Many cases of liver cancer have been reported in databases as adverse drug reaction. However, many unclear points on the mechanism of pathogenesis exist. Therefore, we have attempted to define hepatoma-inducing drugs by FAERS and developed a machine learning prediction model for these groups of drugs using chemical structure descriptors, mutagenicity, and agonist/antagonist activity against nuclear receptors and response pathways. By examining this prediction model, various chemical structures and bioactivity information related to the induction of liver cancer have been estimated.

Doxorubicin hydrochloride (Dox) is a kind of anthracycline antitumor drug with broad spectrum, high efficiency and strong effect, but it has strong toxicity and side effects, especially cardiac toxicity, which greatly limits its application in large dose in clinic. In this study, cardiac toxicity of Dox was studied using fluorescent zebrafish embryos (cmlc2: EGFP) as experimental animals. Zebrafish embryos were exposed to Dox from 4 hour post fertilization (4 hpf), and fetal mortality, heart rate, cardiac morphological changes, and changes in ventricular/atrial myosin heavy chain Myh6/Myh7 gene expression were measured at 48, 72 and 96 hpf, respectively. The results showed that Dox could cause decreased heart rate, increased pericardial edema and decreased cardiac area, and increased Myh6/Myh7 mRNA expression in zebrafish embryos or larva. High dose of Dox causes tube heart in zebrafish larva. These results indicate that Dox has significant cardiotoxicity, and also indicate that zebrafish embryos have the advantage of cardiotoxicity evaluation.

In this study, we examined the inhibitory effect of cold compress on cytotoxicity of MMC using apoptosis and oxidative stress as an indicator.

In the cells exposed to MMC at 37°C, that caused apoptosis significantly increased from 24 hours after exposure. On the other hand, cells exposed to MMC at 23°C did not caused apoptosis. Only cells exposed to MMC at 37°C, the intracellular malondialdehyde concentration significantly increased.

Therefore, apoptosis and oxidative stress were observed in the cytotoxicity caused by MMC, and it was clarified that the cold compress suppressed these.

Despite the recent advances in the life sciences and the remarkable investment in drug discovery research, the success rate of small-molecule drug development remains low. Safety is the second most influential factor of drug attrition in clinical studies; thus, the selection of compounds with fewer toxicity concerns is crucial to increase the success rate of drug discovery. Compounds that promiscuously bind to multiple targets are likely to cause unexpected pharmacological activity that may lead to adverse effects. Therefore, avoiding such compounds during early research stages would contribute to identifying the compounds with a higher chance of success in the clinic. To evaluate the interaction profile against a wide variety of targets, we constructed a small-scale promiscuity panel (PP) consisting of eight targets (ROCK1, PDE4D2, GR, PPARγ, 5-HT_2B, Adenosine A₃, M1 and GABA_A) that were selected from diverse gene families. The validity of this panel was confirmed by comparison with the promiscuity index evaluated from larger-scale panels. The promiscuity assessed using our PP correlated with the occurrence of both in vitro cytotoxicity and in vivo toxicity, suggesting that the small-scale PP is useful to identify compounds with fewer toxicity concerns and would lead to the reduction in drug attrition due to safety issues. In this annual meeting, we also introduce an in silico (QSAR) model which is constructed using the PP data. (Reprinted with permission from Chemical Research in Toxicology. Copyright 2019, American Chemical Society)

The purpose of this study is to develop a workflow to assess the chronic toxic effect of pharmaceuticals on algae with the aim of building a system to support the environmental risk assessment. Algal toxicity prediction results calculated by two QSAR models, KATE and ECOSAR, were evaluated by applicability domain and prediction accuracy. Several pharmaceuticals with specific toxicity mechanisms and high sensitivity to algae were grouped into categories. Based on the results, a workflow was built to assess the chronic toxic effects on algae using QSAR or category approach.

【Aim】

To establish a tumorigenicity test using nude rats, large and capable of transplanting in the same site as humans, but mice are too small to target.

【Method】

HeLa cells were s.c. given to F344/NJcl-rnu/rnu rats with or without Matrigel. For 16 weeks afterward, tumor, histopathology of formed tumor, metastasis were examined.

【Results】

Tumor reached 100% in the 7th week. Matrigel neither affected tumor incidence nor mass. The tumor was solid malignant, and metastasis was observed. Tumor engraftment suggests the possibility of applying this model to evaluating final products in microenvironment.

Anticholinergic organophosphate (OP) agents act on the diverse serine hydrolases, thereby revealing unexpected biological effects. Epidemiological studies indicate a relationship between OP exposure and development of attention-deficit/hyperactivity disorder (ADHD)-like symptoms, whereas no plausible mechanism for the OP-induced ADHD has been established. The present investigation employs EOPF as an OP-probe which is an extremely potent inhibitor of endocannabinoid (EC, anandamide and 2-arachidonoylglycerol)-hydrolyzing enzymes: i.e., fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Ex vivo experiment shows that EOPF treatment decreases FAAH and MAGL activities and conversely increases EC levels in rat brain. Subsequently, EOPF (treated intraperitoneally once at 0, 1, 2, or 3 mg/kg) clearly induces ADHD-like behaviors (in elevated plus-maze test) in both Wistar and spontaneously hypertensive rats. The EOPF-induced behaviors are reduced by a concomitant administration of cannabinoid receptor inverse agonist SLV-319. Accordingly, EC system is a feasible target for OP-caused ADHD-like behaviors in adolescent rats.

