Existing in silico software often fails to predict mutagenicity of our in-house compounds. Therefore, we have developed an AI based prediction system for mutagenicity by using both chemical structure images and Ames test results as a training dataset. We were able to improve the prediction accuracy of in-house compounds with the AI system compared to that of existing in silico software. Furthermore, the AI prediction system can visualize the rationale for the prediction, so it can also be utilized in ICH-M7 expert review. We would like to introduce how this AI-based prediction system works.

The Adverse Outcome Pathway (AOP) related to Wnt signaling activation leading to cancer malignancy has been proposed to OECD, registered as AOP298, and in Coaching and reviewing process. This AOP includes Wnt signaling activation such as Wnt and Frizzled activation, GSK3beta inactivation and activation of beta-catenin, Snail, Zeb, and Twist1, and epithelial-mesenchymal transition (EMT) in AOP scheme consists of Molecular Initiating Event (MIE), Key Events (KEs) and Adverse Outcome (AO). This AOP would be associated to the prediction of malignancy, which is one of the regulatory toxicological endpoints, induced by chemicals, molecules or stimulators activating Wnt signaling.

Aromatic hydrocarbon receptor (AHR) is one of the transcription factors expressed in most cells and tissues in humans, and its transcriptional activation is caused by binding to a ligand. Since AHR ligands are also present in chemicals and drugs in air pollution and in food, it can be said that AHR ligands are closely related to maintaining human health. In this study, ligands, we analyzed the structure-activity relationship of AHR ligands, which are known to be particularly harmful in the air, and natural substances based on molecular structure and toxicity information for a comprehensive understanding of AHR. For the harmful substances in the air, we selected 33 substances known to be present in diesel exhaust. As for natural substances, 14 substances derived from plants were selected. Toxicity information and molecular structure were collected from Biological Test Results and SD files of the public database PubChem. Subsequently, the relationship with the structure / toxicity information was analyzed using ADMET predictor 9.5. As a result, it was found that many of the polycyclic aromatic compounds had more physiological activities of multiple nuclear receptors than the oxidative stress response. On the other hand, the physiological activity data of natural substances was not available, and it was presumed from the structure that there was a response through the AhR transcription activity.

This study was aimed to clarify the relationship between 1-bromopropane (1-BP) and the health effects on peripheral nervous system of male workers. In the direct and indirect contact groups (n=14, 8), the individual concentrations of 1-BP were 4.8±11.1 (0.02-42.5) ppm and 0.3±0.3 (0.01-0.76) ppm. In the control group (n=15), they was not detection. Health effects of 1-BP were not observed in this study.

The environmental risk assessment for human medicinal products in the EU is going to be updated in order to adapt it to the technical progress. Significant changes are foreseen according to the guideline draft of EMA published end of 2018. In the commenting period until mid 2019 intense discussions took place questioning many of the intended changes and suggesting different ones. At the point in time of the compilation of this abstract the submitted comments have not been published yet, which could signify that not only a high number has been received, but also that considerable points have been raised. The time line of the guideline implementation process is unforeseeable. Therefore either the final guideline and its implications for the drug development process or the recent status will be presented. In any case the discontinuation of the option to use marketing data for refinement of the predicted environmental concentration can be expected. Consequently the use of epidemiological data originating from the EU becomes much more important to avoid undesirable leaflet warnings. Also implications to the preclinical toxicity and pharmacokinetic study designs seem likely. It can therefore be assumed that environmentally relevant properties should be considered earlier in drug development.

Gefitinib is a molecular target cancer drug that inhibits tyrosine kinase activity of epidermal growth factor receptor (EGFR). However, accumulating evidence indicates that gefitinib has other targets besides EGFR. In this study, we found that gefitinib-induced suppression of cancer cell migration and invasion is strongly suppressed in p62/SQSTM1 and neighbor of BRCA1 (NBR1) double-deficient but not EGFR-deficient cells. Therefore, our results indicate that gefitinib inhibits cancer cell migration and invasion through the p62 and NBR1-, but not EGFR-dependent mechanisms.

We have established the first open laboratory in Japan to conduct nonclinical safety research in primates for the practical use of genetically modified cells as a part of an AMED development project. We conducted a preliminary toxicity study of new CAR-T cells by producing novel CAR-T from the peripheral blood of cynomolgus monkeys and administering it to donor animals. As a result, new CAR-T cells were detected in all animals, but no appreciable toxicological findings were noted. These results demonstrate the potential use of primate autologous models in assessing the toxicity of CAR-T cells.

Propetamphos (PPT) is a public health organophosphate pesticide universally used to control insects including cockroaches, flies and ants. Methylethylphosphoramidothioate (MEPT) is the major urinary metabolite of PPT. The objective of this study was to develop an effective method for the screening of urinary MEPT using gas chromatography-mass spectrometry, and to assess the exposure levels of MEPT in human populations. Dibutyl phosphate was used as an internal standard, and analyses of MEPT derivatized with pentafluorobenzyl bromide were performed using an Agilent 5975 inert MSD system. The validated method was applied to spot urine samples collected from sprayers (n=15), non PPT-sprayers in the same working population (n=15) and control volunteers (n=80) living in the same area. The calibration curves were obtained with standard-spiked pooled urine. The limit of detection (LOD) was 10 µg/L. Average within-run and between-run precision were 18.4 and 14.2%, and accuracies were 110.6 and 87%, respectively. Urinary MEPT in the PPT sprayers, non-PPT sprayers, and control volunteers were ranged from <LOD–22.3, <LOD–21.9 and <LOD–13.8 µg/L, respectively. Results of the study suggest that study participants including occupational sprayers might expose to low levels of PPT. The present biomonitoring method could be useful for quantitative measurement of urinary MEPT in highly exposed human populations.

