Fluoride nanoparticles (NPs) are expected to be applied to biomedical fields such as bioimaging as fluorescent materials. Interactions of NPs with biomolecules have been investigated, but the mechanisms underlying these interactions remain unclear. In particular, the mechanism of protein aggregation on the surface of NPs is important to control biological phenomena related to the development of neurodegenerative diseases such as Alzheimer's disease. In this study, we investigated the potential of interaction between fluoride NP and amyloid β fragment (Aβ_16–20) peptide, which is a major etiologic agent of Alzheimer's disease, by infrared spectroscopy. Fluoride NPs were synthesized by thermal decomposition and characterized using X-ray diffraction (XRD) and dynamic light scattering (DLS) measurements. The synthesized NPs were CeF₃ with the diameter of approximately 80 nm. The CeF₃ NPs promoted the formation of the β-sheet structure of Aβ_16–20. This phenomenon was attributed to the hydrophobic interactions between NPs and Aβ peptides in an aqueous environment, causing the Aβ peptides to approach each other on the NPs surface and form ordered hydrogen bonds. As a result of examining the effect of coexisting salt on the aggregation state of Aβ peptide, the formation of β-sheet structure of Aβ peptide is promoted in the presence of NH₄⁺ ions, whereas the formation of β sheet structure of Aβ peptide is suppressed in the presence of NO₃^– ions. Molecular dynamics analysis suggested that this phenomenon was induced by electrostatic interactions between lysine residues (amino groups) of Aβ peptides and ions. Our findings may contribute to further comparative studies on the effects of different NPs with different physicochemical properties on the molecular states (conformations and aggregations) of proteins.

With the rapid development of nanotechnology, titanium dioxide (TiO₂) nanoparticles with unique physical and chemical properties and function have been used in a wide range of fields. However, it has been recently reported that inhalation of TiO₂ particles may induce acute inflammation and fibrosis in the lungs, and the detailed mechanism has not been clarified. In this study, we focused on necroptosis, a type of cell death, to elucidate one aspect of the molecular mechanism by which TiO₂ particles induce acute lung inflammation. First, we evaluated the expression of pMLKL, a marker of necroptosis activation, and the localization of TiO₂ particles in the lung tissue of mice intratracheally injected with TiO₂ by immunohistochemistry and dark-field microscopy. The results showed that a large number of pMLKL-positive macrophages that phagocytosed TiO₂ particles were observed after 24 hours of exposure. In contrast, after 72 hours of exposure, the number of pMLKL-positive macrophages and TiO₂ particles decreased. Next, the number of inflammatory cells and chemokine (CCL2 and CCL3) concentrations were measured in bronchoalveolar lavage fluid collected after intratracheal administration of TiO₂ to mice. After 24 hours of exposure, both inflammatory cells and chemokine concentrations were significantly higher than those in the control group, and intraperitoneal administration of a necroptosis inhibitor suppressed these increases. In contrast, after 72 hours of exposure, inflammatory cells and chemokine concentrations decreased compared to those after 24 hours, and the effect of the necroptosis inhibitor was also attenuated. These results suggest that necroptosis of alveolar macrophages phagocytosing TiO₂ particles regulates the acute lung inflammatory response.

Purpose: Our group has been evaluating the function and biocompatibility of nanomaterials for medical applications in the field of orthopedics. We earlier presented on the safety of carbon nanohorns (CNHs) and nano-ferrite particles (NFPs) on osteoblasts. Although the cytotoxicity of CNHs was higher, the osteoblast status differed between the nanomaterials. This study evaluated the cytotoxicity and uptake status of NFPs on osteoblasts under the same conditions as the prior CNH exposure. Methods: The MC3T3-E1 mouse preosteoblastic cell line (MC cells) with (Cal) and without (Non) calcification stimulation was used for NFP cytotoxicity assessment, morphological evaluation of NFP intracellular uptake, and relative quantitative comparisons by flow cytometry (FCM). Results: The cytotoxicity of NFPs was comparable in the Cal and Non groups at 24 h post-exposure, but at 48 h, the Cal group exhibited significantly more cytotoxicity. NFP uptake was evident on optical microscopy in the Cal group only. FCM comparisons showed a slight increase in cellular uptake in the Non group and a significant increase in the Cal group versus each control. Discussion: The cellular response of MC cells to NFPs was considerably different to CNHs. It appears that the intracellular uptake mechanism of CNHs in the non-calcification state of MC cells does not function for NFPs, whereas calcification stimulation induces MC cells to uptake NFPs. These results suggest that the safety of nanomaterials on osteoblasts should be evaluated before and after functional changes caused by calcification stimulation.

