Nrf2 is a key factor in defense against oxidative stress, which is degraded through binding to Keap1. In cancer cells, aberrant Nrf2 activation through Keap1 mutation and the pentose phosphate pathway (PPP) as a source of nucleic acids promote proliferation, and glycolytic intermediates are required for PPP. In this study, we investigated the regulation of Glut1, and enzymes of glycolysis, TCA cycle and PPP by treatment of tBHQ (Keap1 inhibitor), Nrf2 knockdown or overexpression. As a result, we found that Glut1, PGD, TKT and G6PD levels are dependent on the Nrf2 expression level.

Background: Bone tissue destruction is the feature of many symbolic diseases, including osteoporosis, rheumatoid arthritis, and bone metastasis. Receptor activator of nuclear factor-κB ligand (RANKL) has been identified as osteoclast differentiation and activation of an important factor. Atriplex nummularia (AN) is a traditional Chinese medicinal plant used to treat arthralgia and rheumatism, and it also has antioxidant and hepato-protective activities. This study investigates the effects of AN extract (ANE) on the formation of RANKL-activating osteoclasts from RAW264.7 macrophages.

Materials and Methods: Preparation of AN extract, Dry whole plant materials of AN (3.1 kg) were crushed and drenched in 31 L of ethanol for one day and then extracted three times. The extract was collected and concentrated with a vacuum evaporator. The extraction yield was 2.5%. Cell culture and induction of osteoclastogenesis, For differentiation, RAW264.7 macrophage cells were suspended in DMEM supplemented with 10% FBS in the presence of 50 ng/ml RANKL and plated at a density of 1 104 cells/well in a 96-well plate. Multinucleated osteoclasts were observed on differentiation day 4. Cell viability assay, RAW264.7 cells were seeded in 96-well plates, and 5 mg/ml of MTT was loaded to the plate. The cytotoxicity against RAW264.7 cells was determined by measuring the absorbance of the converted dye at 570 nm in an ELISA reader. TRAP staining and activity assay, Cells were stained using the Leukocyte Acid Phosphatase Kit 387-A. To measure TRAP activity, osteoclasts underwent lysis using 0.1% Triton X-100; then TRAP solution was added to the wells of the 96-well plates. The enzyme reaction was performed and an equal volume of 0.3 N NaOH was added to stop the reaction. Absorbance was measured at 405 nm using a Microplate Photometer. Bone resorption Assay, RAW264.7 cells or PBMCs were differentiated into osteoclasts for 7 days on Osteo Assay Surface Multiple Well Plate. Each well was examined for resorption pits by phase-contrast microscope with DP Controller. Western blot analysis, a fixed amount of protein was applied to 11% SDS-polyacrylamide gel electrophoresis. Primary antibodies were added. Following overnight incubation at 4°C, the Western Bright ECL kit was used to develop the signal of the membrane. The signals were detected using Image Lab Software Ver. 2.0.

Results: ANE effectively inhibited RANKL-induced osteoclasts formation without cytotoxicity. ANE was capable of suppressing the activity of mature osteoclasts from RAW264.7 cells. ANE inhibit the bone resorptive activity of mature osteoclasts in a RAW264.7 cells model. ANE significantly inhibited RANKL-induced activation of NFATc1, TRAF6, NF-κB, JNK, ERK, p38 and MMP-9 during osteoclast differentiation from RAW264.7 cells.

Conclusions: ANE has the potential in treating gouty erosions through inhibiting differentiation, formation and bone-resorbing ability of osteoclasts.

Keywords: Atriplex nummularia Extract(ANE), Osteoclastogenesis, RANKL-activating osteoclasts, Nuclear factor-κB ligand (RANKL).

We previously demonstrated that Δ⁹-tetrahydrocannabinol (THC), a major component of the drug-type cannabis plant, up-regulates FA2H expression via PPARα induction in human breast cancer MDA-MB-231 cells (J. Toxicol. Sci., 38: 395, 2013; Toxicology, 326: 18, 2014; Arch. Biochem. Biophys., 662: 219, 2019). However, details of up-regulation mechanisms of FA2H by THC are still unknown. Here, we show that PPARβ/δ-mediated suppression of PPARα activity is functional and THC can release PPARβ/δ-mediated abrogation of PPARα, which results in induction of FA2H.

Cyclin-dependent kinases (CDKs) are regulators of cell cycle progression. Although intestinal epithelial cells are highly proliferative, the role of individual CDK in the cell proliferation has been unclear. In this study, a rat small intestinal epithelial cell line IEC6 was used to understand the role of CDKs in the proliferation and survival of intestinal cells. Based on the results of several experiments, CDK1 and 9 but not CDKs 2, 4, or 6, would be essential for intestinal cell cycle progression and explains the lack of GI toxicity observed with palbociclib.

Menadione (MD, vitamin K₃), the highly toxic substance, is formed as a metabolic intermediate in the process of producing vitamin K₂ from vitamin K₁in vivo. Here, we investigated whether there is a pathway by which MD is reduced and then sulfated by sulfotransferase (SULT) as a reason why MD has not shown any toxicity despite its formation in vivo. Human SULT isoforms involved in the sulfation of the reduced form of MD (MD-OH) were studied. In addition, glucuronidating and sulfating activities in human liver S9 toward MD-OH were investigated to evaluate the contribution of UDP-glucuronosyltransferase (UGT) and SULT in the conjugation reaction following two-electron reduction of MD by NAD(P)H:quinone oxidoreductase 1 (NQO1). In the human liver cytosol, MD-4-O-sulfate was predominantly produced. SULTs 1A1, 1A3, 1B1 and 1E1 showed sulfating activity toward MD-OH. MD-4-O-sulfate was predominantly produced in all isoforms showed MD-OH sulfating activity, but only SULTs 1A3 and 1E1 produced MD-1-O-sulfate. SULT1A1, which is expressed at a high level in the liver, was revealed to be the most important factor in the sulfation of MD-OH in the liver. The contribution of UGT and SULT in the conjugation of MD-OH was examined using human liver S9. The results suggested that at low concentrations of MD, sulfation proceeded predominantly, and at high concentrations, glucuronidation proceeded predominantly.