By acting on receptors in the brain, some chemicals exhibit "signal toxicity" that disrupts intracellular and intercellular signaling, causing unexpected effects. In particular, in brain function, a complex signal network between neurons and glial cells is formed, and disruption of this network can be a critical endpoint of signal toxicity. However, “disruption of brain function” due to signal toxicity is often not accompanied by cell death and accompanying brain tissue degeneration, and cannot be detected by current neurotoxicity test methods. In this study, we used neonicotinoids (NNs) and nicotine, which are agonists of the nicotinic acetylcholine receptor (nAChR), as model compounds, and detected changes in neuronal activity in the somatosensory cortex using a two-photon microscope. The aim was to detect disruption of brain function associated with signal toxicity.

C57BL/6N were orally administered ACE at 20 mg/kg (with reference to LOAEL in general pharmacology test of the central nervous system) and their blood was collected over time. It was analyzed quantitatively using LC-MS/MS. Then, we exposed mice to ACE at 20 mg/kg or 7.1 mg/kg (with reference to NOAEL to rats), and their behavior was observed using openfield (OF) test 1 hour after the administration at the former concentration, and using elevated plus maze (EPM) test 1 hour after the administration at the latter concentration. In addition, C57BL/6J were administered ACE at 30 mg/kg (with reference to NOAEL in the subacute toxicity test) and Nicotine, which is the typical agonist of nAChR, at 1.6 mg/kg (with reference to 1/2 LD50), and the neuronal activity in somatosensory cortex was observed over time by using two-photon microscope.

As a result, ACE absorbed quickly and the Tmax of ACE was around 25 minutes after administration. In comparison, dm-ACE, which is one of its major metabolites, absorbed a bit slower and the Tmax was around 150 minutes. An hour after the administration, the activity of mice decreased, and the anxiety-like behavior increased. In the somatosensory cortex, the firing frequency of nerve cells decreased, and the synchronous firing increased, 1 hour and 2.5 hour after ACE administration. Similarly, the firing frequency tended to decrease, and the synchronous firing increased, 5 minutes and 30 minutes after the administration of Nicotine. This suggest that changes in neuronal activity after administration of ACE are the result of ACE acting as a ligand for nAChR because we can see the same trends between ACE and Nicotine. In summary, it was observed that administration of ACE at a concentration that is originally non-toxic, such as NOAEL, affects the behavior and neural activities. This suggests that cranial nerve activity is responsible for changes in behavior.

Prenatal chemical exposure is a growing health concern as it can cause a lifelong lasting effect on the psychological and physiological health in both pregnant mother and offspring, which supports the Developmental Origins of Health and Disease (DOHaD) hypothesis. In recent years, many reports link prenatal exposure of environmental chemicals to reproductive health, epigenetic modification, and pathological diseases acting on the direct disruption of cells and structures of the nervous system and epigenetic changes. Glyphosate, the main compound of a broadly applied herbicide, is the negative substance on the Organization for Economic Cooperation and Development (OECD) Test Guidelines; however, the safeties of glyphosate or aminomethylphosphonic acid (AMPA), a metabolite in the soil, are unknown. The primary herbicidal function of glyphosate is to inhibit a key plant enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which participates in the biosynthesis of aromatic amino acids (including phenylalanine, tyrosine, and tryptophan) via the shikimate pathway in bacteria, fungi, and plants. Glyphosate may have the potential to modify the animal and human gut microbiota. Previously we have shown that acute exposure of 100 or 250 mg/kg-glyphosate, or 250 mg/kg-AMPA to pregnant rats lead to neuronal and behavioral disorder in developing cerebellum. In this study, we investigated the effects of prenatal chronic glyphosate exposure on the cerebellar cortex in immature offspring. Pregnant rats were exposed to glyphosate-water (approximately 15.625 mg/kg/day; total dosage is 250 mg/kg) from gestational day 4 (G4) until G19. Prenatal chronic glyphosate exposure caused a decrease of Purkinje cells and an increase of microglia in developing cerebellum. Therefore, glyphosate may have some developmental neurotoxicity.

Neonicotinoids, a new class of insecticide, mimic the chemical structure of nicotine to bind to the nicotinic acetylcholine receptor (nAChR) to provide highly systemic insecticidal effects. Due to the difference of nAChR sensitivities between insects and mammal, neonicotinoids have been seen as safer insecticides than organophosphate compounds; however, recently, some reports suggest the developmental neurotoxicity of neonicotinoids.

In this study, we exposed Acetamiprid (ACE), a type of neonicotinoid, in different dosages to rat embryo, and observed cerebellar development in the offspring. 20 mg/kg-, 40 mg/kg-, or 60 mg/kg-ACE were orally administered to Wistar rats on the 15th day of gestation (G15). The male pups underwent perfusion fixation 14 days after birth (P14). The cerebellar slices immunochemistry stained with anti-Calbindin D-28k / Iba1 were observed with a confocal microscope. Then, the samples were subjected to Hematoxylin and Eosin (HE) staining to observe the entire image of the cerebellar vermis with a light microscope.

In ACE 40 mg/kg pups, the alignment of Purkinje Cell (PC) significantly misaligned, and in ACE 60 mg/kg pups, slight misalignment. Some PCs distributed the region under the standard Purkinje layer to be surrounded into the granular layer. ACE 20 mg/kg pups showed no misalignment of PC. In addition, ACE 40 mg/kg- and ACE 60 mg/kg-pups showed the higher cerebellar lobule folding between lobule V and VI in vermis than the control rats, whereas ACE 20 mg/kg-pups was almost as same as the control rats. The folding rates of ACE 40 mg/kg and 60 mg/kg were similar to the cerebellum treated with valproate, which is a well-known HDAC inhibitor and ASD-inducer.

These studies suggest that fetal exposure to high-dose ACE might affect cerebellar development and PC alinement. ACE 40 mg/kg dosage would not induce neuronal overkill but malformation in developing cerebellum, although ACE 60 mg/kg dosage would induce neuronal overkill. It might be related to the degree of microglia activation.