Carbon nanotubes (CNTs) are attracting attention as a next-generation nanomaterial with potential for a wide range of engineering applications. However, it is concerned that its high aspect ratio needle shape and high biodurability may cause asbestos-like toxicity. The aim of this study was to investigate the role of Nrf2 in the effects of multi-walled carbon nanotubes (MWCNTs) on the lung through exposure by pharyngeal aspiration. Nrf2 null or C57BL/6JJcl wild-type male mice were exposed to short or long Nanocyl MWCNT dispersed in Dispersion medium (DM) at 0, 10 and 20 μg per mouse by pharyngeal aspiration. Seven days later, the bronchoalveolar lavage fluid (BALF) of the mice was collected. The total number of cells or macrophages in the BALF was significantly different only between the control group and the long MWCNT 20 μg group in wild-type mice. Histopathological study revealed that exposure to long MWCNT increased granuloma with retention of MWCNT in the lung of wild-type mice, The study suggests that inflammatory responses are induced by exposure to long MWCNT in wild-type mice, but are attenuated by Nrf2 deletion. Length of MWCNTs was also implicated as a factor for induction of inflammation.

Introduction: Recent advances in nanotechnology have led to the development and use of nanomaterials. Despite concerns about human exposure to nanomaterials in various environments, their safety evaluation remains elusive. Amorphous silica nanoparticles (SiO₂NPs) are a particularly widely used nanomaterial, but our laboratory has previously reported that bare SiO₂NPs with hydroxyl groups (OH-SiO₂NPs) may be more injurious to macrophages in vitro and in vivo than SiO₂NPs modified with amino or carboxyl groups. This study aimed to investigate the mechanism of cytotoxicity by OH-SiO₂NPs in vitro.

Methods: We exposed the mouse macrophage cell line RAW264.7 to OH-SiO₂NPs and treated it with inhibitors of various programmed cell death pathways to examine the relationship between cytotoxicity and each cell death pathway. In addition, mRNA expression levels of inflammatory chemokines and cytokines were measured by RT-qPCR.

Results and Discussion: Co-treatment with apoptosis and necroptosis inhibitors improved the cell viability, although sole treatment with apoptosis or necroptosis inhibitor did not. The results with inhibitors of various cell death pathways suggest that apoptosis or necroptosis is partially involved in the mechanism of cell death. Additionally, exposure to SiO₂NPs was found to induce the production of pro-inflammatory cytokines and chemokines by macrophages, however, the relationship between this inflammatory response and cytotoxicity should be further investigated.

Recent epidemiological studies have revealed that fine particulate matter (PM2.5), one of the main causes of air pollution, affect the respiratory and cardiovascular system. It reported that an increase in the concentration of PM2.5 in the atmosphere increases the number of days for hospitalization and the mortality rate after ischemic stroke, suggesting that PM2.5 worsens the prognosis of ischemic stroke. Because PM2.5 contains many substances, it is not clear which component of PM2.5 is involved in worsening the prognosis. In this study, we focused on endotoxin contained in PM2.5 to analyze its effect on the prognosis of cerebral ischemia.

First, we used C3H/HeN (normal TLR4 expression) and C3H/HeJ (mutant TLR4 expression) mice. After intranasal exposure to PM2.5 collected in Yokohama for 7 days, cerebral ischemia was induced by the photothrombotic method, and then motor function after was measured by the rotarod test. Regardless of PM2.5, there was a difference in motor dysfunction between strains. This indicates that TLR4 aggravates the prognosis of cerebral ischemia, and this strain is not suitable for experiments. Therefore, we next used polymyxin B (PMB), which is known to inhibit TLR4 binding of endotoxin. When we treated PM2.5 to macrophages derived from U937, Reduced negative impact. Therefore, PMB is considered to neutralize the action of endotoxin contained in PM2.5. In ICR mice, motor dysfunction after ischemia was exacerbated by PM2.5 exposure, and PMB pretreatment suppressed this effect. The cerebral infarct volume on 7 day after cerebral ischemia did not change in any group.

These results suggest that endotoxin contained in PM2.5 worsens the prognosis of ischemic stroke through potentiation of inflammation. Further study is needed to examine the mechanistic insight by which endotoxin in PM2.5 exacerbates ischemia prognosis.

