Some studies have reported that chemicals and particulates in the environment can aggravate allergic disease or symptoms. Personal care products (PCPs) may contain them, but few studies have examined the effects of PCPs on allergic disease. The purpose of this study was to evaluate the effects of PCPs on atopic dermatitis (AD). AD model mice were exposed to mite extract and a PCP. In the presence of mite extract, the PCP exacerbated the skin lesions or accelerated the pathological advance. These results suggest that the PCP used this experiment can experimentally have an adverse effect on AD.

Subretinal injection is widely used in gene therapy research on age-related macular degeneration and retinitis pigmentosa. Rats, rabbits, and cynomolgus monkeys are often used, but the maximum dose volume and technique must be set in consideration of species. We subretinally administered saline to rats and monkeys and examined IOP fluctuations immediately after dosing. IOP increased up to 2–fold in rats administered 5 μL for 2 seconds, but did not increase in rats administered 5 μL for 1 minute. We also examined the maximum dose volume and IOP changes in monkeys and will present the results.

Rabbits are commonly used in ophthalmic drugs development; however, there are few reports about cardiovascular evaluation in rabbits. In this study, we examined ECG, blood pressure and myocardial biomarkers in rabbits treated with isoproterenol.

An increase of heart rate, ST elevation, negative T, a decrease of blood pressure, high values of troponin T/I, and necrosis of cardiomyocyte were detected. It is considered that the positive ino/chronotropic and vasodilation effect of isoproterenol led myocardial ischemia. These results show the evaluability of cardiovascular effects in rabbits.

Cardiotoxicity, such as arrhythmia, unexpected chrono- or inotropic effects, has been one of the most critical reasons for a termination of drug development. Consortium for Safety Assessment using Human iPS cells (CSAHi) aims to clarify the usefulness of human iPS cell-derived cardiomyocytes (iPS-CMs) in drug safety evaluation. Previously, we have demonstrated that the iPS cell-derived cardiomyocytes with several assay systems could be the powerful tools for detecting these risks. However, it is also known that the predictive accuracy is not fully enough due to immaturity of iPSC derived cells. In this study, we tested the improvement of the evaluating system using the electrically-stimulated iPSC-CMs, which have functionally mature phenotypes.

At present, about 10% of pregnant women in Japan have a hypertensive condition, and are diagnosed with pregnancy-induced hypertension. Among the symptoms of pregnancy-induced hypertension, eclampsia is a serious disease that can have serious consequences for both fetuses and pregnant women. A typical symptom is convulsions, and diazepam (DZ) is used as one of the therapeutic drugs. DZ is one of the benzodiazepines and is used as a sedative, anxiolytic, and anticonvulsant. So far, we have shown that DZ and its metabolites, nordazepam (NZ) and oxazepam (OZ), are detected in the fetus when DZ is administered to pregnant mice. In addition, the amount of DZ and its metabolites that distributed to the fetal brain was also analyzed because DZ and its metabolites are easily transferred to the brain. The results showed that when DZ was administered during pregnancy, the metabolite OZ was distributed to fetuses in a larger amount than DZ. That is, it was considered that OZ rather than DZ had a high possibility of affecting the fetal brain. In this study, we analyzed the effects of OZ on the fetal brain in vitro from a developmental viewpoint. The second trimester is a period in which the division of neural stem cells and the differentiation into neurons and astrocytes occur actively in the cerebral cortex region of the fetus, and the effect of OZ on neurogenesis is feared. Therefore, we isolated and cultured neural stem cells from fetal brain of second trimester mice, added OZ, and analyzed effects on differentiation into neurons and astrocytes by immunostaining and RT-PCR. As a result, it was revealed that when OZ was added to neural stem cells, differentiation into astrocytes was suppressed. This study suggests that the use of DZ during the second trimester may affect the finely programmed neural development because OZ suppresses astrocyte differentiation of neural stem cells.

The drug metabolizing enzyme cytochrome P450 (CYP) is abundant in the liver and plays a very important role in drug metabolism. In particular, the CYP2C family is involved in the metabolism of about 20% of commercially available drugs. However, no detailed analysis has been made as to when expression of the CYP2C family starts in the liver. In this study, we analyzed the expression patterns of the CYP2C family in the liver during the fetal and postnatal period in mice.

The expression levels of mRNA of CYP2C29, 37, 38 and 39 in the liver during mouse development were analyzed by RT-PCR. Furthermore, the expression pattern of CYP2C family proteins was analyzed by Western blotting and immunostaining.

