Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

Triple-negative breast cancer (TNBC) is a highly aggressive cancer for which targeted therapeutic agents are limited. Growing evidence suggests that TNBC originates from breast cancer stem cells (BCSCs), and elucidation of the molecular mechanisms controlling BCSC proliferation will be crucial for new drug development. We have previously reported that the lysosphingolipid sphingosine-1-phosphate mediates the CSC phenotype, which can be identified as the ALDH-positive cell population in several types of human cancer cell lines. In this study, we have investigated additional lipid receptors upregulated in BCSCs. We found that lysophosphatidic acid (LPA) receptor 3 was highly expressed in ALDH-positive TNBC cells. The LPAR3 antagonist inhibited the increase in ALDH-positive cells after LPA treatment. Mechanistically, the LPA-induced increase in ALDH-positive cells was dependent on intracellular calcium ion (Ca2+), and the increase in Ca2+ was suppressed by a selective inhibitor of transient receptor potential cation channel subfamily C member 3 (TRPC3). Moreover, IL-8 production was involved in the LPA response via the activation of the Ca2+-dependent transcriptional factor nuclear factor of activated T cells. Taken together, our findings provide new insights into the lipid-mediated regulation of BCSCs via the LPA-TRPC3 signaling axis and suggest several potential therapeutic targets for TNBC.

DOI： 10.3390/ijms23041967

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.

DOI： 10.1002/jat.4268

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jat.4268

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

Coronavirus disease 2019 (COVID-19) continues to spread across the globe, with numerous clinical trials underway seeking to develop and test effective COVID-19 therapies, including remdesivir. Several ongoing studies have reported hydroxychloroquine-induced cardiotoxicity, including development of torsade de pointes (TdP). Meanwhile, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to serve as a tool for assessing drug-induced cardiotoxicity, such as TdP and contraction impairment. However, the cardiotoxicity of COVID-19 treatments has not been fully assessed using hiPSC-CMs. In this study, we focused on drug repurposing with various modes of actions and examined the TdP risk associated with COVID-19 treatments using field potential using multi-electrode array system and motion analysis with hiPSC-CMs. Hydroxychloroquine induced early after depolarization, while remdesivir, favipiravir, camostat, and ivermectin had little effect on field potentials. We then analyzed electromechanical window, which is defined as the difference between field potential and contraction-relaxation durations. Hydroxychloroquine decreased electromechanical window of hiPSC-CMs in a concentration-dependent manner. In contrast, other drugs had little effect. Our data suggest that hydroxychloroquine has proarrhythmic risk and other drugs have low proarrhythmic risk. Thus, hiPSC-CMs represent a useful tool for assessing the comprehensive cardiotoxicity caused by COVID-19 treatments in nonclinical settings.

DOI： 10.1093/toxsci/kfab079

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.yrtph.2021.104963

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Growing evidence suggests that breast cancer originates from a minor population of cancer cells termed cancer stem cells (CSCs), which can be identified by aldehyde dehydrogenase (ALDH) activity-based flow cytometry analysis. However, novel therapeutic drugs for the eradication of CSCs have not been discovered yet. Recently, drug repositioning, which finds new medical uses from existing drugs, has been expected to facilitate drug discovery. We have previously reported that sphingosine kinase 1 (SphK1) induced proliferation of breast CSCs. In the present study, we focused on the immunosuppressive agent FTY720 (also known as fingolimod or Gilenya), since FTY720 is known to be an inhibitor of SphK1. We found that FTY720 blocked both proliferation of ALDH-positive cells and formation of mammospheres. In addition, we showed that FTY720 reduced the expression of stem cell markers such as Oct3/4, Sox2 and Nanog via upregulation of protein phosphatase 2A (PP2A). These results suggest that FTY720 is an effective drug for breast CSCs in vitro.

DOI： 10.3390/ijms22147259

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.fct.2021.112206

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

The Amino acid Derivative Reactivity Assay (ADRA) is a convenient and effective in chemico test method for assessing covalent binding of test chemicals with protein-derived nucleophilic reagents as a means of predicting skin sensitization potential. Although the original molar-concentration approach to ADRA testing was not suitable for testing multiconstituent substances of an unknown composition, a weight-concentration approach that is suitable for such substances was developed, which also led to the realization that test chemical solutions prepared to molar concentrations higher than the original 1 mM would reduce false negative results as well as enhance predictive capacity. The present study determined an optimal molar-concentration that achieves even higher predictive capacity than the original ADRA. Eight chemicals that were false negatives when tested with 1 mM test chemical solutions were retested with test chemical solutions between 2 and 5 mM, which showed 4 mM to be the optimal molar-concentration for ADRA testing. When 82 chemicals used in the original development were retested with 4 mM test chemical solutions, false negative results were reduced by four. When an additional 85 chemicals used to evaluate the weight-concentration approach to ADRA were retested, the results essentially replicated those obtained with 0.5 mg/ml test chemical solutions and gave 10 fewer false negatives than original ADRA with 1 mM solutions. A comparison of these results for 136 chemicals showed that ADRA testing with 4 mM solutions achieved a four percentage point improvement in accuracy over original ADRA and a two percentage point improvement over DPRA testing.

DOI： 10.1002/jat.4082

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Toxicology  

Predicting drug-induced side effects in the cardiovascular system is very important because it can lead to the discontinuation of new drugs/candidates or the withdrawal of marketed drugs. Although chronic assessment of cardiac contractility is an important issue in safety pharmacology, an in vitro evaluation system has not been fully developed. We previously developed an imaging-based contractility assay system to detect acute cardiotoxicity using human iPS cell-derived cardiomyocytes (hiPSC-CMs). To extend the system to chronic toxicity assessment, we examined the effects of the anti-hepatitis C virus (HCV) drug candidate BMS-986094, a guanosine nucleotide analogue, which was withdrawn from phase 2 clinical trials because of unexpected contractility toxicities. Additionally, we examined sofosbuvir, another nucleotide analogue inhibitor of HCV that has been approved as an anti-HCV drug. Motion imaging analysis revealed the difference in cardiotoxicity between the cardiotoxic BMS-986094 and the less toxic sofosbuvir in hiPSC-CMs, with a minimum of 4 days of treatment. In addition, we found that BMS-986094-induced contractility impairment was mediated by a decrease in calcium transient. These data suggest that chronic treatment improves the predictive power for the cardiotoxicity of anti-HCV drugs. Thus, hiPSC-CMs can be a useful tool to assess drug-induced chronic cardiotoxicity in non-clinical settings.

DOI： 10.2131/jts.46.359

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

The amino acid derivative reactivity assay (ADRA), which is an in chemico alternative to the use of animals in testing for skin sensitization potential, offers significant advantages over the direct peptide reactivity assay (DPRA) in that it utilizes nucleophilic reagents that are sensitive enough to be used with test chemical solutions prepared to concentrations of 1 mm, which is one-hundredth that of DPRA. ADRA testing of hydrophobic or other poorly soluble compounds requires that they be dissolved in a solvent consisting of dimethyl sulfoxide (DMSO) and acetonitrile. DMSO is known to promote dimerization by oxidizing thiols, which then form disulfide bonds. We investigated the extent to which DMSO oxidizes the cysteine-derived nucleophilic reagents used in both DPRA and ADRA and found that oxidation of both N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC) and cysteine peptide increases as the concentration of DMSO increases, thereby lowering the concentration of the nucleophilic reagent. We also found that use of a solvent consisting of 5% DMSO in acetonitrile consistently lowered NAC concentrations by about 0.4 μm relative to the use of solvents containing no DMSO. We also tested nine sensitizers and four nonsensitizers having different sensitization potencies to compare NAC depletion with and without 5% DMSO and found that reactivity was about the same with either solvent. Based on the above, we conclude that the use of a solvent containing 5% DMSO has no effect on the accuracy of ADRA test results. We plan to review and propose revisions to OECD Test Guideline 442C based on the above investigation.

DOI： 10.1002/jat.3948

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.yrtph.2019.104482

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The amino acid derivative reactivity assay (ADRA) is an in chemico alternative method that focuses on protein binding as the molecular initiating event for skin sensitization. It is a simple and versatile method that has successfully solved some of the problems of the direct peptide reactivity assay (DPRA). The transferability and within- and between-laboratory reproducibility of ADRA were evaluated and confirmed as part of a validation study conducted at four participating laboratories. The transfer of ADRA technology from the lead laboratory to the four participating laboratories was completed successfully during a two-step training program, after which the skin sensitization potentials of 40 coded chemicals were predicted based on the results of ADRA testing. Within-laboratories reproducibility was 100% (10 of 10), 100% (10 of 10), 100% (7 of 7) and 90% (9 of 10), or an average of 97.3% (36 of 37); between-laboratory reproducibility as calculated on the results of three laboratories at the time was 91.9%. The overall predictive capacity comprised an accuracy of 86.9%, sensitivity of 81.5% and specificity of 98.1%. These results satisfied the targets set by the validation management team for demonstrating transferability, within- and between-laboratory reproducibility, and predictive capacity as well as gave a clear indication that ADRA is easily transferable and sufficiently robust to be used in place of DPRA.

DOI： 10.1002/jat.3834

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative assay for skin sensitization listed in OECD test guideline 442C. ADRA evaluates the reactivity of sensitizers to proteins, which is key event 1 in the skin sensitization adverse outcome pathway. Although the current key event 1 evaluation method is a simple assay that evaluates nucleophile and test chemical reactivity, mixtures of unknown molecular weights cannot be evaluated because a constant molar ratio between the nucleophile and test chemical is necessary. In addition, because the nucleophile is quantified by HPLC, the frequency of co-eluting the test chemical and nucleophile increases when measuring multi-component mixtures. To solve these issues, test conditions have been developed using a 0.5 mg/mL test chemical solution and fluorescence-based detection. Since the practicality of these methods has not been substantiated, a validation test to confirm reproducibility was conducted in this study. The 10 proficiency substances listed in the ADRA guidelines were tested three times at five different laboratories. The results of both within- and between-laboratory reproducibility were 100%, and the results of ultraviolet- and fluorescence-based measurements were also consistent. In addition to the proficiency substances, a new positive control, squaric acid diethyl ester, was tested three times at the five laboratories. The results showed high reproducibility with N-(2-(1-naphthyl)acetyl)-l-cysteine depletion of 37%-52% and α-N-(2-(1-naphthyl)acetyl)-l-lysine depletion of 99%-100%. Thus, high reproducibility was confirmed in both evaluations of the 0.5 mg/mL test chemical and the fluorescence-based measurements, validating the practicability of these methods.

DOI： 10.1002/jat.3834

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.vascn.2019.01.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA-N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC)-was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu2+ promoted NAC oxidation significantly. When 0.25 μm of EDTA was added in the presence of Cu2+ , NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.

DOI： 10.1002/jat.3707

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Toxicology  

Amino acid derivative reactivity assay (ADRA) has previously been developed as an alternative method to direct peptide reactivity assay (DPRA) to evaluate key event 1 in skin sensitization mechanisms. However, when using alternative methods for skin sensitization, integrated approaches to testing and assessment (IATA) that combine the results of multiple tests evaluating different key events are generally required. To verify whether ADRA can be used in IATA, we replaced DPRA with ADRA in five IATA methods combining DPRA, KeratinoSens, and h-CLAT: (i) the “2 out of 3” approach, (ii) the “3 out of 3” approach, (iii) sequential testing strategy (STS), (iv) integrated testing strategy by scoring approach (ITS-SA), and (v) the “ITS by two methods approach” (ITS-2MA). The prediction accuracy of the “2 out of 3” approach using ADRA (1 mM) and ADRA (0.5 mg/mL) was 90.0% and 91.1%, respectively, for human data, and was very similar to that obtained using DPRA (91.1%). The “3 out of 3” approach also showed good predictability (83.2%) using either ADRA (1 mM) or ADRA (0.5 mg/mL) compared to DPRA. Regarding the accuracy of the prediction of sensitization intensity for the human data by the third classification, prediction accuracy using ADRA was almost the same as STS, ITS-SA, or ITS-2MA using DPRA. As a result, this study showed that ADRA can be used as a test method for key event 1 in the evaluation of skin sensitization by combining multiple alternative methods.

DOI： 10.2131/jts.44.585

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Publishing type：Research paper (scientific journal)   Publisher：Food Safety Commission, Cabinet Office  

DOI： 10.14252/foodsafetyfscj.2018017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

4-Benzylphenol (CAS No. 101-53-1), a structural analog of bisphenol F, has estrogenic activity in vitro and in vivo, as is the case with bisphenol F. 4-Benzylphenol is used in plastics and during organic synthesis. Since its safety is largely unknown, we conducted toxicity tests as part of screening risk assessment in an existing chemical safety survey program. Based on results of the Ames test and the chromosomal aberration test using Chinese hamster lung cells (OECD TG 471 and 473), 4-benzylphenol was determined to be non-genotoxic in vitro. In a 28-day repeated-dose toxicity study, Crl:CD (SD) rats were administrated 4-benzylphenol by gavage at 0, 30, 150, or 750 mg/kg/day (OECD TG 407). Consequently, body weight was lower in males at 750 mg/kg/day. In the liver, relative organ weights were increased in both sexes at 750 mg/kg/day, and centrilobular hepatocellular hypertrophy was observed in males at 150 and 750 mg/kg/day. In the forestomach, hyperkeratosis and hyperplasia of squamous cells were observed in males at 150 and 750 mg/kg/day, and in females at 750 mg/kg/day. Based on these results, we identified the NOAEL for 4-benzylphenol as 30 mg/kg/day, with a hazard assessment value (D-value) of 0.05 mg/kg/day corresponding to hazard class 3.

DOI： 10.1016/j.yrtph.2018.04.017

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Publishing type：Research paper (scientific journal)   Publisher：Food Safety Commission, Cabinet Office  

DOI： 10.14252/foodsafetyfscj.2017023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

2-(2'-Hydroxy-3',5'-di-tert-butylphenyl)benzotriazole (HDBB), the Benzotriazole UV-stabilizer (BUVSs) known as UV-320, is widely used in plastic materials for protection against UV-irradiation. Previously, we reported that oral ingestion of HDBB induce hepatotoxicity including hepatocyte hypertrophy and necrosis in rats and, males was more susceptible compared with females in young rats while no sex-related difference was observed in preweaning rats. Phenotypes observed in our previous study imply involvement of peroxisome proliferator-activated receptor (PPAR) alpha in HDBB hepatotoxicity, however, direct evidence that HDBB can activate PPAR alpha has not been provided and the mechanism which underlying the gender difference of HDBB hepatotoxicity was not clearly elucidated. Here, we conduct transcriptome analysis using microarray expression profiles in the livers of rats administered HDBB. PPAR alpha agonist activity of HDBB was elucidated by comparison with gene expression data of typical PPAR alpha agonist, i.e. clofibrate, WY-14643, gemfibrozil, and fenofibrate, from TG GATEs database. Moreover, we analyzed for PPAR alpha mRNA expression in the liver of developing male and female rats. PPAR alpha mRNA expression level was higher in males than in females on postnatal days (PNDs) 28 and 35, whereas no sex-related difference was found on PNDs 7 and 22. These results suggest that HDBB exerts its hepatotoxicity through the PPAR alpha signal pathway and the sex-related difference in PPAR alpha expression may contribute to the sex-related difference in susceptibility to hepatotoxicity.