There have been many reports on central nervous system diseases and compound side effects caused by abnormal neurotransmitter release. The release of glutamate and GABA regulates the balance between excitation (E) and inhibition (I), and it is known that a disturbance in the E/I balance can cause diseases such as epilepsy and schizophrenia. If neurotransmitter release and extracellular potential (spikes) can be measured simultaneously, it is possible to clarify the relationship between changes in electrical activity and neurotransmitter release, making it an effective means for evaluating compound side effects and elucidating disease mechanisms. In this study, we aimed to develop a microelectrode array (MEA) capable of simultaneously measuring extracellular potential and the release of glutamate and hydrogen peroxide(H₂O₂), which are important factors in seizure phenomena. As a result of developing an enzyme-modified carbon nanotube (CNT)-MEA, the detection limit for glutamate was found to be at the level of 1 nM, and a dose-dependent response was detected at the level of 1 μM. H₂O₂ was successfully detected at the nM level. Next, using the enzyme-modified CNT-MEA, we successfully measured mouse acute brain slices and were able to simultaneously measure glutamate release and extracellular potential, as well as H₂O₂ and extracellular potential, and detect changes in neurotransmitter release and extracellular potential due to drug administration. The simultaneous measurement of neurotransmitter release and extracellular potential using the CNT-MEA developed in this study is expected to be applied as a new toxicity evaluation method that enables risk assessment and elucidation of mechanisms of action of compounds.

Drug-induced liver injury (DILI) is a major cause of the discontinuation of drug development, and a reliable method for the evaluation of DILI risk is highly needed. In this study, we have sought to develop a novel DILI evaluation method based on a read-across approach, in which the toxicity of an untested substance is predicted from the toxicity of its similar substances. From 287 drugs (213 DILI-positive and 74 DILI-negative) selected from the FDA's DILIrank data set, 10% were randomly selected as test drugs, and the rest were used as a reference set for similar drug selection. For the 287 drugs, HepG2 cell-based cytotoxicity assays and high-content analyses were performed, and inhibitory effects on human drug-metabolizing enzymes were determined. Using molecular descriptors calculated with alvaDesc, Euclidean distances between the drugs were calculated and neighbor (similar) drugs for each test drug were selected from the reference set. A test substance was judged as positive if the ratio of positive drugs in the neighbor drugs was higher than the positive rate of the entire data set. The sensitivity, specificity, and consistency rate were 0.714, 0.750, and 0.724, respectively. We further screened the neighbors for the 6 false- positive and 2 false-negative drugs by creating a decision tree using the in vitro assay data, and found that 6 substances were correctly predicted. These results suggest that read-across is useful for DILI prediction and that its accuracy can be improved by using in vitro assay results.

To elucidate the mechanism of renal injury induced by the cisplatin (CDDP), we used immortalized cells (S1, S2, S3 cells) derived from mouse proximal tubule S1, S2, and S3 regions. We found that S3 cells are sensitive to CDDP, and one of the reasons for this is that S3 cells markedly increase the amounts of reactive oxygen species (ROS) by exposure to CDDP. In this study, we aimed to elucidate the mechanisms involved in the vulnerability of S3 cells to CDDP by comparing the cellular responses after the addition of CDDP and paraquat (PQ), a ROS-generating agent. Comparing intracellular free Fe²⁺ levels, S3 cells showed higher values than other cells and CDDP significantly increased the amounts of Fe²⁺ in S3 cells. Intracellular ROS are known to oxidize unsaturated fatty acids contained in cell membrane phospholipids. When we examined the amount of lipid peroxide after exposure to CDDP and PQ, we found that the amounts of lipid peroxide was remarkably high in S3 cells. We next compared the expression levels of glutathione peroxidase 4 (GPX4), which can reduce lipid peroxide. The GPX4 expression levels by exposure to PQ increased in all cells, but decreased only in S3 cells by exposure to CDDP. Ferroptosis, a cell death associated with iron-dependent lipid oxidation, was investigated to determine whether the vulnerability of S3 cells to CDDP. The results showed that ferrostatin-1, an inhibitor of ferroptosis, inhibited CDDP-induced cell death in S3 cells. Taken together, ferroptosis was suggested to be partly responsible for the vulnerability of S3 cells to CDDP.

[Background] Excessive phosphorus intake causes tubular damage with calcification and fibrosis in the tubular interstitium. The changes are known to correlate with decreased renal function, however, the mechanism by which fibrosis occurs is still unknown. In this study, we attempted a histopathological analysis of kidney fibrosis induced by feeding the short-term high-phosphate diet. [Material and Methods] 5-week-old male SD rats were treated with UNx and fed 0.3% or 1.5% KH2PO4-containg diet for 21 days. In the control group, SD rats were sham-operated and fed 0.3% KH2PO4 under the same conditions. Urine was sampled before the day of sacrifice and blood and kidney were sampled at necropsy. [Results] Blood and urine biochemical examinations showed decreased Ccr, increased U-ALB and urinary L-FABP in the UNx+1.5% group. Histopathological findings were observed as follows in the same group: moderate to severe regenerated tubules and inflammatory cell infiltration, slight to severe tubular dilatation and interstitial fibrosis, very slight to moderate calcification. IHC staining revealed CD44 expression in the renal tubular epithelium and increased α-SMA-positive cells in the interstitial area. [Discussion] Severe renal tubular damage with calcification and interstitial fibrosis was induced by UNx and fed 1.5% KH2PO4 in SD rats. Furthermore, CD44 was inferred to be expressed in the regenerating tubular epithelium, indicating that its expression is associated with progression of fibrosis. These results suggested that a high-phosphorus diet may promote the worsening of CKD.