Analysis at the gene level showed that only mCYP2C37 was expressed in the fetal liver. Expression of mCYP2C29 and mCYP2C39 was observed in the liver, 14 days after birth. mCYP2C38 was the first to begin to be expressed, 28 days after birth. As a result of analysis at the protein level, expression of CYP2C protein was hardly observed in the fetal period. In the neonatal period, CYP2C protein expression was extremely low. On the other hand, from the 28th day after birth, it was revealed that the expression level of CYP2C protein rapidly increased.

As described above, this study revealed that the CYP2C family, which plays a central role in drug metabolism, is hardly expressed during the fetal and neonatal period. Therefore, it is considered that almost no drug metabolism by CYP2C occurs in the liver of the fetus or the neonate. In other words, fetuses and newborns cannot metabolize CYP2C substrate drugs, so it is necessary to pay attention to the toxicity of these drugs.

This study examined developmental exposure effect of ethanol (EtOH) on hippocampal neurogenesis in rats. Maternal animals were administered 0, 10 or 12.5% (v/v) EtOH via drinking water from gestational day 6 to postnatal day (PND) 21, and offspring were examined on PND 21 and PND 77. EtOH facilitated proliferation of type-2a progenitor cells and inhibited differentiation into granule cells as a high dose effect on PND 21, suggesting relation with enhanced CALB2⁺ interneuron signal and suppressed RELN signal, respectively. Effect on synaptic plasticity was observed as a late effect.

We examined the exposure effect of α-glycosyl isoquercitrin (AGIQ), an antioxidant, from developmental stage on neuroinflammation-induced brain injury produced by neonatal injection of lipopolysaccharide (LPS) in rats. LPS-injected animals revealed abnormal behaviors, increased oxidative stress in the brain, neuroinflammation and decreased mature granule cells in the dentate gyrus from early postnatal life. AGIQ exposure improved LPS-induced changes. Thus, AGIQ ameliorated the LPS-induced brain injury and abnormal behaviors by anti-inflammatory effects from early period of treatment.

Microarray analysis revealed that maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin reduces its expression. However, the role of the pituitary death associated protein-like 1 (Dapl1) has not been fully elucidated. We constructed Dapl1-null mice to evaluate the function. In PND49, the steroidogenic acute-regulatory protein in testis and serum testosterone level were elevated in Dapl1-null mice. On the contrary, pituitary follicle stimulating hormone-β (FSHβ) was suppressed by Dapl1-ablation. These results suggest that Dapl1 is an important factor regulating the steroidogenesis.

We conducted comet assay in the rabbit corneal epithelial cells with exposured known genotoxic compounds in order to establish an in vivo genotoxicity evaluation system for ophthalmic drugs. Ethidium bromide, paraquat, methyl methanesulfonate (MMS), acrylamide and 4-nitroquinoline 1-oxide (4-NQO) were instilled once onto the eyes in male Japanese white rabbits. As the result, ethidium bromide, MMS and 4-NQO induced DNA fragmentation. These results suggest that this method can be used as an in vivo genotoxicity test in the ophthalmic drug development.

Leucine zipper-like transcriptional regulator 1 (LZTR1) encodes a substrate adaptor of E3 ubiquitin ligase complex. Mutations in LZTR1 have been identified in glioblastoma and RASopathies. Here, we demonstrate that LZTR1 facilitates the polyubiquitination and degradation of RAS via the ubiquitin-proteasome pathway. The degradation of RAS was observed in cells expressing HRAS, NRAS, KRAS, MRAS, RIT1, RRAS2 as well as oncogenic RAS mutants and inhibited the activation of ERK1/2 and cell growth. Taken together, these results show that LZTR1 functions as a RAS killer protein.

Objects: 1,2-Dichloropropane (DCP), one of the halogenated alkanes, is recently reported as the cause of the occupational cholangiocarcinoma (OCC) in printing workers and is classified as a Group 1 carcinogen (carcinogenic to humans) by the International Agency for Research on Cancer (IARC). While immunohistochemical studies on specimens from cholangiocarcinoma cases exposed to DCP showed positive γ-H2AX expression, one of the marker proteins related to DNA damage, in not only neoplasm of the bile duct but also in non-neoplastic biliary epithelial cells, the mechanism of DCP-induced carcinoma is unclear. Considering that alkaline condition is physiologically normal in the bile duct, we tested whether exposure to DCP induces expression of γ-H2AX in human cholagiocytes at alkaline conditions.