【Purpose】Living organisms are controlled by the circadian rhythms. It has become clear that these circadian differences affect the frequency of diseases. Additionally, time-dependent differences in the pharmacokinetics of medications are also reported. Therefore, it is now notable to take care of chronobiology-based medication. Our research, which we describe as “chronotoxicology” has focused on the relationship between injection timings and the degree of onset metal toxicity. We recently investigated that the chronotoxicity of seven metals, including Ni and Cu, showed specific patterns of diurnal variation (Yoshioka et al, J Toxicol Sci, 43 (2), 129-134, 2018). These results present the possibility that each elements or compounds can exhibit a unique chronotoxicity. As for medication, since reports about chronotoxicity were limited, it is important to investigate the chronotoxicity of various medicinal drugs for improving the quality of life.

Cisplatin (CDDP) has using abroad in the way of platinum complex on anticancer drugs. However, it is known that CDDP exhibit serious side effects, such as acute renal damage (AKD). Although the diurnal variation of CDDP-induced nephrotoxicity has been reported, mechanisms of these differentiations were not elucidated. In this study, we investigated the circadian variations in CDDP-induced toxicity focused on toxicity and its mechanism using mouse model.

【Method】(1) For the mortality assays, 7-week-old ICR mice were divided into six groups of five animals each. Each group was administered 20 mg/kg CDDP by an intraperitoneal injection at one of six different time points (clock time; 10:00, 14:00, 18:00, 22:00, 2:00, or 6:00), described here as zeitgeber times (ZT); ZT2, ZT6, ZT10, ZT14, ZT18, or ZT22, respectively. Injections were performed under red light during the dark period (ZT14, ZT18, or ZT22). Surviving numbers of mice was monitored until 14 days after the injection (2) We measured expression levels of OCT2 (intracellular incorporation transporter) in kidney from six different times points. (3) For analysis of circadian variations in the renal injury, the mice received intraperitoneal injections of 20 mg/kg CDDP at ZT2 or ZT14. After 72 h, the animals were euthanized and bled for plasma collection. Kidneys were harvested from each of these animals. Using plasma, we determined creatinine and blood urea nitrogen (BUN). In addition, we performed qPCR, western blotting, and histological analysis in the each kidney.

【Results and Discussion】(1) We showed, mice were tolerant at ZT22 and ZT2 10:00, whereas at ZT6, ZT10, ZT14, and ZT18, mice indicated higher susceptibility to CDDP-induced toxicity These results suggest that injection timings affected the severity of CDDP-induced toxicity. (2) Expression levels of OCT2 was no significant change in all 6 groups. This result indicated that the chronotoxicity of CDDP would be occurred after incorporation in the kidney. (3) We showed that the levels of nephrotoxicity indicators (BUN, creatinine, renal levels of inflammatory cytokines, necrosis, and DNA adducts) significantly increased as ZT14 (dark phase) but not at ZT2 (light phase). These results suggest that the chronotoxicology of CDDP might provide valuable information regarding the importance of injection timings for toxicity evaluation tests and undesirable side effects.

In this study, we aimed to elucidate the mechanism involved in the induction of oncostatin M (OSM) by methylmercury in microglia. We found that methylmercury induced OSM expression by activation of STAT3 and cJun, which are subunit of known transcription factors for OSM expression, in mouse microglial cell line BV2. Moreover, knockdown of up-stream kinases (JAK1 or ASK1/JNK) for STAT3 or cJun also attenuated methylmercury-induced OSM expression. These results suggest that methylmercury may induce OSM expression via JAK1/STAT3 and ASK1/JNK/cJun pathways in microglia.

Although methylmercury (MeHg) results in neuronal cell death, the detailed mechanism has yet to be fully understood. In this study, we report a contribution of endoplasmic reticulum (ER) stress to MeHg-induced neuronal apoptosis in vivo. We found that ER stress derived signals by MeHg administration were detected in the brains. Interestingly, MeHg-induced ER stress signals (unfolded protein response) was specifically observed in neurons of the somatosensory cortex that have been reported to be injured by MeHg. These results indicated that ER stress is implicated in the neuronal loss by MeHg.

Methylmercury (MeHg) is known to cause neural degeneration, which is called Minamata disease. Although one of the major symptoms of the disease is sensory disturbance, the sensitivities to MeHg on several sensory modalities such as cold, hot, pain, and pressure have not been investigated. Wistar rats were exposed to MeHg (6.7 mg/kg/day) fifth a week for two weeks. Behavioral analysis was performed from the beginning day of MeHg exposure for 70 days to quantify the sensory disturbance. The results suggest that each sensory modality seems to have different sensitivity to MeHg neurotoxicity.

Arsenic is an environmental pollutant that causes atherosclerosis. In this study, we found that arsenite inhibits the fibrinolytic activity through NRF2-dependent suppression of t-PA expression in vascular endothelial cells, whereas arsenite upregulated the expression of PAI-1 and tissue factor in macrophage-like THP-1 cells. These results suggest that arsenite decreases the fibrinolytic activity in blood vessels through disruption of the coagulation-fibrinolytic system in vascular endothelial cells and macrophages, resulting in a promotion of atherosclerosis progression.

Diphenylarsinic acid (DPAA) is a non-natural organic arsenic compound detected in the well water in Kamisu, Ibaraki, Japan. People having used this well water developed cerebellar symptoms. In the present study we evaluated the aberrant activation of cultured normal human cerebellar astrocytes (NHA) by prolonged exposure to DPAA. NHA were exposed to 10 μM DPAA for 96, 144, 192, 240, or 288 h, and protein expression and glutathione secretion were evaluated. Exposure to 10 μM DPAA for 96 h little activated NHA, but it for 288 h could induce significant aberrant activation.

Methylmercury (MeHg) exhibits neurotoxicity and caused Minamata disease. Sensory neuron is highly susceptible to MeHg. In this study, we found that the dorsal root ganglion (DRG) were much more sensitive to than anterior horn cells and Schwann cells. Among these cell types, methylmercury most highly accumulated in DRG. The constitutive expression level of LAT-1 was higher and that of MRP-2 was lower in DRG. These results suggest that the susceptibility of DRG to MeHg is due to the constitutive expression levels of LAT-1 and MRP-2, which enables MeHg to accumulate highly within the cells.