Predicting GI toxicity such as diarrhea and constipation is important to develop oral drugs having higher safety and effectiveness. However, drug-induced GI toxicity is not easy to predict because its mechanism is complicated by multiple physiological factors (e.g., fluid/electrolyte balance, GI hormone), and no effective in vitro prediction system has yet been developed. In the present study, we propose an in vitro system for prediction of drug-induced GI toxicity with organoid technology that can reflect physiological functions by mechanistically analyzing the effect of drug exposure on serotonin (5-HT) disposition and fluid regulation in the GI tract, using metformin as a model drug which clinically causes diarrhea. Studies using Caco-2 cells and Xenopus oocytes indicated that metformin inhibits SERT-mediated 5-HT uptake. Administration of metformin into the rat intestinal lumen significantly increased the GI fluid volume and simultaneously elevated 5-HT and Cl^- levels in the GI tract. Taken together, these results suggested that metformin increases the luminal 5-HT levels by inhibiting SERT-mediated intestinal uptake, promoting fluid secretion due to increased luminal Cl^- levels. Consistent with these observations, in vitro swelling assay using rat colon-derived organoids showed that exposure to 5-HT and forskolin, a Cl^- channel activator, significantly increased water secretion. In conclusion, our proposed method for predicting drug-induced GI toxicity using colon-derived organoids can be a useful system that can reflect the mechanism of GI toxicity in vivo.

Inflammatory bowel disease is a serious problem caused by chronic inflammation of the intestinal tract, and the causes of this phenomenon are not fully understood. To explore the mechanisms of the disease, animal models with stable lesion with a short-term experimental period are needed. Although the DSS (Dextran Sulfate Sodium salt)-induced colitis model is widely used, the severity and incidence of the induced disease varies depending on conditions such as DSS concentration, duration, and rearing environment. In this study, we explored the inflammatory factors by examining appropriate concentrations and duration. In addition, inflammatory profile between histopathological findings and gene expression was also examined. Seven-week-old male C57BL/6j mice were given DSS (MP Biomedicals, LCC) at 1.25, 2.5% for 2 weeks. and 5 % for 1 week in drinking water. Some mice in 2.5 % DSS showed severe symptoms or died, while others had no colonic lesions. In the 5% DSS group, a stable effect on intestinal lesions and elevated inflammation-related parameters was observed from day 3, and apparent inflammatory findings were observed in the colon of all animals on 7 days. Gene expression analysis revealed a significant increase in TNF alpha and IL1beta after 3 days, followed by a decrease after 7 days. However, MIP-2 and MRP8 increased significantly from day 3 and continued to increase through day 7. These findings suggest that 5% DSS in drinking water causes stable lesions and enhances inflammation.

Acetaminophen (N-acetyl-p-aminophenol; APAP) is one of the most widely used analgesic and antipyretic drugs. In order to investigate the toxicity of APAP, we used zebrafish embryos or larvae as model animals to test the effect of APAP on zebrafish embryos or larvae. Zebrafish embryos were exposed to 0 (control), 0.01, 0.1, 1, and 5 mM APAP from 4 hour post fertilization (hpf) to detection. Morphological changes in zebrafish were observed every 24 hpf, and behavioral changes were detected from 18 to 120 hpf. Histological changes, transcriptional analysis, and expression changes of thyroid-related genes were performed at 72 hpf. Experimental data showed that APAP caused pericardial edema in zebrafish embryos or larvae (42.7%, P＜0.05), and pigmentation decreased (66.6%, P＜0.05). Behavioral analyzes showed that APAP (0.1-5 mM) exposure also resulted in a significant increase in coiling behavior in early-developing embryos (18-32 hpf). However, when tested at 120 hpf, APAP (0.1 and 1 mM) increased the swimming distance of zebrafish larvae, while 5 mM APAP decreased the swimming distance of zebrafish larvae (100%, P＜0.05). KEGG discovery of retinol metabolism, Fanconi anemia pathway, and tyrosine metabolism. Differentially expressed lncRNAs were identified to affect pigment development (fabp4b, cyp7a1, rpe65a) as well as pigment production in the thyroid (tyrp1b, fabp4b). RT-PCR assay also showed that APAP treatment led to a decrease in zebrafish thyroid peroxidase (tpo) and thyroid hormone receptor β (thrβ) mRNA expression (P ＜ 0.05). The deleterious effects of APAP on zebrafish embryos may be due to its disruptive effect on the functional regulation of the thyroid hormone (THs) system. It is suggested should use APAP with caution.