Methods: Highly differentiated immortalized human cholangiocyte cell line (MMNK-1) were treated with DCP. Immunostaining of γ-H2AX was performed. Since pH of the bile juice is usually more alkalinized (pH7.5-8) than intercellular fluid (pH 7.4) or cytoplasm (pH7.0-7.4), we tested the effect of medium with pH set at 6-8.5 on 500μM DCP-induced expression of γ-H2AX in MMNK-1 cells. We also examined the effect of DCP concentration (50μM-5mM) and exposure time (1, 4, 8hr).

Results and Conclusion: Alkaline conditions enhanced γ-H2AX expression induced by exposure to DCP in MMNK-1 human cholangiocytes. These results suggest that exposure to DCP induces DNA damage especially under the alkaline circumstances in bile ducts.

Permethrin, a pyrethroid chemical, is widely used as a pesticide because of its rapid insecticidal activity. Although permethrin is considered to exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are unclear. In this study, we investigated the effects of low-level, chronic permethrin exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. We exposed mice to the acceptable daily intake level of permethrin (0.05 mg/kg/day) in drinking water during the prenatal and postnatal periods. We then examined the effects on the central nervous system in adult male offspring. In the permethrin exposure group, we detected behavior that displayed the impairment in learning and memory. Immunohistochemical analysis revealed an increase in doublecortin- (an immature neuron marker) positive cells in the hippocampal dentate gyrus in the permethrin exposure group compared with the control group. Additionally, in the permethrin exposure group there was a decrease in astrocyte number in the hilus of the dentate gyrus, and remaining astrocytes were often irregularly shaped. These results suggest that exposure to permethrin at low levels in early life affects the formation of the neural circuit base and behavior after maturation. Therefore, in the central nervous system of male mice, low‐level, chronic permethrin exposure during the prenatal and postnatal periods has effects that were not expected based on the known effects of permethrin exposure in mature animals.

MAS-related G protein coupled receptor-X2 (MRGPRX2) is a Gi- or Gq-coupled receptor expressed in human mast cells and associated with drug-induced pseudo-allergic reactions. Dogs are highly susceptible to drug-induced anaphylactoid reactions caused by various agents including fluoroquinolones; however, the distribution and physiological function of canine MRGPR family genes, including MRGPRX2, remain largely unknown. In the present study, we clarified the distribution of dog MRGPR family genes (D, F, G, and X2) by quantitative PCR and in situ hybridization. We also investigated the stimulatory effects of compound 48/80 and several fluoroquinolones (ciprofloxacin [CPFX], gatifloxacin [GFLX], levofloxacin [LVFX], and pazufloxacin [PZFX]) on HEK293 cells transiently transfected with dog MRGPR family genes to identify the physiological function. Dog MRGPRX2 and MRGPRG were distributed in a limited number of tissues, including the skin (from the eyelid, abdomen, and cheek), whereas MRGPRD and MRGPRF were extensively expressed in almost all tissues examined. Histochemical and in situ hybridization analyses revealed that MRGPRX2 was expressed in dog connective tissue-type mast cells in the skin. Intracellular Ca²⁺ mobilization assay revealed that HEK293 cells expressing dog MRGPRX2 or human MRGPRX2, but not dog MRGPRD, MRGPRF and MRGPRG, responded to histamine releasing agents. Our results suggest that dog MRGPRX2 is the functional orthologue of human MRGPRX2 and plays an essential role in drug-induced anaphylactoid reactions in dogs.

[Aim] We generated a transgenic mouse carrying HLA-B*57:01 to reproduce abacavir-induced idiosyncratic toxicity. Here, to observe clear toxic phenotype, we eliminated immunosuppressive factors (PD-1 and CD4⁺ T cells) in the mouse.

[Results] Depleting CD4⁺ T cells increased memory CD8⁺ T cells by exposure to abacavir, and exacerbated PD-1 expression. In addition, depletion of CD4⁺ T cells in HLA-B*57:01-Tg/PD-1^-/- dramatically increased memory CD8⁺ T cells and induced skin redness. These results infer blockade of immunosuppression should be important in reproducing HLA-mediated immunotoxicity.

[Aim] We investigated drug reactions in keratinocytes (KCs) by focusing on the interaction between HLA-B*57:01 and abacavir (ABC) using our originally produced HLA-B*57:01 transgenic mice (B*57:01-Tg).