Glyphosate is the most widely used herbicide in the world including Japan. Its safety for humans has been believed, because its herbicidal action is the inhibition of the shikimate pathway absent in animals but critical to the plants. In 2015, IARC (International Agency for Research on Cancer) decided to classify the glyphosate as Category 2A (probably carcinogenic to humans), but its carcinogenicity have been controversial. Recently, various adverse health effects of glyphosate have been reported. Glyphosate and its-based formulations induced microbiome disruption, endocrine disruption, excitatory action to NMDA receptors, metal chelating activities, and DNA methylation etc. To consider the glyphosate’s potential as a human carcinogen and adverse health effects, it must be to focus on studies suggesting mechanisms that would go largely undetected in traditional toxicology studies, such as microbiome and endocrine disruptions, DNA methylation.

In addition, glyphosate-based formulations frequently showed higher toxicity than active gradient glyphosate, because the additives, such as surfactant, surprisingly increase their toxicity. In several reports, the glyphosate formulations showed about a hundred times higher toxicity than glyphosate. The safety standard of pesticides, ADI (Acceptable Daily Intake), is calculated from NOAEL (No Observed Adverse Effect Level) of the active gradient, such as glyphosate. But, human exposures of pesticides are derived from the pesticide-based formulations, not the active gradient. The calculation method of ADI includes a fundamental contradiction.

Dinotefuran has been shown to be transferred to offspring via the placenta or milk, which raises concerns about transgenerational effects. Here, ICR mice were given 0, 0.02 and 0.2% of dinotefuran diluted in water from GD 0 to PND 50, and the dams and their offspring were evaluated, focusing on the immunotoxicity. Some dosed groups showed a slight decrease of the WBC in maternal blood, and a tendency of increase of the proportion of Gr-1⁺ cells in the bone marrows of the mothers and the offspring. No statistically significant changes were observed in all of the parameters.

There are concerns that industrial chemicals cause developmental neurotoxicity (DNT), leading to neurobehavioral outcomes, such as learning disabilities and cognitive impairment. Compared to the adult brain, the fetal brain is inherently more susceptible to compound-induced toxicity. Thus, exposure to these substances during early development may lead to adverse neurological effects manifested at a later phase of life. Because current DNT guideline (OECD TG426) requires a lot of animals and costs, it is necessary to establish an in vitro DNT method with more accurate and better predictability. Due to complex processes of cell development and maturation in human brain, human iPS cells (hiPSCs) are expected to provide a novel human cell-based assay systems as alternative in vitro testing approaches. Pyrethroids has been reported to show DNT in rodents. In the present study, we evaluated chemicals with DNT risk using microelectrode array (MEA) system and searched a suitable functional endpoint. Treatment with pyrethroids, such as deltamethrin, inhibited spike numbers and network bursts using MEA recordings of hiPSC-derived neurons. These data suggest that pyrethroids induce neurotoxicity by inhibiting neural network. Taken together, MEA recordings could be an effective tool for elucidating chemical-induced neurotoxicity.

We are exposed to numerous metals on a daily basis through the environment, lifestyle, and dietary habits. The metal exposome can be defined as the measure of all the exposures of environmental metals in a lifetime and how those exposures relate to health; however, the full characterization of the metal exposome throughout the whole lifespan remains a challenge. Therefore, in this study, we attempt modeling a metal exposome specialized for electrophilic metals with cultured cells and mice.

Complement component 8γ (C8γ) is a member of lipocalin and its function is unclear. We have reported C8γ can bind to triphenyltin (TPT) and may affect the toxicity of TPT. Here, we evaluated the affinity of several substances to C8γ. By utilizing RI-labeled TPT, K_d value to C8γ was estimated as 56.2 nM. Competitive assay revealed tributyltin (TBT) can also bind to C8γ and mono- or di-substituted tin compounds showed weaker affinity. Triphenylarshine did not bind to C8γ and triphenyllead showed higher affinity than TPT. These results may shed light on a part of detoxification mechanisms by C8γ.

We investigated whether Cr(III) -induced EPO production could prevent insulin resistance in HepG2 cells. HepG2 cells were cultured in DMEM medium supplemented with palmitic acid to induce insulin resistance. The addition of Cr(III) suppressed the increase in the expression of G-6-Pase and PCK1 mRNA and the decrease in pAKT/AKT, which are indicators of insulin resistance, and prevented insulin resistance. Pretreatment with siEPO inhibited the prevention of insulin resistance. These results suggest that EPO production by Cr(III) is involved in the prevention of insulin resistance in HepG2 cells.

Methylmercury (MeHg) is one of the most toxic environmental pollutants and causes serious health hazards worldwide. Recently, we demonstrated that oleanolic acid-3-glucoside (OA3Glu) has anti-MeHg activity in vitro and in vivo. In this study, we examined the therapeutic effects of OA3Glu on MeHg-induced neurotoxicity in vivo. In MeHg-exposed group, impaired motor function and changes in the center of gravity were observed and these conditions were improved in OA3Glu treatment group. Furthermore, amplitude of synaptic potential of the MeHg exposure group was decreased in cerebrum and cerebellum and improved these of the OA3Glu treatment group. In conclusion, it is suggested that OA3Glu improves the neurotransmission dysfunction and the movement disorder associated with MeHg exposure.

In this study we examined the mechanism involved in the induction of TNF-α expression by methylmercury in microglia. Exposure of mouse microglia BV-2 to methylmercury resulting in induction of TNF-α expression and activation of ASK1/p38 pathway. Reactive oxygen species (ROS) are known to activate this pathway. Methylmercury induced ROS production in mitochondria, and quenching of this production suppressed activation of p38 and induction of TNF-α expression by methylmercury. These results suggest that methylmercury may induce TNF-α expression by ASK1/p38 pathway via mitochondrial ROS in microglia.