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

DOI： 10.14869/toxpt.43.1.0_P-204

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

DOI： 10.14869/toxpt.43.1.0_S7-2

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Language：English   Publisher：The Japanese Society of Toxicology  

Perfluoroalkyl carboxylic acids (PFCAs) are global environmental contaminants that are the cause of concern due to their possible effects on wildlife and human health. Since few studies have investigated the toxicity of long-chain PFCAs, we have performed combined repeated dose toxicity studies with the reproduction/developmental toxicity screening tests. We previously examined perfluoroundecanoic acid (C11), perfluorododecanoic acid (C12), and perfluorooctadecanoic acid (C18). We herein reported our results for perfluorotetradecanoic acid (PFTeDA; C14) and perfluorohexadecanoic acid (PFHxDA: C16). Male and female rats were administered PFTeDA at 1, 3 or 10 mg/kg/day or PFHxDA at 4, 20 or 100 mg/kg/day by gavage, and each female was then mated with a male in the same dose group after 14 days. Males were dosed for a total of 42 days and females were dosed throughout the gestation period until day 5 after parturition. PFTeDA and PFHxDA caused hepatocyte hypertrophy and/or fatty changes in the liver at the middle and high doses. PFTeDA also induced follicular cell hypertrophy in the thyroid at the middle and high doses. The only reproductive/developmental effect observed was an inhibited postnatal body weight gain in pups in the 10 mg/kg/day PFTeDA group. Based on these results, the NOAELs for the repeated dose and reproductive/developmental toxicity were concluded to be 1 and 3 mg/kg/day for PFTeDA and 4 and 100 mg/kg/day for PFHxDA, respectively. Our current and previous results indicate that the toxicity of PFCAs decreases with increases in the carbon chain length from 12 to 18.

DOI： 10.2131/fts.2.177

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Other Link： http://search.jamas.or.jp/link/ui/2016381693

Language：English   Publisher：The Japanese Society of Toxicology  

To obtain information on the possible repeated-dose oral toxicity of β-bromostyrene and its reversibility, Crl: CD (SD) rats were administered β-bromostyrene through gavage at 0, 30, 125, and 500 mg/kg/day once for 28 days, followed by a 14-day recovery period. In the 500 mg/kg group, decrease in spontaneous movement was observed in all males and females on the first dosing day, and one female rat died on Day 3. There were no significant changes in body weight or food consumption. An increase in urine volume and decrease in urine osmolality were observed in males receiving 125 mg/kg and above, and an increase in urine volume was observed in females receiving 500 mg/kg. On blood biochemical examination, increases in total cholesterol, phospholipids, triglycerides, total protein, albumin, inorganic phosphorus, and/or chlorine were observed in the 125 and/or 500 mg/kg groups. Histopathologically, eosinophilic bodies of tubular cells and/or renal tubular degeneration were observed in the kidneys of males in the 125 and 500 mg/kg groups. In the thyroid, hypertrophy of follicular cells was observed in females receiving 125 mg/kg and above and males receiving 500 mg/kg. Furthermore, centrilobular hepatocellular hypertrophy was observed in both sexes receiving 500 mg/kg. These changes observed at the end of the dosing period disappeared or were reduced after the recovery period. Based on these results, the no-observed-adverse-effect-level of β-bromostyrene was judged to be 30 mg/kg/day for both sexes.

DOI： 10.2131/fts.2.191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

Recent studies have shown. that epigenetic alterations correlate with carcinogenesis in various tissues. Identification of these alterations might help characterize the early stages of carcinogenesis. We comprehensively analyzed DNA methylation and gene expression in livers obtained from rats exposed to nitrosodiethylamine (DEN) followed by a promoter of hepatic carcinogenesis, phenobarbital (PB). The combination of DEN and PB induced marked increases in number and area of glutathione S-transferase-placental form (GST-P)-positive foci in the liver. In the liver of rats that received 30 mg/kg of DEN, pathway analysis revealed alterations of common genes in terms of gene expression and DNA methylation, and that these alterations were related to immune responses. Hierarchical clustering analysis of the expression of common genes from public data obtained through the Toxicogenomics Project-Genomics Assisted Toxicity Evaluation system (TG-GATEs) showed that carcinogenic compounds clustered together. MBD-seq and GeneChip analysis indicated that major histocompatibility complex class Ib gene RT1-CE5, which has an important role in antigen presentation, was hypomethylated around the promoter region and specifically induced in the livers of DEN-treated rats. Further, immunohistochemical analysis indicated that the co-localization of GST-P and protein homologous to RT1-CE5 was present at the foci of some regions. These results suggest that common genes were altered in terms of both DNA methylation and expression in livers, with preneoplastic foci indicating carcinogenic potential, and that immune responses are involved in early carcinogenesis. In conclusion, the present study identified a specific profile of DNA methylation and gene expression in livers with preneoplastic foci. Early epigenetic perturbations of immune responses might correlate with the early stages of hepatocarcinogenesis.

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We previously reported a toxicogenomics-based prediction model for hepatocarcinogens in which the expression patterns of signature genes following repeated doses of either genotoxic or non-genotoxic compounds were similar. Based on the results of our prediction model, we hypothesized that repeated doses of non-genotoxic carcinogens might have initiating potential. Here, we conducted a two-stage hepatocarcinogenesis study in rats exposed to the initiating agent nitrosodiethylamine (DEN), and hepatotoxic compounds thioacetamide (TAA), methapyrilene (MP) and acetaminophen (APAP) for 1-2 weeks followed by the liver tumor promoter phenobarbital (PB). The duration of initial treatment was determined based on positive results from our prediction model. Combined treatment of 3 or 30 mg/kg of genotoxic DEN and PB induced marked increases in altered hepatocellular foci and a DEN dose-dependent increase in the number and area of glutathione S-transferase-placental form (GST-P)-positive foci. A low number of altered hepatocellular foci were also observed in rats treated with TAA at a dose of 45 mg/kg. MP at a dose of 100 mg/kg induced a very low number of foci, but APAP did not. Hierarchical clustering analysis using gene expression data revealed that 2-week treatment with TAA at a dose of 30 mg/kg and MP at 45 mg/kg induced specific expression of DNA damage-related genes, similar to 1-week treatment with DEN at a dose of 30 mg/kg. These results suggest that TAA and MP induce DNA damage, which partially supports our hypothesis. Although this study does not indicate whether tumor growth in response to these compounds can be assessed in this model, our results suggest that cumulative treatment with non-genotoxic TAA might have initiating potential in the liver.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

Perfluoroalkyl acids (PFAAs) are environmental contaminants that have received attention because of their possible effects on wildlife and human health. In order to obtain initial risk information on the toxicity of perfluoroundecanoic acid (PFUA), we conducted a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD test guideline 422). PFUA was administered by gavage to rats at 0 (vehicle: corn oil), 0.1, 0.3 or 1.0 mg/kg/day. At 1.0 mg/kg/day, body weight gain was inhibited in both sexes, and there was a decrease in fibrinogen in both sexes and shortening of the activated partial thromboplastin time in males. An increase in blood urea nitrogen and a decrease in total protein in both sexes and increases in alkaline phosphatase and alanine transaminase and a decrease in albumin in males were observed at 1.0 mg/kg/day. Liver weight was increased in males at 0.3 mg/kg/day and above and in females at 1.0 mg/kg/day, and this change was observed after a recovery period. In both sexes, centrilobular hypertrophy of hepatocytes was observed at 0.3 mg/kg/day and above and focal necrosis was observed at 1.0 mg/kg/day. In reproductive/developmental toxicity, body weight of pups at birth was lowered and body weight gain at 4 days after birth was inhibited at 1.0 mg/kg/day, while no dose-related changes were found in the other parameters. Based on these findings, the no observed adverse effect levels (NOAELs) for the repeated dose and reproductive/developmental toxicity were considered to be 0.1 mg/kg/day and 0.3 mg/kg/day, respectively.

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Hypothyroidism induced by xenobiotic treatment was analyzed for possible underlying mechanism(s) on the basis of different responses of the thyroid gland and the liver, using a newly-created database of repeated-dose toxicity of 500 chemicals. Two mechanisms are proposed: direct inhibition of thyroid hormone biosynthesis in the thyroid gland, and stimulated degradation of thyroid hormone by induction of hepatic drug-metabolizing enzymes. In the database there were 10 chemicals inducing hypertrophy/hyperplasia of follicular cells in the thyroid gland and having data on thyroid glands. On the basis of the chemical structure and information available in the literature, we judged three chemicals to be typical thioamide derivatives that act directly on the thyroid gland, and the others as non-thioamide derivatives that were unlikely to have any direct action on the thyroid gland. All these chemicals were classified into two groups using the ratios of relative weight increase rate of thyroid gland versus that of the liver. These values were at least 1.7, but 3.2 or more in the most of the cases for thioamide derivatives, and 1.2 or less for non-thioamide derivatives. This background analysis suggests the feasibility of parameter-supported speculation on the possible underlying mechanism when new repeated-dose toxicity data on hypothyroidism becomes available.

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In order to develop guidelines for setting acute reference doses (ARfDs) for pesticides in Japan, we conducted simulations of ARfD settings based on evaluation reports for 201 pesticides assessed by the Food Safety Commission (FSC) in Japan over the last 8 years. Our conceptual principles were based on the concepts written by Solecki et al. (2005) and were adapted for toxicological data required in Japan. Through this process, we were able to set the ARfDs for over 90% of the 201 pesticides tested. The studies that provided the rationale for ARfD setting were primarily reproductive and developmental toxicity studies, acute neurotoxicity studies, and pharmacology studies. For approximately 30% of the pesticides simulated in the present study, it was not necessary to establish ARfDs. Some of the simulated ARfDs resulting from their endpoints may be conservative estimates, because the evaluation reports were written for acceptable daily intake settings. Thus, it was sometimes difficult to distinguish acute toxic alerts from repeated toxicities. We were unable to set an ARfD for 14 pesticides because of insufficient data on acute toxicities. This could be improved by more complete recordkeeping. Furthermore, we categorized the 201 pesticides by mechanism of action or chemical structure. Our simulation indicates that the conceptual framework presented here can be used as a basis for the development of guidelines on ARfD settings for pesticides in Japan.

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The concern over endocrine disruptors prompted international establishment of a strategic framework for the identification of the estrogenic compounds. OECD has launched the Conceptual Framework tool box containing various screening and testing methods including the uterotrophic assay. The (anti)estrogenicity of 36 chemicals suspected to be estrogen-receptor interactive by in silico and/or in vitro screening in the Extended Scheme for Endocrine Disruptor Screening and Testing of the Ministry of Health, Labour and Welfare, Japan, were monitored by the uterotrophic assay using C57BL/6J ovariectomized adult female mice after a 7-day exposure by oral gavage (po) and subcutaneous injection (sc). Ethynyl estradiol was used as reference for agonist and antagonist detection. In addition, Bisphenol A (sc) and Genistein (po) were tested for the comparison to rat assays. Among the 36, 2-[Bis(4-hydroxyphenyl)methyl]benzylalcohol, 2,2',4,4'-Tetrahydroxybenzophenone, 2,4-Dihydroxybenzophenone, 3,3',5-Triiodothyroacetic acid, New fuchsin and alpha-Naphtholbenzein, showed both estrogenic agonistic and antagonistic activities; first two showed U-shaped dose-response in antagonistic studies. N,N-Diphenyl-p-phenylenediamine, 2,2'-Dihydroxy-4,4'-dimethoxybenzophenone, n-Butyl 4-hydroxybenzoate, and Reserpine were agonistic by sc. Benzo at pyrene, Benz [a] anthracene, Dibenz [a,h] anthracene, 2-(2H-Benzotriazol-2-yl)-4,6-di(t-pentyl)phenol. Rosemarinic acid, meta-Thymol, 6-Gingerol, Colchicine, Malachite green base, Fenbuconazole, and Lead acetate were antagonistic. The rest, i.e. n-Heptyl 4-hydroxybenzoate, Tetrazolium violet, Pravastatin sodium salt. Physostigmine, salicylate (1:1), Nordihydroguaiaretic acid, o-Cresolphthalein, 1,3-Dinitrobenzene, C.I. Pigment orange, Tetrabromobisphenol-A, 2-Hydroxy-4-methoxybenzophenone, Ethylparaben, Propyl p-hydroxybenzoate, Kaempferol, 2-(2-Benzotriazolyl)-p-cresol and Phenolphthalein were negative for both effects. Taking together with in silico/ in vitro screening, the result suggested that the ovariectomized mouse uterotrophic bioassay has sufficient performance comparable to rat for the screening of (anti)estrogenicity of various chemicals.

DOI： 10.2131/jts.37.879

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An important technology used in toxicogenomic drug discovery research is the microarray, which enables researchers to simultaneously analyze the expression of a large number of genes. To build a database and data analysis system for use in assessing the safety of drugs and drug candidates, in 2002 we conducted a 5-year collaborative study in the Toxicogenomics Project (TGP1) in Japan. Experimental data generated by such studies must be validated by different laboratories for robust and accurate analysis. For this purpose, we conducted intra- and inter-laboratory validation studies with participating companies in the second collaborative study in the Toxicogenomics Project (TGP2). Gene expression in the liver of rats treated with acetaminophen (APAP) was independently examined by the participating companies using Affymetrix GeneChip microarrays. The intra- and inter-laboratory reproducibility of the data was evaluated using hierarchical clustering analysis. The toxicogenomics results were highly reproducible, indicating that the gene expression data generated in our TGP1 project is reliable and compatible with the data generated by the participating laboratories.

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Three female Crl:CD(SD) rats/group were dosed with single wall carbon nanotube (SWCNT) or multi wall carbon nanotube (MWCNT) four times by gavage at a total of 50 mg/kg bw or 200 mg/kg bw (four equally divided doses at one-hour intervals). Acute oral doses of SWCNT and MWCNT caused neither death nor toxicological effects, and thus the oral LD50 values for SWCNT and MWCNT were considered to be greater than 50 mg/kg bw and 200 mg/kg bw, in rats respectively. Five or ten Crl:CD(SD) rats/sex were dosed with SWCNT once daily by gavage at a dose of 0 (control), 0.125, 1.25 or 12.5 mg/kg bw/day for 28 days with a 14-day recovery period (0 and 12.5 mg/kg bw/day groups). Six or twelve Crl:CD(SD) rats/sex were dosed with MWCNT once daily by gavage at a dose of 0 (control), 0.5, 5.0 or 50 mg/kg bw/day for 28 days with a 14-day recovery period (0 and 50 mg/kg bw/day groups). Based on no toxicological effects, the no observed adverse effect levels (NOAELs) of repeated dose toxicity of SWCNT and MWCNT were considered to be 12.5 mg/kg bw/day and 50 mg/kg bw/day (the highest dose tested), respectively. It was suggested that SWCNT and MWCNT dosed by gavage reached the gastro-intestinal tract as agglomerates and were mostly excreted via feces.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Most exposure levels of flavor in food are considered to be extremely low. If at all, genotoxic properties should be taken into account in safety evaluations. We have recently established a (quantitative) structure-activity relationship, (Q)SAR, combination system, which is composed of three individual models of mutagenicity prediction for industrial chemicals. A decision on mutagenicity is defined as the combination of predictive results from the three models. To validate the utility of our (Q)SAR system for flavor evaluation, we assessed 367 flavor chemicals that had been evaluated mainly by JECFA and for which Ames test results were available. When two or more models gave a positive evaluation, the sensitivity was low (19.4%). In contrast, when one or more models gave a positive evaluation, the sensitivity increased to 47.2%. The contribution of this increased sensitivity was mainly due to the result of the prediction by Derek for Windows, which is a knowledge-based model. Structural analysis of false negatives indicated some common sub-structures. The approach of improving sub-structural alerts could effectively contribute to increasing the predictability of the mutagenicity of flavors, because many flavors possess categorically similar functional sub-structures or are composed of a series of derivatives. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.fct.2012.02.009

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To obtain initial information on the possible repeated-dose oral toxicity of fullerene C60, Crl:CD(SD) rats were administered fullerene C60 by gavage once daily at 0 (vehicle: corn oil), 1, 10, 100, or 1,000 mg/kg/day for 29 days, followed by a 14-day recovery period. No deaths occurred in any groups, and there were no changes from controls in detailed clinical observations, body weights, and food consumption in any treatment groups. Moreover, no treatment-related histopathological changes were found in any organs examined at the end of the administration period and at the end of the recovery period. Blackish feces and black contents of the stomach and large intestine were observed in males and females at 1,000 mg/kg/day in the treatment group. There were no changes from controls in the liver and spleen weights at the end of the administration period, but those weights in males in the 1,000 mg/kg/day group increased at the end of the recovery period. Using liquid chromatography-tandem mass spectrometry, fullerene C60 were not detected in the liver, spleen or kidney at the end of the administration period and also at the end of the recovery period. In conclusion, the present study revealed no toxicological effects of fullerene C60; however, the slight increases in liver and spleen weights after the 14-day recovery period may be because of the influence of fullerene C60 oral administration. In the future, it will be necessary to conduct a long-term examination because the effects of fullerene C60 cannot be ruled out.

