice. No foci and adenomas showed nuclear accumulation of ß-catenin and expression of GS in CARKO mice, similar to both genotypes of mice treated with OX and ACI. Interestingly, hepatocellular carcinoma induced in ACI-treated WT mice showed nuclear accumulation of ß-catenin and was positive for GS. Our results indicated that ß-catenin mutations were not involved in early-stage hepatocarcinogenesis induced by PROTOX inhibitors in mice, although activation of ß-catenin and CAR is important in PB-induced tumorigenesis. The significant differences in molecular profiles suggested involvements of multiple mode of actions for hepatocarcinogenesis induced by PROTOX inhibitors.

In vivo reporter gene mutation and micronucleus assays in gpt delta mice treated with a flame retardant decabromodiphenyl ether.

Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, have been widely used as additive flame retardants. Recently, the use of brominated flame retardants has been restricted or prohibited under various legislative acts because of the persistence, bioaccumulation potential, and toxicity of these compounds. However, there are also additional concerns regarding environmental contamination and human exposure to PBDEs resulting from informal recycling technology. Decabromodiphenyl ether (decaBDE), one type of PBDE, has carcinogenic potential in the livers of rodents. Although one study has shown that decaBDE exerts genotoxic effects, the other in vitro and in vivo studies were negative for such effects. Thus, it remains unknown whether genotoxic mechanisms are involved in decaBDE-induced hepatocarcinogenesis in rodents. In this study, to explore the genotoxicity of decaBDE in mice, particularly in the context of carcinogenesis, we performed micronucleus assays in the bone marrow and reporter gene mutation assays in the liver using gpt delta mice treated with decaBDE at carcinogenic doses for 28days. Our results demonstrated negative results in micronucleus tests and reporter gene mutation assays. Thus, decaBDE did not exert genotoxic effects at carcinogenic target sites and did not show positive results in conventional in vivo genotoxicity tests in mice for 4-week treatment. Overall, comprehensive evaluation using in vivo genotoxicity data in rats and our data indicated that nongenotoxic mechanisms may be responsible for decaBDE-induced hepatocarcinogenesis.

A crucial role of constitutive androstane receptor (CAR) in liver tumor development by imazalil in mice.

To clarify the major pathway of liver tumor development induced by imazalil (IMA), an imidazole fungicide, male constitutive androstane receptor (CAR)-knockout (CARKO) and wild-type (WT) mice were treated with IMA at 500 ppm in the diet up to 27 weeks after initiation by diethylnitrosamine. After 27 weeks of treatment, neither altered foci nor adenomas were significantly increased in CARKO mice, whereas both eosinophilic altered foci and adenomas were increased in WT mice. After 4 or 13 weeks of IMA treatment, liver hypertrophy was observed at the tumor-inducible dose without differences among genotypes or durations. Analysis of hepatic drug metabolite enzymes, performed after administration of multiple doses during a 1-week period, indicated that pregnane X receptor might be involved in liver hypertrophy because IMA markedly elevated Cyp3a11 and Cyp2b10 expression levels in a dose-dependent manner in both genotypes. Our results demonstrated that the CAR pathway was the main mechanism of liver tumor development induced by IMA. The carcinogenic pathway was different from that of liver hypertrophy.

Involvement of Mouse Constitutive Androstane Receptor in Acifluorfen-Induced Liver Injury and Subsequent Tumor Development.