Our previous study suggested that TNF-α may be involved in the selective damage of sensory neurons by methylmercury (MeHg). To examine this possibility, toxicity of MeHg was compared between wild type (WT) mice and TNF-α deficient (KO) mice. Survival, weight loss, mercury accumulation, and hind limb extension symptoms were evaluated after oral administration of MeHg. The results indicate that TNF-α is involved at least in the hind limb extension symptoms caused by MeHg.

Our previous study demonstrated that cadmium (Cd) triggers renal toxicity through the disruption of transporting system. In this study, we investigated the effect of Cd on the expression level of ABCB1 (ATP binding cassette transporter subfamily B member 1) transporter in HK-2 human proximal tubular cells. Treatment of HK-2 cells with Cd up to 40μM for 6 h showed little cytotoxicity; however, Cd treatment for 24 h showed approximately 80% cytotoxicity. ABCB1 mRNA level was significantly increased by Cd treatment for 6 h in HK-2 cells. The protein level of P-glycoprotein (ABCB1 gene product) was also increased by Cd treatment for 6 and 12 h. These results suggested that the increase in not only gene expression of ABCB1 but also protein level of P-glycoprotein by Cd may induce some abnormalities in transport system in the kidney.

Cadmium is an environmental pollutant and risk factor for atherosclerosis. In the atherosclerotic intima, dermatan sulfate chains accumulate and accelerate the oxidation of LDL cholesterol. Biglycan is the major dermatan sulfate proteoglycan synthesized and secreted by vascular endothelial cells. The purpose of this study is to investigate the effect of cadmium on the synthesis of biglycan in vascular endothelial cells. Confluent cultures of vascular endothelial cells were exposed to cadmium with or without of [³H]glucosamine and [³⁵S]sulfate. Proteoglycans extracted from the cell layer and conditioned medium were separated by anion exchange chromatography. The radioactivity of [³H]glucosamine was increased in biglycan-containing peak but that of [³⁵S]sulfate was not affected by cadmium. The fractions of this peak from the conditioned medium were collected and further analyzed by size exclusion chromatography. Cadmium elongated the length of dermatan sulfate chains and upregulated the expression of CHSY1, an elongation enzyme of dermatan sulfate chains, mediated by protein kinase C, while cadmium did not affect the expression of biglycan mRNA and core protein expression. In addition, CHSY1 overexpression increased the leakage of lactate dehydrogenase by cadmium from vascular endothelial cells. These results suggest that cadmium-induced CHSY1 contributes to the progression of atherosclerosis by not only elongating the dermatan sulfate chains of biglycan but also exacerbating the cytotoxicity of cadmium.

trans-Fatty acids (TFAs) are fatty acids possessing at least one carbon-carbon trans double bond. The intake of TFAs contained in processed foods, such as elaidic acid, has been associated with various diseases including atherosclerosis. However, the underlying disease etiology remains unknown.

Here, we found that TFAs promote DNA damage-induced apoptosis through feedforward amplification of the mitochondrial ROS generation and JNK activation, and that the amplification is mediated by Sab, which is localized at the mitochondrial outer membrane. Our study provides a novel mechanistic insight into TFA-related diseases.

We have previously reported that formation of pyridoxal (PL) from MPN by CYP1A2, was the rate-limiting step in a major metabolic pathway of MPN in mice. In this study, the effects of CYP1A inhibitors and inducers on convulsion which was a main symptom of “Gin-nan food poisoning” and on MPN and its metabolite concentrations in plasma and urine in mice were investigated. Treatment of mice with the inhibitor resulted in a increase in frequency of convulsion and in AUC of MPN, but in a decrease in AUC of PNA which was a further metabolite of PL. In contrast, treatment of mice with the inducers produced the opposite results of inhibitors.

The mechanism of acetamide (AA)-induced hepatocarcinogenesis remains uncertain. Our previous study showed that AA induced histopathological changes implying chromosome aberrations (CA) in the liver of rats. In this study, to clarify possible involvement of CA in hepatocarcinogenesis, we performed micronucleus (MN) test and comet assay using rats. As a result, AA induced CA in the liver, but not direct DNA damage. Moreover, the immunohistochemical analysis showed damaged DNA and abnormal nuclear envelope in MNs, suggesting that chromothripsis may contribute to AA hepatocarcinogenesis.

[Introduction]

Tetrabromobisphenol A(TBBPA) is the most widely used type of brominated flame retardant, which is employed as additives in the manufacturing of office and home electronic equipment. Information about neurotoxicity is limited, despite TBBPA is suspected to be stable in the environment with possible widespread human exposures. In this study, we investigated the effects of TBBPA on emotional behavior in mice.

[Method]

Male ddY mice（8 weeks old）were orally administered TBBPA 500 mg/kg bw daily for two weeks, and the effects of TBBPA on anxiety- and depression-like behavior were examined in behavioral tests. The behavioral tests consist of the open field test, elevated plus-maze test, and forced swim test.

[Results and Discussion]

In the open field test, there was no difference in the number of entries into the center area, the amount of time spent in center area, and total distance between vehicle- and TBBPA-treated groups. In the elevated plus-maze test, administration of TBBPA did not alter the number of open arm entry and time spent in open arm. These results suggest that TBBPA did not affect anxiety-like behavior in mice. In the forced swim test, TBBPA administration resulted in an increase in the immobility time.

[Conclusions]

These results suggest that TBBPA, a brominated flame retardant, did not affect anxiety-like behavior but caused depression-like behavior in mice.

We have suggested that the suppression by dioxin of prolactin level was inherited by next generation females. To confirm its toxicological significance, we measured the licking, a nursing behavior and the amount of breast milk produced by the next generations F1 and F2 female during breastfeeding. We found that maternal exposure to TCDD reduces milk amount significantly in F2. Similarly, that in F1 tends to be reduced. Thus, maternal exposure to TCDD reduces decreases prolactin level in the offspring, which in turn causes heritable toxicity such as the nursing behavior and the breast milk level in the offspring.