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Male and female rats were given perfluorooctadecanoic acid (PFOdA) by gavage at 40, 200 or 1,000 mg/kg/day, and each female was mated with a male in the same dose group after 14-day administration. Males were dosed for 42 days and females were dosed throughout the gestation period until day 5 of lactation. One female given 1,000 mg/kg/day was euthanized on day 18 of gestation due to a moribund condition; however, no other treatment-related clinical signs of toxicity were observed. Body weights fell at 1,000 mg/kg/day from day 28 through the administration period in males and throughout gestation and lactation in females. Red blood cell count, hemoglobin level and hematocrit were decreased at 200 and 1,000 mg/kg/day in males and activated partial thromboplastin time was prolonged at 1,000 mg/kg/day in females. Histopathological examination revealed hepatic changes, such as centrilobular hypertrophy and necrosis, in males given 200 and 1,000 mg/kg/day and in females given 1,000 mg/kg/day. Pancreatic zymogen granule was decreased in both sexes at 1,000 mg/kg/day. As for reproductive and developmental toxicity, there were decreases in the number of corpora lutea, implantation, total number of pups born and the number of live pups on postnatal days 0 and 4 at 1,000 mg/kg/day. At this dose, birth weights of pups were decreased and postnatal body weight gain was inhibited. Based on these findings, the NOAEL of PFOdA was considered to be 40 mg/kg/day for repeated dose toxicity and 200 mg/kg/day for reproductive/developmental toxicity.

DOI： 10.2131/jts.37.63

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

DOI： 10.14869/toxpt.39.1.0.P-240.0

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

DOI： 10.14869/toxpt.39.1.0.P-237.0

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DOI： 10.14869/toxpt.39.1.0.S7-1.0

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

DOI： 10.14869/toxpt.39.1.0.P-126.0

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DOI： 10.14869/toxpt.39.1.0.P-236.0

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Other Link： http://search.jamas.or.jp/link/ui/2013205085

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DOI： 10.14869/toxpt.39.1.0.P-238.0

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DOI： 10.14869/toxpt.39.1.0.P-166.0

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Other Link： http://search.jamas.or.jp/link/ui/2013205015

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DOI： 10.14869/toxpt.39.1.0.P-233.0

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Other Link： http://search.jamas.or.jp/link/ui/2013205082

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DOI： 10.14869/toxpt.39.1.0.P-230.0

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DOI： 10.14869/toxpt.39.1.0.P-239.0

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

To assess the repeated-dose toxicity of fullerene C60 by oral exposure, an OECD guideline test (TG 407) was conducted with administration of fullerene C60 at 0 (vehicle: corn oil), 1, 10, 100, or 1,000 mg/kg/day for 29 days in rats. No changes were observed in clinical observations, body weights, and food consumption in any treatment groups. Moreover, no treatment-related histopathological changes were found in any organs examined. Blackish feces and black contents of the stomach and large intestine were observed in males and females at 1,000 mg/kg/day in the treatment group. No significant changes in any organ weights were observed at the end of the administration period, but the only organ weights in liver and spleen in the 1,000 mg/kg/day group of male were increased at the end of the recovery period (14 days after the treatment period). The causal relationships between the possible absorption of fullerene C60 and those weight changes were suggested, but the pathological findings as indirect influences, such as swelling and congestion, were not also observed in the those organs. Using LC-MS/MS spectrometry analysis, fullerene C60 were not detected in the liver, spleen or kidney at the end of the administration period and also at the end of the recovery period. In conclusion, the present study revealed no toxicological effects of fullerene C60; however, with the prospective exposure by increased uses in future because of low toxic substance, more long-term exposure study is necessary to clarify the effects of fullerene C60 via oral exposure.

DOI： 10.14869/toxpt.39.2.0.AP-166.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.taap.2011.07.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Aluminium ammonium sulfate (AAS) was tested for reproductive/developmental toxicity in a two-generation study. Male and female rats were continuously given AAS in drinking water at 0, 50, 500 or 5000 ppm. Water consumption was decreased in all AAS-treated groups, and the body weight of parental animals transiently decreased in the 5000 ppm group. In either generation, no compound-related changes were found in estrous cyclicity, sperm parameters, copulation, fertility and gestation index, number of implantations and live birth pups, sex ratios of pups or viability during the preweaning period. Male and female F1 pups in the 5000 ppm group showed a lower body weight on postnatal day 21, while there were no differences in the birth weight of F1 and F2 pups between the control and AAS-treated groups. Preweaning body weight gain in F2 males and females indicated a similar decreasing tendency at 5000 ppm. In F1 and F2 weanlings, the weight of the liver, spleen and thymus decreased at 5000 ppm, but no histopathological changes were found in these organs. In F1 females in the 5000 ppm group, vaginal opening was delayed slightly. There were no compound-related changes in male preputial separation or in other developmental landmarks. In behavioral tests conducted for F1 animals at 4-6 weeks of age, no compound-related changes were found in spontaneous locomotor activity and performance in a water-filled multiple T-maze. In conclusion, the NOAEL of AAS for two-generation reproductive/developmental toxicity was considered to be 500 ppm in rats. Considering the aluminium content in the basal diet, the total ingested dose of aluminium from drinking water and food in this 500 ppm group was calculated to be 5.35 mg Al/kg bw/day. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.fct.2011.04.035

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

In a two-generation reproductive toxicity study, male and female rats were given aluminium sulfate (AS) in drinking water at 0, 120, 600 or 3000 ppm. AS reduced water consumption in all treatment groups, and body weight was transiently decreased in the 3000 ppm group. In the F1 and F2 pups, preweaning body weight gain was inhibited at 3000 ppm, and the liver and spleen weight was decreased at weaning. At this dose, vaginal opening was slightly delayed. There were no compound-related changes in other reproductive/developmental parameters, including developmental neurobehavioral endpoints. The data indicated that the NOAEL of AS in this two-generation study is 600 ppm for parental systemic toxicity and reproductive/developmental toxicity. The total ingested dose of aluminium from drinking water and food (standard rat diet, containing 25-29 ppm of aluminium) combined for this 600 ppm group was calculated to be 8.06 mg Al/kg bw/day. (c) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.reprotox.2010.11.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We propose new uncertainty factors (UFs) and a new subdivision of default factors in chemical risk assessment using a probabilistic approach based on the latest applicable information. Rounded values of 150 for mice, 100 for hamsters and rats, and 40 for rabbits, monkeys and dogs for inter- and intra-species differences (UF(AH)) were derived from the probabilistic combination of two log-normal distributions. Further calculation of additional UFs when chronic data (UF(S)) or NOAEL (UF(L)) are lacking was conducted using available log-normal distribution information. The alternative UF(S) and UF(L) values of 4 are considered to be appropriate for both cases where data are lacking. The default contributions of inter-species difference (UF(A)) and intra-species difference (UF(H)) to the UF(AH) of 100 for hamsters and rats as an example are considered to be 25 and 4, respectively. The UF(A) of 25 was subdivided into 25(0.6) (i.e., 7.0) for pharmacokinetics (PK) (UF(A.PK)) and 25(0.4) (i.e., 3.6) for pharmacodynamics (PD) (UF(A.PD)), and the UF(H) of 4 was evenly subdivided into 4(0.5) (i.e., 2) (UF(H.PK) and UF(H.PD)), to account for chemical-specific difference data between humans and laboratory animals for PK and/or PD. These default UFs, which come from actual experimental data, may be more appropriate than previous default UFs to derive tolerable daily intake values. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yrtph.2010.06.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Biotechnology advances have provided novel methods for the risk assessment of chemicals. The application of microarray technologies to toxicology, known as toxicogenomics, is becoming an accepted approach for identifying chemicals with potential safety problems. Gene expression profiling is expected to identify the mechanisms that underlie the potential toxicity of chemicals. This technology has also been applied to identify biomarkers of toxicity to predict potential hazardous chemicals. Ultimately, toxicogenomics is expected to aid in risk assessment. The following discussion explores potential applications and features of the Japanese Toxicogenomics Project.

DOI： 10.1002/mnfr.200900169

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：128  

Tens of thousands of existing chemicals have been widely used for manufacture, agriculture, household and other purposes in worldwide. Only approximately 10% of chemicals have been assessed for human health hazard. The health hazard assessment of residual large number of chemicals for which little or no information of their toxicity is available is urgently needed for public health. However, the conduct of traditional toxicity tests which involves using animals for all of these chemicals would be economically impractical and ethically unacceptable. (Quantitative) Structure-Activity Relationships [(Q)SARs] are expected as method to have the potential to estimate hazards of chemicals from their structure, while reducing time, cost and animal testing currently needed. Therefore, our studies have been focused on evaluation of available (Q)SAR systems for estimating in vivo repeated toxicity on the liver. The results from our preliminary analysis showed the distribution for LogP of the chemicals which have potential to induce liver toxicity was bell-shape and indicating the possibility to estimate liver toxicity of chemicals from their physicochemical property. We have developed (Q)SAR models to in vivo liver toxicity using three commercially available systems (DEREK, ADMEWorks and MultiCASE) as well as combinatorial use of publically available chemoinformatic tools (CDK, MOSS and WEKA). Distinct data-sets of the 28-day repeated dose toxicity test of new and existing chemicals evaluated in Japan were used for model development and performance test. The results that concordances of commercial systems and public tools were almost same which below 70% may suggest currently attainable knowledge of in silico estimation of complex biological process, though it possible to obtain complementary and enhanced performance by combining predictions from different programs. In future, the combinatorial application of in silico and in vitro tests might provide more accurate information which support regulatory decisions. At the same time, an appropriate strategy to use (Q)SAR for of the efficiency and accuracy in chemical management is necessary.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Drug-induced renal tubular injury is one of the major concerns in preclinical safety evaluations. Toxicogenomics is becoming a generally accepted approach for identifying chemicals with potential safety problems. In the present study, we analyzed 33 nephrotoxicants and 8 non-nephrotoxic hepatotoxicants to elucidate time- and dose-dependent global gene expression changes associated with proximal tubular toxicity. The compounds were administered orally or intravenously once daily to male Sprague-Dawley rats. The animals were exposed to four different doses of the compounds, and kidney tissues were collected on days 4, 8, 15, and 29. Gene expression profiles were generated from kidney RNA by using Affymetrix GeneChips and analyzed in conjunction with the histopathological changes. We used the filter-type gene selection algorithm based on t-statistics conjugated with the SVM classifier, and achieved a sensitivity of 90% with a selectivity of 90%. Then, 92 genes were extracted as the genomic biomarker candidates that were used to construct the classifier. The gene list contains well-known biomarkers, such as Kidney injury molecule 1, Ceruloplasmin, Clusterin, Tissue inhibitor of metallopeptidase 1, and also novel biomarker candidates. Most of the genes involved in tissue remodeling, the immune/inflammatory response, cell adhesion/proliferation/migration, and metabolism were predominantly up-regulated. Down-regulated genes participated in cell adhesion/proliferation/migration, membrane transport, and signal transduction. Our classifier has better prediction accuracy than any of the well-known biomarkers. Therefore, the toxicogenomics approach would be useful for concurrent diagnosis of renal tubular injury. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.tox.2009.09.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

A large-scale transcriptome database of rat liver (TG-GATEs) has been established by the Toxicogenomics Project in Japan. In the present study, we focused on 8 hepatotoxic compounds within TG-GATES, i.e., clofibrate, omeprazole, ethionine, thioacetamide, benzbromarone, propylthiouracil, Wy-14,643 and amiodarone, which induced coagulation abnormalities. Aspirin was selected as a reference compound that directly causes coagulation abnormality, but not through liver toxicity. In blood chemical examinations, for all the coagulopathic compounds there was little elevation of aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT), suggesting no severe cell death by treatment with the compounds. We extracted 344 probe sets from the data for these 8 typical drugs, which induced this phenotype at any time from 3 to 28 days of repeated administration. Principal component analysis using these probe sets clearly separated dose- and time-dependent clusters of the treated groups from their controls, except aspirin and propylthiouracil, both of which were considered to cause coagulopathy not due to their hepatotoxicity but due to their direct effects on the blood coagulation system. Reviewing the extracted genes, changes in lipid metabolism were found to be dominant. Genes related to blood coagulation were generally down-regulated by these drugs except that vitamin K epoxide reductase complex subunit 1 (Vkorc1) like 1, a paralogous gene of Vkorc1, was up-regulated. As expected, expression changes of these genes were least prominent in aspirin or propylthiouracil-treated liver. We concluded that these probe sets could be a good starting point in developing mechanism-based biomarkers for diagnosis or prognosis of hepatotoxicity-related coagulation abnormalities in the early stage of drug development.

DOI： 10.2131/jts.34.281

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For assessing carcinogenicity in animals, it is difficult and costly, an alternative strategy has been desired. We explored the possibility of applying a toxicogenomics approach by using comprehensive gene expression data in rat liver treated with various compounds. As prototypic non-genotoxic hepatocarcinogens, thioacetamide (TAA) and methapyrilene (MP) were selected and 349 commonly changed genes were extracted by statistical analysis. Taking both compounds as positive with six compounds, acetaminophen, aspirin, phenylbutazone, rifampicin, alpha-naphthylisothiocyanate, and amiodarone as negative, prediction analysis of microarray (PAM) was performed. By training and 10-fold cross validation, a classifier containing 112 probe sets that gave an overall success rate of 95% was obtained. The validity of the present discriminator was checked for 30 chemicals. The PAM score showed characteristic time-dependent increases by treatment with several non-genotoxic hepatocarcinogens, including TAA, MP, coumarin, ethionine and WY-14643, while almost all of the non-carcinogenic samples were correctly predicted. Measurement of hepatic glutathione content suggested that IMP and TAA cause glutathione depletion followed by a protective increase, but the protective response is exhausted during repeated administration. Therefore, the presently obtained PAM classifier could predict potential non-genotoxic hepatocarcinogenesis within 24 h after single dose and the inevitable pseudo-positives could be eliminated by checking data of repeated administrations up to 28 days. Tests for carcinogenicity using rats takes at least 2 years, while the present work suggests the possibility of lowering the time to 28 days with high precision, at least for a category of non-genotoxic hepatocarcinogens causing oxidative stress. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.tox.2008.05.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for diagnosis of hepatic phospholipidosis, we extracted 78 probe sets of rat hepatic genes from data of 5 drugs, amiodarone, amitriptyline, clomipramine, imipramine, and ketoconazole, which actually induced this phenotype. Principal component analysis (PCA) using these probes clearly separated dose- and time-dependent clusters of treated groups from their controls. Moreover, 6 drugs (chloramphenicol, chlorpromazine, gentamicin, perhexiline, promethazine, and tamoxifen), which were reported to cause phospholipidosis but judged as negative by histopathological examination, were designated as positive by PCA using these probe sets. Eight drugs (carbon tetrachloride, coumarin, tetracycline, metformin, hydroxyzine, diltiazem, 2-bromoethylamine, and ethionamide), which showed phospholipidosis-like vacuolar formation in the histopathology, could be distinguished from the typical drugs causing phospholipidosis. Moreover, the possible induction of phospholipidosis was predictable by the expression of these genes 24 h after single administration in some of the drugs. We conclude that these identified 78 probe sets could be useful for diagnosis of phospholipidosis, and that toxicogenomics would be a promising approach for prediction of this type of toxicity. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.taap.2008.01.036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC TOXICOLOGICAL SCIENCES  

The present sandy was conducted as a model case of the toxicogenomics approach for analyzing toxicological mechanisms and toxicity assessments in the early stage of drug development by comparing with classical toxicology data. Methapyrilene (MP) 100 mg/k produced obvious histopathological changes in liver of rats by single or repeated dose up to 28 days with significant elevation of ALT and AST. In the middle dose groups (30 mg/kg MP), no apparent changes were noted in blood biochemical data by single closing or repeated closing up to one week, and no obvious histopathological changes were observed except a slight hypertrophy in the hepatocytes. Comprehensive gene expression changes were analyzed using Affymetrix GeneChip(R) and differentially expressed probe sets were statistically extracted. These contained many genes related to "glutathione metabolism", "apoptosis". "MAPK signaling pathway" and "regulation of cell cycle", which were all thought to be involved in the development of presently observed phenotypes. In the high dose groups, TGP1 scores (developed in our system in order to overview the responsiveness of drugs to multiple marker gene lists) for these categories were markedly increased from the early time point. after single dose and kept their high expression throughout the repeated dose period. In the middle dose groups, the increment of the scores were noted not only at the dine points when apparent pathological changes emerged. but also at the earlier stage of repeated dosing and even after single dosing. We conclude that toxicogenomics would enable a more sensitive assessment at the earlier time point than classical toxicology evaluation.