Itai-itai disease, developing based on proximal renal tubular injury due to cadmium (Cd) toxicity, often showed severe anemia associated with low serum levels of erythropoietin (EPO), which is mainly produced from kidneys. We observed levels of blood EPO and renal EPO induction in rats exposed to Cd and investigated the relationship between them to clarify the kinetics of EPO in Cd toxicity. Wistar rats were injected s.c. with saline or Cd at 2 mg/kg per week for 3 or 8 months. Then they were put under hypobaric hypoxia (0.65 or 0.55 MPa) for 3 hours to enhance EPO induction or stayed in normoxia. After that, peripheral blood and kidneys were taken. Serum samples were obtained from the blood to measure EPO concentration. Total RNA and paraffin block samples were prepared from the kidneys to observe EPO mRNA expression by real time PCR and highly sensitive in situ hybridization as well as hematoxylin-eosin staining, respectively. The Cd-injected rats showed injured renal tubules and anemia. The serum EPO levels were elevated by hypoxia, and tended to be higher in the Cd-injected rats than the saline-injected, although they were insufficient to compensate anemia. The renal EPO mRNA expression was enhanced by hypoxia, but the enhancement levels were smaller in the Cd-injected rats than the saline-injected. These results indicate that there was a discrepancy between renal EPO induction and blood EPO levels in the Cd-injected rats, suggesting that Cd induces EPO production from other organs, such as liver, to compensate the decreased capacity to produce EPO in kidneys.

Uranium is a nephrotoxic heavy metal. The chronic ingestion of naturally occurring uranium in contaminated groundwater resulted in renal dysfunction related to renal tubular injury. Children are susceptible to renal damage from uranium exposure. The uranium dynamics in kidney in the developmental period, however, is poorly understood. In the present study, the dynamics of uranium distribution, localization and the surrounded tissue alteration were examined in Wistar male prepubertal rats exposed to uranium by combination of in situ determination of elements performed by μPIXE (particle induced X-ray emission) or SR-μXRF (X-ray fluorescence spectrometry using high energy synchrotron radiation) and pathological observation. The chemical form of the localized uranium in kidney was assessed by X-ray absorption fine-structure with microprobe (μXAFS).

A single injection of uranyl acetate to prepubertal rats (0.5 - 4 mg/kg, s.c.) caused tubular lesions in the S3 segment of the proximal tubules, the distal portion of the proximal tubules. μPIXE and SR-μXRF were performed for the areas covering the outer cortex to the medulla, where the S3 segment of the proximal tubules distributed. Uranium concentrated areas were detected in the epithelium of the S3 segment of the proximal tubules at the initial phase of uranium exposure. The uranium levels were 300-fold more than the mean renal uranium concentration. Regenerating and damaged tubules were observed in the restricted regions surrounded the uranium concentrated areas at the recovery phase. The time course of localized quantity of uranium, its oxidative condition, and elemental composition after uranium exposure will be presented.

In the present study, we found that a zinc complex bis(1,4-dihydro-2-methyl-1-phenyl-4-thioxo-3-pyridiolato)zinc(II), named Zn-06, exhibited strong cytotoxicity in vascular endothelial cells. The characteristics of the structure was investigated, and the results showed that the tetracoordinate structure of the Zn-06 molecule, which contains two sulfur and two oxygen atoms coordinated with the zinc atom, facilitated its accumulation within vascular endothelial cells and the whole structure of the zinc complex was involved in its cytotoxicity in the cells. (J. Toxicol. Sci., 44(2):113-120, 2019)

Recentrly, a high incidence rate of bladder cancer were reported among employees in a chemical factory , where using aromatic amines. We investigated the whole-animal biodistribution of OCA using autoradiography after percutaneous administration in rats. [¹⁴C]OCA was percutaneously administered for 8 and 24 hours by covering with a lint cloth soaked in [¹⁴C]OCA solution at a dose of 50 mg/748kBq/4 mL/kg. After the completion of administration, and whole-body autoradioluminograms were prepared. The bladder had high level of radioactivity at 8 hr, indicating that OCA can be absorbed from the skin and transferred to bladder. The urinary excretion reached 86% of administered OCA during 8-24 hours.

We examined early changes in the urinary bladder administered aromatic amines with similar structure to o-toluidine. Male F344 rats were administered 0.5% or 1% 4-chloro-o-toluidine (4-CT), 1% o-acetotoluidine (o-AT) and other 2 chemicals in the diet for 4 weeks. 1% 4-CT and o-AT caused severe mucosal damage with urothelial hyperplasia and significant increase in γ-H2AX formation, similar to the results in o-toluidine. In contrast, 0.5% 4-CT and other chemicals did not induce such lesions. Genes related to cell cycle and DNA damage showed similar expression patterns in 1% 4-CT and o-AT groups.

Thyrotoxic and hepatotoxic chemicals, 4- or 5-methyl-2-mercaptobenzimidazole (4- or 5-MeMBI) and 2-mercaptobenzimidazole (MBI) caused decrease in CYP3A2 activity at 48 h, which was lessened at 96 h, in primary cultured rat hepatocytes. CYP1A1/2 activity was induced by 4-MeMBI and 5-MeMBI, but not by MBI. In SIRC rabbit cells, 4-MeMBI and 5-MeMBI were more cytotoxic than MBI. It is considered from these results 4-MeMBI, 5-MeMBI and MBI can cause metabolic interaction of chemical toxicity by changing drug metabolizing activity, and that cytotoxicity of MBI is intensified by methyl substitution.