Acifluorfen (ACI), a protoporphyrinogen oxidase (PROTOX) inhibitor herbicide, promotes the accumulation of protoporphyrin IX (PPIX), and induces tumors in the rodent liver. Porphyria is a risk factor for liver tumors in humans; however, the specific mechanisms through which ACI induces hepatocarcinogenesis in rodents are unclear. Here, we investigated the mode of action of ACI-induced hepatocarcinogenesis, focusing on constitutive androstane receptor (CAR, NR1I3), which is essential for the development of rodent liver tumors in response to certain cytochrome P450 (CYP) 2B inducers. Dietary treatment with 2500 ppm ACI for up to 13 weeks increased Cyp2b10 expression in the livers of wild-type (WT) mice, but not in CAR-knockout (CARKO) mice. Microscopically, ACI treatment-induced cytotoxic changes, including hepatocellular necrosis and inflammation, and caused regenerative changes accompanied by prolonged increases in the numbers of proliferating cell nuclear antigen-positive hepatocytes in WT mice. In contrast, these cytotoxic and regenerative changes in hepatocytes were significantly attenuated, but still observed, in CARKO mice. ACI treatment also increased liver PPIX levels similarly in both genotypes; however, no morphological evidence of porphyrin deposition was found in hepatocytes from either genotype. Treatment with 2500 ppm ACI for 26 weeks after initiation with diethylnitrosamine increased the incidence and multiplicities of altered foci and adenomas in hepatocytes from WT mice; these effects were significantly reduced in CARKO mice. These results indicated that prolonged cytotoxicity in the liver was a key factor for ACI-induced hepatocarcinogenesis, and that CAR played an important role in ACI-induced liver injury and tumor development in mice.

Constitutive active/androstane receptor, peroxisome proliferator-activated receptor α, and cytotoxicity are involved in oxadiazon-induced liver tumor development in mice.

Oxadiazon (OX) is a protoporphyrinogen oxidase-inhibiting herbicide that induces porphyria and liver tumors in rodents. Although porphyria is generally considered to be a risk factor for liver tumor development, the mechanisms through which OX mediates tumor development are unclear. Therefore, in this study, we investigated the mechanisms of tumor development by focusing on constitutive active/androstane receptor (CAR), which is essential for the development of tumors in response to several chemicals. After 1, 4, or 13 weeks of dietary treatment with 1000 ppm OX, hepatic Cyp2b10 expression was induced in wild-type (WT) mice. However, this effect was blocked in CAR-knockout (CARKO) mice. Hepatic Cyp4a10 expression, indicative of peroxisome proliferator-activated receptor α (PPARα) activation, and cytotoxic changes in hepatocytes were also observed in both groups of mice. After initiation by diethylnitrosamine, 26-week treatment with OX resulted in an increase in proliferative lesions, including foci and adenomas, in both genotypes, and the incidence and multiplicity of proliferative lesions in CARKO mice were higher than those in control mice but lower than those in WT mice. These results suggested that CAR, PPARα activation, and cytotoxicity were involved in the development of liver tumors. Moreover, porphyrin was not apparently involved in OX-induced tumor development.

Dose-dependent difference of nuclear receptors involved in murine liver hypertrophy by piperonyl butoxide.

Nuclear receptors play important roles in chemically induced liver hypertrophy in rodents. To clarify the involvement of constitutive androstane receptor (CAR) and other nuclear receptors in mouse liver hypertrophy induced by different doses of piperonyl butoxide (PBO), wild-type and CAR-knockout mice were administered PBO (200, 1,000, or 5,000 ppm) in the basal diet for 1 week. Increased liver weight and diffuse hepatocellular hypertrophy were observed at 5,000 ppm for both genotypes, accompanied by increased Cyp3a11 mRNA and CYP3A protein expression, suggesting that CAR-independent pathway, possibly pregnane X receptor (PXR), plays a major role in the induction of hypertrophy. Moreover, wild-type mice at 5,000 ppm showed enhanced hepatocellular hypertrophy and strong positive staining for CYP2B in the centrilobular area, suggesting the localized contribution of CAR. At 1,000 ppm, only wild-type mice showed liver weight increase and centrilobular hepatocellular hypertrophy concurrent with elevated Cyp2b10 mRNA expression and strong CYP2B staining, indicating that CAR was essential at 1,000 ppm. We concluded that high-dose PBO induced hypertrophy via CAR and another pathway, while lower dose of PBO induced a pathway mediated predominantly by CAR. The dose-responsiveness on liver hypertrophy is important for understanding the involvement of nuclear receptors.

