Hitomi Hayashi

Prediction model of potential hepatocarcinogenicity of rat hepatocarcinogens using a large-scale toxicogenomics database.

The present study was performed to develop a robust gene-based prediction model for early assessment of potential hepatocarcinogenicity of chemicals in rats by using our toxicogenomics database, TG-GATEs (Genomics-Assisted Toxicity Evaluation System developed by the Toxicogenomics Project in Japan). The positive training set consisted of high- or middle-dose groups that received 6 different non-genotoxic hepatocarcinogens during a 28-day period. The negative training set consisted of high- or middle-dose groups of 54 non-carcinogens. Support vector machine combined with wrapper-type gene selection algorithms was used for modeling. Consequently, our best classifier yielded prediction accuracies for hepatocarcinogenicity of 99% sensitivity and 97% specificity in the training data set, and false positive prediction was almost completely eliminated. Pathway analysis of feature genes revealed that the mitogen-activated protein kinase p38- and phosphatidylinositol-3-kinase-centered interactome and the v-myc myelocytomatosis viral oncogene homolog-centered interactome were the 2 most significant networks. The usefulness and robustness of our predictor were further confirmed in an independent validation data set obtained from the public database. Interestingly, similar positive predictions were obtained in several genotoxic hepatocarcinogens as well as non-genotoxic hepatocarcinogens. These results indicate that the expression profiles of our newly selected candidate biomarker genes might be common characteristics in the early stage of carcinogenesis for both genotoxic and non-genotoxic carcinogens in the rat liver. Our toxicogenomic model might be useful for the prospective screening of hepatocarcinogenicity of compounds and prioritization of compounds for carcinogenicity testing.

Concomitant apoptosis and regeneration of liver cells as a mechanism of liver-tumor promotion by β-naphthoflavone involving TNFα-signaling due to oxidative cellular stress in rats

β-naphthoflavone (BNF) is a strong inducer of cytochrome P450 1A enzymes, and exerts liver tumor-promoting activity through enhancement of oxidative stress responses in rats. This study investigated the role of the tissue environment surrounding hepatocellular preneoplastic lesions in the early tumor-promotion stage by BNF, using enzymatically modified isoquercitrin (EMIQ) as an anti-oxidative chemopreventive agent. Male F344 rats were fed a diet containing BNF (0.5%) for 6 weeks, with or without EMIQ (0.2%) in the drinking water, 2 weeks after initiation with N-diethylnitrosamine, and were subjected to two-thirds partial hepatectomy 1 week after starting BNF-promotion. BNF-treatment increased concentrations of liver thiobarbituric acid-reactive substances, single liver cells expressing glutathione S-transferase placental form or heme oxygenase (HO)-1, and concomitant apoptosis and proliferation of liver cells. Transcript upregulation of anti-oxidative enzymes (Aldh1a1 and Nqo1), cell cycle-related molecules (Cdc20 and Cdkn2b) and inflammation-related molecules including proinflammatory cytokines (Ccl2, Col1a1, Il6, Nos2 and Serpine1) was also evident. Furthermore, BNF increased HO-1-expressing Kupffer cells and liver cells expressing tumor necrosis factor receptor 1 (TNFR1) and the TNFR1-associated death domain. Most of these BNF-induced fluctuations disappeared or were suppressed by EMIQ in conjunction with suppression of tumor-promotion. Tnf transcript levels with BNF were also suppressed by EMIQ. These results suggest that BNF-induced oxidative stress causes single liver cell toxicity, allowing subsequent concomitant apoptosis and regeneration involving inflammatory responses including TNFα-signaling, contributing to tumor promotion. Kupffer cells may act to protect against inflammatory stimuli induced as a result of oxidative cellular stress by BNF, causing proinflammatory cytokine level fluctuations.

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.

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.

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.

Long-lasting immunosuppressive effects of tacrolimus-loaded micelle NK61060 in preclinical arthritis and colitis models

AIM Tacrolimus (TAC) is an important drug for inflammatory diseases. However, TAC has several limitations, such as variable trough concentrations among individuals and a high medication frequency. In this study, we created NK61060, a novel micellar TAC formulation, to circumvent these disadvantages. MATERIALS & METHODS Immunosuppressive activity of NK61060 was determined in the collagen-induced arthritis rat model, mannan-induced arthritis mouse model and dextran sodium sulfate-induced colitis mouse model. The pharmacokinetics and toxicology of NK61060 were evaluated in those models. RESULTS In arthritis and colitis models, NK61060 exhibited superior immunosuppressive activity compared with that of TAC. Pharmacokinetic and toxicological analyses indicated that NK61060 had a wider safety margin and could be administered at a reduced medication frequency. CONCLUSION NK61060 mitigates the trough concentration variability and the medication frequency and it may be a safer and more effective option for use in clinical settings. Further studies are needed to determine its clinical usefulness.