Diphenylarsinic acid (DPAA) was a pentavalent arsenic detected in the well water, inducing neurological symptoms in nearby residents. DPAA could induce aberrant intracellular activation of cultured rat cerebellar astrocytes including induction/activation of antioxidant proteins, MAP kinases, and transcription factors in cultured rat cerebellar astrocytes. Here we evaluated protective effects of major heavy metal ion chelating agents against DPAA-induced aberrant astrocyte activation, and demonstrated that DMSA and DPEN, but not BAL and DMPS, were possible agents for the therapeutic use.

Molybdenum has redox potential which is utilized as a cofactor of oxidoreductases including Xanthine oxidoreductase. Exess intake of molybdenum causes hepatic and neuronal toxicities. Therefore, a fine tuning of molybdenum trafficking is required. However, the storage mechanism of molybdenum remains unclear. In this study, we showed that molybdenum interacts with melanin by our in vivo and cell-free system. Melanin might be a potential storage of molybdenum.

Organotins, which are persistent marine pollutants, are potent endocrine disruptors acting as agonists for peroxisome proliferator activated receptor (PPAR) γ. Previously, we identified PPAR in Leucoraja erinacea (LePPAR), a kind of ray in cartilaginous fish, suggesting that organotins possibly have some adverse effects to such an oldest jawed vertebrate via PPAR, too. Here, to extend our knowledge concerning risk assessment of organotins in cartilage fish, we investigated the binding mode of organotins to LePPAR by comparing it to that of human PPARγ.

The purpose of study is to identify hepatic enzyme(s) responsible for the decrease in the formation of mefenamic acid-quinoneimine (MFAQI), which may be a reactive metabolite of mefenamic acid. The CYP1A2-mediated MFAQI formation was significantly decreased by human liver cytosol. Superoxide dismutase 1 (SOD1) was identified, by purification from cytosol, as an enzyme decreasing MFAQI. Cytotoxicity by MFA in CYP1A2-overexpressed HepG2 cells was enhanced by knockdown of SOD. Thus, we found that SOD1 has a role to decrease MFAQI production, which may be due to scavenging of superoxide.

NSAIDs containing carboxyl groups may be conjugated with CoA as well as glucuronic acid, and their conjugates are likely related with toxicities. In this study, we clarified that, among 26 acyl-CoA synthetase (ACS) isoforms, ACSL1 was the highest expressing isoform in the human livers. Propionic acid-class NSAIDs were selectively conjugated with CoA by ACSL1. Protein binding abilities of CoA-conjugates of these NSAIDs were higher than those of their acylglucuronides (AG). Thus, CoA-conjugates produced by ACSL1 may be related with propionic acid-class NSAIDs-induced toxicity rather than AG.

We examined how each cytokine is involved in P450 down-regulation in clinical sepsis model by cecal ligation and puncture (CLP). CLP significantly down-regulated CYP1A2, CYP2C29, and CYP3A11 in IL-1α/β KO and TNFα KO mice as well as that in wild-type mice. In contrast, CLP showed no significant effect on CYP1A2 and CYP3A11 in IL-6 KO mice. CYP2C29 was also decreased by CLP in IL-6 KO mice, although the reduced level was less than WT mice. The respective P450 activities were reflected in P450 gene expressions. These results suggest that IL-6 released by CLP down-regulates P450 gene expression.

The sex- and age-differences in the susceptibilities to xenobiotics including drugs are well known in experimental animals and humans. As the causes of these different susceptibilities, qualitative and quantitative differences in drug metabolizing enzymes and transporters in various organs, especially the liver, are thought. In this study, to elucidate the mechanism of androgen-dependent changes in the gene expression of drug metabolizing enzymes, we established an androgen receptor (AR)-stably expressed cell line from HepG2 cells and evaluated whether AR is involved in regulating the gene expression.

In this study, to elucidate the effects of gestational arsenic exposure on the F1 reproductive system, we examined gene expression of epigenetic-related genes and spermatogenesis-related gene in F1 male mice gestationally exposed to arsenic. We found that gestational arsenic exposure probably induced DNA hypermethylation in the testis of F1 mice. Our results suggested that altered DNA methylation affected spermatogenesis through disruption of spermatogonia differentiation in the testis of gestationally arsenic exposed F1 mice.

Metallothionein (MT) is a small, cysteine-rich protein active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Mouse MT-1 gene transcription is regulated by metal response element (MRE)-binding transcription factor-1 (MTF-1), which is strongly recruited to the promoter in response to zinc. MTF-1 shows the highest affinity to MREd among 5 MREs (MREa-MREe) in MT1 promoter. Several studies showed that epigenetics, such as methylation of CpG, is also involved in the MT1 gene expression. Here, we examined whether the specific CpG sites involved in regulation of MT1 expression using repoter vector, pCpGfree-basic-Lucia, which is devoid of CpG. Twenty-nine CpG sites are in the MT1 promoter spanning bases －264 to +10 (relative to the transcription start site). Complete CpG methylation by M.SssI (CG → ^5mCG) inhibited MT1 promoter-mediated Lucia expression. The inhibitory effect of M.SssI-methylation was not observed in MREd/MREe-deleted reporter vector. The inhibitory effect was remained in MREa/MREb/MREc-deleted reporter vector. Similar results were observed in point mutated reporter vectors. To clarify the effect of partial CpG methylation, we used HhaI (GCGC → G^5mCGC)-methylated reporter vectors. Point mutation analysis revealed that methylation of CpG site near MREd/MREe is strongly inhibit MT1 gene expression. Our results suggest that methylation/demethylation of the CpG near MREd/MREe is important for regulation of the MT-1 gene expression.

