Eriko Taniai

No effect of sustained systemic growth retardation on the distribution of Reelin-expressing interneurons in the neuron-producing hippocampal dentate gyrus in rats

Reelin signaling plays a role in neuronal migration and positioning during brain development. To clarify the effect of systemic growth retardation on the distribution of Reelin-expressing interneurons in the hilus of the hippocampal dentate gyrus, pregnant rats were fed a synthetic diet with either a normal (20% casein) or low (10% casein) protein concentration from gestational day 10 to postnatal day (PND) 21 at weaning. Male offspring were immunohistochemically examined at PND 21 and on PND 77. Protein-restricted offspring displayed systemic growth retardation through PND 77 and had decreased absolute brain weights and an increased number of external granular cells in the cerebellar cortex, suggestive of retarded brain growth at weaning. However, maternal protein restriction did not change the cellular distribution of immunoreactivity for Reelin, Calbindin-D-28K, or glutamic acid decarboxylase 67 or of NeuN-positive postmitotic neurons in the dentate hilus either at PND 21 or PND 77, which suggests that the population of γ-aminobutyric acid-ergic interneurons involving synthesis of Reelin was not affected. Furthermore, as well as the distribution of hilar neurons expressing neurogenesis-related FoxG1, cell proliferation and apoptosis in the subgranular zone were unaffected through PND 77. These results suggest that systemic growth retardation caused by maternal protein restriction does not affect neuronal migration and postnatal neurogenesis of the dentate gyrus resulting in unaltered distribution of Reelin-synthesizing interneurons.

Ochratoxin A induces karyomegaly and cell cycle aberrations in renal tubular cells without relation to induction of oxidative stress responses in rats

Ochratoxin A (OTA) is a renal carcinogen that induces karyomegaly in target renal tubular cells of the outer stripe of the outer medulla (OSOM). This study was performed to clarify the relationship between oxidative stress and the karyomegaly-inducing potential involving cell cycle aberration of OTA in the OSOM. Rats were treated with OTA for 28 days in combination with enzymatically modified isoquercitrin (EMIQ) or α-lipoic acid (ALA) as antioxidants. OTA increased the mRNA levels of the antioxidant enzyme-related genes Gpx1, Gpx2, Gstm1 and Nfe2l2, but did not increase the levels of Gsta5, Keap1, Nqo1, Hmox1, Aldh1a1, Por, Prdx1 and Txn1. OTA also did not change the levels of thiobarbituric acid-reactive substances, glutathione disulfide/reduced glutathione, and the immunoreactive tubular cell distribution of nuclear factor erythroid 2-related factor 2 in the OSOM. Co-treatment with EMIQ or ALA did not cause any changes in these parameters. As previously reported, OTA increased cell proliferation activity, apoptosis and immunohistochemical cellular distributions of molecules suggestive of induction of DNA damage and cell cycle aberrations involving spindle checkpoint disruption and cell cycle arrest. However, co-treatment with EMIQ or ALA did not suppress these changes, and ALA co-treatment increased the cell proliferation activity induced by OTA. These results suggest that OTA facilitates cell cycling involving cell cycle aberrations and apoptosis as a basis of the mechanism behind the development of karyomegaly and subsequent carcinogenicity targeting the OSOM, without relation to induction of oxidative stress. On the other hand, ALA may promote the OTA-induced proliferation of carcinogenic target cells.

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.

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.

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.

Aberrant activation of M phase proteins by cell proliferation-evoking carcinogens after 28-day administration in rats.

We have previously reported that hepatocarcinogens increase liver cells expressing p21(Cip1), a G1 checkpoint protein and M phase proteins after 28-day treatment in rats. This study aimed to identify early prediction markers of carcinogens available in many target organs after 28-day treatment in rats. Immunohistochemical analysis was performed on Ki-67, p21(Cip1) and M phase proteins [nuclear Cdc2, phospho-Histone H3 (p-Histone H3), Aurora B and heterochromatin protein 1α (HP1α)] with carcinogens targeting different organs. Carcinogens targeting thyroid (sulfadimethoxine; SDM), urinary bladder (phenylethyl isothiocyanate), forestomach (butylated hydroxyanisole; BHA), glandular stomach (catechol; CC), and colon (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and chenodeoxycholic acid) were examined using a non-carcinogenic toxicant (caprolactam) and carcinogens targeting other organs as negative controls. All carcinogens increased Ki-67(+), nuclear Cdc2(+), p-Histone H3(+) or Aurora B(+) carcinogenic target cells, except for both colon carcinogens, which did not increase cell proliferation. On the other hand, p21(Cip1+) cells increased with SDM and CC. HP1α responded only to BHA. Results revealed carcinogens evoking cell proliferation concurrently induced cell cycle arrest at M phase or showing chromosomal instability reflecting aberration in cell cycle regulation, irrespective of target organs, after 28-day treatment. Therefore, M phase proteins may be early prediction markers of carcinogens evoking cell proliferation in many target organs.

Spermatocytic Seminoma With Neuroectodermal Differentiation and Sertoli Cell Tumor in a Dog

Two distinct nodules developed in a cryptorchid testis of an 8-year-old male West Highland White Terrier. One nodule was a Sertoli cell tumor. The other was a spermatocytic seminoma with focal primitive neuroectodermal differentiation: formation of Homer–Wright rosettes and perivascular pseudorosettes, with immunoreactivity for S-100 protein, neuron-specific enolase, synaptophysin, neurofilament–68 kDa, microtubule-associated protein 2, and vimentin. The dog was alive and healthy 2 years after castration.

Neuroendocrine carcinoma of the apocrine glands of the anal sac in a dog

A perianal subcutaneous tumor involving the anal sac developed in an 8-year-old male mixed Labrador Retriever dog. Histologically, this tumor showed typical features of the solid-type carcinoma of the apocrine glands of the anal sac. However, neoplastic cells were immunoreactive for cytokeratin 8, chromogranin A, vasoactive intestinal peptide, neuron-specific enolase, and synaptophysin, and negative for S-100 protein, α-smooth muscle actin, vimentin, glucagon, insulin, somatostatin, carcinoembryonic antigen, serotonin, and parathyroid hormone–related protein. Considering the distribution of chromogranin A–positive cells within the anal sac apocrine glands, this tumor was diagnosed as neuroendocrine carcinoma originating from the apocrine glands of the anal sac.