Essential role of constitutive androstane receptor in Ginkgo biloba extract induced liver hypertrophy and hepatocarcinogenesis.

Ginkgo biloba extract (GBE) is commonly used as a herbal supplement. The National Toxicology Program (NTP) study of GBE reported clear evidence of hepatocarcinogenicity in mice. To clarify the mode of action (MOA) for hepatocarcinogenesis by GBE, we investigated the involvement of the constitutive androstane receptor (CAR) in hepatocarcinogenesis induced by GBE using CAR-knockout (CARKO) and wild type (WT) mice. We used the same lot of GBE that was used for the NTP study. In 1-week GBE dietary treatment, hepatocellular DNA replication was increased in WT mice but not in CARKO mice. In 4- or 13-week treatment, greater hepatic Cyp2b10 induction and hepatocellular hypertrophy were observed in WT mice, whereas these effects of GBE were much smaller in CARKO mice. In a two-stage hepatocarcinogenesis model initiated by diethylnitrosamine, 27-week treatment with GBE resulted in an increase of eosinophilic altered foci and adenomas in WT mice. By contrast, foci and adenomas were clearly less evident in CARKO mice. These results indicate that GBE-induced hepatocarcinogenesis is mainly CAR-mediated. Since CAR-mediated MOA for hepatocarcinogenesis in rodents is considered to be qualitatively implausible for humans, our findings would be helpful to evaluate the carcinogenic characterization of GBE to humans.

Role of p53 in the progression from ochratoxin A-induced DNA damage to gene mutations in the kidneys of mice.

Carcinogenic doses of ochratoxin A (OTA) cause increases of mutant frequencies (MFs) of the red/gam gene (Spi(-)) in the kidneys of p53-deficient gpt delta mice, but not in p53-proficient mice. Here, we investigated the role of p53 in the progression from OTA-induced DNA damage to gene mutations. To this end, p53-proficient and -deficient mice were administered 5 mg/kg OTA for 3 days or 4 weeks by gavage. After 3 days of administration, comet assays were performed and there were no differences in the degrees of OTA-induced DNA damage between p53-proficient and -deficient mice. However, the frequencies of γ-H2AX-positive tubular epithelial cells in p53-deficient mice were significantly higher than those in p53-proficient mice, implying that p53 inhibited the progression from DNA damage to DNA double-strand breaks (DSBs). Evaluation of global gene expression and relevant mRNA/protein expression levels demonstrated that OTA increased the expression of Cdkn1a, which encodes the p21 protein, in p53-proficient mice, but not in p53-deficient mice. Moreover, in p53-deficient mice, mRNA levels of cell cycle progression and DSB repair (homologous recombination repair [HR])-related genes were significantly increased. Thus, G1/S arrest due to activation of the p53/p21 pathway may contribute to the prevention of DSBs in p53-proficient mice. In addition, single base deletions/insertions/substitutions were predominant, possibly due to HR. Overall, these results suggested that OTA induced DSBs at the carcinogenic target site in mice and that p53/p21-mediated cell cycle control prevented an increase in the formation of DSBs, leading to gene mutations.

Involvement of constitutive androstane receptor in liver hypertrophy and liver tumor development induced by triazole fungicides.

We clarified the involvement of constitutive androstane receptor (CAR) in triazole-induced liver hypertrophy and tumorigenesis using CAR-knockout (CARKO) mice. Seven-week-old male CARKO and wild-type (WT) mice were treated with 200 ppm cyproconazole (Cypro), 1500 ppm tebuconazole (Teb), or 200 ppm fluconazole (Flu) in the diet for 27 weeks after initiation by diethylnitrosamine (DEN). At weeks 4 (without DEN) and 13 (with DEN), WT mice in all treatment groups and CARKO mice in Teb group revealed liver hypertrophy with mainly Cyp2b10 and following Cyp3a11 inductions in the liver. Teb also induced Cyp4a10 in both genotypes. Cypro induced slight and duration-dependent liver hypertrophy in CARKO mice. At week 27, Cypro and Teb significantly increased eosinophilic altered foci and/or adenomas in WT mice. These proliferating lesions were clearly reduced in CARKO mice administered both compounds. The eosinophilic adenomas caused by Flu decreased in CARKO mice. The present study indicates that CAR is the main mediator of liver hypertrophy induced by Cypro and Flu, but not Teb. In contrast, CAR played a crucial role in liver tumor development induced by all three triazoles.