We used human SH-SY5Y cells to investigate the effects of silver nanoparticles widely used in well-known products on amyloid-beta expression, a key molecule in Alzheimer’s disease. The present study showed that silver nanoparticles of 10 nm in diameter induced the protein level of amyloid precursor protein upregulation through oxidative stress and after amyloid-beta increment in SH-SY5Y cells. Our results provide basic evidence that the adverse effects of silver nanoparticles on the central nervous system.

We evaluated the effects of multiple dose of nSP50 (amorphous silica nanoparticles of 50 nm in diameter) on acute toxicities, when mice were pretreated and challenged with PBS or nSP50 to identify their inverse biological effects mediated by acquired immunity. As a result, the plasma level of hepatic damage markers in nSP50-pretreated mice was significantly increased compared to those in PBS-pretreated mice. On the other hand, it wasn’t observed in immunodeficiency mice. These results suggest nSP50-inducible hepatic damages would worsen by pre-treatment of nSP50 via acquired immune system.

We evaluated the effect of silver nanoparticles in 10 nm diameter (nAg10) on forskolin-induced BeWo syncytia. RT-PCR analysis found that the forskolin-induced up-regulation of Syncytin-1/2, syncytium-promoting molecules, were suppressed. Moreover, co-treatment of nAg10 with forskolin significantly reduced the forskolin-induced increase in nucleus size. These results suggest that nAg10 might inhibit the formation of the syncytium cells.

Many studies have examined carbon nanomaterials (CNMs) as biomaterials for medical application. However, CNMs introduced into the living body may accumulate in lymph nodes to exert unknown cellular effects. This study evaluated the cellular response of human lymphatic endothelial cells (HLECs) derived from lymph nodes to CNMs.

Four CNMs (untreated carbon nanohorns [CNHs], oxidized CNHs [oxCNHs]), carbon black [CB], and multi-walled carbon nanotubes [MWCNTs]) were assessed for cellular responses in HLECs in terms of cytotoxicity, intracellular uptake, and inflammatory reactions.

In cytotoxicity tests, cell viability was significantly reduced in the MWCNT group at concentrations of 50 and 100 μg/mL after 48 h exposure as compared with the dispersant-only control group. Regarding cellular uptake, transmission electron microscopy and fluorescence microscopy revealed that all CNMs were localized in lysosomes for degradation. Quantitative evaluation of cellular uptake showed the highest levels for the MWCNT group (MWCNT>CB>oxCNH>CNH). In inflammatory response assessments, exposure to MWCNTs at a concentration of 100 μg/mL significantly increased the expression of IL-6, CCL5, CXCL8, and TNF versus the control group.

The above results suggested that whereas all CNMs were taken up by HLECs, none except MWCNTs showed short-term effects on the lymphatic system. Further evaluation of the impact of CNMs accumulated in cells is needed.

We tested whether AMPA receptor activation in the substantia nigra causes Parkinson’s syndrome in rats. AMPA injection into the substantia nigra induced movement disorder and nigral dopaminergic neurodegeneration. AMPA-induced increase in nigral intracellular Zn²⁺ and AMPA-induced nigral neurodegeneration, which were accelerated with aging, were suppressed by co-injection of intracellular Zn²⁺ chelators. The present data suggest that AMPA receptor activation in the substantia nigra aggravates Parkinson’s syndrome in aged rats via nigral intracellular Zn²⁺ dysregulation.

We examined the preventive effect of Ninjin-yoei-to (NYT) on amyloid β_1-42 (Aβ_1-42)-induced hippocampal neuron death in vivo. Pre-administration of NYT for 2 weeks prevented Aβ_1-42-induced neuron death. Aβ_1-42-induced increase in intracellular Zn²⁺ in dentate granule cells was blocked by NYT intake for 2 weeks. The present study suggests that pre-administration of NYT prevents neuron death in the hippocampus by blocking Aβ_1-42-induced intracellular Zn²⁺ toxicity via induction of metallothioneins, intracellular Zn²⁺-binding proteins. NYT may be effective for preventing Alzheimer’s disease.

Acrylamide (ACR) is an electrophile which has been used extensively in industry and is also formed unintentionally in food substances cooked or processed at high temperatures, such as potato chips or coffee through Maillard reaction. Acrylamide has been recognized as a potent neurotoxin which is known to cause neuropathy or encephalopathy in humans and experimental animals. As a measure of protection against neurotoxicity, the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has been identified to be a master regulator of the cellular defense system which activates antioxidant and cytoprotective genes. However, knowledge about mechanistic roles of Nrf2 in acrylamide-induced neurotoxicity remains poorly understood. This study therefore sought to investigate and clarify the roles of Nrf2 in ACR-induced neurotoxicity.

Forty-eight male Nrf2-knockout (Nrf2-KO) mice from the C57BL/6JJcl background, aged 10-weeks together with their age and sex-matched wild-type (WT) counterparts were each divided into four groups of twelve and daily exposed to ACR at 0, 67, 110 or 200 ppm in drinking water for 28 days. Following exposure of mice to ACR, Landing Foot spread test, an assessment of motor function and a major endpoint marker of neurotoxicity as well as immunohistochemistry for noradrenaline transporter (NAT) and serotonin transporter (SERT) antibody in the dorsal and ventral medial prefrontal cortex were performed.

Nrf2-KO mice showed exacerbated impairment of motor functions evidenced by the increased hindlimb splay relative to WT mice at the same exposure levels. Immunohistochemistry results showed severe degeneration of both noradrenergic and serotonergic axons characterized by a dose-dependent decrease in the density of immunoreactive axons in Nrf2-KO mice relative to WT mice. Moreover, body weight, whole brain weight and cerebellum weight were significantly reduced in Nrf2-KO mice compared to the WT mice. The results suggest increased susceptibility to ACR-induced neurotoxicity in mice lacking the Nrf2 gene.