  Amphetamine-type stimulants are widely abused worldwide. In Japan, methamphetamine (METH) abuse is particularly high and continues to be the most abused drug. It is reported that maternal METH abuse led to lower birth weight and impaired motor growth. However, despite the fact that most METH abusers are male, the impact of paternal abuse on the next generation is unclear. Therefore, we used a mouse model to examine the effects of paternal METH abuse on the next (F1) and subsequent (F2) generations.

  Male ICR mice (6-week-old) were administered METH or saline for 21 days and then mated with naïve female mice to obtain F1 mice. Body weight, righting reflex, the cliff avoidance test, and the wire-hanging test were performed to evaluate the growth of pups. Six-week-old F1 mice were subjected to spontaneous locomotor activity (LA) and the elevated plus-maze test, and 7-week-old F1 mice to the passive avoidance test. The striatum and hippocampus samples were collected from 7-week-old F1 mice, and the gene expression levels were measured by qPCR. Nine-week-old male F1 mice were mated with females to obtain F2 mice. F2 mice were tested in the same manner as F1 mice.

  Both F1 and F2 mice of the METH group showed significant growth retardation compared to the respective control group. LA of both F1 and F2 mice was significantly lower in the METH group than in the control group. In the passive avoidance test, F1 mice of the METH group showed a decrease in memory performance, but F2 mice did not. In the brain of F1 and F2 mice, changes in Comt and Gabra3 expressions were observed. These results suggest that paternal METH abuse may affect certain gene expressions, resulting in developmental delay in the following generations.

Thyroid hormone (TH) is an important regulator of brain development and TH insufficiency causes neurodevelopmental deficits. Ammonium perchlorate (AP), used as an oxidizing agent in the manufacture of solid rocket fuel, explosives, etc., inhibits the uptake of iodine, which is essential for TH synthesis, and decreases TH release. Since perchlorate accumulates in breast milk, its environmental contamination may expose infants to high concentrations. The present study investigated the maternal exposure effect of AP on offspring hippocampal neurogenesis as a highly sensitive endpoint of developmental neurotoxicity. Maternal rats were administered AP in drinking water at doses of 0, 300, and 1,000 ppm from gestation day 6 to day 21 post-delivery at weaning, according to the OECD Test Guideline 426. At the end of exposure, both dams and offspring showed thyroid follicular cell hyperplasia at 300 ppm and 1000 ppm, and offspring showed decreased serum T3 and T4 levels at 1,000 ppm. In the neurogenic niche, AP decreased the numbers of type-1 neural stem cells and type-2a and type-2b neural progenitor cells by suppressing their proliferation, suppressed ARC-dependent synaptic plasticity, and decreased the number of PVALB⁺ interneurons at the end of exposure, similar to the previously reported changes for antithyroid drugs. In contrast, RELN⁺ interneurons were inconsistently decreased in number, suggesting an inhibition of stem cell quiescence or maintenance. At the adult age on postnatal day 77, all the changes were disappeared, suggesting a transient fashion of disruptive neurogenesis.

The chemical-induced maternal hypothyroidism has been concerned to adversely affect the growth and brain development of offsprings. In 2018, therefore, the assessment of thyroid-related endpoints was added to related OECD test guidelines (TG). However, it is not fully understood the correlation between developmental toxicity and changes in maternal thyroid-related endpoints in pregnant mothers with mild hypothyroidism observed as chemical-induced thyroid dysfunction. In the present study, we conducted the prenatal developmental toxicity test according to the OECD TG414 and investigated the effects of various degree of maternal hypothyroidism on the fetal development in mice. Pregnant ICR mice were treated with propylthiouracil (PTU) containing food at the concentrations of 0, 2, 10, 50, 250 ppm from gestation day (GD) 6 to GD18, and dams and fetuses were analyzed at GD18. A significant decrease in the serum T3 and T4 levels and a significant increase in the serum TSH level were observed in 50 and 250 ppm PTU-treated dams. Moreover, histological abnormalities in the thyroid gland were observed in dams treated with more than 10 ppm of PTU. These results suggest that histological abnormalities in the thyroid are more sensitive biomarker to detecting hypothyroidism than changes in serum thyroid-related hormones. In addition, fetus exposed to maternal hypothyroidism did not show any developmental toxicity in the current experimental condition. Our findings will be useful in the development of management strategies for the safe use of chemicals that may induce hypothyroidism.