Acrylamide induces specific DNA adduct formation and gene mutations in a carcinogenic target site, the mouse lung.

Acrylamide (AA) is a contaminant in heated foods and is carcinogenic in multiple organs of rodents. There have been many reports regarding AA-induced DNA modification and genotoxicity. However, the data are insufficient to understand fully the relationship between the two events. A recent report demonstrated carcinogenicity in the mouse lung. The lung is advantageous for investigation of AA-induced genotoxicity because DNA adduct levels are relatively high in this organ. In the present study, reporter gene mutation assays and quantitative analyses of specific DNA adducts were performed in the lungs of mature gpt delta mice treated with AA at doses of 100, 200 and 400 p.p.m. in drinking water for 4 weeks. N7-GA-Gua was detected in all AA-treated mice in a dose-dependent manner. gpt mutant frequencies (MFs) were significantly increased in the middle- and high-dose groups. In the analysis of mutation spectra, significant increases in GC-TA transversions and single base deletion mutations were observed in the high-dose group. Spi(-) MFs were significantly increased in the high-dose group. Analysis of Spi(-) mutants revealed significant increases in the frequencies of single base deletion mutation in runs of G/C and A/T. Analyses of immature mice under the same experimental conditions showed that there were no differences of susceptibility to AA-induced genotoxicity in the two age classes. The overall data clearly show the causal relationship between AA-induced DNA adducts and the gene mutations at carcinogenic target sites.

No effect of high fat diet-induced obesity on spontaneous reporter gene mutations in gpt delta mice.

A large number of epidemiological studies have demonstrated that obesity is a risk factor for several human cancers. Several animal studies using rodents with diet-induced or genetic obesity have also demonstrated that obesity can promote tumor development. However, the effects of obesity on the early stages of carcinogenesis, and especially on the spontaneous occurrence of somatic gene mutations, remain unclear. To investigate the effects of obesity on the rate of spontaneous gene mutations, we performed reporter gene mutation assays in liver, kidney, and colon, organs in which obesity appears to be associated with cancer development on the basis of epidemiological or animal studies, in mice with high fat diet (HFD)-induced obesity. Six-week-old male and female C57BL/6 gpt delta mice were fed HFD or standard diet (STD) for 13 or 26 weeks. At the end of the experiments, reporter gene mutation assays of liver, kidney, and colon were performed. Final body weights and serum leptin levels of male and female mice fed HFD for 13 or 26 weeks were significantly increased compared with corresponding STD-fed groups. Reporter gene mutation assays of liver, kidney, and colon revealed that there were no significant differences in gpt or Spi- mutant frequencies between STD- and HFD-fed mice in either the 13-week or 26-week groups. These results indicate that HFD treatment and consequent obesity does not appear to influence the spontaneous occurrence of somatic gene mutations.

The Kidneys of Infant Mice are not Sensitive to the Food Mycotoxin Contaminant Nivalenol.

Nivalenol (NIV) is a trichothecene mycotoxin produced by Fusarium fungi that frequently contaminates agricultural commodities. Dietary administration of NIV to adult mice affects the renal glomeruli, but data about NIV toxicity in human infants are limited. To evaluate the effects of NIV on infant kidneys, 3-week-old male ICR-derived glomerulonephritis (ICGN) and ICR mice were administered 0, 4, 8 or 16 ppm NIV in diet for 4 weeks, and their renal status was compared with age-matched or adult ICR mice. In ICGN mice, the number of glomeruli showing mesangial expansion and α-smooth muscle actin (SMA)-positive mesangial cells was higher with 16 ppm NIV compared with controls. No other significant differences were observed in ICGN mice. In infant ICR mice, the IgA serum concentrations were significantly elevated without glomerular morphological changes in the 16 ppm NIV group. There was no difference in NIV sensitivity in the kidneys of infant ICGN and ICR mice. These data suggest that the kidneys in infant mice are not sensitive to nivalenol under the present conditions.