In conclusion, Nrf2 is able to attenuate the effects of ACR-induced neurotoxicity in mice and thus remains a crucial target for the preventive modulation of neurotoxicity.

To investigate the mechanisms by which environmental electrophile acrylamide (ACR) induces degeneration of bioaminergic neurons, differentiated noradrenergic neural cells 1C11^NE or serotonergic neural cells 1C11^5-HT were treated with ACR or conditioned medium from ACR-exposed BV2 microglia for 24h. ACR lower than 1 mM didn’t decrease 1C11^NE or 1C11^5-HT cell viability, nor increase LDH release. However, cell treatment with conditioned medium from BV2 cells exposed to ACR at 1 mM significantly decreased the neurite length of 1C11^NE or 1C11^5-HT.

Conclusion: microglia play a critical role in ACR-induced retraction of noradrenergic neurons and serotonergic neurons.

The purpose of this study is to investigate whether airborne particle exposure affects the prognosis of ischemic stroke.

Mice were intranasally administered airborne particles for 7 days and then subjected to focal cerebral ischemia by photothrombosis (PT). In airborne particle treated mice, Rotarod score as well as Hanging-wire and mNSS scores deteriorated, indicating decreased motor ability. Furthermore, mortality after PT increased by airborne particle exposure.

We have demonstrated for the first time that airborne particle exposure aggravates the outcome of ischemic stroke.

[Introduction] Brain disease and drug toxicity are often caused by transmission disorder between brain regions. Drug discovery in the central nervous system also is aimed at improving transmission disorder. A method to measure the action potential (AP) propagation between brain regions in detail is effective for drug efficacy and toxicity assessment. However, detection of AP propagation in detail between brain regions requires the measurement device with high spatiotemporal resolution. In this research, we attempted to detect the propagation pattern between brain regions and the change of pattern by drug administration using CMOS-Microelectrode Array(MEA), which has over 10,000 electrodes with an electrode pitch 11.72μm.

[Methods] The brain slices (300μm) were acutely isolated from adult male C57/BL6NCrSlc mice aged 6-7 weeks and placed in oxygenated (95% O2 and 5% CO2), ice-cold artificial cerebrospinal fluid (ACSF). Spontaneous activities in brain slice were recorded with CMOS-MEA chip (Sony Semiconductor Solutions Corporation). 4-aminopyridine (4-AP) 10 μM and kainic acid (KA) 1 μM were administered to examine the change in propagation pattern due to drug administration, respectively.

[Results] Detailed pattern of AP propagation between DG, CA1, CA2, and CA3 regions in hippocampus were detected in convulsants administration. Interestingly, AP propagation between the hippocampus and the cerebral cortex was also measured. This is a feature of our CMOS-MEA, which has a large measurement area that can detect not only the hippocampus region but also other areas at the same time. 4-AP and KA increased the number of synchronous burst firings, but the AP propagation pattern was different.

[Conclusion] Using CMOS-MEA measurement method, we detected AP propagation pattern between the brain slice regions, and found that the propagation pattern was different depending on mechanisms of action of drugs. This CMOS-MEA measurement method combined with brain slice is expected to be a new drug efficacy and toxicity assessment method based on the propagation pattern between brain regions.

In vitro microelectrode array (MEA) assays using rat primary neurons or human induced pluripotent stem cell (hiPSC)-derived neurons are expected to predict the seizure liability of new drugs. We have previously reported that spike data from cultured neurons can be used to distinguish the responses to convulsants from negative control, and to separate the responses by mechanism of action of convulsants. However, to approach in vitro to in vivo extrapolation (IVIVE), it is necessary to determine the toxic dose of drugs that reflects in vivo brain. In this study, we compared the responses of spike data in rat primary neurons or hiPSC-derived neurons to the drug concentration in rat CSF during seizure. Firstly, dose-dependent MEA data to 4-aminopyridine (4-AP) and pentylenetetrazol (PTZ) administration, and to acetaminophen and aspirin as negative control were obtained. Next, dose-dependent significant difference between the responses of convulsants and the negative control was examined using PCA. Finally, we compared the in vitro concentration of the convulsants, which is significantly different from responses of the negative control, to the in vivo CSF concentrations during seizure. As a result, CSF concentration of 4-AP at the pre-convulsive state and during seizure were 2.14 μM and 5.13 μM, respectively. MEA data of 4-AP showed a significant difference from negative control at 3 μM or more, which was consistent with the result of in vivo CSF concentration. CSF concentration of PTZ at the pre-convulsive state and during seizure were 468 μM and 705 μM, respectively. In the MEA data of PTZ, a significant difference was obtained at 300 μM or more, which almost coincided with the result of in vivo CSF. These results suggest that comparison of in vitro MEA data with in vivo CSF concentration is an effective method to approach IVIVE.

Seizure is one of a severe toxicity for drug developments. Recently, many reports suggested a usefulness of in vitro microelectrode array (MEA) systems to detect seizure risks using the well-known seizure-inducible compounds with mechanism of action. On the other hands, there are few information about the usage of the in vitro MEA systems to detect the seizure risks with compounds whose pharmacological action is not directly associated with inducing seizure. In fact, these compounds, which do not have a theoretical risk for seizure based on their pharmacological actions, sometimes induce tonic/chronic convulsions in in vivo toxicity studies. Thus, we are now trying to assess the seizure risks of in-house compounds whose pharmacological action is not directly associated with seizure, and to confirm a usefulness of the in vitro MEA systems in more realistic cases. Neuronal activity of iPSC-derived neuron and human primary astrocyte (Neucyte) with these compounds was recorded by in vitro MEA systems (Axion Inc.) and some parameters were calculated. Using these parameters, PCA or cluster analysis were conducted to classify the test compounds into seizure-inducible or non-seizure-inducible compounds. In vitro MEA systems categorized some in-house compounds into seizure-inducible compound. These seizurogenic responses in neural activity were observed only in higher concentrations but not in lower concentrations. Further, the compounds categorized into the seizure-inducible compound induced the convulsions in in vivo toxicity studies which had been conducted previously, indicating that the results of the in vitro MEA were consistent with that of the in vivo studies. This study provided that in vitro MEA systems are useful to detect seizure risks even the case of the compounds whose pharmacological action is not directly suggested with seizure risks.