[Results] ABC exposure increased mRNA expression of IFN-γ and immunoproteasome (IP) subunits induced by IFN-γ in KCs derived from B*57:01-Tg. In addition, ABC exposure increased protein expression of IP subunits in skin tissue of B*57:01-Tg. These results imply that increased IP expression by ABC exposure in KCs expressing HLA-B*57:01 might produce specific antigens and induce immunotoxicity.

We aimed to elucidate the possible intervention of inorganic arsenic compounds (AS) exposure with allergic asthma development. A mouse model of asthma was developed by intranasal administration of mite extract (ITEA Inc). AS (10 and 100 ppm) were orally administered in drinking water during the experimental period. Eosinophils, cytokine levels, IgE-positive B cells and serum IgE level were analyzed. AS exposure significantly heightened the number of eosinophils, cytokine levels, number of IgE-positive B cells and serum IgE levels compared to the control group. Our findings imply that AS exposure exacerbated the response in a mouse model of allergic asthma.

Seafood contaminants, tributyltin (TBT) and triphenyltin (TPT), are known to have immunotoxicity with thymic atrophy, but their mode of action remains unclear. We here examined the effects of TBT and TPT on thymic morphology and T cell differentiation in mice at a dose without nonspecific toxicity. TPT induced thymic atrophy with adipogenesis via PPARγ activation and reduced peripheral naive T cells. In contrast, TBT induced thymic atrophy without adipogenesis and PPARγ activation. Both act as PPARγ agonists, but our results suggest that their mechanisms of thymic atrophy are much different.

【Purpose】Mitochondrial toxicity is paid attention as a factor of drug-induced liver injury. For the assessment of mitochondrial toxicity induced cell death in vitro, sugar resource substitution from glucose in the culture medium to galactose helps maintain higher mitochondrial activity in cultured hepatocytes. However, mitochondrial permeability transition (MPT)-induced toxicity can’t be detected by only substituting sugar resource. Recently, we reported that lipopolysaccharide-induced mitochondrial reactive oxygen species (mtROS) sensitized MPT-induced cell death by diclofenac in vivo. Based on this, we aim to build a comprehensive cell-based assay system for MPT-inducers by loading mtROS in vitro.

【Methods】To load mtROS, primary mouse hepatocytes cultured in either glucose or galactose media were incubated in 1% O2 for 4 hr, then 40% O2 for 20 hr (designated H/R). During this time, the drug was exposed for total 24 hr. Cell death was evaluated by lactate dehydrogenase leakage.

【Result・Discussion】Three well-known MPT-inducers (benzbromarone, flutamide, troglitazone) significantly induced cell death only in combination with H/R and galactose. Such enhancement of cell death was not observed in hepatocytes lacking cyclophilin D (regulator of MPT pore opening). Drugs which have other mitochondrial toxicity mechanisms caused highest cell death in H/R and galactose. In conclusion, MPT could be evaluated with a new cell-based assay combining galactose and H/R. This cell-based system is useful for comprehensive assessment of the MPT inducibility of parent drugs and drug metabolites.

Iron ion is an essential element involved in various metabolic processes including oxygen transport, DNA synthesis, and energy transduction. Excess iron due to the defect of iron balance in a disease such as β-thalassemia, activates nuclear factor-erythroid 2-related factor 2 (Nrf2) and induces the expression of Nrf2 target genes. Nrf2 is a transcription factor responsible for the regulation of cellular oxidative response in mammals. The activation of Nrf2 by excess iron ion is mediated by reactive oxygen species (ROS) formation, which can lead to the induction of oxidative stress. Therefore, efforts to prevent oxidative stress caused by excess iron ion are necessary. In this study, we aimed to investigate the effects of iron chelators on iron ion-induced oxidative stress. We confirmed that exposure of excess iron ion to Hep3B cells for 12 hours increased intracellular ROS and Nrf2 levels, indicating the elevation of oxidative stress. It also upregulated the expression of Nrf2 target genes, such as heme oxygenase 1 (HO-1) and NAD(P)H Quinone Dehydrogenase 1 (NQO1), and heavy metal-binding proteins, such as metallothionein (MT) I and II. Next, we treated the Hep3B cell with iron chelator (2,2'-Dipyridyl) for 6 hours under the exposure of iron ion. As expected, dipyridyl diminished the induction effect of iron ion on Nrf2 levels and NQO1 expression. Interestingly, the expression of HO-1, MT-I, and MT-II was induced by dipyridyl treatment. We are now investigating the mechanism underlying elevated HO-1, MT-I, and MT-II expression by dipyridyl under the exposure of iron ion.