In vivo genotoxicity of Ginkgo biloba extract in gpt delta mice and constitutive androstane receptor knockout mice.

The National Toxicology Program study of Ginkgo biloba extract (GBE), a herbal supplement, reported concerns regarding genotoxicity and clear evidence of hepatocarcinogenicity and liver hypertrophy in mice. To clarify the genotoxicity of GBE in vivo, we performed reporter gene mutation assay using gpt delta mice. We also used a combined liver comet assay and bone marrow micronucleus assay using C3H-derived constitutive androstane receptor knockout (CARKO) and wild-type mice. No remarkable increases in gpt or Spi(-) mutation frequencies were observed in DNA extracted from the livers of gpt delta mice that had been exposed to GBE up to 2000 mg/kg bw/day. In the comet and micronucleus assays, no statistically significant increases in positive cells were observed at doses up to 2000 mg/kg bw/day of GBE in either mouse genotype. The present study provides clear evidence that GBE is not genotoxic in vivo. Our results indicate that GBE-induced hepatocarcinogenesis in mice occurs through a non-genotoxic mode of action.

Inhibitory potential of postnatal treatment with cyclopamine, a hedgehog signaling inhibitor, on medulloblastoma development in Ptch1 heterozygous mice.

Medulloblastomas (MBs) are thought to be derived from granular cell precursors in the external granular layer (EGL) of the developing cerebellum. Heterozygous patched1 (Ptch1) knockout mice develop MBs that resemble those in humans when the sonic hedgehog (Shh) signaling pathway is activated. The present study was conducted to evaluate postnatal effects of a Shh signaling inhibitor, cyclopamine, on the development of MBs in Ptch1 mice. Ptch1 and wild-type mice were treated daily with subcutaneous cyclopamine at 40 mg/kg or vehicle from postnatal day (PND) 1 to PND14, and the subsequent development of MBs and preneoplastic lesions was examined up to week 12 (W12). Proliferative lesions in the cerebellum, MBs, and preneoplastic lesions were only detected in Ptch1 mice. Cyclopamine treatment resulted in a statistically significant reduction in the incidence and/or area of proliferative lesions at PND14 and 21. The trend of decreasing preneoplastic lesions persisted up to W12. At PND7, cyclopamine treatment reduced the width and proliferation of the EGL regardless of genotype. These results indicate that inhibition of Shh signaling during cerebellar development has prolonged inhibitory potential on MB development in Ptch1 mice. This inhibitory potential might be related to inhibition of EGL proliferation, including preneoplastic MB cells.

Oral administration of pentachlorophenol induces interferon signaling mRNAs in C57BL/6 male mouse liver.

Pentachlorophenol (PCP) was monitored for transcriptome responses in adult mouse liver at 2, 4, 8 and 24 hr after a single oral administration at four dose levels, 0, 10, 30 and 100 mg/kg. The expression data obtained using Affymetrix GeneChip MOE430 2.0 were absolutized by the Percellome method and expressed as three dimensional (3D) surface graphs with axes of time, dose and copy numbers of mRNA per cell. We developed the programs RSort, for comprehensive screening of the 3D surface data and PercellomeExploror for cross-referencing and confirmed the significant responses by visual inspection. In the first 8 hr, approximately 100 probe sets (PSs) related to PXR/SXR and Cyp2a4 and other metabolic enzymes were induced whereas Fos and JunB were suppressed. At 24 hr, about 1,200 PSs were strongly induced. We cross-referenced the Percellome database consisting of 111 chemicals on the liver transcriptome and found that about half of the PSs belonged to the metabolic pathways including Nrf2-mediated oxidative stress response networks shared with some of the 111 chemicals. The other half of the induced genes were interferon signaling network genes (ISG) and their induction was unique to PCP. Toll like receptors and other pattern recognition receptors, interferon regulatory factors and interferon alpha itself were included but inflammatory cytokines were not induced. In summary, these data indicated that functional symptoms of PCP treatment, such as hyperthermia and profuse sweating might be mediated by the ISG rather than the previously documented mitochondrial uncoupling mechanism. PCP might become a hint for developing low molecular weight orally available interferon mimetic drugs following imiquimod and RO4948191 as agonists of toll-like receptor and interferon receptor.