Recently, there has been a concern about health hazards caused by mycotoxins. They are metabolized by drug-metabolizing enzymes (DME) in livestock and human bodies. The resultant metabolites are at risk of being converted to the active forms by endogenous or exogenous hydrolases. Thus, metabolic studies of mycotoxins are essential for their risk assessment. Our group has successfully generated recombinant yeasts expressing DME, and established a technology to produce metabolites of drugs, food factors, and various toxins. Our technology may contribute to risk assessment of mycotoxins.

Acrylamide (AA) is carcinogenic in lungs and Harderian glands of mice. To clarify the relationship between AA-induced DNA adduct, gene mutation and carcinogenesis, we examined N7-GA-Gua formation and gene mutation in the lungs, harderian glands and livers in the gpt delta mice. As a result, AA at the carcinogenic dose significantly increased gpt MFs only at the carcinogenic sites, suggesting that mutagenicity of AA might contribute to its carcinogenicity. However, the common observation of N7-GA-Gua formation at the same levels in any sites independently of the duration of the exposure may imply involvements of other factors than DNA modifications in AA-induced mutagenicity and carcinogenicity.

The Bohai Bay is a semi-closed bay located in the western part of the Bohai Sea in China. Due to the discharge of industrial wastewater into the Bahai Sea, which caused potential water pollution, we need to evaluate the degree of pollution. In this study, sediment samples were collected near a factory outlet around the Bohai Bay and extracted by alcohol. Zebrafish embryos were used to monitor PAHs in the sediment extract (SE). Chemical analysis was carried out by GC-MS. The results showed that mortality was significantly increased in the 5 mg/mL SE group at 24 hpf (p < 0.001), and in the 0.5 mg/mL and 1 mg/mL SE groups at 96 hpf (p < 0.01). By 72 hpf, the hatching rate of zebrafish embryos was significantly inhibited in 0.5 mg/mL, 1 mg/mL and 5 mg/mL SE groups, respectively (p < 0.05). Interestingly, SE could significantly reduce pigmentation in the eyes of zebrafish embryos (p < 0.05). SE inhibited the thyroid-related genes Tpo, Thrβ and Dio2 mRNA expression. However, SE significantly increased Dio3 mRNA expression by 1.67 to 5.09 fold (p < 0.05). Chemical analysis showed that SE mainly contained polycyclic aromatic hydrocarbons such as fluorene, phenanthrene, fluoranthene, and fluorene. Our results indicate that zebrafish embryos may be useful for biomonitoring PAHs pollution in the sediments.

Pulmonary fibrosis is considered to as a disease caused by persistent lung injury and inflammation. Cigarette smoke (CS) exposure is known to be associated with development of inflammatory lung diseases. The present study investigated whether the inflammatory response by CS repeated exposure may be involved in exacerbation of bleomycin (BLM)-induced lung injury in rats. Rats were exposed nose-only inhalation to CS (0, 100, 200 or 300 μg/L) for 4 weeks (4 hours/day, 7 days/week), and received intratracheal BLM (single dose on first day). The following eight experimental groups (n=6) were evaluated: vehicle control (VC), BLM, CS 100, 200, 300 and BLM + CS 100, 200, 300. After 4 weeks, in the lung tissues of BLM groups exhibited histopathological changes, including alveolar macrophage aggregation, chronic inflammation and fibrosis. These changes were exacerbated in the lungs tissues of BLM + CS groups in a concentration-dependent manner of CS. The expression of inflammatory cytokines was markedly increased in the bronchoalveolar lavage fluid (BALF) of BLM + CS groups compared to BLM or CS alone, and similar was observed in the lung tissues. On the other hand, there were no significant differences in the levels of these cytokines in the serum of BLM and CS groups compared with VC group. These results suggest that repeated exposure to CS produces an inflammatory response that may contribute to the development of BLM-induced pulmonary fibrosis.

This research was supported by a grant (19183MFDS463) from the Ministry of Food and Drug Safety in 2020.

As toxicities of chemicals depend on their pharmacokinetics and pharmacodynamics, it is important to consider their pharmacokinetics to complement missing toxicity data. We collected existing values of PBPK model parameters for environmental chemicals, and classified them into 4 categories based on intermolecular forces. For each category, probability distribution of these parameters and their correlation tendencies with physicochemical properties were analyzed. Results suggested useful applicability of PBPK modeling to justify pharmacokinetic similarity in each categorical chemicals.

Environmental electrophiles modify thiol groups of sensor proteins, leading to activation of redox signaling such as the Keap1/Nrf2 pathway. While activation of Nrf2 by single exposure has been established well, effect of combination of other electrophiles on the activation has not been evaluated. Here, we examined whether or not atmospheric electrophiles such as 1,2- naphthoquinone and 1,4-naphthoquinone could enhance a phytochemical (E)-2-hexenal-mediated Nrf2 activation in HepG2 cells.

We recently found that TNF receptor 3 (TNFR3) is involved in the cell death caused by methylmercury. In this study, we investigated the involvement of TNFR3 to methylmercury-caused neurotoxicity in mouse brain. The motor ability of mouse was decreased by methylmercury, while this decrease was partially recovered by TNFR3 knockout. Moreover, in TNFR3 knockout mouse, the increase in astrocyte and microglia by methylmercury was also suppressed. These results suggest that TNFR is involved in the central nervous system disorder caused by methylmercury.

Recently, several studies have reported the adverse effects of microplastics (MPs) on microalgae. Nylon powders, which are one of the MPs, are used in personal care products included in cosmetics. In previous reports, we revealed that nylon6 (Ny6) powders inhibit algal cell-growth of Pseudokirchneriella subcapitata by electrostatically adsorption. However, it is considered that nylon powders released in natural environment are deteriorated by weathering, such as UV irradiation. Here, we examined the effects of nylon powders (Ny6 and nylon12: Ny12) irradiated by UVc on algae cells P. subcapitata.