Maternal hypothyroidism can adversely affect neuronal development in the child. Therefore, it is an urgent need to establish a risk assessment strategy for chemicals that may induce hypothyroidism. However, the relationship between brain development and mild maternal hypothyroidism is not fully understood. Here, we evaluated the effects of two different levels of perinatal hypothyroidism on brain development of offsprings by using the transgenic mice expressing the luciferase (Luc) under the control of neuronal differentiation marker promoter. To induce mild or severe hypothyroidism, pregnant mice were fed propylthiouracil (PTU) containing food at 10 or 250 ppm from gestation day 6 to lactational day (LD)13, and at 5 or 125 ppm from LD13 to LD21, respectively. In vivo imaging showed higher Luc activity in the brain of 10 ppm group than that in control from postnatal day (P)4 to P16. It was significant at P4 and P7. These results suggest that the mild maternal hypothyroidism may affect brain development of offspring. In the 250 ppm group, Luc activity in the brain was significantly higher at P10 and P13. These results suggest that the effects on brain development may be diverged by the severity of perinatal hypothyroidism. Our findings will provide useful information about management strategies of chemicals that may induce hypothyroidism.

Imidacloprid (IMI) is a widely used neonicotinoid insecticide; however, its neurotoxic potential in mammalian brain remains unknown. The present study investigated the developmental exposure effect of IMI on postnatal neurogenesis in the hippocampal dentate gyrus (DG) of rat offspring. Dams were exposed to IMI (83, 250, and 750 ppm in diet) from gestation day 6 until day 21 post-delivery on weaning, and offspring were maintained without IMI exposure until adult age on postnatal day 77. On weaning, 750 ppm IMI decreased the numbers of DCX⁺ cells, TUBB3⁺ cells, and PCNA⁺ proliferating cells in the neurogenic niche, FOS⁺ or p-ERK1/2⁺ granule cells, and RELN⁺ interneurons in the DG hilus, suggesting suppressed proliferation of late-stage neural progenitor cells and decreased synaptic plasticity of newborn granule cells through suppressing RELN signaling. In adult age, IMI decreased the number of GFAP⁺ type-1 neural stem cells from 250 ppm and downregulated Pcna at 750 ppm, suggesting a late effect of suppressed RELN signaling on stem cell quiescence or maintenance at weaning. Immunohistochemical and gene expression data in the DG suggested a shift from an induction of both neuroinflammation and oxidative stress on weaning to a state of increased sensitivity to oxidative stress in adult age. Moreover, 750 ppm IMI sustained to decrease hippocampal acetylcholinesterase level. The results suggest that developmental IMI exposure persistently affected hippocampal nicotinergic signaling and neurogenesis involving neuroinflammatory and oxidative stress responses at high doses in rats.

Clothianidin [CLO] is a neonicotinoid insecticide. Although CLO has been considered to have low toxicity to mammals, recent studies have claimed that CLO is toxic to mammal. The problem seems to lie in the fact that most studies have used only male animals, and few showed data comparing behavioral effects of CLO between males and females. It is known that sex differences have the possibility of leading to different toxic responses. The main objective of this study is to discuss the delayed behavioral effects of CLO exposure during post-natal period, adolescence or maturity and the differences between males and females. CLO which was dissolved in acetone solution at a dose of 80mg/kg was administered to male and female mouse. The pups were divided into four groups: 2wk-CLO, 6wk-CLO, 10wk-CLO, Vehicle. Subsequently, mouse behavioral battery test (BBT) was conducted with the open field test, light/dark transition test, and contextual/cued fear conditioning test, at 13~15 or 28~30 weeks of age. In first BBT at 13~15 weeks of age, no significant behavioral abnormalities were observed. However, in second BBT at 28~30 weeks of age, female specific behavioral abnormalities were observed. In 2wk-CLO group, the time spent in the center region in open field test and the freezing rate in cued fear conditioning test was significantly low. In 6wk-CLO group, the freezing rate in contextual fear conditioning test was significantly low. these results indicate that female-specific behavioral abnormalities have become apparent over time by CLO administration.

Ozone gas has been widely used as an infection control including COVID-19. Safety standard level (0.1 ppm) is set in most developed countries according to the results of preclinical and clinical studies in a healthy donor. Therefore, another safety standard level of ozone gas for the patient with respiratory disorders assumed to exist in a medical facility is required. The objective of this study is to investigate the detail adverse effects of 0.1 ppm ozone gas exposure in a mouse model of allergic asthma by focusing on both antigen-dependent and -independent immune responses including type 2 helper T cells (Th2) and type 2 innate lymphoid cells (ILC2). A mouse model of asthma was generated by repetitive intranasal sensitization and challenge of dermatophagoides farinae (Df) in female BALB/c mice. Ozone gas (0.1 ppm) was inhaled for continuous 5 days (6 hours/day) just before the end of the experiment. Ozone gas exposure significantly decreased the percutaneous oxygen saturation (SpO₂). Inflammatory responses (related Th2 cytokine) and gene expression (IL13, IL33) in lung tissue, more over serum IgE levels were enhanced. This data indicates the safety standard level ozone gas (0.1ppm) has toxic effects for asthma patients. In the next step, reversibility of adverse effects of ozone gas was evaluated in a group with 2 weeks recovery phase after the final exposure to ozone gas. There was no impact of ozone gas exposure on SpO₂, Th2 immune reactions (IL-4 and IL-5 production by T cells), and gene expression of lung tissue. Taken together, exposure to 0.1 ppm ozone gas significantly worsened the antigen-dependent and -independent asthma symptoms, however, the adverse effects of 0.1 ppm of ozone gas was temporary and 2 weeks recovery terms can alleviate the these symptoms to the normal.