Astrocyte and neural network activities are closely related. It has been reported that in evoking convulsions, astrocyte oscillation precedes neural activity. If an astrocyte response to a convulsant could be detected in an in vitro culture sample, this would be an effective method for predicting seizure liability of drugs. We investigated whether astrocyte oscillation could be detected using the MEA measurement, and whether a response to a convulsant could be detected based on astrocyte oscillations. Rat hippocampal astrocytes and neurons were seeded on MEA plates (Alpha med scientific), and a sample containing only astrocytes and a sample having a different ratio of neurons were constructed. To detect the astrocyte response, 4-AP was administered and the evoked response was measured. Picrotoxin, chlorpromazine, and pilocarpine were administered to co-cultured samples of human iPS cell-derived neurons and astrocytes (Axol) in order to examine whether astrocyte responses to convulsants having different mechanism of action were detected. Oscillation was detected by 4-AP administration in the astrocyte-only sample. In addition, the frequency intensity was increased in a dose-dependent manner with 4-AP. In an astrocyte co-cultured sample with a small percentage of neurons, it was observed that neural activity was induced after astrocyte oscillation. In samples with a high proportion of neurons, oscillation of astrocytes and neurons was observed almost simultaneously. From these results, it was found that astrocyte oscillation could be detected by MEA, and that astrocytes induced neural activity in advance. In a co-culture system of human iPS cell-derived astrocytes and neurons, picrotoxin, chlorpromazine and pilocarpine dose-dependently increased the frequency intensity below 1 Hz, which reflects astrocyte oscillation. The response of cultured astrocytes was detected for the first time using MEA. In addition, the response of astrocytes to convulsants was detected in a dose-dependent manner. The present study demonstrated the efficacy of a method for predicting seizure liability of drugs based on astrocyte responses.

In recent years, the number of patients with nonalcoholic steatohepatitis (NASH) has been increasing. A new research tool to reproduce the NASH pathology has been demanded. We therefore generated organoids derived from liver tissue of NASH model mice. To generate liver organoids, we used different stages of NASH model mice (NASH A, NASH B, NASH C). As a result, we demonstrated that liver organoids derived from NASH model mice can reproduce the fibrotic pathology of NASH disease. It was also suggested that the gene whose expression increased could become a new biomarker of NASH.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide, although its pathogenesis remains to be elucidated. Recently, we revealed that hepatic Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol re-absorber from bile to the liver expressed on the bile canalicular membrane, is an NAFLD exacerbation factor. Indeed, transgenic mice with hepatic expression of human NPC1L1 under a liver-specific promoter (L1-Tg mice) developed steatosis with a high-fat diet (HFD) containing cholesterol within a few weeks, and this steatosis was prevented and recovered by ezetimibe, an NPC1L1-selective inhibitor used as a lipid-lowering drug.¹ Unlike the livers of wild-type mice that have little expression of hepatic Npc1l1, the livers of L1-Tg mice displayed time-dependent changes that indicated steatosis formation. In an early stage of the steatosis characterized by mild accumulation of hepatic cholesterol and triglycerides (TG), between WT and L1-Tg mice fed a HFD for 2 weeks, there were no significant differences in the hepatic expression of Pparα, Acox1, Cd36, Srebf1, and Srebf2; however, the hepatic ability to secrete VLDL-TG decreased in L1-Tg mice.² These results suggest that hepatic NPC1L1 exacerbates diet-induced steatosis, which was accompanied by decreased hepatic ability of VLDL-TG secretion. Our findings provide a deeper understanding of L1-Tg mice as a promising NAFLD animal model that is able to re-absorb biliary-secreted cholesterol similar to humans.

1) Toyoda et al, FASEB Bioadv. 2019 1(5):283-295.

2) Toyoda et al, Lipids Health Dis. 2019 18:234.

NASH causes liver cirrhosis and cancer. In order to elucidate its underlying mechanism, we have developed a NASH model of mice fed a CDAA-HF-T (-) diet. In this study, 6-week-old male C57BL/6J mice were fed the diet for 14 months. As a result, multiple giant nodules were observed in the livers of all mice at the end of month 12, and hepatocellular carcinomas and hemangiosarcomas were induced at the end of month 14. In conclusion, this model is useful to reveal details of NASH-associated hepatocarcinogenesis in mice.

The purpose of this study was to identify chemical and structural features associated with drug-induced liver injury (DILI) using in silico methods. The model drugs’ data were obtained from the US FDA’s Liver Toxicity Knowledge Base and literature. The 2D chemical descriptors were calculated with Dragon 7 software. In the decision tree analysis of 432 DILI and 220 no-DILI drugs, 64% and 71% of DILI and no-DILI drugs, respectively, were correctly classified by AMW. By individual analyses of each ATC class 1 group, highly accurate discriminant models were obtained for classes A, C, M and S.

[Background and Purpose] Diclofenac, a major NSAID, has hepatotoxicity and rarely induces Reye’s syndrome, which is characterized by increase in serum ALT and small lipid droplets in liver. In 1990’s, mitochondrial permeability transition (MPT), a type of mitochondrial injury, was proposed as an inducible factor in Reye’s syndrome. However, there is no report that MPT is involved in drug-induced Reye’s syndrome. Therefore, we aimed to clarify the relationship between MPT and Reye’s syndrome and liver injury induced by diclofenac using cyclophilin D knockout mice, which is deficient in MPT. [Method] (1) Fasted female mice (C57B6/J) were treated with sublethal dose of diclofenac. After 6 h, liver injury and lipid accumulation were assessed. (2) Female mice hepatocytes were incubated with oleate and diclofenac. After 24 h, lipid accumulation was assessed. [Results and Discussion] (1) Diclofenac increased serum ALT level after 6 h. Although tissue injury was not observed, triglyceride content in liver was higher in diclofenac-treated group than control group. Moreover, this increase was suppressed in cyclophilin D knockout mice. (2) Hepatocytes incubated with oleate and diclofenac in non-toxic dose showed lipid accumulation and it was suppressed in cyclophilin D knockout mice. Altogether, it was indicated that diclofenac exacerbated lipid accumulation in MPT-dependent manner. [Conclusion] Our results suggest that MPT is involved in drug-induced lipid accumulation in liver, perhaps by impairing beta-oxidation.