The water flea Daphnia magna, is a branchiopoda crustacean, which is a common inhabitant in fresh water. In general, normal and healthy populations are entirely female because they produce by parthenogenesis. Previously we and others found that females exposed to juvenile hormone produce males, suggesting that juvenile hormone is a key molecule for sex determination. By using genome editing tools, we developed transgenic lines that can detect hormone like activities by expression of fluorescent protein. One line has ecdysone response elements in the promoter region of fluorescent protein and the other has juvenile hormone response elements in its promoter. These transgenic lines enabled us to detect spatiotemporal distribution of ecdysone and juvenile hormone. In addition, these lines also could detect hormone like activities present in aquatic environment. As there was no convenient system for juvenile hormone activities to date, these lines are useful for the evaluation of hormone like activities.

The water fleas (daphnids, such as Daphniamagna and D.pulex) reproduce primarily by cyclic parthenogenesis in which only female offspring are produced. However, the daphnids switch to bisexual reproduction producing dormant resting eggs under the adverse environmental conditions, such as low food availability, low temperature, and hypoxic condition. Although juvenile hormones (JHs) are one of the hormones that control growth and development of arthropods, methyl farnesoate (MeFa: JH in daphnids) induces male offspring production in daphnids via JH-receptor methoprene-tolerant (Met). Therefore, chemicals with JH-like activity probably affect the endocrine system of daphnids, leading the reduction in population density. However, there are no invitro screening tests using juvenile hormone response element (JHRE) currently available for the detection of JH in D.magna. In this study, we found a novel JHRE, located upstream region of gene encoding Vrille [the transcriptional activator of doublesex-1 (Dsx1)] of D.magna and established reporter gene assay using the JHRE to detect JH-like substances in yeast expressing Met and steroid receptor coactivator SRC derived from D.magna.

We found a novel JHRE in the upstream region of the Vrille gene, and established yeast strain transformed JHRE reporter plasmid, D.magna Met expression plasmid, and D.magna steroid receptor coactivator expression plasmid. In this study, we evaluated the JH activity of the various compounds using the yeast reporter gene assay. The reporter gene assay was able to selectively and sensitively detect JH-like compounds.

【Introduction】With the increasing use of nanoparticles in various consumer products, concerns have been raised to the environmental and biological impact of nanoparticles. Indium compounds are known as an important material in flat panel displays and semiconductor compounds, and the Ministry of Health, Labor and Welfare added indium compound to substances regulated by the Ordinance on Prevention of Hazards Due to Specified Chemical Substances (OPHSCS) in 2013. Single particle (SP)-ICP-MS is a new method to analyze nanoparticles that utilizes the features of ICP-MS, such as high sensitivity, high selectivity, rapid measurement, and simple data processing. This technique has been established as a method that can measure the size, size distribution, and particle concentration. The aim of this study was to clarify the presence of indium nanoparticles in serum after intratracheal instillation of indium tin oxide (ITO）nanoparticles.

【Materials and Methods】Male Wistar rats were intratracheally instilled with a single dose of 8.6 mg In/kg BW (4 rats) or 25.8 mg In/kg BW (4 rats) of ITO nanoparticles (mean diameter:18 nm, 99.5% , 20 wt % in H2O), and control rats (2 rats) were instilled with 1ml/kg distilled water. Rats in the test group were euthanized until 40 weeks after the instillation, and then number of indium (In) nanoparticles in serum was determined using SP-ICP-MS (PerkinElmer NexION ICP-MS).

【Results】Indium nanoparticles were detected in serum for all rats treated with ITO nanoparticles, but not in the controls. The ratio of serum Indium particle concentration to all serum Indium concentration was 3 to 6 % in the 8.6 mg In/kg BW group and 2 to 28 % in the 25.8 mg In/kg BW group.

It was considered that some of the ITO nanoparticles instilled to the lungs were transferred to the internal organs by systemic circulation.

To look for a potential alternative to a two-year inhalation study of MWCNT, we now perform a chronic toxicity study designed to intermittently expose MWNT-7 to F344 male rats by the intratracheally instillation method with dose of 0, 0.125 or 0.5 mg/kg BW at every 4 weeks. Aggregation of MWCNT-laden macrophages associated with hyperplasias of type II alveolar cell was prominent in lungs of satellite animals at 0.5- and 1- yr after the beginning of the study. Cell numbers in BALF increased dose- and time- dependently. No lung tumors and pleural mesotheliomas were observed until now.

Multiwall carbon nanotubes (MWCNT) have been shown to induce a robust IL-1β production via NLRP3 inflammasome in macrophages. We explored inhibitors and identified K-NK104, which markedly reduced IL-1β production stimulated by MWCNT, but not by extracellular ATP. MWCNT uptake by cells was repressed, but cellular levels of NLRP3 inflammasome components were unaffected. These findings suggest that K-NK104 inhibits MWCNT-stimulated inflammasome activation and the consequent IL-1β production by repressing its internalization into cells.