Drosophila melanogaster has been used as a human model. We analyzed the effects of exposure by formaldehyde gas (FA) or xylene gas (XY) on Drosophila. By 20 days-exposure to XY, reactive oxygen species and reactive nitrogen species significantly increased. The expression levels of oxidative stress-related genes significantly increased in both gas-exposure groups, and the increase was larger in XY group. The cellular aging was enhanced in both exposure groups by exposure longer than ten days. These suggested that XY and FA may cause the cellular aging in Drosophila with different manner.

Antibacterial cephems are safe drugs used to control various infectious diseases. Meanwhile, cephems cause rare but serious side effects, such as acute tubular necrosis (ATN), due to the cytotoxicity of cephems. However, toxic mechanisms of cephems remain unknown. In this study, we performed comprehensive analysis of the toxic mechanisms, and found that the nuclear accumulation of the ubiquitinated protein-containing aggresomes, called aggresome-like induced structures (ALIS), driven by reactive oxygen species (ROS) is a common feature of cytotoxic cephems, which promotes cephem-induced cytotoxicity.

Recently, we found that the W208A substitution in human UDP-glucuronosyltransferase (UGT)1A7 diminished its function, even though the corresponding residue of UGT1A6, a related active isoform, is Ala. To address this anomaly, we constructed mutants of UGT1A7 to evaluate the role of Trp208 and its surrounding residues on its SN-38 glucuronidation activity. Analyses of the mutations suggest that His210 has a negative effect on UGT1A7 activity and further, that activity of the UGT1A7(W208A) mutant is restored when the surrounding residues are mutated to mimic those of UGT1A6.

Significant individual differences have been noted in the degree of onset and symptoms of sick house syndrome, but the mechanism has not been elucidated. In this study, to this end, we evaluated the expression levels of ADAM33 and other genes in human tracheal and lung tissues, which are known to be involved in airway hyperreactivity and asthma. Real-time RT-PCR revealed that the gene expression level of ADAM33 did not show significant individual differences in trachea. In contrast, the levels of MMP9 and GPR99 (OXGR1) showed remarkable individual differences in trachea and lung. It is possible that these factors play an important role as one of the factors explaining individual differences in the degree of onset of sick house syndrome.

In this study, we found that nuclear receptors LXR-alpha and LXR-beta (originally characterized as regulatory factors involved in cholesterol/bile acid homeostasis) regulate each other’s expression in normal renal-derived cells and normal hepatocyte-derived cells. Since LXR-alpha stimulates fatty acid synthesis, an LXR-beta-specific agonist is assumed to be the basis for an anti-arteriosclerotic drug that only stimulates reverse cholesterol transport. However, our findings show that the development of such an anti-arteriosclerotic drug would require further elucidation of the complex mechanism of LXR-alpha and LXR-beta regulation.

Parthanatos is a newly discovered non-apoptotic cell death mediated by the stress-sensing protein PARP-1. However, there are few effective inducers of parthanatos, and thus we screened and identified novel parthanatos inducers. Among them, antibiotic X possesses interesting properties as a parthanatos inducer that selectively executes parthanatos in cancer cells lacking the tumor suppressor protein STK11 (serine/threonine kinase 11), but not normal cells. Therefore, our results suggest that antibiotic X works as a novel anticancer agent that targets cancer cells expressing loss-of-function mutants of STK11.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) both positively and negatively regulates stress-induced cell death. However, roles of TRAF2 in DNA damage-induced cell death remain unknown. In this study, we assessed the roles of TRAF2 in DNA damage-induced cell death induced by cisplatin, a representative DNA-damaging agent. TRAF2-deficient cells exhibit significant resistance to cisplatin-induced apoptosis, accompanied by the reduction of p53 protein level. Thus, our results suggest that TRAF2 promotes cisplatin-induced apoptosis through p53-dependent mechanisms.

Since vascular endothelial cells can be a target of chemical substances in the blood, the cellular defense systems of the cells are important. Reactive sulfur species (RSS) can contribute to the defense systems. We investigated the regulation of RSS-producing enzyme expression by TGF-β₁, a cytokine that regulates endothelial cell functions, and the intracellular pathways that mediate the regulation. It was found that TGF-β₁ selectively induces the expression of CSE and CBS among RSS-producing enzymes via both the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells.

