Atsushi Sanbuissho

Ethylene Glycol Monomethyl Ether–Induced Toxicity Is Mediated through the Inhibition of Flavoprotein Dehydrogenase Enzyme Family

Ethylene glycol monomethyl ether (EGME) is a widely used industrial solvent known to cause adverse effects to human and other mammals. Organs with high metabolism and rapid cell division, such as testes, are especially sensitive to its actions. In order to gain mechanistic understanding of EGME-induced toxicity, an untargeted metabolomic analysis was performed in rats. Male rats were administrated with EGME at 30 and 100 mg/kg/day. At days 1, 4, and 14, serum, urine, liver, and testes were collected for analysis. Testicular injury was observed at day 14 of the 100 mg/kg/day group only. Nearly 1900 metabolites across the four matrices were profiled using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Statistical analysis indicated that the most significant metabolic perturbations initiated from the early time points by EGME were the inhibition of choline oxidation, branched-chain amino acid catabolism, and fatty acid β-oxidation pathways, leading to the accumulation of sarcosine, dimethylglycine, and various carnitine- and glycine-conjugated metabolites. Pathway mapping of these altered metabolites revealed that all the disrupted steps were catalyzed by enzymes in the primary flavoprotein dehydrogenase family, suggesting that inhibition of flavoprotein dehydrogenase–catalyzed reactions may represent the mode of action for EGME-induced toxicity. Similar urinary and serum metabolite signatures are known to be the hallmarks of multiple acyl-coenzyme A dehydrogenase deficiency in humans, a genetic disorder because of defects in primary flavoprotein dehydrogenase reactions. We postulate that disruption of key biochemical pathways utilizing flavoprotein dehydrogenases in conjugation with downstream metabolic perturbations collectively result in the EGME-induced tissue damage.

On-chip in vitro cell-network pre-clinical cardiac toxicity using spatiotemporal human cardiomyocyte measurement on a chip

To overcome the limitations and misjudgments of conventional prediction of arrhythmic cardiotoxicity, we have developed an on-chip in vitro predictive cardiotoxicity assay using cardiomyocytes derived from human stem cells employing a constructive spatiotemporal two step measurement of fluctuation (short-term variability; STV) of cells repolarization and cell-to-cell conduction time, representing two origins of lethal arrhythmia. Temporal STV of field potential duration (FPD) showed a potential to predict the risks of lethal arrhythmia originated from repolarization dispersion for false negative compounds, which was not correctly predicted by conventional measurements using animal cells, even for non-QT prolonging clinical positive compounds. Spatial STV of conduction time delay also unveiled the proarrhythmic risk of asynchronous propagation in cell networks, whose risk cannot be correctly predicted by single-cell-based measurements, indicating the importance of the spatiotemporal fluctuation viewpoint of in vitro cell networks for precise prediction of lethal arrhythmia reaching clinical assessment such as thorough QT assay.

Practical Application of Toxicogenomics for Profiling Toxicant-Induced Biological Perturbations

A systems-level understanding of molecular perturbations is crucial for evaluating chemical-induced toxicity risks appropriately, and for this purpose comprehensive gene expression analysis or toxicogenomics investigation is highly advantageous. The recent accumulation of toxicity-associated gene sets (toxicogenomic biomarkers), enrichment in public or commercial large-scale microarray database and availability of open-source software resources facilitate our utilization of the toxicogenomic data. However, toxicologists, who are usually not experts in computational sciences, tend to be overwhelmed by the gigantic amount of data. In this paper we present practical applications of toxicogenomics by utilizing biomarker gene sets and a simple scoring method by which overall gene set-level expression changes can be evaluated efficiently. Results from the gene set-level analysis are not only an easy interpretation of toxicological significance compared with individual gene-level profiling, but also are thought to be suitable for cross-platform or cross-institutional toxicogenomics data analysis. Enrichment in toxicogenomics databases, refinements of biomarker gene sets and scoring algorithms and the development of user-friendly integrative software will lead to better evaluation of toxicant-elicited biological perturbations.