DOI： 10.2131/jts.33.37

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To identify candidate biomarker gene sets to evaluate the potential risk of chemical-induced glutathione depletion in livers, we conducted microarray analysis on rat livers administered with phorone (40, 120 and 400 mg/kg), a prototypical glutathione depletor. Hepatic glutathione content was measured and glutathione depletion-responsive gene probe sets (GSH probe sets) were identified using Affymetrix Rat Genome 230 2.0 GeneChip by the following procedure. First, probe sets, whose signal values were inversely correlated with hepatic glutathione content throughout the experimental period, were statistically identified. Next, probe sets, whose average signal values were greater than 1.5-fold compared to those of controls 3 hr after phorone treatment, were selected. Finally, probe sets without unique Entrez Gene ID were removed, ending up with 161 probe sets in total. The usefulness of the identified GSH probe sets was verified by a toxicogenomics database. It was shown that signal profiles of the GSH probe sets in rats treated with bromobenzene were strongly altered compared with other chemicals. Focusing on bromobenzene, time-course profiles of hepatic glutathione content and gene expression revealed that the change in gene expression profile was marked after the bromobenzene treatment, whereas hepatic glutathione content had recovered after initial acute depletion, suggesting that the gene expression profile did not reflect the hepatic glutathione content itself, but rather reflects a perturbation of glutathione homeostasis. The identified GSH probe sets would be useful for detecting glutathione-depleting risk of chemicals from microarray data.

DOI： 10.2131/jts.32.469

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We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for interpretation of plasma triglyceride (TG) decrease, we extracted 218 probe sets of rat hepatic genes from data of 15 drugs that decreased the plasma TG level but differentially affected food consumption. Pathway and gene ontology analysis revealed that the genes belong to amino acid metabolism, lipid metabolism and xenobiotics metabolism. Principal component analysis (PCA) showed that 12 out of 15 compounds were separated in the direction of PC1, and these 12 were separated in the direction of PC2, according to their hepatic gene expression profiles. It was found that genes with either large or small eigenvector values in principal component PC 2 were those reported to be regulated by peroxisome proliferator-activated receptor (PPAR)α or constitutive androstane receptor (CAR), respectively. In fact, WY-14,643, clofibrate, gemfibrozil and benzbromarone, reported to be PPARα activators, distributed to the former, whereas propylthiouracil, omeprazole, phenobarbital, thioacetamide, methapyrilene, sulfasalazine and coumarin did to the latter. We conclude that these identified 218 probe sets could be a useful source of biomarkers for classification of plasma TG decrease, based on the mechanisms involving PPARα and CAR.

DOI： 10.2131/jts.32.387

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The Toxicogenomics project has been constructing a large-scale database of about 150 compounds exposed to rat (single dose, 3, 6, 9, 24 hrs and repeated dose for 3, 7, 14 28 days with 3 dose levels) and rat hepatocytes (2, 8, 24 hr with 3 concentrations) and data of transcriptome in liver using GeneChip, and the related toxicological measures are being accumulated. In the present study, the data of three ligands of peroxisome proliferator activated receptor α (PPARα), i.e., clofibrate, WY-14643 and gemfibrozil in our database were analyzed. Many of the β-oxidation-related genes were commonly induced in vivo and in vitro, whereas expression changes in genes related to cell proliferation, apoptosis, were detected in vivo (single and repeated dose) but not in vitro. Changes in those related to the immune response, coagulation and the stress response were also detectable exclusively in vivo. Using the genes mobilized in two or three PPARα agonists, hierarchical clustering was performed on 32 compounds stored in our database. In the profiling of an in vivo single dose, benzbromarone and aspirin were located in the same cluster of the three PPARα agonists. The clustering of in vitro data revealed that benzbromarone, three NSAIDs (aspirin, indomethacin and diclofenac sodium) and valproic acid belonged to the same cluster of PPARα agonists, supporting the reports that benzbromarone,valproic acid and some NSAIDs were reported to be PPARα agonists. Using the genes commonly up-regulated both in vivo and in vitro, principal component analysis was performed in 32 compounds, and principal component 1 was found to be the convenient parameter to extract PPARα agonist-like compounds from the database.

DOI： 10.2131/jts.31.471

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In The Toxicogenomics Project, about 150 chemicals are administered to rats, and gene expression in the liver analyzed by Affymetrix GeneChip and stored in the database. As the quality of RNA greatly influences the accuracy of gene expression data, conditions of the storage of the sample are very important. Recently, an RNA stabilization solution, RNAlater®, has become commercially available. In this study, the new storage method was compared with the traditional storage method (stored in freezer or liquid nitrogen) under various conditions by looking at the degradation of RNA assessed by its total yield, OD260/280 ratio, 28S/18S ratio, and quantity of β-actin. It was confirmed that RNAlater® preserved the liver tissue sample by maintaining the quality of RNA for one year (in liquid N2 or -80°C), for 3 days (4°C), or for 2 hr (room temperature) without degradation of RNA. Quality of RNA samples dissolved in buffer RLT and stored at -20°C tended to decrease, but samples stored at -80°C were almost equivalent to those stored in liquid nitrogen. In conclusion, we recommend the following procedure for preservation of liver tissue for extraction of RNA: 1) tissues removed should be put into chilled RNAlater® as soon as possible
2) samples in RNAlater® must be stored overnight or longer at 4°C and can be left for as long as 2 weeks without freezing
3) samples in RNAlater® can be stored for at least one year under less than -.20°C and 4) samples dissolved in buffer RLT can be preserved at least for one year under -80°C.

DOI： 10.2131/jts.31.509

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：5  

A large-scale toxicogenomcis database has now been constructed in the Toxicogenomics Project in Japan (TGP). To facilitate the analytical procedures for such large-scale microarray data, we developed a simple one-dimensional score, named TGP1 which expresses the trend of the changes in expression of biomarker genes as a whole. To evaluate the usefulness of the TGP1 score, microarray data of rat liver and rat hepatocytes deposited in the TGP database were scored for three biomarker gene sets, i.e., carcinogenesis-related, PPARα-regulated and glutathione depletion-related gene sets. The TGP1 scoring system gave reasonable results, i.e., the scores for carcinogenesis-related genes were high in omeprazole-, chlorpromazine-, hexachlorobenzene-, sulfasalazine- and Wy-14,643-treated rat livers, that for PPARα-regulated genes were high in clofibrate-, Wy-14,643-, gemfibrozil-, benzbromarone- and aspirin-treated rat livers as well as rat hepatocytes, and for glutathione deficiency-related genes were high in omeprazole-, bromobenzene-, acetaminophen- and coumarin-treated rat liver. We concluded that the TGP1 score is useful for surveying the expression changes in multiple biomarker gene sets for a large-scale toxicogenomics database, which would reduce the time of doing conventional multivariate statistical analysis. In addition, the TGP1 score can be applied to screening of compatible biomarker gene sets between rat liver and rat hepatocytes, like PPARα-regulated gene sets, which will allow us to develop an appropriate in vitro system for drug safety assessment in vivo.

DOI： 10.2131/jts.31.433

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The aim of this study was to compare gene expression profiles in the different kidney regions as the basis for toxicogenomics. Rat kidney was separated into papilla, medulla and cortex, and total RNA was isolated from these and from the whole slice. Gene expression profiling was performed using Affymetrix Rat Genome 230 2.0 Array. When global normalization was applied, the expression of β-actin or GAPDH varied among the regions. It was considered that such a comparison could not be made, especially between papilla and other portions, since the production of total mRNA in the former was relatively low. In fact, ANOVA was performed on the gene expression values with global normalization in papilla, medulla, cortex, and whole slice, and the numbers of genes appeared to be the highest in papilla. It was also observed that many genes showed their maximum or minimum in the whole slice, which was theoretically impossible. To overcome the problems associated with global normalization, the "percelome" normalization (a way to obtain the values directly related to the copies of mRNA per cell) was employed to compare the regions. In applying this procedure, probe sets with regional difference in expression were efficiently extracted by ANOVA. When they were sorted by the fold difference to other regions, the higher rank was occupied by genes characteristic of the functions of kidney, i.e., channels, transporters and metabolic enzymes. Some of them were consistent with the literature and were related to pathophysiological phenomena. Comprehensive comparison of data of gene expression in the renal anatomical area will greatly enhance studies of the physiological function and mechanism of toxicity in kidney.

DOI： 10.2131/jts.31.449

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Transcriptome data from quantitative PCR (Q-PCR) and DNA microarrays are typically obtained from a fixed amount of RNA collected per sample. Therefore, variations in tissue cellularity and RNA yield across samples in an experimental series compromise accurate determination of the absolute level of each mRNA species per cell in any sample. Since mRNAs are copied from genomic DNA, the simplest way to express mRNA level would be as copy number per template DNA, or more practically, as copy number per cell.
Results: Here we report a method (designated the "Percellome" method) for normalizing the expression of mRNA values in biological samples. It provides a "per cell" readout in mRNA copy number and is applicable to both quantitative PCR (Q-PCR) and DNA microarray studies. The genomic DNA content of each sample homogenate was measured from a small aliquot to derive the number of cells in the sample. A cocktail of five external spike RNAs admixed in a dose-graded manner (dose-graded spike cocktail; GSC) was prepared and added to each homogenate in proportion to its DNA content. In this way, the spike mRNAs represented absolute copy numbers per cell in the sample. The signals from the five spike mRNAs were used as a dose-response standard curve for each sample, enabling us to convert all the signals measured to copy numbers per cell in an expression profile-independent manner. A series of samples was measured by Q-PCR and Affymetrix GeneChip microarrays using this Percellome method, and the results showed up to 90% concordance.
Conclusion: Percellome data can be compared directly among samples and among different studies, and between different platforms, without further normalization. Therefore, "percellome" normalization can serve as a standard method for exchanging and comparing data across different platforms and among different laboratories.

DOI： 10.1186/1471-2164-7-64

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC ANALYTICAL CHEMISTRY  

Because concern over endocrine disrupting reactions caused by chemicals to humans and animals is growing, a rapid and reliable screening assay for endocrine disrupting chemicals is required. We have developed an in vitro screening assay based on a hormone receptor mechanism using a surface plasmon resonance (SPR) sensor. The interaction between an estrogen receptor alpha (ER) and an estrogen response element (ERE) is monitored in real time, when ER is injected over the SPR sensor chip on which a DNA fragment containing ERE is immobilized. In the presence of a chemical with estrogenic activity, the ER-ERE interaction is enhanced and the kinetic parameters are altered. We have validated the assay in terms of its specificity, dose dependency, optimal reaction conditions and reproducibility. It has been shown that the assay is very reliable as a rapid and quantitative screening method to judge the estrogenic activities of chemicals.

DOI： 10.2116/analsci.20.611

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Language：Japanese   Publisher：The Japanese Society for Lymphoreticular Tissue Research  

DOI： 10.3960/jslrt1961.36.217

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.toxlet.2016.07.546

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.toxlet.2016.06.1538

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© 2016, National Institute of Health Sciences. All rights reserved. Under the Chemical Substances Control Law in Japan, initial hazard information on existing chemical substances is collected by the Ministry of Health, Labour and Welfare, Japan to assess potential initial risks to human health. This hazard information includes acute toxicity, repeated-dose toxicity, genotoxicity, and/or reproductive/developmental toxicity. In an attempt to disseminate such information more widely, we have been reporting this information. In the present report, a summary of hazard information is presented for the following five existing chemical substances: 2,3,4,4′-tetrahydr oxybenzophenone (CAS: 31127-54-5); 1-propene, tetramer (CAS: 6842-15-5); 4-chlorobenzaldehyde (CAS: 104-88-1); diammonium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate (CAS: 3012-65-5); and 2-nitrop- cresol (CAS: 119-33-5). Additionally, we created a dossier for the clarification and evaluation of each study in English using the International Uniform Chemical Information Database version 5.

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Language：English   Publisher：WILEY-BLACKWELL  

Perfluoroalkyl carboxylic acids (PFCAs) are a series of environmental contaminants that have received attention because of their possible adverse effects on wildlife and human health. Although many toxicological studies have been performed on perfluorooctanoic acid with carbon chain length C8, available toxicity data on PFCAs with longer chains are still insufficient to evaluate their hazard. A combined repeated dose and reproductive/developmental toxicity screening study for perfluorododecanoic acid (PFDoA; C12) was conducted in accordance with OECD guideline 422 to fill these toxicity data gaps. PFDoA was administered by gavage to male and female rats at 0.1, 0.5, or 2.5 mg/kg/day. The administration of PFDoA at 0.5 and 2.5 mg/kg/day for 42-47 days mainly affected the liver, in which hypertrophy, necrosis, and inflammatory cholestasis were noted. Body weight gain was markedly inhibited in the 2.5 mg/kg/day group, and a decrease in hematopoiesis in the bone marrow and atrophic changes in the spleen, thymus, and adrenal gland were also observed. Regarding reproductive/developmental toxicity, various histopathological changes, including decreased spermatid and spermatozoa counts, were observed in the male reproductive organs, while continuous diestrous was observed in the females of the 2.5 mg/kg/day group. Seven of twelve females receiving 2.5 mg/kg/day died during late pregnancy while four other females in this group did not deliver live pups. No reproductive or developmental parameters changed at 0.1 or 0.5 mg/kg/day. Based on these results, the NOAELs of PFDoA were concluded to be 0.1 mg/kg/day for repeated dose toxicity and 0.5 mg/kg/day for reproductive/developmental toxicity. (C) 2014 Wiley Periodicals, Inc.

DOI： 10.1002/tox.21996

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.yrtph.2015.07.002

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.toxlet.2015.08.969

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Language：English   Publisher：TAYLOR & FRANCIS LTD  

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on five acetal flavouring substances uniquely used in Japan: acetaldehyde 2,3-butanediol acetal, acetoin dimethyl acetal, hexanal dibutyl acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal. As no genotoxicity study data were available in the literature, all five substances had no chemical structural alerts predicting genotoxicity. Using Cramer's classification, acetoin dimethyl acetal and hexanal dibutyl acetal were categorised as class I, and acetaldehyde 2,3-butanediol acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal as class III. The estimated daily intakes for all five substances were within the range of 1.45-6.53 mu g/person/day using the method of maximised survey-derived intake based on the annual production data in Japan from 2001, 2005, 2008 and 2010, and 156-720 mu g/person/day using the single-portion exposure technique (SPET), based on the average use levels in standard portion sizes of flavoured foods. The daily intakes of the two class I substances were below the threshold of toxicological concern (TTC) - 1800g/person/day. The daily intakes of the three class III substances exceeded the TTC (90g/person/day). Two of these, acetaldehyde 2,3-butanediol acetal and hexanal glyceryl acetal, were expected to be metabolised into endogenous products after ingestion. For 4-methyl-2-pentanone propyleneglycol acetal, one of its metabolites was not expected to be metabolised into endogenous products. However, its daily intake level, based on the estimated intake calculated by the SPET method, was about 1/15000th of the no observed effect level. It was thus concluded that all five substances raised no safety concerns when used for flavouring foods at the currently estimated intake levels. While no information on in vitro and in vivo toxicity for all five substances was available, their metabolites were judged as raising no safety concerns at the current levels of intake.