Metabolomic data in rat plasma samples administered with hepatotoxicants in Toxicogenomics Informatics Project (TGP2), an industry-government-academia joint research in Japan were analyzed.　The results shown that localization specific changes in necrotic hepatocyte were observed in some metabolites. 5 hepatotoxicants (N-nitrosodiethylamine [DEN], ethionamide [ETH], thioacetamide [TAA], metapyrylene [MP], acetaminophen [APAP]) were orally administered to rats for 7 or 14 consecutive days. Plasma samples collected 24 hours after last dosing were comprehensively analyzed for metabolites using CE-TOFMS, and 223 metabolites were identified and quantified. An increase in glutamine (Gln) concentrations and a decrease in glutamate (Glu) concentrations in plasma were simultaneously observed only in MP-treated rats. A Gln/Glu ratio in plasma increased also in the same group. On the other hand, an increase in urea concentrations in plasma was observed in all hepatotoxicants-treated rats except for low of dose DEN- and TAA-treated rats. In TGP2-archived histopathology data, hepatocyte necrosis is observed in periportal zone in MP-treated rats and the change is observed in perivenous zone in the other hepatotoxicant-treated rats. In detoxication process of ammonia in the liver, ammonia convert to urea in periportal hepatocytes, and glutamate is converted to glutamine as detoxication of ammonia in perivenous hepatocytes. Taken together, a change of ammonia detoxication pattern show from urea synthesis to glutamine synthesis in MP-treated rats and inverse change was observed in the other groups. Interestingly, both changes were simultaneously observed in MP-treated rats. Accordingly, an increase in a Gln/Glu ratio in plasma will be a useful biomarker as prediction of hepatotoxicity localization (hepatocyte necrosis in periportal zone).

The goal of this project was to use RNA-Seq to profile a broad collection of tissues from preclinical species to build a comprehensive tissue expression atlas complementary to human tissue transcriptomic maps for rapid assessment of target distribution. Tissues from the same organ systems clustered together and overall human to nonclinical species correlation was above 60%. These datasets can also complement other information such as functional assay data to support the selection of pharmacologically relevant species for safety evaluation and de-risking studies of new drugs.

Identification of chemical compounds, such as toxic compounds and their metabolites, is of vital importance for toxicological studies. Mass spectrometry has been widely used as one of the most versatile analytical tools. On the other hand, identifying the chemical structures of compounds in complex sample containing a large number of unknown compounds is still quite difficult to accomplish.

Mass spectral similarity networking, also called as molecular networking, is an approach to organize large amount of fragment spectra according to their spectral similarities. We have developed a data analysis framework employing mass spectral similarity network, to organize, classify, structurally annotate and visualize the complex mass spectral dataset containing unknown compounds. Fragment spectra from the sample of interest are processed and organized as a network on base layer. Reference fragment spectra are acquired from public spectral library, which covers great variety of chemical species, and used to create “reference network”. Further, reference network is divided into user-defined sublayers according to chemical/biochemical/toxicological properties.

The multi-layer representation of mass spectral similarity networks effectively visualizes and categorizes the spectra of unknown compounds in sample. Structural information systematically extracted from reference spectral networks provides structural insights into unknown compounds. This versatile method can be applied to spectral dataset containing a wide range of unknown compounds in toxicological studies.

We investigate the association between telomere length (TL) and fetal PCB exposure.

The subjects were 94 mother-child pairs participating in a birth cohort, the Chiba study of Mother and Child Health. Maternal and cord blood (CB) were collected during pregnancy and at birth, respectively. Cord tissue was obtained at birth. TL was assessed by qPCR using genomic DNA extracted from cord tissue. The maternal and cord serum levels of PCB congeners were assessed using GC-MS in cord serum were detected. Serum levels in CB of PCB99 for all or females showed a positive association with TL.

Preventing clinical drug-induced liver injury (DILI) remains a major challenge because DILI develops via multifactorial mechanisms and its prediction at the preclinical stage is not straightforward. Immune and inflammatory reactions are considered important mechanisms of DILI; however, biomarkers from in vitro systems using immune cells have not been comprehensively studied. The aims of this study were 1) to identify promising biomarker genes for predicting DILI in an in vitro coculture model of peripheral blood mononuclear cells (PBMCs) with a human liver cell line, and 2) to evaluate these genes as predictors of DILI using a panel of drugs with different clinical DILI risk. Transcriptome-wide analysis of PBMCs cocultured with HepG2 or differentiated HepaRG cells that were treated with several drugs revealed an appropriate separation of DILI-positive and DILI-negative drugs, from which 12 putative biomarker genes were selected. To evaluate the predictive performance of these genes, PBMCs cocultured with HepG2 cells were exposed to 77 different drugs, and gene expression levels in PBMCs were determined. The MET proto-oncogene receptor tyrosine kinase (MET) showed the highest area under the receiver operating characteristic curve (ROC-AUC) value of 0.81 among the 12 genes with a high sensitivity/specificity (85/66%). However, a stepwise logistic regression model using the 12 identified genes showed the highest AUC value of 0.94 with a high sensitivity/specificity (93/86%). Together, we established a coculture system using PBMCs and HepG2 cells and selected biomarkers that can predict DILI risk. The established model would be useful in detecting the DILI potential of compounds, in particular those that involve an immune mechanism.