In this study, we demonstrated the activation of human and mouse TRPA1s (hTRPA1 and mTRPA1, respectively) by ferulic acid and its 6 analogues. Among them, methyl ferulate (MF) and ethyl ferulate (EF) activated both hTRPA1 and mTRPA1 with hTRPA1 being activated at lower concentration ranges. Mutations at V875G in hTRPA1 and G878V in mTRPA1, which have been shown to be involved in species differences in the TRPA1-activation by menthol, had no effect on the affinity of MF and EF to hTRPA1 and mTRPA1. These results suggest that MF and EF interacts with amino acid residue(s) different from those crucial in the case of menthol.

The aryl hydrocarbon receptor (AhR), a transcription factor, is well known to translocate from cytoplasm to nucleus by binding of its ligands. We have found the increased expression of AhR and its constitutive activation in a human epithelial growth factor (HER) 2-overexpressing breast cancer cell line. Further, HER2 signal activation shows AhR-mediated induction of inflammatory cytokine. Therefore, in the present study, we evaluated AhR-dependent alterations in open chromatin regions by ATAC-seq method to elucidate the mechanism of malignant transformation of HER2-positve breast cancer cells.

Convulsion causes significant impact on drug development but there is no good biomarker. The aim of this study was to investigate the potential of heart rate variability (HRV) as novel biomarker for drug-induced convulsion in monkeys. Telemetry-implanted monkeys were dosed with convulsants, and HRV was analyzed for 3 hours after dosing by multivariate statistical process management. As a result, frequency and duration of alarms were tended to be increased by picrotoxin and penthylentetrazole. It suggests HRV is useful for predicting drug-induced convulsions in monkeys.

Liver-derived L-FABP is reported to be a potential of disease biomarker for NAFLD/ NASH and viral hepatitis in human patients. CCl4 was administrated subcutaneously once to hL-FABP Tg mice at the dose levels of 2.5, 5 and 10 mL/kg. After 24, 48 and 72 hours of administration, the hepatotoxicity and levels of serum hL-FABP were evaluated. hL-FABP was reached the maximum levels at 24 hours after dosing and then decreased over time with dose-dependently, however, there was no difference in hepatological features at all levels. And the protein expression of hL-FABP in the liver was enhanced over time. The mechanism of these changes will be analyzed.

Drug-induced vasculitis is difficult to monitor in clinical trials, as the underlying mechanism has not been elucidated and no specific/sensitive biomarkers are known to date. So, it is one of the major challenges in drug development. We conducted in vivo MRI, a powerful imaging technique in clinic, to clarify whether drug-induced vasculitis in rats can be detected.

Rats were administered fenoldopam mesylate and 11.7 T MRI were performed. The mesenteric arteries were evaluated ex vivo/in vivo MRI and histopathological evaluation. Vasculitis was detectable by ex vivo and in vivo MRI.

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Since 3D organoid culture takes much cost and labor, the establishment of cheaper and Matrigel-free culture method which can keep the characteristics of 3D organoids is demanded. In this study, we tried to induce 2D cells from 3D bladder cancer organoids (named “2.5D organoid”) and identified the most suitable medium components. The 2.5D organoids grew faster than the 3D ones and showed the similar characteristics of original 3D organoids, suggesting that our established 2.5D organoid culture method might become a reasonable and useful tool in various cancer and toxicology research fields.

There is increasing concern about transient exposure in disaster and emergency. However, conventional toxicology testing methods may not address this situation. Thus, we assessed the toxic effects of a history of transient exposure in childhood stages by exposing male rats to tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) at their neonatal and adult stages. Rats receiving TDCIPP on both stages had significantly increased adrenal gland weight compared with rats receiving TDCIPP at adult stage only.

Isometric contractile force was recorded using cell-sheet-tissues. The drug effects were evaluated at 3 different electrical stimulation conditions as follows; spontaneous beat, 1 Hz pacing, and spontaneous beat after preconditioning (72hr stimulation at 2 Hz for maturation purpose). Drugs which were difficult to measure under spontaneous beat could be evaluated under 1 Hz pacing without beating arrest, or exhibited large increase of contractile force after preconditioning. Taken together, this method can be a very useful tool for preclinical study.