Dose-response involvement of constitutive androstane receptor in mouse liver hypertrophy induced by triazole fungicides.

To clarify the dose-response relationship between constitutive androstane receptor (CAR) activity and induction of cytochrome P450 2B (CYP2B) expression and hypertrophy by triazole fungicides in mouse liver, three dose levels of cyproconazole (Cypro), tebuconazole (Teb), fluconazole (Flu), and phenobarbital (PB), a typical CYP2B inducer, were administrated in diet to male wild-type (WT) and CAR-knockout (CARKO) mice for one week. In WT mice, all compounds dose-dependently induced liver weight increases and hepatocellular hypertrophy accompanied by CYP2B expression. In CARKO mice, these effects were not induced by PB, while Cypro or Flu induced these effects only at the highest dose. Dose-dependent liver hypertrophy was detected in CARKO mice treated with Teb, but at the lowest dose the intensity was weakened compared to WT mice. The present results indicate that Cypro and Flu mainly induced CAR-mediated liver hypertrophy, while Teb slightly involved CAR. The involvement of CAR in triazole-induced liver hypertrophy was dose-responsive. In addition, all three triazoles have non-CAR-mediated liver hypertrophy pathways, indicating that the hypertrophy induced by these triazoles differs from that of PB.

Different pathways of constitutive androstane receptor-mediated liver hypertrophy and hepatocarcinogenesis in mice treated with piperonyl butoxide or decabromodiphenyl ether.

The constitutive androstane receptor (CAR) is essential for Cyp2b induction, liver hypertrophy, and hepatocarcinogenesis in response to phenobarbital (PB). Liver hypertrophy with Cyp2b induction is a major mode of action of hepatocarcinogenesis in rodents. However, it remains unclear whether CAR is involved in the response to many other nongenotoxic hepatocarcinogens besides PB. In this study, we investigated CAR involvement in liver hypertrophy and hepatocarcinogenesis of Cyp2b-inducing nongenotoxic hepatocarcinogens, piperonyl butoxide (PBO), and decabromodiphenyl ether (DBDE), using wild-type and CAR knockout (CARKO) male mice. PB was used as the positive control. In the wild-type mice, 4-week treatment with PBO, DBDE, or PB induced hepatocellular hypertrophy with increased Cyp2b10 messenger RNA and Cyp2b protein expression. In CARKO mice, only PBO showed liver hypertrophy with Cyp2b10 and Cyp3a11 induction. After 27-week treatment following diethylnitrosamine initiation, PBO and PB generated many eosinophilic altered foci/adenomas in wild-type mice; however, the lesions were far less frequent in CARKO mice. DBDE increased the multiplicity of basophilic altered foci/adenomas in wild-type and CARKO mice. Our findings indicate that murine CAR plays major roles in hepatocarcinogenesis but not in liver hypertrophy of PBO. DBDE may act via CAR-independent pathways during hepatocarcinogenesis.

Thickened area of external granular layer and Ki-67 positive focus are early events of medulloblastoma in Ptch1⁺/⁻ mice.