DOI： 10.1080/19440049.2015.1067927

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Language：English   Publisher：OXFORD UNIV PRESS  

Toxicogenomics focuses on assessing the safety of compounds using gene expression profiles. Gene expression signatures from large toxicogenomics databases are expected to perform better than small databases in identifying biomarkers for the prediction and evaluation of drug safety based on a compound's toxicological mechanisms in animal target organs. Over the past 10 years, the Japanese Toxicogenomics Project consortium (TGP) has been developing a large-scale toxicogenomics database consisting of data from 170 compounds (mostly drugs) with the aim of improving and enhancing drug safety assessment. Most of the data generated by the project (e.g. gene expression, pathology, lot number) are freely available to the public via Open TG-GATEs (Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System). Here, we provide a comprehensive overview of the database, including both gene expression data and metadata, with a description of experimental conditions and procedures used to generate the database. Open TG-GATEs is available from http://toxico.nibio.go.jp/english/index.html.

DOI： 10.1093/nar/gku955

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Language：English   Publisher：National Institute of Health Sciences  

Under the Chemical Substances Control Law（CSCL）in Japan, initial hazard information for existing chemical substances has been collected by the Ministry of Health, Labour and Welfare, Japan（MHLW） to assess potential initial risks to human health. We have reviewed all collected toxicity information pertaining to acute toxicity, repeated dose toxicity, genotoxicity, and/or reproductive/developmental toxicity and performed hazard assessments. Approximately 150 substances are currently undergoing review and assessment. For clarification and evaluation of each toxicity study, we have created a dossier（a collection of study data containing a detailed summary of the methods, results, and conclusions of each study）in English using the International Uniform Chemical Information Database （IUCLID）version 5. The IUCLID dossier format is widely used and has been accepted as one of the most beneficial formats for providing summarized chemical substance toxicity assessments. In this report, as a contribution to our ongoing hazard assessment activity, we present summary hazard information related to the potential human health effects of the following 5 chemical substances: 4-chlorobenzoyl chloride（CAS: 122-01-0）
benzenesulfonic acid, 4-hydroxy-, tin（2+）salt（CAS: 70974- 33-3）
chlorocyclohexane（CAS: 542-18-7）
1,3-cyclohexanedimethanamine（CAS: 2579-20-6）
and 1,3,5-triazine-2,4,6（1H,3H,5H）-trithione（CAS: 638-16-4）. The IUCLID dossiers created for these 5 chemical substances will be made available via the Japan Existing Chemical Data Base（JECDB）at &lt
http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp&gt
. Additional human health hazard information on existing chemical substances will be provided using the same methodology and website when it is available.

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Language：English   Publisher：Japanese Society of Toxicology  

Recent studies have shown that epigenetic alterations correlate with carcinogenesis in various tissues. Identification of these alterations might help characterize the early stages of carcinogenesis. We comprehensively analyzed DNA methylation and gene expression in livers obtained from rats exposed to nitrosodiethylamine (DEN) followed by a promoter of hepatic carcinogenesis, phenobarbital (PB). The combination of DEN and PB induced marked increases in number and area of glutathione S-transferase-placental form (GST-P)-positive foci in the liver. In the liver of rats that received 30 mg/kg of DEN, pathway analysis revealed alterations of common genes in terms of gene expression and DNA methylation, and that these alterations were related to immune responses. Hierarchical clustering analysis of the expression of common genes from public data obtained through the Toxicogenomics Project-Genomics Assisted Toxicity Evaluation system (TG-GATEs) showed that carcinogenic compounds clustered together. MBD-seq and GeneChip analysis indicated that major histocompatibility complex class Ib gene RT1-CE5, which has an important role in antigen presentation, was hypomethylated around the promoter region and specifically induced in the livers of DEN-treated rats. Further, immunohistochemical analysis indicated that the co-localization of GST-P and protein homologous to RT1-CE5 was present at the foci of some regions. These results suggest that common genes were altered in terms of both DNA methylation and expression in livers, with preneoplastic foci indicating carcinogenic potential, and that immune responses are involved in early carcinogenesis. In conclusion, the present study identified a specific profile of DNA methylation and gene expression in livers with preneoplastic foci. Early epigenetic perturbations of immune responses might correlate with the early stages of hepatocarcinogenesis.

DOI： 10.2131/jts.39.837

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Hydroquinone (HQ) is used in skin bleaching agents, hair dyes, and finger nail treatments. Many skin-lightening cosmetics that contain HQ are currently marketed in Japan. Concerns have been expressed regarding health risks to the general population because the carcinogenicity of HQ was previously suggested in animal studies. HQ induced hepatocellular adenomas and forestomach hyperplasias in mice and renal tubular cell adenomas in male rats. In the present study, the lacZ transgenic mutation assay was conducted according to OECD test guideline 488 to determine whether mutagenic mechanisms were involved in HQ-induced carcinogenesis. Male Muta (TM) mice were repeatedly administered HQ orally at dosages of 0, 25, 50, 100, or 200 mg/kg bw/day for 28 days. Body weight gain was decreased in all treatment groups. No significant differences were observed in mutant frequencies in the liver, stomach, lung, or kidney between HQ-treated mice and the concurrent negative controls, whereas the significant induction of mutations was noted in the positive control, N-ethyl-N-nitrosourea. These results suggest that a mutagenic mechanism is not responsible for HQ-induced carcinogenesis. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.mrgentox.2014.10.009

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We propose a category approach to assessing the testicular toxicity of chemicals with a similar structure to ethylene glycol methyl ether (EGME). Based on toxicity information for EGME and related chemicals and accompanied by adverse outcome pathway information on the testicular toxicity of EGME, this category was defined as chemicals that are metabolized to methoxy- or ethoxyacetic acid, a substance responsible for testicular toxicity. A Japanese chemical inventory was screened using the Hazard Evaluation Support System, which we have developed to support a category approach for predicting the repeated-dose toxicity of chemical substances. Quantitative metabolic information on the related chemicals was then considered, and seventeen chemicals were finally obtained from the inventory as a shortlist for the category. Available data in the literature shows that chemicals for which information is available on the metabolic formation of EGME, ethylene glycol ethyl ether, methoxy- or ethoxyacetic acid do in fact possess testicular toxicity, suggesting that testicular toxicity is a concern, due to metabolic activation, for the remaining chemicals. Our results clearly demonstrate practical utility of AOP-based category approach for predicting repeated-dose toxicity of chemicals. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yrtph.2014.10.011

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Language：English   Publisher：ELSEVIER IRELAND LTD  

Drug-induced liver injury (DILI) is a significant safety issue associated with medication use, and is the major cause of failures in drug development and withdrawal in post marketing. Cytokines are signaling molecules produced and secreted by immune cells and play crucial roles in the progression of DILI. Although there are numerous reports of cytokine changes in several DILI models, a comprehensive analysis of cytokine expression changes in rat liver injury induced by various compounds has, to the best of our knowledge, not been performed. In the past several years, we have built a public, free, large-scale toxicogenomics database, called Open TG-GATEs, containing microarray data and toxicity data of the liver of rats treated with various hepatotoxic compounds. In this study, we measured the protein expression levels of a panel of 24 cytokines in frozen liver of rats treated with a total of 20 compounds, obtained in the original study that formed the basis of the Open TG-GATEs database and analyzed protein expression profiles combined with mRNA expression profiles to investigate the correlation between mRNA and protein expression levels. As a result, we demonstrated significant correlations between mRNA and protein expression changes for interleukin (IL)-1 beta, IL-1 alpha, monocyte chemo-attractant protein (MCP)-1/CC-chemokine ligand (Ccl)2, vascular endothelial growth factor A (VEGF-A), and regulated upon activation normal T cell expressed and secreted (RANTES)/Ccl5 in several different types of DILI. We also demonstrated that IL-1 beta protein and MCP-1/Ccl2 mRNA were commonly up-regulated in the liver of rats treated with different classes of hepatotoxicants and exhibited the highest accuracy in the detection of hepatotoxicity. The results also demonstrate that hepatic mRNA changes do not always correlate with protein changes of cytokines in the liver. This is the first study to provide a comprehensive analysis of mRNA protein correlations of factors involved in various types of DILI, as well as additional insights into the importance of understanding complex cytokine expression changes in assessing DILI. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.tox.2014.07.005

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Language：English   Publisher：WILEY-BLACKWELL  

Nephrotoxicity is a common side effect observed during both nonclinical and clinical drug development investigations. The present study aimed to identify metabolomic biomarkers that could provide early and sensitive indication of nephrotoxicity in rats. Metabolomic analyses were performed using capillary electrophoresis-time-of-flight mass spectrometry on rat plasma collected at 9 and 24 h after a single dose of 2-bromoethylamine or n-phenylanthranilic acid and at 24 h after 7 days of repeated doses of gentamicin, cyclosporine A or cisplatin. Among a total of 169 metabolites identified, 3-methylhistidine (3-MH), 3-indoxyl sulfate (3-IS) and guanidoacetate (GAA) were selected as candidate biomarkers. The biological significance and reproducibility of the observed changes were monitored over time in acute nephrotoxicity model rats treated with a single dose of cisplatin, with the glomerular filtration rate monitored by determination of creatinine clearance. Increased plasma levels of 3-MH and 3-IS were related to a decline in glomerular filtration due to a renal failure. In contrast, the decrease in plasma GAA, which is synthesized from arginine and glycine in the kidneys, was considered to reflect decreased production due to renal malfunction. Although definitive validation studies are required to confirm their usefulness and reliability, 3-MH, 3-IS and GAA may prove to be valuable plasma biomarkers for monitoring nephrotoxicity in rats. Copyright (C) 2013 John Wiley & Sons, Ltd.

DOI： 10.1002/jat.2933

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.toxlet.2014.06.566

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DOI： 10.1016/j.toxlet.2014.06.786

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Language：English   Publisher：WILEY-BLACKWELL  

Historical control data on rodent developmental toxicity studies, performed between 1994 and 2010, were obtained from 19 laboratories in Japan, including 10 pharmaceutical and chemical companies and nine contract research organizations. Rats, mice, and hamsters were used for developmental toxicity studies. Data included maternal reproductive findings at terminal cesarean sections and fetal findings including the spontaneous incidences of external, visceral, and skeletal anomalies. No noticeable differences were observed in maternal reproductive data between laboratories. Inter-laboratory variations in the incidences of fetuses with anomalies appeared to be due to differences in the selection of observation parameters, observation criteria, classification of the findings, and terminology of fetal alterations. Historical control data are useful for the appropriate interpretation of experimental results and evaluation of the effects of chemical on reproductive and developmental toxicities.

DOI： 10.1111/cga.12050

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on four flavoring substances structurally related to menthol (L-menthyl 2-methylbutyrate, DL-menthyl octanoate, DI-menthyl palmitate, and DL-menthyl stearate) uniquely used in Japan. While no genotoxicity study data were available in the literature, all four substances had no chemical structural alerts predictive of genotoxicity. Moreover, they all four are esters consisting of menthol and simple carboxylic acids that were assumed to be immediately hydrolyzed after ingestion and metabolized into innocuous substances for excretion. As menthol and carboxylic acids have no known genotoxicity, it was judged that the JECFA procedure could be applied to these four substances. According to Cramer's classification, these substances were categorized as class I based on their chemical structures. The estimated daily intakes for all four substances were within the range of 1.54-4.71 mu g/person/day and 60-1250 mu g/person/day, using the methods of Maximized Survey-Derived Intake and Single Portion Exposure Technique, respectively, based on the annual usage data of 2001, 2005, and 2010 in Japan. As the daily intakes of these substances were below the threshold of concern applied to class I substances viz., 1800 mu g/person/day, it was concluded that all four substances raise no safety concerns when used for flavoring foods under the currently estimated intake levels. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.fct.2013.11.044

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Language：English   Publisher：Japanese Society of Toxicology  

We previously reported a toxicogenomics-based prediction model for hepatocarcinogens in which the expression patterns of signature genes following repeated doses of either genotoxic or non-genotoxic compounds were similar. Based on the results of our prediction model, we hypothesized that repeated doses of non-genotoxic carcinogens might have initiating potential. Here, we conducted a two-stage hepatocarcinogenesis study in rats exposed to the initiating agent nitrosodiethylamine (DEN), and hepatotoxic compounds thioacetamide (TAA), methapyrilene (MP) and acetaminophen (APAP) for 1-2 weeks followed by the liver tumor promoter phenobarbital (PB). The duration of initial treatment was determined based on positive results from our prediction model. Combined treatment of 3 or 30 mg/kg of genotoxic DEN and PB induced marked increases in altered hepatocellular foci and a DEN dose-dependent increase in the number and area of glutathione S-transferase-placental form (GST-P)-positive foci. A low number of altered hepatocellular foci were also observed in rats treated with TAA at a dose of 45 mg/kg. MP at a dose of 100 mg/kg induced a very low number of foci, but APAP did not. Hierarchical clustering analysis using gene expression data revealed that 2-week treatment with TAA at a dose of 30 mg/kg and MP at 45 mg/kg induced specific expression of DNA damage-related genes, similar to 1-week treatment with DEN at a dose of 30 mg/kg. These results suggest that TAA and MP induce DNA damage, which partially supports our hypothesis. Although this study does not indicate whether tumor growth in response to these compounds can be assessed in this model, our results suggest that cumulative treatment with non-genotoxic TAA might have initiating potential in the liver.

DOI： 10.2131/jts.39.785

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Perfluoroalkyl acids (PFAAs) are environmental contaminants that have received attention because of their possible effects on wildlife and human health. In order to obtain initial risk information on the toxicity of perfluoroundecanoic acid (PFUA), we conducted a combined repeated dose toxicty study with the reproduction/developmental toxicity screening test (OECD test guideline 422). PFUA was administered by gavage to rats at 0 (vehicle: corn oil), 0.1, 0.3 or 1.0 mg/kg/day. At 1.0 mg/kg/day, body weight gain was inhibited in both sexes, and there was a decrease in fibrinogen in both sexes and shortening of the activated partial thromboplastin time in males. An increase in blood urea nitrogen and a decrease in total protein in both sexes and increases in alkaline phosphatase and alanine transaminase and a decrease in albumin in males were observed at 1.0 mg/kg/day. Liver weight was increased in males at 0.3 mg/kg/day and above and in females at 1.0 mg/kg/day, and this change was observed after a recovery period. In both sexes, centrilobular hypertrophy of hepatocytes was observed at 0.3 mg/kg/day and above and focal necrosis was observed at 1.0 mg/kg/day. In reproductive/developmental toxicity, body weight of pups at birth was lowered and body weight gain at 4 days after birth was inhibited at 1.0 mg/kg/day, while no dose-related changes were found in the other parameters. Based on these findings, the no observed adverse effect levels (NOAELs) for the repeated dose and reproductive/developmental toxicity were considered to be 0.1 mg/kg/day and 0.3 mg/kg/day, respectively.

DOI： 10.2131/jts.39.97

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Language：English   Publisher：NATURE PUBLISHING GROUP  

MicroRNAs (miRNAs) are small (similar to 22 nucleotide) noncoding RNAs that play pivotal roles in regulation of gene expression. The value of miRNAs as circulating biomarkers is now broadly recognized; such tissue-specific biomarkers can be used to monitor tissue injury and several pathophysiological conditions in organs. In addition, miRNA profiles of normal organs and tissues are important for obtaining a better understanding of the source of modulated miRNAs in blood and how those modulations reflect various physiological and toxicological conditions. This work was aimed at creating an miRNA atlas in rats, as part of a collaborative effort with the Toxicogenomics Informatics Project in Japan (TGP2). We analyzed genome-wide miRNA profiles of 55 different organs and tissues obtained from normal male rats using miRNA arrays. The work presented herein represents a comprehensive dataset derived from normal samples profiled in a single study. Here we present the whole dataset with miRNA profiles of multiple organs, as well as precise information on experimental procedures and organ-specific miRNAs identified in this dataset.