Human iPSC-derived neurons are expected to be applied to toxicity evaluations in nonclinical studies. Microelectrode array (MEA) measurement system is suitable to evaluate the electrophysiological responses to drugs. In this study, we examined its application for the risk assessment of antibiotic associated encephalopathy (AAE). AAE can be classified into three clinical encephalopathy types: encephalopathy accompanied by seizures; encephalopathy characterized by psychosis; and encephalopathy accompanied by cerebellar signs. We investigated whether the electrophysiological responses to antibiotics in human iPS cell-derived neural network classify into three clinical types. Human iPS cell-derived neurons and astrocytes were co-cultured on MEA. More than 10 antibiotics were cumulative administered in culture samples. We classified the responses to antibiotics by multivariate analysis. Cephalosporins and penicillins, which are classified into type 1 AAE, increased the firing frequency and synchronized activities of the neural network. These results were consistent with seizures of type 1 AAE in clinically reported. Quinolones, macrolides, and procaine penicillin, which are classified into type 2 AAE in clinically reported, showed a different response from type 1. These results suggested that the responses to antibiotics in human iPSC-derived neurons and the classification of antibiotics encephalopathy in clinical are correlated. MEA assay coupled with human iPSC-derived neurons and our multivariate analysis are useful for the risk prediction and risk classification of AAE.

The integrated testing strategy (ITS) developed by Kao covers three alternative models, namely h-CLAT, DPRA and DEREK, as a defined approach for the potency classification (EC3 < 2%; EC3 ≥ 2%; Non-sensitizer) of skin sensitization. To refine the potency estimation, we developed a strategy combining the ITS and read-across using relevant analogues. Moreover, to determine the applicability of the novel strategy, we performed four case studies using cosmetic ingredients with known EC3 values. These case studies showed that our novel strategy was useful to estimate the skin sensitization potency.

We aimed to develop in silico model to quantitatively predict EC3 values of LLNA test in skin sensitization evaluation based on the IATA approach. Using in vitro tests data and physical property values of 120 substances in the Cosmetics Europe database, we constructed the regression model with gradient boosting decision tree. Our model showed the coefficient of determination was 0.7 or more and data of the h-CLAT contributed largely to the model. In conclusion, we constructed the high-performance prediction model of EC3 value of LLNA test with interpretability

Cosmetics applied to the skin are directly exposed to sunlight, and thus the photosafety evaluation of ingredients is essential. Photochemical assays such as UV/VIS spectral analysis and ROS assay (OECD TG495) used as screening method show high sensitivity. However, because of its high false positive rate, the development of follow-up system has been needed. Although we have so far reported some in vitro photo-sensitization tests (e.g. photo-DPRA, photo-KeratinoSens and photo-h-CLAT) based on its adverse outcome pathway (AOP), these tests have not yet been established. This study aimed to obtain photo-DPRA data of 82 chemicals (67 photo-sensitizers and 15 non-photo-sensitizers) used as a reference database, and to clarify the applicability domain.

The protocol has been developed1) based on DPRA (OECD TG442C). The peptide % depletion differences between with- and without UV exposure were calculated and the positive criteria was set according to original DPRA prediction model. Fifty-two chemicals were evaluated as positive, 14 chemicals were judged as negative, 8 chemicals were considered as inconclusive and 8 chemicals were not applicable due to co-elution with Cys peptide. The sensitivity, specificity and accuracy were 81%, 83% and 81%, respectively. Our results showed the utility of photo-DPRA to support the discrimination between photo-sensitizers and non-photo-sensitizers. The data presented here will be used for a reference database when developing an AOP-based approach to assess photo-sensitization potential.

Reference

1.Nishida, H. et al., 2016. Development of Photo-Direct Peptide Reactivity Assay. 55th Annual meeting for Society of Toxicology, Abstract No. 2777

The test method for respiratory sensitization has no established, despite its serious toxicity, but it is known that respiratory sensitizers have high reactivity with lysine. ADRA is a test method using a cysteine derivative (NAC) and a lysine derivative (NAL). The possibility of evaluating them by ADRA was examined. As a result of measurement of reactivity of respiratory sensitizers with NAC and NAL, most of the respiratory sensitizers were correctly predicted. NAL depletion/NAC depletion≧0.9 or NAL depletion≧20% was set as a criterion.

Purpose

In the cosmetics industry, where continuous pressure is given to eliminate animal testing, it is difficult to evaluate systemic toxicity; including developmental toxicity where assessment in two species are recommended for pharmaceuticals. iTTC (internal Threshold of Toxicological Concern) may provide means to account for interspecies differences in toxicokinetics (TK) and the root-to-root extrapolation by using more refined approaches. In this study, we collected the data of developmental toxicity studies and TK studies in two species (rats and rabbits) to set up iTTC values from review report in PMDA database.