Patched1 (Ptch1) encodes a receptor for Sonic hedgehog (Shh) and is major gene related to human medulloblastoma (MB) in the Shh subgroup. MB is thought to arise from residual granule cell precursors (GCPs) located in the external granular layer (EGL) of the developing cerebellum. As the detailed preneoplastic changes of MB remain obscure, we immunohistochemically clarified the derived cell, early events of MBs, and the cerebellar developmental processes of Ptch1(+/-) (Ptch1) mice, an animal model of human MB of the Shh subgroup. In Ptch1 mice, the earliest proliferative lesions were detected at PND10 as focal thickened areas of outer layer of the EGL. This area was composed of GCP-like cells with atypia and nuclei disarrangement. In the latter cerebellar developmental period, GCP-like cell foci were detected at high incidence in the outermost area of the cerebellum. Their localization and morphological similarities indicated that the foci were derived from GCPs in the EGL. There were two types of the foci. A Ki-67-positive focus was found in Ptch1 mice only. This type resembled the GCPs in the outer layer of EGL characterized by having proliferating activity and a lack of neuronal differentiation. Another type of focus, Ki-67-negative, was observed in both genotypes and exhibited many of the same features of mature internal granule cells, suggesting that the focus had no preneoplastic potential. Due to morphological, immunohistochemical characteristics, our results indicate that the focal thickened area of EGL and Ki-67-positive foci are preneoplastic lesions of MB.

Reporter gene mutation in the livers of gpt delta mice treated with 5-(hydroxymethyl)-2-furfural, a contaminant of various foods.

A major product formed during the Maillard reaction is 5-(hydroxymethyl)-2-furfural (HMF), which is present in various foods and beverages such as honey and fruit juice. HMF was shown to be a hepatocarcinogen in female mice using long-term bioassays. Although HMF is not a mutagen in conventional in vitro mutation assays, 5-sulfoxymethylfurfural (SMF), a reactive metabolite of HMF produced following sulfotransferase conjugation, does show mutagenicity. Thus, HMF-induced hepatocarcinogenesis likely involves genotoxic mechanisms. To clarify the mechanisms underlying HMF-induced hepatocarcinogenesis, female B6C3F(1) gpt delta mice were given HMF at carcinogenic doses (188 or 375 mg/kg b.w.) by gavage for 5 days per week for 4 weeks. This treatment produced no significant differences in mutant frequencies (MFs) of gpt and red/gam (Spi(-)) genes among the groups. These results suggest that genotoxicity does not contribute to HMF-induced hepatocarcinogenesis. Parameters related to cell proliferation, such as proliferation cell nuclear antigen-labeling index and Cyclin D1 and E1 mRNA expression, exhibited no significant changes in the livers of HMF-treated groups. In view of the lack of carcinogenicity in rats, HMF may be considered to be a weak carcinogen. These results help us to understand the underlying mechanisms of action of HMF carcinogenesis.

Development of an early induction model of medulloblastoma in Ptch1 heterozygous mice initiated with N-ethyl-N-nitrosourea.

Mice heterozygous for the ptch1 gene (ptch1 mice) are known as a valuable model of medulloblastoma, a common brain tumor in children. To increase the incidence and reduce the time required for tumor development, allowing for evaluation of modifier effects on medulloblastoma in a short time, we attempted to develop an early induction model of medulloblastoma in ptch1 mice initiated with N-ethyl-N-nitrosourea (ENU). Ptch1 mice and their wild-type littermates received a single intraperitoneal injection of ENU (10, 50 or 100 mg/kg) on postnatal day 1 (d1) or 4 (d4), and histopathological assessment of brains was conducted at 12 weeks of age. The width of the external granular layer (EGL), a possible origin of medulloblastoma, after injection of 100 mg ENU on d1 or d4 was measured in up to 21-day-old mice. Cerebellar size was apparently reduced at the 50 mg dose and higher regardless of genotype. Microscopically, early lesions of medulloblastomas occurred with a high incidence only in ptch1 mice receiving 10 mg on d1 or d4, but a significant increase was not observed in other groups. Persistent EGL cells and misalignment of Purkinje cells were increased dose-dependently. Although EGL was strikingly decreased after ENU injection, strong recovery was observed in mice of the d1-treated group. In summary, neonatal treatment with ENU is available for the induction of medulloblastoma in ptch1 mice, and 10 mg of ENU administered on d1 appeared to be an appropriate dose to induce medulloblastoma.