We now trying to development vegetable-originated paper which inhibits carcinoma cell proliferation.

Microplastics (MPs), plastic particles less than 5 mm are common in the environment, and we cannot avoid being exposed to them. It is said that MPs in the environment are degraded by external factors such as ultraviolet rays and waves, resulting in alteration of their properties. However, there is less knowledge of how degraded MPs affect our health. In this study, we focused on cytotoxicity and tried to identify its mechanism to understand the toxicity of MPs.

Polyethylene MPs (PE) with a median diameter of 230 µm were irradiated with ultraviolet light at a wavelength of 172 nm under air to prepare degraded PE (d-PE) which imitate MPs existing in the environment. Next, RAW264.7 cells and THP-1 cells were treated with PE and d-PE to analyze the mechanisms of cytotoxicity using flow cytometry, western blotting, MTT assay and so on.

First, we investigated whether MPs induce cell death. The results showed that there was an activation of programmed cells death in d-PE treated cells, while there was almost no increment of cell death in PE treated cells. In contrast, activation of caspase-3 which is apoptosis marker was not observed, suggesting that d-PE induced cell death other than apoptosis. Alternatively, LC3-II/LC3-I ratio was increased in d-PE treated cells and co-treatment with autophagy inhibitor suppressed the cytotoxicity caused by d-PE. These data show that d-PE induce autophagy dependent cell death. We currently explore the degradation process of autophagy by focusing on the degradation substrates and lysosomal pH to reveal the mechanism of MPs’ cytotoxicity.

Glioblastoma is one of the most malignant brain tumors. No effective treatment against glioblastoma has been established. Recently, drug repositioning approaches have identified existing drugs that show antitumor effects against glioblastoma in vitro. In this study, we investigated the combinations of existing drugs with antitumor effects against glioblastoma that show additive and synergistic effects in combination with each other, with the aim of developing more effective therapies for glioblastoma. We examined the effects of arsenite, ascorbic acid (AA), retinol (Rol), retinoic acid (RA), and valproic acid (VPA) alone or in combination on the viability of human glioblastoma U251 cells. As a result, Rol, a vitamin A (VA) derivative, showed an additive effect when combined with VPA and a synergistic effect when combined with RA. In addition, AA [vitamin C (VC)] showed a synergistic antitumor effect when used in combination with arsenite. Both VA and VC have been reported to exhibit antitumor effects via induction of endoplasmic reticulum (ER) stress. The combination of Rol and RA or arsenite and AA increased the expression of ER stress markers (CHOP and GRP78), suggesting that these combinations may exert antitumor effects against glioblastoma via the induction of ER stress.

Photodynamic therapy (PDT) is a treatment that uses a laser to irradiate tumor-affinity photosensitizer that have accumulated in tumor tissue. This photochemical reaction generates reactive oxygen species (ROS), which causes cell death of tumor cells. We have previously demonstrated that PDT using a photosensitizer sodium talaporfin (TS-PDT) has an antitumor effect on glioblastoma (GBM), the most common and aggressive malignant brain tumor. Recently, it has been reported that reactive sulfur species (RSS), a biological substance with antioxidant properties, attenuates the therapeutic effects of chemotherapy using anticancer drugs, but there are no studies on the relationship between RSS and TS-PDT. In this study, we investigated the effect of TS-PDT on the expression levels of RSS-producing enzymes in human GBM T98G cells and found that the mRNA level of cystathionine-gamma-lyase (CSE) was significantly increased. In addition, knockdown of Nrf2, a transcription factor, significantly suppressed the induction of CSE expression by TS-PDT, suggesting that activation of Nrf2 may be involved in the induction of CSE expression by TS-PDT. Furthermore, propargylglycine, a CSE inhibitor, enhanced the sensitivity of T98G cells to TS-PDT. These results suggest that the induction of CSE via activation of Nrf2 plays a protective role in the antitumor effect of TS-PDT on GBM.