In order to accelerate the social implementation of cellulose nanofibers (CNFs), which are expected to be used in a variety of new materials, it is necessary to evaluate the safety of CNFs. However, there is still little information on the effects of inhalation. In this presentation, after examining the following (1) to (3), we conducted a rat intratracheal instillation test to elucidate the inhalation effect of chemically modified CNFs, compared with multi-walled carbon nanotubes (MWCNTs) (4). (1) CNFs have an intermediate property between gel and sol, and its viscosity changes with time and shear rate (thixotropy). Therefore, we dispersed and prepared slurry CNFs without damaging the physicochemical properties. (2) After intratracheal instillation, CNF samples may cause suffocation or behavioral abnormalities in rodents because the physico-chemical properties of CNFs. Thereafter, we observed the state of the rat for several days and measured injection state of CNF dispersion from feeding needle. The concentration and administration conditions of the CNF samples that could be intratracheally instilled were established. (3) We prepared CNF stained with a chemical dye and analyzed of CNF in each lobe of rat lungs after intratracheal instillation. We verified that the test material was not unevenly distributed in the lungs after intratracheal instillation and was uniformly distributed. (4) We analyzed the number of cells, total protein, lactate dehydratase activity (LDH), cytokines in bronchoalveolar lavage fluid (BALF), and pathological observation during 90 days of intratracheal instillation of CNF samples, and verified the degree of inflammation and the changes over time. These results suggested that the inflammatory response due to the CNF was weaker than that of MWCNTs and decreased over time. This presentation was based on the results obtained from a project commissioned by the New Energy and Industrial Technology Development Organization (NEDO).

Oure previous study revealed that titanate nanosheet (TiNS) exposed monocytes underwent apoptosis following marked vacuolation. The achievements to date showed that TiNS increased lysosome level as well as autophagy-related gene expression, and that apoptosis was suppressed by bafilomycin A1 but not wortmannin, demonstrating that the toxicity is mediated through the function of lysosomes. The present study clarified that TiNS exposure increased cytosolic Ca²⁺ level as well as ATP6V1E1 and MCOLN1 mRNA levels and that lysosomal Ca²⁺ channel agonist accelerated apoptosis of monocytes exposed to TiNS.

Our CSAHi neuron team aims to develop in vitro system using a micro-electrode array (MEA) for detection of seizure. We investigated the versatility of MEA systems using two types of rat neuron cells under multi facilities. Gabazine, Picrotoxin (PTX), 4-AP and Acetoaminophen (APAP) were used. As a result, in all facilities, Gabazine and PTX could be separated from APAP in two samples and in two MEA systems, but not 4-AP in facilities where the number of spikes and firing bursts increased in APAP. MEA is a versatile evaluation system, and the responses to the negative compounds may be important.

　Human induced pluripotent stem cell (iPSC)-derived neurons can be used to predict neurotoxicity and CNS drug efficacy by measuring their activity with recording systems such as multielectrode arrays (MEAs). However, most of the current methods rely on uniform cultures of randomly mixed neurons, which might not be representative of the complexity of brain networks. Here we investigate how network organization affects electrical responses of cultured iPSC-derived neurons.

　Several types of neuronal populations were generated by transcription factor-induced differentiation of iPSCs and were shown to exhibit specific and reproducible spontaneous burst profiles when cultured individually. Then, using micropatterning processes, distinct neuronal populations were arranged in separate areas of a MEA. Co-cultured neuronal clusters synchronized their firing activity over time, indicating that synaptic connections occurred both within and among the neuronal clusters. These micropatterned structures were further evaluated for their response to neurotransmitter receptor blockers and for the functional recovery of connectivity following axonal injury.

　Our results show that monitoring the activity of neuronal clusters allows to evaluate the dynamic changes in neurotransmission induced by network regulation or disruption and their effects on pharmacological responses. Thus, the combination of robust neuronal differentiation methods with microengineering techniques should lead to the development of new in vitro models that are physiologically more relevant to in vivo brain functions.

Adverse effects of CNS are a major safety concern on the drug development. We tried Ca-transient assay using iCell DopaNeurons and XCell Neurons. Cells were cultured in 96well U-bottom plates. Calcium sensitive fluorescence indicator (EarlyTox Cardiotoxicity Kit) and test compounds (4-aminopyridine and so on) were added and Ca-oscillations were measured with FDSS/µCELL. This simple and effective throughput assay detected the effects of neurotoxic substances. Gabazine induced better response in XCell Neurons (Glutamatergic/GABAergic). We will also show our scheme for practical use.

[Background and purpose] Drug-induced convulsion is a serious adverse event in clinical trials; translation of convulsion risk from animals to humans is still a huge challenge because of species differences in susceptibility to drug-induced convulsions. In the present study, we chose a number of reference compounds to induce convulsions in mice, rats, and monkeys, to compare convulsion liability between humans and animals on the basis of plasma concentrations.

[Materials and methods] Mice (DBA/2) and rats (Crl: WI (Han)) were given different classes of reference compounds (a total of 11 compounds: e.g. Pilocarpine, 4-Aminopiridine, Pentylenetetrazole) intraperitoneally or intravenously. Plasma concentrations just after convulsion onset or completion of administration were measured by LC-MS/MS. Monkeys (cynomolgus) were dosed intravenously with a limited number of the reference compounds.

[Results and discussion]

Convulsion was observed in mice, rats, and monkeys with the reference compounds, except for a GAT1 competitive inhibitor which did not induce convulsion in rats. There was a general trend for the unbound plasma levels in monkeys to be closest to those in humans at convulsion. Our results suggest that, when considering the amount of compound available at the drug discovery stage and time, combination approach of appropriate species is recommended for in vivo convulsion screenings to select the compound with low convulsion risk.

We previously reported a rapid bone staining procedure (RAP-B) based on a rapid and nondestructive tissue clearing system (RAP system), preserving the histological structure of tissues even after the specimens were highly cleared. In the present study, we applied the RAP system to immunohistochemical observation. The RAP system improved permeation of antibodies into whole-mount specimens and thick tissue sections. Thus, we concluded that tissue clearing by RAP system could provide the great advance in immunohistochemical analysis.

