Ryohei Ichimura

Antioxidant enzymatically modified isoquercitrin suppresses the development of liver preneoplastic lesions in rats induced by β-naphthoflavone

To investigate the modifying effect of enzymatically modified isoquercitrin (EMIQ) on hepatocellular tumor promotion induced by beta-naphthoflavone (BNF) treatment, male rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) and were fed a diet containing BNF (0.5%) for 6 weeks with or without EMIQ (0.2%) in the drinking water after DEN initiation. One week after the commencement of the administration of BNF, rats were subjected to a two-thirds partial hepatectomy. The number and area of GST-P positive foci, the number of COX2-positive cells and the area of elastica-van Gieson (EVG)-positive connective tissue fibers promoted by BNF were significantly suppressed by the administration of the antioxidant EMIQ. Real-time RT-PCR analysis revealed that EMIQ treatment decreased mRNA expression levels of Gstm1, Serpine1, Cox2 and Nfkbia and increased mRNA expression levels of Yc2 compared with those in the DEN-BNF group. These results suggest that co-administration of EMIQ suppresses the hepatocellular tumor-promoting activity of BNF in rats through the anti-inflammatory effects of EMIQ and restores the cellular redox balance altered by BNF.

Induction of GST-P-positive proliferative lesions facilitating lipid peroxidation with possible involvement of transferrin receptor up-regulation and ceruloplasmin down-regulation from the early stage of liver tumor promotion in rats

To elucidate the role of metal-related molecules in hepatocarcinogenesis, we examined immunolocalization of transferrin receptor (Tfrc), ceruloplasmin (Cp) and metallothionein (MT)-1/2 in relation to liver cell foci positive for glutathione-S-transferase placental form (GST-P) in the early stage of tumor promotion by fenbendazole (FB), phenobarbital, piperonyl butoxide or thioacetamide in a rat two-stage hepatocarcinogenesis model. To estimate the involvement of oxidative stress responses to the promotion, immunolocalization of 4-hydroxy-2-nonenal, malondialdehyde and acrolein was similarly examined. Our findings showed that MT-1/2 immunoreactivity was not associated with the cellular distribution of GST-P and proliferating cell nuclear antigen, suggesting no role of MT-1/2 in hepatocarcinogenesis. We also found enhanced expression of Tfrc after treatment with strong tumor-promoting chemicals. With regard to Cp, the population showing down-regulation was increased in the GST-P-positive foci in relation to tumor promotion. Up-regulation of Tfrc and down-regulation of Cp was maintained in GST-P-positive neoplastic lesions induced after long-term promotion with FB, suggesting the expression changes occurring downstream of the signaling pathway involved in the formation of GST-P-positive lesions. Furthermore, enhanced accumulation of lipid peroxidation end products was observed in the GST-P-positive foci by promotion. Post-initiation treatment with peroxisome proliferator-activated receptor α agonists did not enhance any such distribution changes in GST-P-negative foci. The results thus suggest that facilitation of lipid peroxidation is involved in the induction of GST-P-positive lesions by tumor promotion from an early stage, and up-regulation of Tfrc and down-regulation of Cp may be a signature of enhanced oxidative cellular stress in these lesions.

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.

Disruption of Smad-dependent signaling for growth of GST-P-positive lesions from the early stage in a rat two-stage hepatocarcinogenesis model

To clarify the involvement of signaling of transforming growth factor (TGF)-β during the hepatocarcinogenesis, the immunohistochemical distribution of related molecules was analyzed in relation with liver cell lesions expressing glutathione S-transferase placental form (GST-P) during liver tumor promotion by fenbendazole, phenobarbital, piperonyl butoxide, or thioacetamide, using rats. Our study focused on early-stage promotion (6weeks after starting promotion) and late-stage promotion (57weeks after starting promotion). With regard to Smad-dependent signaling, cytoplasmic accumulation of phosphorylated Smad (phospho-Smad)-2/3 - identified as Smad3 by later immunoblot analysis - increased in the subpopulation of GST-P(+) foci, while Smad4, a nuclear transporter of Smad2/3, decreased during early-stage promotion. By late-stage promotion, GST-P(+) lesions lacking phospho-Smad2/3 had increased in accordance with lesion development from foci to carcinomas, while Smad4 largely disappeared in most proliferative lesions. With regard to Smad-independent mitogen-activated protein kinases, GST-P(+) foci that co-expressed phospho-p38 mitogen-activated protein kinase increased during early-stage promotion; however, p38-downstream phospho-activating transcriptional factor (ATF)-2, ATF3, and phospho-c-Myc, were inversely downregulated without relation to promotion. By late-stage promotion, proliferative lesions downregulated phospho-ATF2 and phospho-c-Myc along with lesion development, as with downregulation of phospho-p38 in all lesions. These results suggest that from the early stages, carcinogenic processes were facilitated by disruption of tumor suppressor functions of Smad-dependent signaling, while Smad-independent activation of p38 was an early-stage phenomenon. GST-P(-) foci induced by promotion with agonists of peroxisome proliferator-activated receptor-α did not change Smad expression, suggesting an aberration in the Smad-dependent signaling prerequisites for induction of GST-P(+) proliferative lesions.

Tumor promotion by copper-overloading and its enhancement by excess iron accumulation involving oxidative stress responses in the early stage of a rat two-stage hepatocarcinogenesis model.