Method

76 pharmaceutics with No observed adverse effect level (NOAEL) values were extracted from the database. Each NOAEL value was converted to dNOAEL (dNOAEL (ng·hr/mL)=NOAEL(mg/kg)·AUC(ng·hr/mL)/Dose in TK study(mg/kg)). 95% tile value of dNOAEL were calculated to set up iTTC for developmental toxicity in two species. Systemic exposure of some cosmetic ingredients were calculated and compared with iTTC as case-study.

Result and discussion

The 95% tile value of dNOAEL values are 77.05 ng·hr/mL and 294.02 ng·hr/mL for rats and rabbits, respectively. Each iTTC was calculated 3.08 ng·hr/mL and 11.76 ng·hr/mL by applied MoS (Margin of Safety) to dNOAEL. As these values can be applicable to the assessment of cosmetic exposure in normal usages, it is strongly expected that risk assessment of the raw material which lacks data for developmental toxicity will be possible by combining iTTC values with the predicted internal exposure in human by the PBPK modeling.

IATA based read-across with considering biological similarities is expected to enhance prediction accuracy of systemic toxicity. To confirm this concept, we compared biological similarities in the liver by microarray analysis and ADME prediction in model categories (Naphthalene and Clorofene group). The results showed differences in cellular key event and exposure level of the liver between category members. The integrated conclusion taking thses results into account well explained the NOAEL and toxicological findings for the liver in each chemical. Thus, the present study suggested that IATA improve the prediction accuracy of read-across.

We previously tried to validate a zebrafish (ZF) embryo developmental toxicity test using 28 positive reference compounds (described on the Reproduction and Developmental Toxicity Test Guideline of the International Conference on Harmonization of Pharmaceutical Regulations), and found that the eight compounds were false negative. In the present study, the concentration of some false-negative compounds in the ZF embryo was measured. Based on the results of the experiments, the restriction of uptake of cytarabine with very low log P (-2.7) might be the cause of false negatives.

The purpose of this study was to develop a new in vitro screening system to evaluate the percutaneous toxicity of chemicals using a 3-dimentsioal reconstructed human epidermis model (3-D model). A pesticide, folpet, LabCyte EPI-MODEL and were used as a test chemical, a 3-D model and a monolayer culture model, respectively. The obtained biochemical, histopathological and molecular biological data have indicated that the layered structure of the epidermis has a protective effect against the skin toxicity of folpet. It is thus suggested that the monolayer culture model is insufficient to evaluate the skin toxicity of chemicals in vitro, and that the 3-D model is useful for such a purpose.

To investigate the effects of choline-deficient methionine-lowered amino acid diet (CDAA) or high fat diet (HF) on SDT fatty (f) rats, which are obese type 2 diabetic animals, on liver and intraperitoneal adipose tissue, 17 weeks old male SDT f and SD rats were fed basal diet (CE2), HF or CDAA ad libitum for 4 weeks. At 21 weeks of age, animals were dissected, collected the blood and organs and were performed various analysis.

In SDT f rats, the induction of fatty liver by CDAA or HF were weaker than those in SD rats, on the other hand, the visceral fat accumulation by HF was more apparent. The mechanism of these changes will be analyzed.

In this study, we aimed to develop a new In silico method for predicting the inhibitory activity of chemical substances against drug-metabolizing enzymes using chemical structure information. We used In vitro experimental values of 218 substances selected from the HESS database as learning data. All models by random forest showed ROC-AUC achieved 0.8 or more. We have developed high-efficiency and high-performance In silico prediction models for the inhibitory activity of chemical substances to drug-metabolizing enzymes.

To establish a novel and more objective read-across method, we have examined a possible use of molecular descriptors. Euclidean distances among substances were calculated using molecular descriptors and multiple thresholds were set. Substances, which had ≥ 5 substances (source) within a given threshold, were used as target substances, and the toxicity of the target and source substances were compared. The results obtained indicate that it is necessary to appropriately select a set of descriptors and a threshold value, according to the types of substances and toxicological findings of interest.

In this study, we focused on developing a Drug-Induced Liver Injury (DILI) prediction method by machine learning from chemical structure information of drugs using large-scale adverse drug reaction data. We constructed the higher accurate prediction model by the random forest using the dataset combining the Japanese Adverse Drug Event Report (JADER) database and Drug Induced Liver Injury Rank (DILIrank), compared with using JADER alone. These results suggest that our model is useful to classify DILI-causative drugs with high accuracy using large-scale adverse drug reaction data.