[Purpose] MicroRNAs (miRNA) have received much attention as potential biomarkers of drug-induced liver injury. We recently reported that plasma miR-143-3p and miR-218a-5p were increased in the early stage of severe cholestasis in rats. In this study, we investigated whether these miRNAs increase in a severity-dependent manner and pathophysiological roles of them.

[Method] Male SD rats were orally administered different doses of 4,4-methylenedianiline or α-naphthylisothiocyanate to induce cholestasis. Rats were also orally administered acetaminophen or thioacetamide to induce hepatocellular injury. The miRNA expression levels were measured by RT-qPCR. Effects of miR-218a-5p on proliferation were investigated in an immortalized human cholangiocyte line MMNK-1. Target genes of miR-218a-5p were predicted by in silico analysis, and expression levels of target mRNAs after overexpression of miR-218-5p were determined.

[Result and discussion] Plasma miR-218a-5p and miR-143-3p levels were dose-dependently increased in cholestatic rats. Proliferation of MMNK-1 cells was significantly suppressed after overexpression of miR-218a-5p. The mRNA levels of GNAI2, PPP1CB and PPP2R5A, which were potential targets, were decreased by overexpression of miR-218a-5p in MMNK-1 cells. In conclusions, our data suggest that 1) miR-218a-5p decreases cholangiocyte proliferation by inhibiting the expression of GNAI2, PPP1CB and PPP2R5A thereby associated with pathogenesis of cholestasis; and 2) miR-218a-5p was leaked into plasma probably from damaged cholangiocytes in a severity-dependent manner.

L-type Fatty Acid Binding Protein(L-FABP), which is located in the cytoplasm of renal proximal tubular cells, responses to ischemia and oxidative stress and is excreted in urine. We have reported L-FABP is also very useful as a toxicological renal biomarker in animal models of acute kidney injury (AKI). The purpose of this research was to evaluate urinary L-FABP with other 7 urine biomarkers (Clusterin, KIM-1, NGAL, Osteopontin, NAG, β2M and Cystatin C) in adenine-induced CKD rat model. Adenine was administered orally to male Sprague-Dawley rats at doses of 0, 40, 70 or 100 mg/kg /day for 7 days, or at 0, 25, 100 or 250 mg/kg/day for 28 days. Serum and urinary biomarkers were measured on Days 1, 8, 15, 22 or 29, and the histopathological examination was performed in the kidney on Days 8, 15 or 29.

As a result, on Day 1 (next day of the first dosing), urinary L-FABP and Clusterin elevated at adenine of 70 mg/kg, but conventional renal biomarkers as blood urea nitrogen (BUN) and serum creatinine (SCr) were not changed. By Day 8, Kim-1 and NAG elevated. On Day 28, L-FABP, NGAL, Kim-1, Osteopontin, NAG and Clusterin were significantly increased in a dose-dependent manner with accompanying severe proximal tubule damages, but other urinary biomarkers were not changed. In addition, BUN and SCr elevated at more than 100 mg/kg after Day 8.

These results suggest that the urinary L-FABP is one of the most sensitive biomarker for detecting proximal tubule damage in adenine-induced CKD model rats. Furthermore, urinary L-FABP and other novel biomarkers would predict the CKD progression.

In this study, the conditions for long term culture and extended canaliculi formation were examined in human-induced pluripotent stem cell-derived hepatocytes (hiPSC-hep) in order to evaluate for the repeated dose toxicity and the cholestasis toxicity.

When hiPSC-hep from vendor A were cultured in long-term culture medium from vendor B, the gene expression levels of CYPs and ALB were maintained for 28 days. In addition, culture condition for forming equally the extended-canaliculi on the entire culture surface was successfully established.

Neurotoxicity is one of the most common causes of drug development failures during clinical phases. In order to predict toxicity in the early phases, a number of in vitro assay models are being developed using neurons generated from human iPSCs with various different methods. In this study we investigated the usefulness of neurons generated using the Quick-Tissue^TM technology, a method that enables rapid differentiation. Calcium imaging of 3D spheroids revealed neuronal responses to representative drugs, suggesting the potential suitability of these neurons to efficient toxicity assays.

Neuron plates, neuronal networks cultured on microelectrode arrays, are widely used for toxicological assays. Human iPSC-derived neurons are extensively tested for this purpose to evaluate species-specific effects. In-house preparation of human neuron plates is costly, while using outside parties for neuron plate testing of proprietary compounds could have information security risks. To overcome these problems, we have developed transportable human neuron plates by optimizing culture conditions. We believe this technology can contribute to reduce the cost and risk of neurotoxicity testing.

Intratracheal instillation (ITI) method is attracting attention as an alternative for aerosol inhalation toxicity test. In this study, the acute inhalation toxicity of sulfuric acid between ITI and whole-body inhalation was compared. Sulfuric acid was administered to rats by ITI. General condition and body weight were evaluated until 14 days after administration, and then pathological examination were conducted. LD₅₀ of sulfuric acid by ITI was estimated to be 7 to 20 mg/kg. The acute inhalation toxicity by ITI was considered to more than twice as strong as that by whole-body inhalation.

Miosis has been evaluated with dedicated devices under non-physiological condition. In this study, versatile video camera was used to examine the effects of illuminance and observation clock hour on the miotic response. Miosis score (pupil/cornea diameter ratio) of SD rats at ≤5 lx was 2-3 times higher than that at 400 lx. Observation clock hour had no effect on miosis score. In a positive control group, miosis score was decreased dose-dependently at ≤5 lx, but not at 400 lx. These results suggest that illuminance for miosis evaluation should be optimized for the purpose of each experiment.