[Introduction]

We have been developing a method for predicting seizure liability of drugs using in vitro human iPSC-derived neurons. However, it is necessary to approach the in vitro to in vivo extrapolation (IVIVE). As one approach to IVIVE, an assessment method using a human brain organoid that mimics the three-dimensional structure is considered to be effective. In this study, we attempted to detect the response to convulsants by MEA measurement using human cerebral cortical organoids.

[Methods]

Human cerebral organoids were prepared using the STEMdiff Cerebral Organoid kit from STEMCELL Technologies. After 6 months of culture, the human cerebral organoids were placed on the MEA (Alpha med scientific), and spontaneous activity was recorded at 37℃, 5% CO2. Next, strychnine and 4-Aminopyridine (4-AP) were administered to detect response to convulsants.

[Result]

Spontaneous activity waveforms of brain organoids were observed in low frequency components. Almost no spike components as detected in the cultured neural network were detected. After strychnine administration, the shape and frequency of the slow wave changed in a dose-dependent manner. Wavelet analysis revealed that the frequency intensity from the θ wave to the β wave component significantly increased in a dose-dependent manner. Also in the 4-AP administration, an increase in the frequency intensity from the θ wave to the β wave component was observed. An increase in the number of synchronized burst firings specific to 4-AP was detected.　It was found that responses to convulsants with different mechanisms of action could be detected in human brain organoids.

[Conclusion]

We succeeded in detecting electrophysiological activities of human cerebral cortical organoids by analyzing low frequency components in MEA data. In addition, it has been found that the component from the θ wave to the β wave is enhanced by the administration of convulsants. This is a result that enables comparison with in vivo brain waves. This study demonstrated that MEA measurement using human brain organoids may be a method that can approach in vitro to in vivo extrapolation in prediction of seizure liability of drugs.

Because Juzen-taiho-to may be administered for long-term due to characteristic of a traditional Chinese medicine and be used with other medicines, drug-drug interactions should be considered. In this study, drug-drug interactions resulted from enzyme inhibition of Juzen-taiho-to in dog liver microsomal cytochrome P-450 (CYP) were examined. Juzen-taiho-to most likely inhibited CYP 1A activity in major CYPs assessed in this study such as CYP 1A, 2D and 3A. However, it is hardly considered Juzen-taiho-to cause drug-drug interactions even on CYP 1A considering digestion and absorption.

Drug-Induced Liver Injury (DILI) is one of the major causes of attrition in clinical trials and drug withdrawal after marketing. Thus, it is considered important for pharmaceutical companies to predict hepatotoxicity early in non-clinical studies. On the other hand, many hepatotoxic compounds are idiosyncratic as well as intrinsic, which complicates prediction of DILI in nonclinical studies. In recent years, it has been reported that compounds which show both high lipophilicity and high daily dose are associated with a risk of hepatotoxicity (Rule of Two), and that multiple disorders of liver functions, such as dual inhibition of both BSEP and mitochondrial respiration, increase the risk of hepatotoxicity.

This study has aimed to extract features related to hepatotoxicity and help selecting candidate compounds by clarifying the profiles of the current physicochemical and in vitro evaluation systems (lipophilicity, daily dose, BSEP inhibition, mitochondrial respiratory inhibition, reactive metabolites) for 11 proprietary compounds that induced the elevation of blood liver enzymes in the clinical trials.

Among the 11 compounds, 8 compounds showed either high lipophilicity or high daily dose (daily dose > 100 mg), but none met both. Although the rule of two was not applied to these compounds, it was suggested that each parameter was involved in hepatotoxicity. On the other hand, 2 compounds each showed either dual inhibition of mitochondrial function and BSEP, or inhibition of mitochondrial function and production of reactive metabolites, and 1 compound showed inhibition of BSEP and production of reactive metabolites, suggesting that multiple disorders of in vitro parameters are related to hepatotoxicity. These results support recent literature and are useful for future prediction of clinical hepatotoxicity.

Drug-induced liver injury (DILI) is one of highest causes of drug attrition. The plasma Cmax total at efficacious doses is reported to be a major driver for DILI predictions (Shah et al, Toxicol Sci 2015). Recently, scoring system combining physicochemical properties and Cmax-corrected IC50 of in vitro assay endpoints was proposed for DILI prediction (Aleo et al, Chem Res Toxicol 2019). Because it is challenging to estimate human plasma Cmax in early phase of drug development, we assessed the use of multiple in vitro assay endpoints without Cmax-correction and compare predictive power with published physicochemical property-based DILI risk classification (BDDCS; Benet et al, AAPSJ 2011) to propose potential workflow to prioritize drug candidates in early drug discovery phase.

We assessed the utility of HepG2- and human hepatocyte spheroids-based cytotoxicity assay, HepaRG-based GSH depletion assay and liver humanized mice-derived human hepatocytes-based bile acid transport inhibition assay. Eighty licensed drugs were evaluated. The results showed in vitro assays in combination were able to predict DILI with sensitivity range of 54-61%; specificity range of 77-84%. Most of the false positive drugs showed the Cmax values < 1 µM; sufficiently high margins. In vitro assays and BDDCS detected different DILI drugs as positive with overwrap. Our data indicated in vitro assays and/or BDDCS could contribute to drug candidate prioritization in early drug development, and correction of assay results with human exposure might be effective to improve predictive power when available.

This study tried to elucidate a pathogenesis of the induction of non-obese (Asiatic) non-alcoholic steatohepatitis (NASH). In this study, 6-week-old male C57BL / 6J mice were fed a control diet or experimental diets; CDAHFD Met0.1, CDAHFD Met0.6, for 3 months. Histopathological changes and inflammatory marker were markedly increased in CDAHFD Met0.1 than in CDAHFD Met0.6. In RNA-Seq. analysis, Rho GTP signal and oxidative phosphorylation were markedly changed in CDAHFD Met0.1.