DOI： 10.1038/sdata.2014.5

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Language：Japanese   Publisher：FOOD HYGIENE & SAFETY  

Basic principles for simulation of acute reference dose (ARfD) setting were defined based on the work of Solecki et al. (2005). The principles are: (1) Appearance of acute toxicity within 24 h after oral administration. (2) Rationale for setting toxicity that appears or could appear after single oral administration. (3) ARfD setting is assumed to be necessary for all pesticides. (4) ARfD setting is not necessary when the value is at or above the cutoff level. (5) The setting basically applies to the general population. (6) ARM is set based on the lowest NOAEL among all the available study data concerning endpoints for acute effects. (7) Effects of exposure during critical periods should be considered as endpoints for ARfD setting. (8) The approach for the safety coefficient is the same as that for acceptable daily intake. (9) If available, human data are acceptable as an endpoint for ARfD setting.

DOI： 10.3358/shokueishi.54.331

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A 28-day repeated dose toxicity test and reproduction/developmental toxicity test for N,N'-diphenyl-p-phenylenediamine (DPPD) were conducted in [Crl:CD(SD)] SPF rats. Male and female rats were dosed with DPPD by gavage for 28 days at 0, 100, 300, or 1000 mg/kg bw/day or for a total of 42-46 days at 0, 8, 50, or 300 mg/kg bw/day. No significant adverse effects were observed in the repeated dose toxicity study up to 1000 mg/kg bw/day in both sexes. In the reproduction/developmental toxicity study, two females showed piloerection, hypothermia, and pale skin; one died and the other showed dystocia on day 23 of pregnancy at 300 mg/kg bw/day. Another female delivered only three live pups at 300 mg/kg bw/day. A significantly prolonged gestation period was observed at 50 and 300 mg/kg bw/day. The NOAELs of repeated dose toxicity and reproduction/developmental toxicity were considered to be 1000 and 8 mg/kg bw/day, respectively. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.fct.2013.02.029

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Language：English   Publisher：OXFORD UNIV PRESS  

Editor's Highlight: Current blood biomarkers lack sensitivity, are detectable only late after injury occurs, and lack predictive power. The present study identified novel circulating liver-specific mRNAs in plasma from rats treated with various hepatotoxicants to validate circulating liver-specific mRNAs as biomarkers for drug- induced liver injury. Liver-specific mRNAs (Alb, Ambp, Apoh, and Gc) were quantified by real-time RT-PCR in plasma from rats with single dosing of seven hepatotoxicants. Time course analysis within 24 h after dosing revealed the plasma levels of Alb and Gc mRNAs increased substantially earlier than the widely used biomarker alanine aminotransferase, suggesting that patterns of changes in circulating liver-specific mRNAs indicate the progression of liver injury. These results strongly support the reliability and usefulness of the four circulating liver-specific mRNAs as biomarkers for drug-induced liver injury.Circulating liver-specific mRNAs such as albumin (Alb) and -1-microglobulin/bikunin precursor (Ambp) have been reported to be potential biomarkers for drug-induced liver injury (DILI). We identified novel circulating liver-specific mRNAs and quantified them, together with the two previously reported mRNAs, in plasma from rats treated with various hepatotoxicants to validate circulating liver-specific mRNAs as biomarkers for DILI. Among six genes selected from the database, high liver specificity of apolipoprotein h (Apoh) and group-specific component (Gc) mRNAs were confirmed by reverse transcription (RT)-PCR and the copy numbers of these mRNAs elevated in plasma from rats treated with thioacetamide. Liver-specific mRNAs (Alb, Ambp, Apoh, and Gc) were quantified by real-time RT-PCR in plasma from rats with single dosing of seven hepatotoxicants. There were noticeable interindividual and intercompound variabilities in the severity of liver injury. The levels of four mRNAs increased almost in parallel and correlated with changes in the alanine aminotransferase (ALT) values and the hepatocellular necrosis scores at 24h after dosing. It was noteworthy that the magnitude of the increases in mRNA levels was greater than that in the ALT value. Time course analysis within 24h after dosing revealed that the timing of the increase was different among mRNA species, and the plasma levels of Alb and Gc mRNAs increased substantially earlier than the ALT values, suggesting that patterns of changes in circulating liver-specific mRNAs indicate the progression of liver injury. These results strongly support the reliability and usefulness of the four circulating liver-specific mRNAs as biomarkers for DILI.

DOI： 10.1093/toxsci/kfs340

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We tested a category approach to predict the hepatotoxic effects of repeated doses of allyl esters using a new database for repeated-dose toxicity. Based on information on hepatotoxic mechanism of allyl acetate, the category was defined as allyl esters that are hydrolyzed to allyl alcohol. Allyl alcohol is readily oxidized to acrolein in the liver, causing hepatotoxicity. Seventeen marketed allyl esters were obtained and grouped into category by identifying or predicting allyl alcohol formation. Allyl esters with a saturated straight alkyl carboxylic acid moiety (allyl acetate, hexanoate and heptanoate as tested species, and allyl butyrate, pentanoate, octanoate, nonanoate and decanoate as untested species) are likely similar in rate of ester hydrolysis, thereby defining subcategory 1. NOAEL and LOAEL for the hepatotoxic effects were estimated at 0.12 and 0.25 mmol/kg/d for the untested species, based on those of allyl acetate. The remaining nine allyl esters with other alkyl or aromatic carboxylic acid moieties were placed in subcategory 2: their hepatotoxicity levels were not predictable due to an unclear match between their degree of structural complexity and rate of hydrolysis. Our results demonstrate the usefulness of the category approach for predicting the hepatotoxicity of untested allyl esters with saturated straight alkyl chains. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yrtph.2012.12.001

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The available toxicity information for boron was reevaluated and four appropriate toxicity studies were selected in order to derive a tolerable daily intake (TDI) using newly proposed uncertainty factors (UFs) presented in Hasegawa et al. (2010). No observed adverse effect levels (NOAELs) of 17.5 and 8.8mgB/kg/day for the critical effect of testicular toxicity were found in 2-year rat and dog feeding studies. Also, the 95% lower confidence limit of the benchmark doses for 5% reduction of fetal body weight (BMDL05) was calculated as 44.9 and 10.3mgB/kg/day in mouse and rat developmental toxicity studies, respectively. Measured values available for differences in boron clearance between rats and humans and variability in the glomerular filtration rate (GFR) in pregnant women were used to derive chemical specific UFs. For the remaining uncertainty, newly proposed default UFs, which were derived from the latest applicable information with a probabilistic approach, and their subdivided factors for toxicokinetic and toxicodynamic variability were applied. Finally, overall UFs were calculated as 68 for rat testicular toxicity, 40 for dog testicular toxicity, 247 for mouse developmental toxicity and 78 for rat developmental toxicity. It is concluded that 0.13mgB/kg/day is the most appropriate TDI for boron, based on rat developmental toxicity. © 2012 Elsevier Inc.

DOI： 10.1016/j.yrtph.2012.10.013

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Language：English   Publisher：ELSEVIER IRELAND LTD  

Renal papillary injury is a common side effect observed during nonclinical and clinical investigations in drug development. The present study aimed to identify genomic biomarkers for early and sensitive detection of renal papillary injury in rats. We hypothesized that previously identified genomic biomarkers for tubular injury might be applicable for the sensitive detection of papillary injury in rats. We selected 18 genes as candidate biomarkers for papillary injury based on previously published studies and analyzed their expression profiles by RT-PCR in each kidney region, namely the cortex, cortico-medullary junction, and papilla in various nephrotoxicity models. Comparative analysis of gene expression profiles revealed that some genes were commonly upregulated or downregulated in the renal papilla, reflecting papillary injuries induced by 2-bromoethylamine hydrobromide, phenylbutazone, or n-phenylanthranilic acid. By applying receiver operator characteristics analysis, six candidate biomarkers were identified and their usefulness was confirmed by using an independent data set. The three top-ranked genes, Timp1, Igf1, and Lamc2, exhibited the best prediction performance in an external data set with area under the curve (AUC) values of greater than 0.91. An optimized support vector machine model consisting of three genes achieved the highest AUC value of 0.99. In conclusion, even though definitive validation studies are required for the establishment of their usefulness and reliability, these identified genes may prove to be the most promising candidate genomic biomarkers of renal papillary injury in rats. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.tox.2012.09.012

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Crl:CD(SD)rats were given 3-cyanopyridine by gavage at 0, 5, 30 or 180 mg/kg/day. Males were dosed for 42 days beginning 14 days before mating, and females for 40-53 days beginning 14 days before mating to day 3 of lactation, including throughout the mating and gestation periods. General toxicity, mainly liver damage, was observed in males at >= 30 mg/kg/day and in females at >= 5 mg/kg/day. Sertoli cell vacuolation was observed at 180 mg/kg/day, and spermatocyte damages were observed at >= 30 mg/kg/day. Effects on estrous cycles, corpora lutea and implantations, and unsuccessfully mated females, despite additional mating, were observed at 180 mg/kg/day. Delayed initiation of delivery, dystocia, and deaths or mori-bundities of pregnant females were observed at 180 mg/kg/day, and only two pregnant rats delivered live pups at that dose. The NOAEL for reproductive/developmental toxicity was concluded to be 30 mg/kg/day. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.reprotox.2012.10.014

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Hypothyroidism induced by xenobiotic treatment was analyzed for possible underlying mechanism(s) on the basis of different responses of the thyroid gland and the liver, using a newly-created database of repeated-dose toxicity of 500 chemicals. Two mechanisms are proposed: direct inhibition of thyroid hormone biosynthesis in the thyroid gland, and stimulated degradation of thyroid hormone by induction of hepatic drug-metabolizing enzymes. In the database there were 10 chemicals inducing hypertrophy/ hyperplasia of follicular cells in the thyroid gland and having data on thyroid glands. On the basis of the chemical structure and information available in the literature, we judged three chemicals to be typical thioamide derivatives that act directly on the thyroid gland, and the others as non-thioamide derivatives that were unlikely to have any direct action on the thyroid gland. All these chemicals were classified into two groups using the ratios of relative weight increase rate of thyroid gland versus that of the liver. These values were at least 1.7, but 3.2 or more in the most of the cases for thioamide derivatives, and 1.2 or less for non-thioamide derivatives. This background analysis suggests the feasibility of parametersupported speculation on the possible underlying mechanism when new repeated-dose toxicity data on hypothyroidism becomes available.

DOI： 10.2131/jts.38.291

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In order to develop guidelines for setting acute reference doses (ARfDs) for pesticides in Japan, we conducted simulations of ARfD settings based on evaluation reports for 201 pesticides assessed by the Food Safety Commission (FSC) in Japan over the last 8 years. Our conceptual principles were based on the concepts written by Solecki et al. (2005) and were adapted for toxicological data required in Japan. Through this process, we were able to set the ARfDs for over 90% of the 201 pesticides tested. The studies that provided the rationale for ARfD setting were primarily reproductive and developmental toxicity studies, acute neurotoxicity studies, and pharmacology studies. For approximately 30% of the pesticides simulated in the present study, it was not necessary to establish ARfDs. Some of the simulated ARfDs resulting from their endpoints may be conservative estimates, because the evaluation reports were written for acceptable daily intake settings. Thus, it was sometimes difficult to distinguish acute toxicalerts from repeated toxicities. We were unable to set an ARfD for 14 pesticides because of insufficient data on acute toxicities. This could be improved by more complete recordkeeping. Furthermore, we categorized the 201 pesticides by mechanism of action or chemical structure. Our simulation indicates that the conceptual framework presented here can be used as a basis for the development of guidelines on ARfD settings for pesticides in Japan.

DOI： 10.2131/jts.38.205

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Language：English   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.tox.2012.04.005

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Language：English   Publisher：ELSEVIER IRELAND LTD  

Drug-induced renal tubular injury is a major concern in the preclinical safety evaluation of drug candidates. Toxicogenomics is now a generally accepted tool for identifying chemicals with potential safety problems. The specific aim of the present study was to develop a model for use in predicting the future onset of drug-induced proximal tubular injury following repeated dosing with various nephrotoxicants. In total, 41 nephrotoxic and nonnephrotoxic compounds were used for the present analysis. Male Sprague-Dawley rats were dosed orally or intravenously once daily. Animals were exposed to three different doses (low, middle, and high) of each compound, and kidney tissue was collected at 3, 6, 9, and 24 h after single dosing, and on days 4, 8, 15, and 29 after repeated dosing. Gene expression profiles were generated from kidney total RNA using Affymetrix DNA microarrays. Filter-type gene selection and linear classification algorithms were employed to discriminate future onset of proximal tubular injury. We identified genomic biomarkers for use in future onset prediction using the gene expression profiles determined on day 1, when most of the nephrotoxicants had yet to produce detectable histopathological changes. The model was evaluated using a five-fold cross validation, and achieved a sensitivity of 93% and selectivity of 90% with 19 probes. We also found that the prediction accuracy of the optimized model was substantially higher than that produced by any of the single genomic biomarkers or histopathology. The genes included in our model were primarily involved in DNA replication, cell cycle control, apoptosis, and responses to oxidative stress and chemical stimuli. In summary, our toxicogenomic model is particularly useful for predicting the future onset of proximal tubular injury. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.tox.2012.03.014

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.toxlet.2012.03.665

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Phospholipid accumulation manifests as an adverse effect of cationic amphiphilic drugs in particular. Detection, however, by histopathology examination is time-consuming and may require repeated administration of compounds for several weeks. To eliminate compounds with potential for inducing phospholipidosis from the discovery pipeline, we have identified and validated a set of biomarkers for predicting the phospholipidosis-inducing potential utilizing a comprehensive rat transcriptome microarray database created by the Japanese Toxicogenomics and Toxicogenomics Informatics Projects (TGP/TGP2) together with in-house data. The set of biomarkers comprising 25 Affymetrix GeneChip probe sets was identified using genetic algorithm optimization on 24-h time-point microarray data from rats treated with single doses of hepatotoxic compounds including amiodarone, clomipramine, haloperidol, hydroxyzine, imipramine, and perhexiline. The set of novel biomarkers represents an early time-point gene-expression pattern characteristic for a condition eventually leading to phospholipidosis. This implies significant advantages in terms of time and resources over currently published biomarkers derived using repeated-dosing late time-point data. The biomarker set was validated by 11 independent compounds. Accuracy, sensitivity, and specificity values were 82%, 67%, and 100%, respectively and the area under the receiver operating characteristic curve was 0.97. These results show that the biomarker set possesses a high classification accuracy for novel compounds. Pathway analysis was carried out for the biomarkers and the detection of pathways related to lipid-metabolism was statistically significant. These pathways most probably reflect lipid metabolism changes associated with phospholipidosis supporting the validity of our novel biomarkers. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.tox.2012.02.015

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To obtain initial information on the possible repeated-dose oral toxicity of fullerene C60, Crl:CD(SD) rats were administered fullerene C60 by gavage once daily at 0 (vehicle: corn oil), 1, 10, 100, or 1,000 mg/kg/day for 29 days, followed by a 14-day recovery period. No deaths occurred in any groups, and there were no changes from controls in detailed clinical observations, body weights, and food consumption in any treatment groups. Moreover, no treatment-related histopathological changes were found in any organs examined at the end of the administration period and at the end of the recovery period. Blackish feces and black contents of the stomach and large intestine were observed in males and females at 1,000 mg/kg/day in the treatment group. There were no changes from controls in the liver and spleen weights at the end of the administration period, but those weights in males in the 1,000 mg/kg/day group increased at the end of the recovery period. Using liquid chromatography-tandem mass spectrometry, fullerene C60 were not detected in the liver, spleen or kidney at the end of the administration period and also at the end of the recovery period. In conclusion, the present study revealed no toxicological effects of fullerene C60
however, the slight increases in liver and spleen weights after the 14-day recovery period may be because of the influence of fullerene C60 oral administration. In the future, it will be necessary to conduct a long-term examination because the effects of fullerene C60 cannot be ruled out.