To predict human cardiac adverse effects in vitro, electrophysiological studies and calcium imaging with human induced pluripotent stem cells derived cardiomyocyte (hiPSC-CM) has been used. However, these systems generally use rigid cell- culture dish, dynamic motions are suppressed. The gelatin hydrogel fibrous nonwoven (GHFN) has both flexibility and deformation- recovery property. These nature are expected to give dynamic and stable motion of CM. We achieved simultaneous measurement of calcium transient and dynamic motion from hiPSC-CM. Drug response of iPSC-CM on GHFN will be reported.

We previously showed that human hepatocytes (PXB-cells^®) freshly isolated from chimeric mice with humanized livers (PXB-mice^®) could be maintained expressing major hepatic genes up to 3 weeks.

In this study, we evaluated whether an in vitro model using PXB-cells can be used to predict the risk of drug-induced steatosis. PXB-cells were treated with valproic acid (VPA) in a medium supplemented with fatty acids. VPA induced lipid accumulation in PXB-cells at 3 days after treatment. It was suggested that PXB-cells would be useful in predicting the risk of drug-induced steatosis.

Introduction

Giemsa stained vaginal smear specimens are prepared for checking the estrous cycles of rodents in non-clinical studies. Methanol, a deleterious substance, is used to fix the specimens after drying. Since the management of deleterious substances has been strengthened in recent years, we tried using ethanol instead to eliminate the use of deleterious substances.

Materials and methods

The vaginal smears of rats and mice were fixed with ethanol for 20 seconds after drying. Then we stained the specimens with Giemsa solution.

Results

There was no problem with the stainability of the specimens, and no difference from the methanol-fixed specimens with was seen.

We compared the toxicokinetic parameters from tail vein microsampling (0.05 mL) and from conventional jugular vein sampling (0.2 mL) in rats. Six drugs of different types in modalities, administration route or distribution were administered. Furthermore, we evaluated the tail vein microsampling method under the anemic conditions in case of failure to collect sufficient amount of blood (0.05 mL) in toxicity studies. In that case, conventional jugular vein sampling (0.2 mL) was also applied and evaluated. The results will be reported in the poster presentation.

Blood microsampling (MS) was introduced in rats treated with phenacetin for 4 weeks and examined whether MS affect the toxicological parameters induced by phenacetin. The findings indicative of hemolytic anemia were observed in hematological parameters and histopathology in the liver, spleen and bone marrow both in rats with and without MS except for the hematocrit value, which was decreased only in rats without MS. In conclusion, MS had limited effect on the toxicological parameters induced by phenacetin and satellite animals for toxicokinetics could be eliminated in the toxicity study.

The implementation of the ICH S7B and E14 guidelines has been successful in preventing the introduction of potentially torsadogenic drugs to the market, but it has also unduly constrained drug development by focusing on only IKr block and QT prolongation as essential determinants of proarrhythmia risk.

Namely, the present surrogate markers such as IKr block or QT prolongation are highly sensitive but not very specific for predicting ventricular proarrhythmia risk; and there are clinically important drugs that block IKr but not proarrhythmic at therapeutic plasma concentrations. In response to these background, the comprehensive in vitro proarrhythmia assay (CiPA) initiative proposed the necessities of effects on multiple human cardiac current, in silico model based on electrophysiologic activity within a heart cell, and human stem-cell derived cardiomyocyte assays to confirm findings of in vitro and in silico assays.

We has started hERG and peak Nav1.5 current screening since 2017, Cav1.2 current screening since 2018, and late Nav1.5 current screening since 2019 using a SyncroPatch 384PE (Nanion Technologies GmbH). The SyncroPatch 384PE is a high throughput patch clamp instrument recording from up to 384 wells simultaneously. We will present the validation results of hERG, Cav1.2, peak Nav1.5, and late Nav1.5 current screening in this poster. When compared with the IC50 of these ion channel inhibitors and publication data, high correlative relationship was found. We would like to discuss how we should correspond to a novel strategy in conformity with a new paradigm.

We surveyed the current situation of rat LM test methods using published papers and NDA dossiers. [Results and discussion] Several methods with various LM category and complexity of task were used in JAS. These methods in JAS were slightly different from academic research, and bridging between them seemed insufficient. In a survey of launched drugs, there still seemed to be methods with low detectability even if existing confounding factors which affect test results were considered. We concluded that further investigations were needed to establish recommended test methods.

Effect of nose-only inhalation exposure on estrous cycle in female rats was examined to obtain basic data for reproductive toxicity study by inhalation exposure. Female rats were exposed to air for 6 hours/day, 7 days/week, for 4 weeks. Estrous cycle was determined for pre-exposure period and during exposure period. As a result, prolonged estrous cycle was observed in half of animals. Estrous cycle in the animals recovered to the pre-exposure level within 2 or 3 weeks. It was considered that restraint stress by nose-only inhalation exposure resulted in the prolonged estrous cycle.

DILI is one of the major reasons for drug discontinuation and withdrawal from the market, therefore highly predictive toxicity model is desired.　The development of liver toxicity is mediated by a variety of mechanisms, making it difficult to predict from a single biomarker. In these circumstances, we attempted to improve in vitro model for DILI detection by conducting HCA using 3D culture of human primary liver cells as a study to construct a liver toxicity model. Cells were exposed to multiple drugs causing DILI and were comprehensively evaluated using various toxicity biomarkers.