To investigate liver tumor promotion mechanisms of copper (Cu)- and iron (Fe)-overloading, immunolocalization of metal-related biomolecules and lipid peroxidation end products was examined in preneoplastic liver cell foci that expressed glutathione S-transferase placental form (GST-P) in early-stage tumor promotion over 6 weeks in a rat two-stage hepatocarcinogenesis model. Gene expression and concentrations of thiobarbituric acid-reactive substance (TBARS) in the liver were also analyzed. Cu-overloading alone exerted a weak promoting activity, which was enhanced by Fe-overloading. By Cu-overloading, GST-P(+) foci that co-expressed transferrin receptors or downregulated ceruloplasmin increased, suggesting preneoplastic lesion-specific enhancement of oxidative cellular stress. Cu-overloading also increased transcripts of antioxidant enzymes (Gstm3 and Gst Yc2 subunit), cell proliferation, and numbers of single liver cells expressing GST-P or heme oxygenase-1 (HO-1) in the liver, suggesting that oxidative stress induces single-cell toxicity, with the ensuing regeneration contributing to tumor promotion. Fe-overloading increased liver TBARS and HO-1-expressing Kupffer cells, the latter suggesting protection against inflammatory stimuli causing fluctuating proinflammatory cytokine mRNA levels. By co-overloading of Cu and Fe, Cu-overload-related single liver cell toxicity and regeneration increased, as did cytokine imbalances involving increased cyclooxygenase-2-producing Kupffer cells and accumulation of malondialdehyde within GST-P(+) foci. These results suggest an involvement of oxidative stress responses in Cu-induced tumor promotion and Fe-induced enhancement by increasing cytokine imbalances and GST-P(+) foci-specific lipid peroxidation.

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.

Prior attenuation of KiSS1/GPR54 signaling in the anteroventral periventricular nucleus is a trigger for the delayed effect induced by neonatal exposure to 17alpha-ethynylestradiol in female rats

Neonatal exposure to 17alpha-ethynylestradiol (EE) causes delayed effect, a late-occurring irreversible damage to reproductive functions characterized by the early onset of age-matched abnormal estrous cycling. To clarify the involvement of a hypothalamic key cycling regulator KiSS1/GPR54 in the delayed effect, we investigated artificially induced LH surges and KiSS1 mRNA expression in the anteroventral periventricular nucleus (AVPV) of cycling young adult rats neonatally exposed to EE, and compared these parameters to those in about 5 months old middle-aged rats. KiSS1 mRNA expression, the number of KiSS1-positive cells and KiSS1/ERα co-expressing cells in the AVPV decreased in both EE-exposed and middle-aged rats. The peak area and levels of LH surge dose-dependently decreased in EE-exposed rats, and reduction was more evident in middle-aged rats. These results indicate that the prior attenuation of KiSS1 and consequent depression of LH surges plays a key role in the onset of abnormal estrous cycling in the delayed effect.

The impact of neonatal exposure to 17alpha-ethynylestradiol on the development of kisspeptin neurons in female rats

Neonatal exposure to 17alpha-ethynylestradiol (EE) at relatively low doses leads to delayed effects characterized by the early onset of age-related anovulation. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV), located at the anterior hypothalamus, are proposed to play key roles in appearance of these delayed effects after maturation. To understand the initial changes, we investigated Kiss1 mRNA expression in the anterior and posterior hypothalamus before weaning in female rats that received neonatal exposure to EE at various doses (0.002-2000μg/kg). The level of Kiss1 mRNA in the anterior hypothalamus was decreased from 0.002μg/kg which did not induce delayed effects. In the posterior hypothalamus, Kiss1 mRNA expression did not differ among the groups except 2000μg/kg group. These results suggest that neonatal exposure to EE affects the development of kisspeptin neurons and kisspeptin neurons in the AVPV are highly susceptible to neonatal EE treatment.

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.

Neonatal exposure to SERMs disrupts neuroendocrine development and postnatal reproductive function through alteration of hypothalamic kisspeptin neurons in female rats

Selective estrogen receptor modulators (SERMs) are a class of therapeutic chemicals which present tissue-specific estrogen receptor modulating activity. Neonatal exposure to SERMs has been reported to adversely affect central nervous system development, however, mechanism and involvement of hypothalamic kisspeptin neurone in this impairment remains undetermined. To clarify this uncertainty, neonates from female Donryu rats were subcutaneously injected with raloxifene (RLX) at 0.1, 1, and 10mg/kg or tamoxifen (TMX) at 10mg/kg on postnatal day 0, and then hypothalamic KiSS1 mRNA expression and gonadotropin levels were investigated during young adulthood and estrous cycling was monitored until middle age. Treatment with RLX or TMX at 10mg/kg significantly depressed luteinizing hormone surge levels and KiSS1 mRNA expression in the anteroventral periventricular nucleus (AVPV), the control center of estrous cyclicity. The 10mg/kg TMX group also showed decreased levels of follicle-stimulating hormone and KiSS1 mRNA expression in the arcuate nucleus (ARC). Early cessation of normal estrous cycling was observed in the 10mg/kg RLX group, while the estrous cycle in the 10mg/kg TMX group had ceased by the start of the analysis. The same dose of tamoxifen or raloxifene had either weak-estrogenic or anti-estrogenic activity on the uterus, respectively; however, treatment in adulthood with both SERMs did not affect KiSS1 mRNA expression in either the AVPV or ARC in the present study. These results indicate that neonatal exposure to SERMs could disrupt neuroendocrine development and postnatal reproductive function through the alteration of kisspeptin neurons.