Sayaka Kemmochi

Sustained production of Reelin-expressing interneurons in the hippocampal dentate hilus after developmental exposure to anti-thyroid agents in rats.

To detect molecular evidence reflecting a permanent disruption of neuronal development due to hypothyroidism, distribution of Reelin-producing cells that function in neuronal migration and positioning was analyzed in the hippocampal dentate hilus using rats. From gestation day 10, maternal rats were administered either 6-propyl-2-thiouracil (PTU) at 3 or 12ppm (0.57 or 1.97mg/kg body weight/day) or methimazole (MMI) at 200ppm (27.2mg/kg body weight/day) in the drinking water and male offspring were immunohistochemically examined at the end of exposure on weaning (postnatal day 20) and at the adult stage (11-week-old). Offspring with MMI and 12ppm PTU displayed evidence of growth retardation lasting into the adult stage. On the other hand, all exposure groups showed a sustained increase in Reelin-expressing cells in the dentate hilus until the adult stage in parallel with Calbindin-D-28K-expressing cells at weaning and with glutamic acid decarboxylase 67-positive cells in the adult stage, confirming an increase in gamma-aminobutyric acid (GABA)ergic interneurons. At the adult stage, NeuN-positive postmitotic mature neurons were also increased in the hilus in all exposure groups, however, the increased population of Reelin-producing cells at this stage was either weakly positive or negative for NeuN, indicative of immature neurons. At weaning, neuroblast-producing subgranular zone of the dentate gyrus showed increased apoptosis and decreased cell proliferation suggestive of impaired neurogenesis. The results suggest that sustained increases of immature GABAergic interneurons synthesizing Reelin in the hilus could be a signature of compensatory regulation for impaired neurogenesis and mismigration during the neuronal development as a hypothyroidism-related brain effect rather than that secondary to systemic growth retardation.

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.

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.

Lac color inhibits development of rat thyroid carcinomas through targeting activation of plasma hyaluronan-binding protein

Coccid-derived natural food colorants contain active ingredients that potentiate inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In the present study, we examined the effect of lac color (LC) and cochineal extract (CE), representative coccid-derived colorants containing laccaic acid and carminic acid as active ingredients, in an intracapsular invasion model of experimental thyroid cancers using rats. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSIc rats were fed a powdered diet containing 5.0% LC or 3.0% CE during promotion with 0.15% sulfadimethoxine (SDM) in the drinking water for 13 weeks. Capsular invasive carcinomas (CICs) and lung metastases were decreased by LC treatment and accompanied by transcript downregulation on angiogenesis and PHBP-related tissue proteolysis in CICs. In contrast, CE upregulated angiogenesis-related genes in CICs. PHBP was expressed in capsular macrophages and thyroid proliferative lesions with increased intensity in CICs, and LC decreased PHBP-expressing CICs. The size of CICs and their proliferation activity, however, were unchanged compared with those treated with SDM alone. Suppression of cancer by invasion by LC was more evident after an eight-week treatment, exhibiting a profound decrease in tenascin-C-positive early invasive foci and marked reductions in capsular inflammation and fibrosis. These results suggest that LC and CE exerted dissimilar effects on CIC development, the former suppressing the initial step of neoplastic cell invasion into the capsule by targeting PHBP activity of macrophages and neoplastic cells on tissue proteolysis involving inflammatory responses and angiogenesis, and the latter promoting angiogenesis of developed CICs at later stages.

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.