[Introduction] Peripheral neuropathies including axonal disorders and myelin disorders cause the changes of axonal conduction velocity (ACV). If the change of ACV to drug administration can be evaluated in vitro cultured peripheral neurons, it may be a new platform for toxicity assessment of peripheral neuropathies. However, measurement of ACV requires high spatiotemporal resolution measurement. In this study, we attempted to detect the ACV in cultured dorsal root ganglion (DRG) neurons and the changes of ACV to drug administration using CMOS-MEA consisting of more than 10,000 electrodes with an electrode pitch 11.72μm.

[Method] Rat DRG neurons dissociated from 10 weeks-old male rat were seeded at 1×105 cells/cm2 on CMOS-MEA coated with PEI and laminin. To suppress the proliferation of non-neural glial cells, the cells were treated with 10 µM Uridine and 10 µM 2′-Deoxy-5-fluorouridine for 3 days. The spontaneous activities were measured from 2 weeks to 6weeks in vitro (WIV), and 30 µM 4-AP, which induces hyperexcitation, was administered to the sample. In addition, 10nM vincristin, a type of anticancer drug which causes peripheral neuropathy, was exposed overnight.

[Results] The axonal conduction in spontaneous activities, in which the action potential generated from the soma was conducted through the axon of single neuron, were detected with more than 100 electrodes from 2 WIV. A complex conduction to the branched axons and expansion of propagation path were observed with the passage of culture days. Furthermore, it was also found that the ACV was different between the axon origin and the end section. Next, in order to detect the changes of ACV to drug administration, a 4-AP was administrated. As a result, ACV was significantly increased compared to before administration. It is considered that the ACV increased due to the increase in the membrane potential.　On the other hand, vincristine reduced the ACV by about 20%.

[Conclusion] The ACV and spatial temporal pattern of ACV in cultured rat DRG neurons were detected by CMOS-MEA with high spatiotemporal resolution, and the change of ACV by 4-AP and vincristin was detected. This ACV measurement method is considered to be effective as an in vitro toxicity assessment of peripheral neuropathies.

Purpose: Long-term exposure to estrogens increases the incidence of gynecologic cancers (breast, ovarian, and endometrial cancers) in humans. During the carcinogenic processes, estrogens acquire DNA-damaging activity via metabolic activation. Our group has studied the carcinogenic mechanisms of estrogens using an animal model for estrogen-induced breast cancer. To understand the precise mechanisms, it is quite essential to measure the metabolic profile of estrogens. In this study, we developed a rapid and simultaneous quantification method for three estrogens (estrone, E1; 17ß-estradiol, E2; 17α-estradiol, αE2) using a high-resolution mass spectrometer equipped with ultra-high-performance liquid chromatography (UHPLC-MS).

Methods: UHPLC-MS analysis of E1, E2, and αE2 was performed on a quadrupole time-of-flight MS (Triple TOF6600) and UHPLC (Nexera XR). Estrogens were quantified using a high-resolution multiple reaction monitoring as follows: E1 (m/z 269.1>145.069), E2/αE2 (m/z 271.1>145.069).

Results and discussion: Our method achieved a fast analysis (run time, 5 min), and good sensitivity and linearity as follows: E1 (1-1000 pg/mL, r²=0.9987), E2 (2-1000 pg/mL, r²=0.9991), αE2 (1-1000 pg/mL, r²=0.9989). Recovery test using a charcoal-stripped serum spiked 10-1000 pg/mL of estrogens showed a suitable recovery efficiency from 85.6% to 106.6%. Since the serum level of estrogen ranges from several to several hundred pg/mL, our method would be applicable for direct measurement of estrogen profiles without chemical derivatization.

We conducted a questionnaire survey to clarify the state of efforts and concerns in microsampling and to disseminate information for the dissemination of microsampling by organizing the information necessary for the effective utilization of microsampling technology in drug development in the future.

The survey was conducted for 60 member companies and received responses from 44 companies. We summarized the responses to the survey and discussed the widespread use of microsampling for TK assessment.

The INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) has been operational since 2005. A Global Editorial Steering Committee (GESC) helps coordinate overall objectives of the project. Development of harmonized terminology for each rodent organ system or non-rodent species is the responsibility of the Organ Working Groups (OWG) or Non-rodent Working Groups (NRWG) respectively, drawing upon experts from North America, Europe and Japan.

Great progress has been made with 15 rodent organ systems published to date-Respiratory, Hepatobiliary, Urinary, Central/Peripheral Nervous Systems, Male Reproductive and Mammary, Zymbals, Clitoral and Preputial Glands and Hematolymphoid System in Toxicologic Pathology and the Integument and Soft Tissue, Female Reproductive System, Digestive System, Cardiovascular System, Skeletal System, Special Senses and Endocrine System in the Journal of Toxicologic Pathology as supplements and on a web site-www.goReni.org. Recommendations of the Apoptosis/Necrosis Working Group have been published. There are 5 non-rodent working groups-non-human primate, dog, minipig, rabbit and fish-with draft manuscripts in progress. A new group has been formed to address terminology in non-rodent ocular toxicity studies. A comprehensive review of all rodent systems to standardize terminology common to organ systems was completed and terminology updated in goRENI. INHAND guides offer terminology, diagnostic criteria, differential diagnoses and guidelines for recording lesions in toxicity and carcinogenicity studies. The guides provide representative photo-micrographs of morphologic changes, information regarding pathogenesis, and key references.

INHAND GESC representatives attend meetings with representatives of FDA Center for Drug Evaluation and Research (CDER), Clinical Data Interchange Standards Consortium (CDISC), and National Cancer Institute (NCI) Enterprise Vocabulary Services (EVS) to assist with incorporating INHAND terminology as preferred terminology for SEND (Standard for Exchange of Nonclinical Data) submissions to the FDA. Interest in INHAND nomenclature, based on input from industry and government scientists, is encouraging wide acceptance of this nomenclature.