DOI： 10.2131/jts.37.353

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Language：English   Publisher：Japanese Society of Toxicology  

Three female Crl:CD(SD) rats/group were dosed with single wall carbon nanotube (SWCNT) or multi wall carbon nanotube (MWCNT) four times by gavage at a total of 50 mg/kg bw or 200 mg/kg bw (four equally divided doses at one-hour intervals). Acute oral doses of SWCNT and MWCNT caused neither death nor toxicological effects, and thus the oral LD50 values for SWCNT and MWCNT were considered to be greater than 50 mg/kg bw and 200 mg/kg bw, in rats respectively. Five or ten Crl:CD(SD) rats/sex were dosed with SWCNT once daily by gavage at a dose of 0 (control), 0.125, 1.25 or 12.5 mg/kg bw/day for 28 days with a 14-day recovery period (0 and 12.5 mg/kg bw/day groups). Six or twelve Crl:CD(SD) rats/sex were dosed with MWCNT once daily by gavage at a dose of 0 (control), 0.5, 5.0 or 50 mg/kg bw/day for 28 days with a 14-day recovery period (0 and 50 mg/kg bw/day groups). Based on no toxicological effects, the no observed adverse effect levels (NOAELs) of repeated dose toxicity of SWCNT and MWCNT were considered to be 12.5 mg/kg bw/day and 50 mg/kg bw/day (the highest dose tested), respectively. It was suggested that SWCNT and MWCNT dosed by gavage reached the gastro-intestinal tract as agglomerates and were mostly excreted via feces.

DOI： 10.2131/jts.37.463

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Language：English   Publisher：Japanese Society of Toxicology  

The concern over endocrine disruptors prompted international establishment of a strategic framework for the identification of the estrogenic compounds. OECD has launched the Conceptual Framework tool box containing various screening and testing methods including the uterotrophic assay. The (anti)estrogenicity of 36 chemicals suspected to be estrogen-receptor interactive by in silico and/or in vitro screening in the Extended Scheme for Endocrine Disruptor Screening and Testing of the Ministry of Health, Labour and Welfare, Japan, were monitored by the uterotrophic assay using C57BL/6J ovariectomized adult female mice after a 7-day exposure by oral gavage (po) and subcutaneous injection (sc). Ethynyl estradiol was used as reference for agonist and antagonist detection. In addition, Bisphenol A (sc) and Genistein (po) were tested for the comparison to rat assays. Among the 36, 2-[Bis(4-hydroxyphenyl) methyl]benzylalcohol, 2,2',4,4'-Tetrahydroxybenzophenone, 2,4-Dihydroxybenzophenone, 3,3',5-Triiodothyroacetic acid, New fuchsin and alpha-Naphtholbenzein, showed both estrogenic agonistic and antagonistic activities
first two showed U-shaped dose-response in antagonistic studies. N,N-Diphenyl- p-phenylenediamine, 2,2'-Dihydroxy-4,4'-dimethoxybenzophenone, n-Butyl 4-hydroxybenzoate, and Reserpine were agonistic by sc. Benzo [a] pyrene, Benz [a] anthracene, Dibenz [a,h] anthracene, 2-(2H-Benzotriazol-2-yl)-4,6-di(t-pentyl)phenol, Rosemarinic acid, meta-Thymol, 6-Gingerol, Colchicine, Malachite green base, Fenbuconazole, and Lead acetate were antagonistic. The rest, i.e. n-Heptyl 4-hydroxybenzoate, Tetrazolium violet, Pravastatin sodium salt, Physostigmine, salicylate (1:1), Nordihydroguaiaretic acid, o-Cresolphthalein, 1,3-Dinitrobenzene, C.I. Pigment orange, Tetrabromobisphenol- A, 2-Hydroxy-4-methoxybenzophenone, Ethylparaben, Propyl p-hydroxybenzoate, Kaempferol, 2-(2-Benzotriazolyl)-p-cresol and Phenolphthalein were negative for both effects. Taking together with in silico/ in vitro screening, the result suggested that the ovariectomized mouse uterotrophic bioassay has sufficient performance comparable to rat for the screening of (anti)estrogenicity of various chemicals.

DOI： 10.2131/jts.37.879

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An important technology used in toxicogenomic drug discovery research is the microarray, which enables researchers to simultaneously analyze the expression of a large number of genes. To build a database and data analysis system for use in assessing the safety of drugs and drug candidates, in 2002 we conducted a 5-year collaborative study in the Toxicogenomics Project (TGP1) in Japan. Experimental data generated by such studies must be validated by different laboratories for robust and accurate analysis. For this purpose, we conducted intra- and inter-laboratory validation studies with participating companies in the second collaborative study in the Toxicogenomics Project (TGP2). Gene expression in the liver of rats treated with acetaminophen (APAP) was independently examined by the participating companies using Affymetrix GeneChip microarrays. The intra- and inter-laboratory reproducibility of the data was evaluated using hierarchical clustering analysis. The toxicogenomics results were highly reproducible, indicating that the gene expression data generated in our TGP1 project is reliable and compatible with the data generated by the participating laboratories.

DOI： 10.2131/jts.37.791

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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DOI： 10.1016/j.toxlet.2011.05.351

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.toxlet.2011.05.961

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Language：Japanese   Publisher：(一社)日本毒性学会  

【背景・目的】Ames試験で陰性となった化合物の発がん性はがん原性試験等を実施しなければ見極めることができず、費用や期間の面から簡便かつ短期間で実施可能な評価方法の確立が望まれている。本研究ではトキシコゲノミクスプロジェクトにおいて構築されたデータベース（TG-GATEs）を用いて、細胞障害性の非遺伝毒性肝発がん化合物のリスク評価マーカの探索および、予測モデルの構築を行い、その有用性を検討した。【方法】6週齢の雄性ラットに各種化合物（低・中・高用量）を単回・反復投与し、投与開始後3・6・9・24時間後、あるいは4・8・15・29日目に屠殺した。その後、Affymetrix社製のGeneChipを用いて肝臓の網羅的遺伝子発現データを取得し、次の手順で判別分析を行った。(i)「Ames試験陰性、且つラットで細胞障害性肝発がんを誘発することが知られている6化合物の反復29日の最高投与量」を正例とし、「ラットで肝発がんを誘発しないことが知られている54化合物の単回3～24時間および反復4～29日の最高投与量」を負例に設定した。(ii) 正負例間での遺伝子発現値の倍率変化、Mann-WhitneyのU統計量、SVM Marginを指標として遺伝子の総合ランキングを算出した。(iii) 抽出した遺伝子を使用し、SVMによりモデルを構築した。モデルの検証には5-fold Cross Validationを用い、ROC解析により最適遺伝子数を決定した。【結果】ランキング上位の9遺伝子を使用した際にモデルの判別性能が最大となり、感度は約99%、特異度は約97%であった。選抜した遺伝子には発がんとの関連性が報告されている<I>Cd276</I>や<I>Cdh13</I>などの既知遺伝子や、細胞増殖・細胞死・DNA修復関連の遺伝子が含まれていた。また、これら遺伝子は、細胞障害性の遺伝毒性肝発がん物質でも共通の発現変動を示した。【結論】我々の構築した予測モデルは、化合物の肝発がんリスクを早期に評価することが可能であり、創薬の初期段階において有用であると考えられた。

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Language：English   Publisher：Wiley-Blackwell  

Biotechnology advances have provided novel methods for the risk assessment of chemicals. The application of microarray technologies to toxicology, known as toxicogenomics, is becoming an accepted approach for identifying chemicals with potential safety problems. Gene expression profiling is expected to identify the mechanisms that underlie the potential toxicity of chemicals. This technology has also been applied to identify biomarkers of toxicity to predict potential hazardous chemicals. Ultimately, toxicogenomics is expected to aid in risk assessment. The following discussion explores potential applications and features of the Japanese toxicogenomics project.

DOI： 10.1002/9783527634705.ch3b

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薬剤誘発性腎障害は，創薬において最も注意すべき副作用の一つである．従って，創薬研究の初期段階に，医薬品候補化合物の腎障害誘発リスクをいち早く見極め，腎毒性リスクのない化合物を効率的に選び出すことが肝要である．今回，トキシコゲノミクス・インフォマティクスプロジェクトにおいて構築された大規模トキシコゲノミクスデータベース（TG-GATEs）を使用して，薬剤誘発性腎尿細管障害の診断マーカーの探索を実施した．【方法】6週齢の雄ラットに，薬剤（低，中，高用量）を1日1回，最長28日間反復投与し，投与開始後，4，8，15，29日目に屠殺して腎臓を採取した．腎臓の病理検査を実施すると共に，網羅的遺伝子発現解析を実施した．【結果及び考察】病理組織検査の結果に基づき，反復投与により尿細管障害が認められる21化合物の高用量群サンプルを正例，障害の認められない41化合物の低用量群サンプル及び腎毒性の陰性対照である8化合物の高用量群サンプルを負例とし，filter-typeの遺伝子選択法を適用してマーカー候補遺伝子（feature gene）を抽出した後，これを用いて線形判別器を構築した．5-fold cross validationによる判別精度検証を実施した結果，偽陽性率が約10%のとき，検出力は90%となった．次に，学習に用いなかった高用量，中用量群サンプルをテストセットとして構築した判別器を適用し，毒性予測を行った．その結果，多くの化合物において，病理変化が見られる時間よりも早い時点，あるいは，低い用量で毒性を検出できた．以上，今回構築した腎毒性の判別モデルは，薬剤誘発性腎障害の有無を高感度に検出する有用な手段になり得ると考えられた．

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【背景・目的】トキシコゲノミクス・インフォマティクスプロジェクト（TGP2）は国立医薬品食品衛生研究所、（独）医薬基盤研究所および製薬企業13社によって進められており、TGP第一期で取得した約150化合物のラット肝遺伝子発現データ及び関連する毒性学的データを用いて安全性バイオマーカーの探索を行っている．本発表では、TGPデータベースを用い、ストレス応答性転写因子Nrf2制御下遺伝子のラット肝における発現変化について報告を行う．【方法】150化合物を投与開始時６週齢の雄性SDラットにそれぞれ単回および28日間反復投与を行い計8時点で肝臓を採取した．遺伝子発現解析には、Affymetrix GeneChip Rat 230 2.0を用いた．【結果】文献情報に基づきNrf2制御下遺伝子のリスト化を行い、酸化ストレスの既知マーカー、グルタチオン代謝関連遺伝子、プロテアソーム構成遺伝子等を含む93プローブセットを解析対象とし、それらの発現変化が顕著であった単回投与後24時間のラット肝のGeneChipデータを用いて解析を行った．発現変化が大きい化合物には、フルタミドやクマリン、ニメスリド等、酸化ストレスや反応性代謝物の生成が報告されている化合物が含まれていた．また、ニメスリドのようにラット28日間反復投与試験では顕著な肝障害が認められなかったものの臨床では肝障害報告がある化合物についても、Nrf2制御下遺伝子は単回投与で大幅に変動していることから、Nrf2制御下遺伝子の発現変化を調べることにより酸化ストレスや反応性代謝物を介した肝障害リスクを短期で捉えることができる可能性が示唆された．

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薬剤誘発性腎障害は，創薬において最も注意すべき副作用の一つである．従って，創薬研究の初期段階に，医薬品候補化合物の腎障害誘発リスクをいち早く見極め，安全な化合物を効率的に選び出すことが肝要である．今回，トキシコゲノミクス・インフォマティクスプロジェクトにおいて構築された大規模トキシコゲノミクスデータベース（TG-GATEs）を使用して，単回投与毒性試験において活用可能な薬剤誘発性腎尿細管障害の予測マーカーの探索を実施した．【方法】6週齢の雄性ラットに，種々の化合物（低，中，高用量）を1日1回，最長28日間反復投与し，投与開始後，1，4，8，15，29日目に屠殺した．採取した腎臓の病理検査を実施すると共に，Affymetrix社製のGeneChipを用いて網羅的遺伝子発現解析を実施した．【結果】反復投与時（投与後4 から29日目）の病変の有無によりアンカリングした投与後1日目の遺伝子発現データ（10/14化合物が単回投与後翌日の時点では病変なし）に対してfilter-typeの遺伝子選択法を適用し，マーカー候補遺伝子（feature gene）を抽出した後，これを用いて線形判別器を構築した．5-fold cross validationによる判別精度検証を実施した結果，偽陽性率が約10%のとき，検出力は約93%となった．feature geneには，Kim1，Timp1，Clusterin等良く知られた既知の腎尿細管障害マーカーに加え，新規のマーカー候補遺伝子も幾つか含まれていた．また，これらの既知マーカーを単独で判別に用いた時の予測精度と比較して，今回構築した複数個の遺伝子を用いた判別器で大幅に高い予測精度が得られた．【結論】今回構築した腎毒性の判別モデルは，薬剤誘発性腎尿細管障害の有無を早期に予測する有用な手段になり得ると考えられた．

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We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for the diagnosis of elevated total bilirubin (TBIL) and direct bilirubin (DBIL), we extracted 59 probe sets of rat hepatic genes from the data for seven typical drugs, gemfibrozil, phalloidin, colchicine, bendazac, rifampicin, cyclosporine A, and chlorpromazine, which induced this phenotype from 3 to 28 days of repeated administration in the present study. Principal component analysis (PCA) using these probes clearly separated dose-and time-dependent clusters in the treated groups from their controls. Eighteen more drugs in the database, reported to elevate TBIL and DBIL, were estimated by PCA using these probe sets. Of these, 12 drugs, that is methapyrilene, thioacetamide, ticlopidine, ethinyl estradiol, alpha-naphthylisothiocyanate, indomethacin, methyltestosterone, penicillamine, allyl alcohol, aspirin, iproniazid, and isoniazid were also separated from the control clusters, as were the seven typical drugs causing elevation of TBIL and DBIL. The principal component 1 (PC1) value showed high correlation with TBIL and DBIL. In the cases of colchicine, bendazac, chlorpromazine, gemfibrozil, and phalloidin, the possible elevation of TBIL and DBIL could be predicted by expression of these genes 24 h after single administration. We conclude that these identified 59 probe sets could be useful to diagnose the cause of elevation of TBIL and DBIL, and that toxicogenomics would be a promising approach for prediction of this type of toxicity.

DOI： 10.1177/0960327109104528

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【目的】トキシコゲノミクス・インフォマティクスプロジェクトにおいてSVM等の判別器と病理所見を用いた遺伝子発現プロファイルの分類によって毒性マーカー遺伝子が同定されつつある。これらマーカー遺伝子のパスウェイ等のコンテキストを調べるため、大規模トキシコゲノミクスデータベースを使用して、網羅的な解析を検討した。 【方法】各サンプルを表現型ではなく実験条件によって分類、比較した。実験条件で分類することで、例えば、投与量と投与時間を固定して化合物を変えることによって、遺伝子発現の変化の原因を化合物の違いに絞ることができる。さらに、遺伝子単位ではなくパスウェイ単位で比較し、有意差を計算することで毒性の特異性とは関係のない遺伝子の影響を減らすことができる。例えば、高投与量と低投与量のサンプルを比較した場合、 タンパク合成系の遺伝子の多くに発現変動が観察されるが、パスウェイ単位で比較することでその数の重みは軽減される。パスウェイの有意差の算出にはGSEA法を用いた。GSEA法では、遺伝子の発現差によるランキングとSN比を用いて、２つのサンプル間の特定の遺伝子セット（パスウェイ）の有意差を算出する。また、これらの遺伝子セットのプロファイルに基づいたサンプル間の関係の可視化についても検討した。 【結果】上記方法によっていくつかの毒性マーカー候補が有意差のある遺伝子セット上に観察され、そのコンテキストを推測することができた。

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DOI： 10.1016/j.toxlet.2008.06.567

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One expected result from toxicogenomics technology is to overcome the barrier because of species-specific differences in prediction of clinical toxicity using animals. The present study serves as a model case to test if the well-known species-specific difference in the toxicity of coumarin could be elucidated using comprehensive gene expression data from rat in-vivo, rat in-vitro, and human in-vitro systems. Coumarin 150 mg/kg produced obvious pathological changes in the liver of rats after repeated administration for 7 days or more. Moreover, 24 h after a single dose, we observed minor and transient morphological changes, suggesting that some early events leading to hepatic injury occur soon after coumarin is administered to rats. Comprehensive gene expression changes were analyzed using an Affymetrix GeneChip (R) approach, and differentially expressed probe sets were statistically extracted. The changes in expression of the selected probe sets were further examined in primary cultured rat hepatocytes exposed to coumarin, and differentially expressed probe sets common to the in-vivo and in-vitro datasets were selected for further study. These contained many genes related to glutathione metabolism and the oxidative stress response. To incorporate human data, human hepatocyte cultured cells were exposed to coumarin and changes in expression of the bridging gene set were examined. In total, we identified 14 up-regulated and 11 down-regulated probe sets representing rat-human bridging genes. The overall responsiveness of these genes to coumarin was much higher in rats than humans, consistent with the reported species difference in coumarin toxicity. Next, we examined changes in expression of the rat-human bridging genes in cultured rat and human hepatocytes treated with another hepatotoxicant, diclofenac sodium, for which hepatotoxicity does not differ between the species. Both rat and human hepatocytes responded to the marker genes to the same extent when the same concentrations of diclofenac sodium were exposed. We conclude that toxicogenomics-based approaches show promise for overcoming species-specific differences that create a bottleneck in analysis of the toxicity of potential therapeutic treatments.