Rapid deposition of glomerular IgA in BALB/c mice by nivalenol and its modifying effect on high IgA strain (HIGA) mice

To clarify the underlying mechanisms of IgA nephropathy (IgAN) induced by nivalenol (NIV), a trichothecene mycotoxin, we examined the time and dose relationships of glomerular deposition of IgA by NIV in BALB/c mice (Experiment 1), and also evaluated the modification of NIV on spontaneous IgAN in an inbred murine model, a high IgA strain (HIGA), during its early stage of pathogenesis (Experiment 2). In Experiment 1, female BALB/c mice were given a diet containing 0, 12, or 24 ppm concentration of NIV for 4 or 8 weeks. An increase in serum IgA levels was found at 24 ppm from 4 weeks. At week 8 of treatment, dose-dependent increases in serum IgA levels and glomerular deposition of IgA and IgG were observed without accompanying histopathological glomerular changes. On the other hand, in Experiment 2, control HIGA mice exhibited rather high levels of serum IgA as compared with BALB/c mice from 4 weeks of experiment as well as glomerular deposition of IgA and IgG and mesangial proliferation as revealed at week 8. NIV at 24ppm further increased serum IgA in this strain; however, it did not enhance glomerular immunoglobulin deposition or histopathological lesion. These results suggest that NIV-induced increase of serum IgA levels may be primarily responsible for glomerular immunoglobulin deposition; however, NIV does not enhance glomerular IgA deposition that may lead to exacerbation of predisposed IgAN in the short term, irrespective of the further elevation of serum IgA from the high basal levels.

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.

Protective Effect of Stachybotrys microspora Triprenyl Phenol-7on the Deposition of IgA to the Glomerular Mesangium in Nivalenol-induced IgA Nephropathy Using BALB/c Mice

Activators of tissue proteolysis including Stachybotrys microspora triprenyl phenol (SMTP)-7 are a new class of agents that are expected to be effective for amelioration of chronic tissue destructive diseases. The present study was performed to examine whether SMTP-7 is effective for the amelioration or protection of early-stage IgA nephropathy (IgAN) induced by nivalenol (NIV) in female BALB/c mice. In Experiment 1, mice were administered NIV at 24 ppm in diet for 8 weeks, and during the NIV treatment, they were intraperitoneally injected with SMTP-7 (10 mg/kg) three times a week. In Experiment 2, mice were injected similarly with SMTP-7 during the last 4 weeks of a 16-week NIV treatment. Immunofluorescence analysis revealed an inhibitory effect of SMTP-7 on the glomerular deposition of IgA in Experiment 1; however, it was ineffective in Experiment 2. On the other hand, SMTP-7 did not affect the serum concentration of IgA in both experiments. These results suggest that SMTP-7 has a potential to decrease the progression of IgAN induced by NIV through inhibition of local accumulation of IgA in the glomerular mesangium, while it was ineffective for suppression of IgA production. On the other hand, SMTP-7 was found to be ineffective for already deposited IgA, suggesting that SMTP-7 may not be effective for ameliorating advanced IgAN.

No Modifying Effect of Antioxidant N-Acetyl-L-Cysteine on Fenofibrate-induced Hepatocarcinogenesis in Rats

To clarify the modifying effect of N-Acetyl-L-Cysteine (NAC), which has antioxidative ability, on hepatocarcinogenesis promoted by fenofibrate (FF), a peroxisome proliferator-activated receptor (PPAR) alpha agonist , male F344/N rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) as an initiator followed by administration of a diet containing 3,000 ppm of FF for 16 weeks. Two-thirds partial hepatectomy was performed 1 week after the FF treatment. Additionally, NAC treatments for 14 weeks from 2 weeks after the FF treatment were performed. Although the expression level of tumor protein p53 (Tp53) mRNA decreased in the DEN+FF+NAC group as compared with that in the DEN+FF group, no significant differences between the DEN+FF and DEN+FF+NAC groups were observed in the number of hepatocellular altered foci and activities of hepatocellular proliferation. In addition, the results of an antioxidant enzyme assay and measurement of the amounts of total glutathione in the liver revealed no significant difference between the DEN+FF and DEN+FF+NAC groups; although no significant differences were observed in many genes between the DEN+FF and DEN+FF+NAC groups, only glutathione peroxidase 2 (Gpx2) mRNA increased in the DEN+FF+NAC group as compared with the DEN+FF group. The results under the present experimental conditions indicate no obvious modifying effect of NAC on liver tumor promotion by FF in rats.