Seizure is one of the serious toxicities in drug development. Recently, in vitro study using cultured neurons and microelectrode array (MEA) systems showed the potential to detect a seizure risk of compounds inducing seizure in vivo, by using parameters based on neuronal activities. However, these analytical methods are not well established. The purpose of this study was to　investigate the analytical method focusing on a multivariate analysis using a number of parameters calculated by MEA systems, in order to categorize test compounds into seizurogenic or non-seizurogenic. As a result, cluster analysis was able to classify the seizurogenic compounds and non-seizurogenic compounds.

Various animal cell-based approaches have been proposed as alternatives for developmental toxicity test. Since species differences significantly affect teratogenic responses, human cell-based assays are desired to more precisely evaluate human teratogens. In this study, we focused on signaling pathways which play a vital role in embryogenesis. We report that human iPS cell-based signaling reporter systems are promising approaches for the screening of teratogenic chemicals.

Diabetes mellitus (DM), a common metabolic disorder, has increasingly raised great global health concerns. The complications of DM severely cost the finances of individuals and nations’ economies. Diabetic nephropathy is one of the most common long-term complications in patients with DM, in which microvascular changes often aggravate the illness and lead to chronic kidney disease. Extracts of Sigesbeckia Orientalis have been identified to have the excellent anti-oxidative activity and used in modern traditional Chinese medicine. In consideration of only few studies examining their effects on diabetic kidney, this study aims to explore the protective effects of the extracts on diabetic nephropathy and their underlying molecular mechanisms. Murine animals were first injected with streptozotocin (STZ) to induce diabetes mellitus, followed by orally given with the extracts for a 3-week treatment. After the treatment, the histological and morphometric analysis of animals in each group was performed. The results of our study provided the protective evidence of the extracts from Sigesbeckia Orientalis on diabetic nephropathy, with respect to the histological demonstration and immunohistochemistry staining for the expression patterns of 8-hydroxyguanosine (8-OHdG), relative proteins involved in nuclear factor kappa B (NF-κB) inflammatory pathway and endothelial nitric oxide synthase (eNOS) simultaneously.

The transplantation model has an ectopic problem, the chemical carcinogenesis model takes longer time. Using a Tg-rasH2 mouse model with two-stage carcinogenesis model, we examined usefulness of this combination. Tg-rasH2 ♀ mice given ENU, and then BHT weekly for 5 weeks. For 2, 2, 9 mice at 5, 7 and 9 weeks after the first dosing BHT, tumor and PD-L1 were examined. At each necropsy, tumors were observed in 2, 2 and 9 cases. Tumor formation was 9 weeks in Umemura’s report (2006), but now it was observed 5 weeks with PD-L1 expression. The result suggests usefulness of this model.

[Introduction]

Anticoagulant rodenticides (ARs) have been used for control of rodents’ population. ARs inhibit vitamin K epoxide reductase (VKOR) playing role in producing clotting factors, which results in lethal hemorrhage in rodents. However, ARs-spread have caused accidental death to non-target species. Thus, it is necessary for proper usage of ARs to evaluate sensitivity of wild species. In Japan, ARs were used in Bonin (Ogasawara) islands. Bonin fruit bat (Pteropus pselaphon) is the only endemic mammalian species in the islands, however, ARs-sensitivity of fruit bats has been unclear. For the assessment, we performed pharmacokinetics analysis of ARs with Egyptian fruit bats (Rousettus aegyptiacus).

[Materials & Methods]

Animals; four female adult egyptian fruit (EF) bats and female Sprague Dawley (SD) rats (10 weeks old) were used in the study. Chemicals; we used two ARs (warfarin and diphacinone). Both of them were classified in the first generation ARs and have been commonly used in Japan. Methods; 4 mg/kg of warfarin sodium disloved in saline water was orally administrated to the EF bats and SD rats. Blood was taken from wing vein of EF bats and tail vein of SD rats. Prothrombin time (PT) was measured by CoaguCheck XS (Roche). Warfarin and its metabolites was extracted by liquid-liquid distribution with ether and their concentration was measured by HPLC-MS/MS (Shimadzu). After two month of warfarin administration, 4 mg/kg of diphacinone disolved in corn oil was also administrated to EF bats.

[Results and Discussion]

After warfarin administration, prolonged PT was observed in both EF bats and SD rats, however, EF bats recovered to normal value earlier than SD rats. EF bats showed higher clearance of warfarin than SD rats and produced higher amounts of warfarin-metabolites than SD rats. These results suggested EF bats were less sensitive to warfarin than SD rats due to their high warfarin excretion ability. On the other hand, PT prolonged earlier in EF bats after diphacinone exposure, although plasma concentration of diphacinone was significantly lower in EF bats than in SD rats. These results indicated fruit bats showed different sensitivity to warfarin and diphacinone, although they have same mode of action.

[Conclusion]

This study revealed Egyptian fruit bats were relatively tolerant to warfarin but susceptible to diphacinone compared to rats. Further study with Bonin fruit bats is required for safety usage of ARs.

Accurate hazard identification is the first step of risk assessment which is an effective tool of risk management. In line with UN Sustainable Development Goals, risk management of hazardous chemical is one of the key elements. Objective is to develop a novel approach on hazard identification.

Chemicals used in Oil and Gas company were evaluated their reproductive toxicity by regulatory agency classification (The Globally Harmonized System of Classification and Labelling of Chemicals, GHS). Secondly, known or presumed reproductive toxicants (GHS Category 1) were further analyzed with carcinogenicity and germ cell mutagenicity for prioritization purpose.