Role of connexin 32 in acetaminophen toxicity in a knockout mice model

Gap junctional intercellular communication (GJIC), by which glutathione (GSH) and inorganic ions are transmitted to neighboring cells, is recognized as being largely involved in toxic processes of chemicals. We examined acetaminophen (APAP)-induced hepatotoxicity clinicopathologically using male wild-type mice and mice lacking the gene for connexin32, a major gap junction protein in the liver [knockout (Cx32KO) mice]. When APAP was intraperitoneally administered at doses of 100, 200, or 300mg/kg, hepatic centrilobular necrosis with elevated plasma aminotransferase activities was observed in wild-type mice receiving 300mg/kg, and in Cx32KO mice given 100mg/kg or more. At 200mg/kg or more, hepatic GSH and GSSG contents decreased significantly and the effect was more severe in wild-type mice than in Cx32KO mice. On the other hand, markedly decreased GSH staining was observed in the hepatic centrilobular zones of Cx32KO mice compared to that of wild-type mice. These results demonstrate that Cx32KO mice are more susceptible to APAP hepatotoxicity than wild-type mice, and indicate that the distribution of GSH of the centrilobular zones in the hepatic lobules, rather than GSH and GSSG contents in the liver, is important in APAP hepatotoxicity. In conclusion, Cx32 protects against APAP-induced hepatic centrilobular necrosis in mice, which may be through the GSH transmission to neighboring hepatocytes by GJIC.

Evaluation of primary and secondary responses to a T-cell-dependent antigen, keyhole limpet hemocyanin, in rats

To develop a rat T-cell-dependent antibody response (TDAR) model evaluating both primary and secondary antibody responses, keyhole limpet hemocyanin (KLH) was used to immunize rats twice during a 14-day course of study, a pattern closely linked to that of a short-term general toxicity study. Female rats of four representative strains (e.g., Sprague-Dawley, Wistar, Fischer, and Lewis) were immunized twice with intravenous administrations of KLH (300 µg/rat) on Days 5 and 9 during a 14-day treatment regimen with cyclophosphamide (CPA) at 1, 3, or 6 mg/kg/day. The primary and secondary immunizations of KLH markedly elevated serum anti-KLH IgM and IgG levels in all strains on Days 9 and 15. Remarkable higher levels of anti-KLH IgG (≈ 1000 µg/ml) were noted in all strains, which were more than 4-times compared with those of anti-KLH IgM levels at Day 9, indicating that predominant IgG reactions were induced by the dual immunizations. A large inter-individual variability in KLH-specific IgM and IgG production was observed in all strains. However, levels of the KLH-specific antibodies were considered sufficient for the evaluation, even in Sprague-Dawley and Wistar rats reported as strains with a wide range of variability since immunosuppression of CPA on responses in both anti-KLH IgM and IgG were observed in all strains to the same extent. In addition, the sensitivity of the KLH-ELISA assay system detecting the immunosuppressive effects of CPA was comparable to other assay systems with PFC assay or ELISA using SRBC. The results here demonstrated that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in rats when exposed to potential immunomodulatory drugs. Furthermore, the design of the presented TDAR study would support comprehensive evaluation together with the outcome of the conventional general toxicity study.

Chondrotoxicity and toxicokinetics of novel quinolone antibacterial agents DC-159a and DX-619 in juvenile rats

Quinolone antibacterial agents are extensively utilized in antimicrobial chemotherapy. However, they have been reported to induce arthropathy in juvenile animals, and the mechanism has not been clarified. Recently, we have demonstrated that Dusp1, Tnfrsf12a, Ptgs2, Fos, Mt1a, Plaur, Mmp3, Sstr1 and Has2 genes change in the articular cartilage of juvenile rats with a single oral administration of ofloxacin (OFLX), suggesting that these genes are involved in the induction of OFLX-induced chondrotoxicity. In the present study, to compare the chondrotoxic potential between new synthesized quinolones DC-159a and DX-619, and OFLX, they were orally administered by gavage at a dose level of 300 or 900 mg/kg/day to male juvenile Sprague-Dawley (SD) rats, 3 weeks of age, for 7 days. Then the distal humerus and femur were subjected to microscopic examination. Moreover, concentrations of these quinolones in the femoral articular cartilage were measured in male juvenile SD rats following a single oral administration at 100, 300 or 900 mg/kg. Furthermore, gene expression of Dusp1, Tnfrsf12a, Ptgs2, Fos, Mt1a, Plaur, Mmp3, Sstr1 and Has2 was investigated in the articular cartilage of the distal femur in male juvenile SD rats treated with 900 mg/kg of DC-159a or DX-619 by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis. In a microscopic examination, no changes in the articular cartilage were observed in any animal administered DC-159a or DX-619. On the contrary, cavity formation and chondrocyte cluster in the cartilage of distal humerus and femur were noted in animals receiving OFLX at 300 mg/kg/day or more. In toxicokinetic analysis, the maximum concentration (C(max)) value in the articular cartilage (cartilage C(max)) of DC-159a or DX-619 at 900 mg/kg was lower than that of OFLX at 300 mg/kg. However, the area under the cartilage concentration-time curve (cartilage AUC)(0-24h) value of DC-159a or DX-619 at 900 mg/kg was higher than that of OFLX at 300 mg/kg. In qRT-PCR analysis, up-regulated Dusp1, Fos and Mt1a, and down-regulated Sstr1 and Has2 genes were seen in the femoral articular cartilage of rats given DX-619 or DC-159a at 900 mg/kg. However, Tnfrsf12a, Ptgs2, Plaur and Mmp3 genes, which were up-regulated in the distal femoral articular cartilage exposed to OFLX, did not increase or slightly increased. In conclusion, the penetration of DC-159a or DX-619 into the cartilage was low compared with that of OFLX, and no obvious changes in Tnfrsf12a, Ptgs2, Plaur and Mmp3 genes were observed in the articular cartilage of juvenile rats treated with DC-159a or DX-619, which was likely to be responsible for non-chondrotoxic potentials of DC-159a and DX-619.