[Purpose] Drug-induced liver injury is a major problem in drug development and clinical drug therapy. Halothane (HAL), an inhaled anesthetic, induces severe and idiosyncratic liver injury. It has been known that disordered hepatic calcium homeostasis is an early feature of HAL-induced liver injury in guinea pigs. Even though some underlying mechanisms in HAL-induced hepatotoxicity have been understood, there are no reports of the involvement of ryanodine receptors (RYR) in HAL-induced liver injury.

[Methods] Female BALB/cCrSlc mice were treated with 50 μg/kg of ryanodine (RYA, a RYR agonist, i.p.) 1 h before HAL (15 mmol/kg, i.p.) treatment. Dantrolene sodium (DAN, an inhibitor of RYR) was orally given at a dose of 80 mg/kg 2 h after HAL (20 mmol/kg, i.p) treatment. The mice were dissected to collect plasma and livers, and then the parameters were determined.

[Results and Discussion] RYA co-treated with HAL significantly elevated plasma ALT and AST levels, while resulted in severe hepatic inflammation, necrosis and neutrophil infiltration. In contrast, DAN treatment showed a dose-dependent manner to suppress the liver injury induced by HAL. The hepatic mRNA levels of RYR1/2/3 were increased at different time points in HAL-induced hepatotoxicity. Among the hepatic expression levels of inflammatory and apoptosis factors , RYA showed an enhanced effect and DAN showed a protective effect on HAL-induced liver injury.

[Conclusions] We found a new insight into the effect of RYR which might be as a novel factor involved in the analysis of the mechanism on HAL-induced liver injury.

[Backgrounds]

Bromocriptine has been used to treat type 2 diabetes in the US but this apparent mechanism is well unknown. We examined the hypoglycemic effect of bromocriptine, focusing on gluconeogenesis in hepatocytes.

[Methods]

The expression levels of G6Pase and PEPCK1 mRNA were measured in both the liver isolated from rats treated orally with bromocriptine for 2 weeks and the HepG2 cells treated with bromocriptine.

[Results and Discussion]

G6Pase and PEPCK1 mRNA levels were decreased in both the rat liver and HepG2 cells. Bromocriptine may directly inhibit gluconeogenesis in hepatocytes.

CAR and PXR are nuclear receptors highly expressed in the liver and activated by numerous chemicals. CAR activation by its activators such as phenobarbital induces hepatocyte proliferation and liver carcinogenesis in rodents. In contrast, it has been unknown whether PXR activation promotes liver carcinogenesis. In this study, to investigate the influence of PXR activation on liver carcinogenesis, we treated mice with the PXR activator PCN following the tumor initiator diethyl nitrosamine for 35 weeks. We have found that PXR activation didn't promote liver cancer in mice.

Cisplatin (CDDP) is an anticancer drug that causes severe kidney damage, especially in the S3 region of the proximal tubule. We showed that cytotoxicity to CDDP, carboplatin (CAR), and oxaliplatin (OXA) is the highest in the S3-derived cells. The S3-specific toxicity could not be explained by accumulation of Pt. Since the highest amount of Pt uptake was found 15 minutes after the exposure, the cytotoxicity by 15 min-exposure to CDDP, CAR, and OXA was examined. As a result, even the 15-min exposure to these drugs caused the highest cytotoxicity in S3 cells, suggesting the importance of shout-term cellular responses.

To investigate effects of high fat diet on kidney in obese type 2 diabetes model, 4-week-old male db/db and KK-Ay mice were fed CE-2 (BD: Basal Diet), Quick Fat (QF: High fat diet) or Western Diet (WD: QF+2% cholesterol added) ad libitum for 8 weeks. C57BL/6J mice were used as a control group. At 13 weeks of age, animals were dissected, collected the blood and organs and were performed various analysis.

In both diabetic strains, urinary protein content and fatty degeneration in the proximal tubular epithelium were prominent in the both fat diet groups as compared to BD and these changes were remarkable in KK-Ay mice. These enhancements were considered to relate to apparent insulin resistance in KK-Ay mice.

A selective BRAF protein kinase inhibitor, vemurafenib, exhibits anti-tumorigenic activity in patients with metastatic melanoma. However, adverse reactions such as an acne-like rash and dry skin appear in patients treated with vemurafenib. Such cutaneous disorders have been associated with the dysfunction of sebaceous glands and pilosebaceous units, allowing us to speculate that vemurafenib may influence sebum production in sebaceous glands. In this study, we demonstrated that vemurafenib enhanced the production of triacylglycerol (TG), a major component in sebum, in insulin-differentiated hamster sebocytes (HamSeb). However, vemurafenib suppressed 5α-dihydrotestosterone (5α-DHT)-induced TG production in HamSeb. Regarding the molecular mechanism(s) of the bidirectional regulation of TG production, both insulin and 5α-DHT activated the signal pathway of mTOR, which is a pivotal mediator for sebaceous lipogenesis, in HamSeb. In addition, an mTOR inhibitor, KU0063794, inhibited both insulin- and 5α-DHT-augmented TG production. Furthermore, vemurafenib-augmented TG production was decreased by KU0063794 in insulin-differentiated HamSeb, whereas there was no change in the vemurafenib-suppressed TG production in 5α-DHT-treated HamSeb. Therefore, these results suggest that vemurafenib bidirectionally regulates sebum production dependent on sebogenesis factor species, leading to adverse reactions such as an acne-like rash and dry skin. Furthermore, the side effects of vemurafenib may be associated with the activation of the mTOR pathway in sebaceous glands.