DOI： 10.1177/0960327107087910

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

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DOI： 10.1016/j.toxlet.2006.06.015

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DOI： 10.1016/j.toxlet.2006.07.277

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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A 52-week study of oral-repeated-dose toxicity for the extraction powder of Gymnema sylvestre (GS), Indian-native genus, Metaplexis japonica, was conducted in both genders of Wistar rats. The rats were administered a graded dose of GS at 0.01, 0.10 and 1.00% of basal powder diet, along with a group fed solely with the basal powder diet without GS, for 52 weeks. General conditions were recorded daily. Body weights and food consumptions were recorded weekly up to 12 weeks, and thereafter at longer intervals. At 26 weeks, for an intermediate examination, and 52 weeks, for the final examination, animals were subjected to hematology, serum chemistry, and pathological examination. None of the animals died in the period up to 52 weeks. No exposure-related changes in body-weight, in the food consumption, in the hematological examinations, or in the serum biochemical examinations were recognized. No histopathological alterations were seen. Thus, it was concluded that there was no toxic effect in rats treated with GS at up to 1.00% in the diet for 52 weeks. The no-observable-effect level from this study is 1.00% GS, ie., 504 mg/kg/day for male and 563 mg/kg/day for female as mean daily intake, for 52 weeks.

DOI： 10.3358/shokueishi.45.8

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Other Link： http://search.jamas.or.jp/link/ui/2004271189

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Because concern over endocrine disrupting reactions caused by chemicals to human and animals is growing, a rapid and reliable screening assay for endocrine disrupting chemicals is required. We have developed an in vitro screening assay based on a hormone receptor mechanism using a surface plasmon resonance (SPR) sensor. When a DNA fragment containing a sequence of the estrogen response element (ERE) is immobilized on a sensor chip of a SPR sensor and an estrogen receptor a(ER) is injected over the sensor chip, the interaction of ER and ERE can be monitored in real time. In the presence of a chemical with estrogenic activity, the ER-ERE interaction is enhanced and the kinetic parameters are altered. We have validated the assay in terms of its specificity, dose dependency, optimal reaction conditions and reproducibility. It has been shown that the assay is very reliable as a rapid and quantitative screening method to judge the estrogenic activities of chemicals.

DOI： 10.2116/bunsekikagaku.51.389

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Toluene is a widely abused inhaled solvent. This study was designed to determine whether toluene abuse affects the reproductive functions or general health of males. Seven-week-old male Sprague-Dawley rats were exposed to toluene vapor inhalation (0, 4000, or 6000 ppm; 2 h/day) daily for 5 weeks. Exposure-related suppression of body weight gain and food consumption were observed. Salivation and lacrimation were observed during exposure periods and intensified with repeated exposure. Rats exposed to 6000 ppm toluene had decreased spleen and thymus weights, as well as suppressed lymphocyte counts. In 6000 ppm group, the epididymal sperm counts, sperm motility, sperm quality and in vitro penetrating ability to zona-free hamster eggs were significantly reduced, while no exposure-related changes in the testes weight or spermatogenesis within testes were detected. Tail-less sperm heads were seen within zona-free eggs incubated with sperm from rats exposed to 6000 ppm toluene, but not control rats. No significant changes were observed in serum luteinizing hormone, follicle-stimulating hormone, or testosterone levels following 1 month of exposure to 6000 ppm toluene. These results indicate that high concentrations of toluene may directly target sperm in the epididymis and disrupt sperm maturation. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0300-483X(99)00120-1

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A 13-week oral repeated dose toxicity study of haematococcus color, a food additive mainly composed of astaxanthin, was conducted in male and female F344 rats. Rats were randomly divided into 4 groups each consisting of 10 males and 10 females and given CRF-1 powder diet containing 0%, 0.025%, 0.075%, and 0.25% haematococcus color, correspond to 0%, 0.5%, 1.5%, and 5% as the product. None of the animals died during the administration period. There were no exposure-related changes in body weight gain or food consumptions. Serum biochemical examinations showed dose-related increase in cholesterol, but the differences were slight and not defined as an adverse effect. No effects related to treatment were noted in hematological examinations and organ weights, and no abnormalities that could be ascribed to exposure to heamatococcus color were observed in histopathological examinations. In conclusion, ingestion of haematococcus color in the diet for 13 weeks does not cause any toxicological changes in F344 rats.

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A subchronic toxicity study of pecan nut color was performed using F344 rats of both genders. Pecan nut color, a flavonoid extrcated from the coat of pecan nuts (Carya pecan ENGL. et GRAEBN.), is permitted for use as a food color by the Ministry of Health and Walfare, Japan. The product containing 60% dextrin (added in the process of manufacture) was used in the experiment. Rats were administered pecan nut color at concentrations of 0% (basal diet, control group), 0.5%, 3.0% and 5.0% in the basal powder diet for 90 days. Rats in the vehicle control group were given dextrin at the concentration of 3.0% in the same manner as used for the experimental groups. No significant changes related to pecan nut color or dextrin were found in body weight or food consumption throughout the observation period, or in hematology, serum chemistry or organ weights at autopsy. Furthermore, neither gross nor histological examination revealed any adverse effect or significant difference from the control. IL is concluded that feeding of diet containing pecan nut color at a concentration of 5.0% (1,287 mg/kg/day for males and 1,344 mg/kg/day for females as mean intake of pecan nut color without dextrin) for 90 days has no adverse effect in rats.

DOI： 10.3358/shokueishi.39.6_375

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A control survey was conducted to check the accuracy of automated analyzers used in the evaluation of clinical chemistry parameters in nonclinical toxicology studies. Pooled serum samples from male Sprague- Dawley rats were delivered refrigerated to each facility 98 laboratory facilities throughout Japan within 18 hours after sample preparation and analyzed. Commercially available normal human serum samples from a single lot were also analyzed at the same time. Survey results were divided into three categories. (1) Parameters with small coefficient of variation (CV) values for both rat and human serum samples included protein, glucose, cholesterol (CHO), urea nitrogen (UN), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and inorganic phosphate (IP). Definition of normal values in rats should be straight forward for these parameters. (2) Parameters with large CV values, but with a relatively good correlation between rat and human values include triglycerides (TG), glutamic oxaloacetic transaminase/aspartate aminotransferase (GOT/AST), glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT), and alkaline phosphatase (ALP). Measurements based on different principles gave different mean values, and this values contributed to the increase in CV values. Assessment of normal values would require a consideration of the measurement principles. (3) Parameters with large CV values only in rat serum samples included albumin (albumin/globulin ratio: A/G ratio), creatinine (CRE), and total bilirubin(BIL). Reactivity was different in rat albumin (ALB), depending on the reagents used. This difference needs to be corrected with values available by electrophoresis, or adjusted by rat ALB values, because of the lack of an appropriate measurement method. The enzyme method gave low values for rat CRE, which suggests the need for further examination of this method. The BIL values were extremely low in rat samples. It seems to be necessary to select appropriate methods to measure clinical pathology parameters correctly for rats. There was no deviation in values due solely to the mechanical operations of the analytical equipment. Non-standard initial settings of the equipment (equipment originally intended for human samples, but now applied to animal samples) was the main cause of the wide range of analytical values seen.

DOI： 10.2131/jts.22.25

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A survey was conducted to determine the accuracy and quality control of automated haematology analysers used in non-clinical toxicity studies. Pooled blood samples from male Sprague-Dawley rats were distributed to 98 laboratory facilities throughout Japan, the samples being delivered under refrigeration to each facility within 18 h of sample preparation. At each facility, the samples were analysed within 4 h of receipt. Commercially available normal human blood samples from a single lot were also analysed at the same time. Most haematological results were within the mean ± 3SD (standard deviation), but some facilities gave either high or low values consistently for both human and rat samples. No facility gave high or low values to certain parameters sporadically, which suggests no problem with the accuracy of the equipment. However, it was suspected that there would be some problem in comparing analytical values determined in a unique way by specific equipment design. The use of certain equipment resulted, in rat haematocrit values in particular, being either too high or too low. In these cases, it was deemed necessary to make some adjustments or calibration changes. There were also platelet values with a 'plus drift' which was apparently due to contamination with, or failure to identify small red blood cells (RBC). There was no deviation in values which could be attributed solely to the mechanical operation of any of the analytical equipment. Non-standard, initial setting up of the equipment (originally intended for human use, but now used for a variety of animal species) has been recognised as the main cause for a wider range of the analytical values seen. The results of this survey suggest that it may be necessary to review equipment calibration at each facility, and to re-establish the historical background data. © 1996 Springer-Verlag London Limited.

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Male and female Sprague-Dawley rats were exposed to toluene vapor at 600 and 2000 ppm for 6 h/day, and effects on their fertility were investigated. Females were exposed from 14 days before mating until day 7 of gestation. Males were exposed for a total of 90 days, including the mating period
treatment was begun 60 days before pairing, and toxicity with respect to testicular and reproductive functions was examined. In females of the 2000 ppm-treated group, salivation and lacrimation that may have been caused by CNS depression were observed starting 20 days after exposure. Although no abnormalities were seen in mating behavior or fertility, fetal mortality and the number of dams with dead fetuses increased in the 2000 ppm group. In the males exposed to 2000 ppm toluene for 90 days, an increase in kidney weights and a decrease in thymus weights were observed. Basophilic changes and necrosis of kidney tubules were greater at the higher exposure level. Additionally, decreases in the weights of the epididymides and spermatic count were observed, indicating toxicity of toluene to the male reproductive system in vivo for the first time. In conclusion, embryo-fetal toxic effects were apparent in female rats exposed to toluene before and during the early stage of pregnancy. Subacute exposure to a high level (2000 ppm) of toluene vapor elicited mild toxic changes in the kidneys, thymus, and reproductive organs of males. Toxic effects on fertility and reproduction were thus demonstrated not only in females but also in males exposed to toluene vapor in the present study.

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Language：English   Publisher：SPRINGER VERLAG  

A survey was conducted to determine the accuracy and quality control of automated haematology analysers used in non-clinical toxicity studies. Pooled blood samples from male Sprague-Dawley rats were distributed to 98 laboratory facilities throughout Japan, the samples being delivered under refrigeration to each facility within 18 h of sample preparation. At each facility, the samples were analysed within 4 h of receipt. Commercially available normal human blood samples from a single lot were also analysed at the same time. Most haematological results were within the mean +/- 3SD (standard deviation), but some facilities gave either high or low values consistently for both human and rat samples. No facility gave high or low values to certain parameters sporadically, which suggests no problem with the accuracy of the equipment. However, it was suspected that there would be some problem in comparing analytical values determined in a unique way by specific equipment design. The use of certain equipment resulted, in rat haematocrit values in particular, being either too high or too low. In these cases, it was deemed necessary to make some adjustments or calibration changes. There were also platelet values with a 'plus drift' which was apparently due to contamination with, or failure to identify small red blood cells (RBC). There was no deviation in values which could be attributed solely to the mechanical operation of any of the analytical equipment.
Non-standard, initial setting up of the equipment (originally intended for human use, but now used for a variety of animal species) has been recognised as the main cause for a wider range of the analytical values seen. The results of this survey suggest that it may be necessary to review equipment calibration at each facility, and to re-establish the historical background data.

DOI： 10.1007/BF00368455

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Language：English   Publisher：Japanese Society of Toxicology  

Toluene is a widely used solvent in industry which is the subject of abuse among the younger generation. A teratogenicity study of toluene by inhalation exposure was carried out in Sprague-Dawley rats and the effects on dams, fetuses and offspring were assessed. Pregnant females were exposed to 600 or 2000 ppm toluene for 6 h/day from day 7 to day 17 of pregnancy. The control group inhaled conditioned clean air under the same exposure conditions. Maternal exposure to 2000 ppm toluene caused significant toxic effects such as body weight suppression of dams and offspring, high fetal mortality and embryonic growth retardation, but no external, internal or skeletal anomalies were observed in the fetuses of any treated group. In addition, there were no differences in the results of pre- and postweaning behavioral tests of the offspring. However, no toxic or teratogenic changes which could be related to toluene exposure were apparent in the 600 ppm group. Further studies are warranted with toluene at higher concentrations applied during the period of organogenesis.

DOI： 10.2131/jts.20.109

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Language：English  

The conductance caused by translocation of various phosphonium cations across phospholipid (from soybean) bilayer membrane was measured. Phosphonium cations used were tetraphenylphosphonium (TPP+) and triphenylalkylphosphonium cations formulated as (Phe)3−P+-(CH2)nCH3 (n = 0–5). The conductance was dependent on voltage applied externally to the membrane in accordance with a theory developed by previous authors. Using the theory, values of βki were determined, where β and ki are a linear partition coefficient and a rate constant of transmembrane ion transport, respectively. Since βki depended on the phosphonium ion concentration, values extrapolated to infinite dilution, (βki)0, were determined. Temperature dependence of (βki)0 allowed us to estimate the activation energy of transport, Ea. For TPP+, thermodynamic values obtained were consistent with values calculated by Flewelling and Hubbell [(1986) Biophys. J. 49, 541–552]. When (Phe)3−P+-(CH2)nCH3 (n = 0–5) were used, Ea depended on the odd or even of n. This “odd and even” pattern was observed in a variety of phenomena such as solubility in water, equivalent ionic conductivity in water, and 31P NMR chemical shift. © 1994, American Chemical Society. All rights reserved.

DOI： 10.1021/bi00180a027

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The concentration dependence and mutually competitive inhibition in the membrane transport of the lipophilic cations tetraphenylphosphonium (TPP+) and its homologues were studied by use of a planar phospholipid bilayer. The current‐voltage characteristics were analyzed by use of a model developed by Ketterer et al. (J. Membr. Biol. 1971, 5, 225–245). The conductance at 0 mV [G(0)], which represents the transport ability, increased with an increase in concentration, and the G(0) values revealed saturation at higher concentrations. The relationship between G(0) and concentration was well fitted to a Michealis‐Menten‐type equation with a saturable component {maximum conductance [G(0)max] and concentration yielding one‐half of G(0)max [Km]}. The G(0)max and Km values were calculated to be 4.1 x 10−8−242 x 10−8 S/cm2 and 13–489 μM, respectively, depending on the lipophilic cations used. The G(0) values normalized by the corresponding G(0)max values were plotted against the concentrations normalized by the corresponding Km values. Normalized curves for TPP+ homologues were all superimposed on a single curve. The mechanism for the saturation of G(0) values may involve the adsorption of TPP+ homologues to a planar phospholipid bilayer, because the Km values calculated in the present study were comparable to those for adsorption to phosphatidylcholine liposomes. We further determined the mutual inhibition of membrane transport by these homologues. Tetraphenylmethylphosphonium at 1 mM, a concentration threefold higher than the Km value for this compound, reduced the G(0) for other TPP+ homologues by 38–55%, whereas reduction by 10 mM tetraethylammonium was minimal. The type of inhibition was classified as mutually competitive. In experiments on membrane transport, it is often assumed that the observation of saturation and mutually competitive inhibition indicates the presence of a carrier. The present paper, however, presents facts contradicting this assumption: greater caution should be taken in interpreting transport data to indicate carrier‐mediated transport. Copyright © 1993 Wiley‐Liss, Inc., A Wiley Company

DOI： 10.1002/jps.2600820107

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