We found substantial disagreement in hazard classification between agencies. An inconsistent hazard classification for the same chemical may result in inconsistent risk assessment outcomes. As misjudgement of hazard identification e.g. under estimation, may result in the potential health risks, the most stringent classification was adopted. It is also found that only limited agencies provided data/ evidence based justification of classifications.

We develop a novel and sustainable approach for hazard identification of chemicals used in Oil and Gas company by using publicly available regulatory databases. It is a cost minimum approach and applicable not only for health hazard, but also for physical chemical hazard and environmental hazard. The approach is not limited to Oil and Gas company use but broader, e.g. small and medium industries which may have limited toxicological expertise and funding.

In November 2018, the European Medicines Agency (EMA) drafted a new EU Guideline on the Environmental Risk Assessment (ERA) of Medicinal Products for Human Use. The drafted new EU Guideline recommends a step-wise assessment consisting of a screening Phase I and testing Phase II (Tiers A and B). The step-wise assessment was already outlined in the current EU Guideline released in 2006, but some changes have been proposed in trigger values in assessment of persistence, bioaccumulation, and toxicity (PBT), and testing strategy in the sediment, terrestrial, and groundwater compartments in Phase II assessments in the drafted new EU Guideline; furthermore, the drafted new EU Guideline requires a tailored assessment for active substances with a specific mode of action such as antibiotics and endocrine active substances (EAS). The Japanese ERA Guidance, released by the Ministry of Health, Labour and Welfare (MHLW) in 2016, refers to the EU Guideline for the step-wise assessment; therefore, it is important to understand the concept of the new EU Guideline for development of a time-and-cost effective ERA scheme that suits environmental conditions in Japan. This poster presentation discusses the proposed changes of testing strategy in the drafted new EU Guideline, and possible influence on ERA in Japan by comparison of the ERA scheme between Japan and EU.

We evaluated the risk of issues in experimental procedures in typical GLP toxicity studies using a risk-based approach and examined its application in process-based quality assurance (QA) inspection.

Risk assessment was performed similarly to a healthcare failure mode and effects analysis. As typical toxicity studies, we selected a 4-week repeated dose toxicity study (4W) and a bacterial reverse mutation assay (Ames test). The experimental procedures in these studies were identified and possible issues were listed. We evaluated the degree of severity and the occurrence of possible issues, and criticality was calculated based on the severity and occurrence. The risk of possible issues was evaluated based on criticality. We examined whether the testing department could detect possible issues classified as high risk.

Many issues in both studies were classified as low risk. Almost all high-risk cases were issues involving computerized systems (4W) or records (Ames test). It was considered that the QA inspection did not need to be performed for all experimental procedures in each study because almost all high-risk cases would be detectable by the testing department.

We conclude that process-based QA inspection is applicable to all experimental procedures in both studies. However, it is necessary to assess the possible issues at each facility, because the possible issues of each facility differed according to the implementation system, test articles, and other factors. We consider that a risk-based approach is useful for determining the applications of process-based inspection.

We analyzed the gap between the GXP data integrity guidance issued by the Medicines & Healthcare products Regulatory Agency (MHRA) and the current state of GLP data management in Japan. As a result, the gaps that had a strong impact were related to governance systems, blank format management, and computerized system management. Among them, the gap for blank format management and computerized system management was thought to require significant costs and tasks to fully address the gap.

【Introduction】Nanomaterials (NMs) have the same component as a constituent unit but have unique characteristics such that they exhibit different functions and activities depending on the form. Therefore, it is expected to be applied to new materials having superior properties that have not been seen before, and active research and development are being promoted internationally. However, it is difficult to predict toxicity based on structure-activity relationships from constituents such as low-molecular-weight chemicals, and a comprehensive approach to toxicity evaluation and safety that uses the morphological characteristics of nanoparticles as descriptors is required. In this study, we collected test data of physicochemical properties and harmfulness information for six types of titanium dioxide nanoparticles (TiO₂ NPs), and searched and scrutinized data necessary for statistical analysis. Furthermore, we analyzed the similarity of physical property values and examined the usefulness of this method for toxicity evaluation by using multivariate analysis for the relationship between physicochemical properties and hazard information.

【Method】Sources of information on the physicochemical properties and hazard test data on repeated dose toxicity by inhalation and intratracheal administration routes for six types of TiO₂ NPs were provided by the OECD NM safety assessment test document (Dossier) and eNanoMapper DB. SIMCA-P was used for statistical analysis.

【Result and Discussion】

Physicochemical properties of about 168 items for which was collected information were classified by hierarchical clustering analysis, and their similarities were shown by Dendrogram. Combinations of items showing similarity in physicochemical properties were indicated by PCA(Principal component analysis), and the relevance of hazard information could be linked to physical property items showing toxicity by OPLS(Orthogonal partial least squares). From the results obtained in this analysis, it was possible to clarify the slight differences in physicochemical properties associated with toxicity using a vast data collection on the physicochemical properties of NM. Therefore, it is suggested that the various statistical methods used in this analysis can be useful tools for evaluating the properties of NM, and are expected to be applied to toxicity evaluation methods based on the properties of NM.

In order to assess the environmental impact of human medicines, information of their concentrations in the rivers are necessary. In the present study, the concentrations of acetaminophen and sulpyride in the 20 rivers were calculated using a watershed model (AIST-SHANEL). Then, we compared the estimated concentrations of both medicines with the measured concentrations. Further, we examined the parameters that is significant for the calculation results. The maximum concentrations of both medicines calculated by the model was lower than the predicted no-effect concentrations (PNEC).