These results indicate that there is a signal that specifically changes in non-obese NASH progression.

We have previously reported that perfluorooctanesulfonate (PFOS) causes autophagy-induced apoptosis in renal tubular epithelial cells (RTCs) through a ROS-dependent mechanism. Herein, we extend our findings and provide the therapeutic potency of L-carnitine in PFOS-treated RTCs. L-carnitine reversed the effects of PFOS on autophagy induction and then the blocked autophagy flux. In addition, L-carnitine downregulated the protein level of p47PHOX at a 1-hr treatment of PFOS, which might be ascribable to the reduction of cytosolic ROS stained by DCFH-DA. Moreover, L-carnitine not only reduced the ROS levels in mitochondria stained by MitoSox, but also rescued the impaired function of mitochondria from PFOS, as the evidence of the increased ATP synthesis and oxygen consumption rate, and the reduced proton leakage in seahorse XF stress test. PFOS also increased IRE1a expression, a ER stress marker, which can be attenuated by 4-PBA, an ER stress inhibitor and L-carnitine pretreatment. The inhibition of ER stress by L-carnitine was associated with the decreased levels of Beclin and LC3BII, and then Bax and cleaved PARP and caspase 3. Collectively, these results indicate that through eliminating oxidative stress and ERK activation, L-carnitine decreases cell autophagy and concomitantly increased cell viability in RTC cells. This study does not only provide the potential mechanism of PFOS-mediated renal tubular cell apoptosis, but also sheds light on new strategies of using L-carnitine for the prevention and treatment of renal tubular cell apoptosis caused by PFOS.

Measurement of the renal resistive index (RRI) is one of the standard diagnostic procedures for assessing kidney disability clinically. To establish a practical RRI measurement procedure in cynomolgus monkeys (Macaca fascicularis) which utilized in conventional toxicology studies, RRI was measured by ultrasonography in the spine position in conscious and ketamine-immobilized monkeys. The RRI of conscious monkeys and ketamine-immobilized monkeys could be measured consistently without excessive abdominal or thoracic movement. Consequently, the variability of the RRI in conscious monkeys was comparable to that in ketamine-anesthetized monkeys. No sex difference in RRI was noted between the two conditions. The mean values and standard deviations (SD) of the RRI of 48 healthy monkeys (n = 24/sex) were 0.55 ± 0.07 and 0.50 ± 0.05, under conscious and ketamine-immobilized conditions, respectively. The RRI of ketamine-immobilized monkeys was significantly lower than that of conscious monkeys, correlating with the decreased blood pressure and heart rate. In a monkey model of cisplatin-induced acute renal injury, which was characterized histopathologically by minimal to mild renal tubular necrosis and regeneration, the RRI was increased beyond the cut off value (mean + 2SD, 0.68) associated with the progression of renal pathogenesis. The present results suggest that ultrasonographic measurement of the RRI in conscious monkeys would be a useful tool in conventional toxicology studies evaluating drug-induced renal injury.

Compensation is a physiological response that occurs during chemical exposure to maintain homeostasis. Since compensatory responses are not considered adverse effects, it is important to understand its detailed mechanisms. Although the kidney is a major target organ for toxicity, there is controversy whether the compensation is contributed by hyperplasia or by hypertrophy, and there is limited information to apply for chemical risk assessment. In this study, compensatory mechanisms of the kidney were investigated in unilateral nephrectomy (UNx) model using adult male and female F344 rats. In residual kidneys of male and female rats after UNx, 5-bromo-2'-deoxyuridine-labeling indices and mRNA expression of cell cycle-related genes were increased, while there were no fluctuations in mRNA expression of transforming growth factor-β1, which contributes to hypertrophy in renal tubules. Pathway analysis using microarray data for mRNA revealed forkhead box M1 (FOXM1) as an upstream regulator of cell proliferation in residual kidneys of both sexes. Microarray for microRNAs (miRNAs) demonstrated that 9 miRNAs were downregulated in residual kidneys, and mRNA/miRNA integrated analysis indicated that miRNAs were associated with the expression of factors downstream of FOXM1. Overall results suggested that FOXM1-mediated hyperplasia contributed to compensatory mechanisms in the kidney and that miRNAs regulated downstream FOXM1 signaling. These results will be beneficial for evaluating nephrotoxicity in chemical risk assessment and for developing new biomarkers to predict nephrotoxicity.

Allergic contact dermatitis is caused by substances such as plants, animals, medicines, metals and foods, and the skin barrier dysfunction causes the skin symptoms such as drying, itching and red flare. It was generally difficult to monitor the status of drying and itching. In order to quantify the skin function and scratching behavior and to determine the status of symptoms, we tried to develop the evaluation method for the drying and itching by atopic dermatitis model in hairless mice.

To produce symptoms similar to atopic dermatitis, female Hos: HR-1 hairless mice (Japan SLC Inc.) were fed a special diet (HR-AD) and applied the mite antigen ointment (Biostir AD®) to induce itching and drying. Before and after induction, skin moisture content, TEWL (transepidermal water loss) and viscoelasticity were measured by a Cutometor MPA580 (skin function measurement device), and scratching behavior was analyzed using the animal motion analysis system (ANIMA). Skin inflammation scoring, IgE concentration in the blood, cytokine (multiplex method) in the auricle related to inflammatory response, and histopathological findings were quantitatively evaluated the correlation with skin function and scratching. In addition, the improvement effect of tacrolimus on the skin symptoms was investigated by this quatification method.

In this conference, we will report not only this study result also the result comparing with “the skin toxicity evaluation of atopic dermatitis in NC / Nga mice” published last year.