Gene set-level network analysis using a toxicogenomics database

Toxicogenomics data sets on rat livers covering 118 compounds were subjected to inference of a gene set-level, not individual gene-level, network structure. Expression changing levels for 58 gene sets was used for network inference with a Gaussian graphical model algorithm. The established network contained reasonable relationships, such as ones between the blood glucose level and glycolysis-related genes or the blood transaminase level and cellular injury-related genes, indicating that the gene set-level network inference successfully highlighted biological pathway-level interactions. In addition, the robustness of the inferred network structure was investigated using microarray data on bromobenzene-treated rat livers, where the gene set-level activation exhibited time-dependent propagation through neighbored nodes (i.e. gene sets) on the network, indicating that the network structure was robust and comparable with an external microarray data set. Accumulating such robust gene sets with toxicity-associated subnetwork structures would lead to a better understanding of the molecular mechanisms of drug-elicited toxicities.

Structural and numerical chromosome aberration inducers in liver micronucleus test in rats with partial hepatectomy.

The liver micronucleus test is an important method to detect pro-mutagens such as active metabolites not reaching bone marrow due to their short lifespan. We have already reported that dosing of the test compound after partial hepatectomy (PH) is essential to detect genotoxicity of numerical chromosome aberration inducers in mice [Mutat. Res. 632 (2007) 89-98]. In naive animals, the proportion of binucleated cells in rats is less than half of that in mice, which suggests a species difference in the response to chromosome aberration inducers. In the present study, we investigated the responses to structural and numerical chromosome aberration inducers in the rat liver micronucleus test. Two structural chromosome aberretion inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and two numerical chromosome aberration inducers (colchicine and carbendazim) were used in the present study. PH was performed a day before or after the dosing of the test compound in 8-week old male F344 rats and hepatocytes were isolated 4 days after the PH. As a result, diethylnitrosamine and 1,2-dimethylhydrazine, structural chromosome aberration inducers, exhibited significant increase in the incidence of micronucleated hepatocyte (MNH) when given either before and after PH. Colchicine and carbendazim, numerical chromosome aberration inducers, did not result in any toxicologically significant increase in MNH frequency when given before PH, while they exhibited MNH induction when given after PH. It is confirmed that dosing after PH is essential in order to detect genotoxicity of numerical chromosome aberration inducers in rats as well as in mice. Regarding the species difference, a different temporal response to colchicine was identified. Colchicine increased the incidence of MNH 4 days after PH in rats, although such induction in mice was observed 8-10 days after PH.

Toxicogenomic investigation on rat testicular toxicity elicited by 1,3-dinitrobenzene

Rats were treated with a single oral dose of 10, 25 and 50mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli-germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity.

Mixed Germ Cell Tumor with Embryonal Carcinoma, Choriocarcinoma, and Epithelioid Trophoblastic Tumor in the Ovary of a Cynomolgus Monkey

A seven-year-old female cynomolgus monkey had a mass in the left ovary with metastasis to the lung and the right ovary. The mass of these organs showed three different characteristics, and its immunohistochemical profiles were consistent with embryonal carcinoma (EC), choriocarcinoma (CC), and epithelioid trophoblastic tumor (ETT). The EC was characterized with sheets and glandlike structures with large pleomorphic, single-nucleated epithelial cells that were immunohistochemically positive for α-fetoprotein, octamer-4, and CD30, and with multinucleated giant cells resembling syncytiotrophoblasts. The CC also represented biphasic proliferation of the cytotrophoblast positive for cytokeratin 7 (CK7), which showed negative immunoreactivity for all three of the above antibodies, and it was syncytiotrophoblast positive for human chorionic gonadotropin. The ETT showed numerous floating cells in an abundant eosinophilic extracellular matrix with vacuolated or eosinophilic cytoplasm and was immunohistochemically positive for CK7, p63, and α-inhibin, which features nodule or cordlike structures. Collectively, this neoplasm was identified as a mixed germ cell tumor with EC, CC, and ETT. To our knowledge, this is the first report of EC in nonhuman primates as a component of mixed germ cell tumor.