[Purpose] Methylation of adenosine at N⁶ position (m⁶A modification), which is one of the common post-transcriptional modifications in mammals, is relevant with the onset or progression of several diseases. The methylation is catalyzed by methyltransferase like (METTL) 3-METTL14 (METTL3/14) complex, the demethylation is catalyzed by fat mass and obesity-associated protein (FTO) or AlkB homolog 5 (ALKBH5), and m⁶A is recognized by reader proteins, resulting in the changes in mRNA stability and translational efficiency. The purpose of this study is to clarify the impact of m⁶A modification on the expression of antioxidant enzyme hemeoxygenase-1 (HO-1) in the lung, a tissue susceptible to oxidative stress caused by smoking or air pollutants, aiming to elucidate the significance of m⁶A modification in the biological defense response to oxidative stress.

[Results and Discussion] In human lung adenocarcinoma-derived A549 cells, HO-1 mRNA level and activity were significantly reduced by the knockdown of METTL3/14. RNA immunoprecipitation using anti-m⁶A antibody revealed that the coding region of HO-1 mRNA was highly, and its 3’-untranslated region was moderately subjected to m⁶A modification. Reporter assay revealed that m⁶A in the coding region of HO-1 mRNA contributes to the regulation of HO-1 expression. HO-1 mRNA stability was reduced by the knockdown of METTL3/14, suggesting that some reader proteins function to increase HO-1 expression by facilitating mRNA stability. In conclusion, we found that m⁶A modification upregulates HO-1 expression and suppresses oxidative stress. This study provided new insights into the involvement of epitranscriptomic regulation in oxidative stress-related diseases.

[Purpose]

Abemaciclib (ABM) induces lung injury through the epithelial-mesenchymal transition (EMT). Recently, we found that ABM induced both EMT and cell cycle arrest in human alveolar epithelial cell line, A549. In this study, we focused on the cell cycle-regulating factors, which are associated with a pharmacological effect of ABM, and examined the association of EMT with ABM-induced cell cycle arrest.

[Methods]

A549 cells were transfected with siRNA for cyclin D1, Rb protein (Rb), and CDK4/6, using Lipofectamine for 24 h, then treated with ABM for 24 h. The mRNA and protein expression levels of α-smooth muscle actin (α-SMA), an EMT marker, were evaluated by real-time PCR and western blot analysis, respectively. Cell cycle phase in the treated cells was analyzed by flow cytometry.

[Results and Discussion]

Knockdown of cyclin D1 induced cell cycle arrest and significant increases in mRNA/protein expression levels of α-SMA. In addition, knockdown of Rb partially suppressed ABM-induced cell cycle arrest, while had no effect on ABM-induced EMT. Thus, the mechanisms underlying ABM-induced cell cycle arrest and EMT would have different pathways. Under the studies using siRNA and p53-deficient cell lines, p53 would be specifically involved in ABM-induced EMT. These findings would provide a significant basic knowledge to specify the differences between ABM-induced pharmacological effects and side effects through the EMT.

The polypeptide antibiotics, including polymyxin B and colistin, are effective antibiotics against multidrug-resistant bacteria such as multidrug-resistant Pseudomonas aeruginosa (MDRP). However, the clinical use of colistin is limited due to its high toxicity and severe side effects. In particular, colistin frequently causes nephrotoxicity, yet the underlying mechanisms remain unclear. In this study, we found that both polymyxin B and colistin as polypeptide antibiotics frequently cause acute renal failure (ARF), stimulated the extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) pathways in kidney tubular cells, and thereby upregulated macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP-1), leading to infiltration of macrophages into kidney. Thereafter, the kidney-infiltrated macrophages were exposed to the polypeptide antibiotics, which initiated activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Interestingly, blockade of the NLRP3 activation clearly ameliorated the pathology of ARF induced by the polypeptide antibiotics. These results therefore suggest that a combination of the distinct cellular responses to the polypeptide antibiotics in kidney tubular cells and macrophages play a key role in the pathogenesis of colistin-induced ARF.

It has been reported that environmental chemicals such as benzopyrene (BP) are involved in the development of cancers. On the other hand, little is known about the effects of chemicals on malignant change of cancer, in which a primary tumor acquire a metastatic capacity or a dormant tumor re-activate a proliferative machinery. Recently, cellular senescence, an irreversible arrest of the cell cycle, has been attracted attention as it has been reported that senescence contribute to malignant change of cancer although it is known to be tumor suppressive. In this study, we attempted to analyze the effects of BP, which is known to be carcinogenic due to DNA damage, on cellular senescence and malignant change of cancer. MCF7 which is the estrogen receptor (ER)-positive breast cancer cell line was exposed to BP for 3 days to induce senescence. Cellular senescence was evaluated by senescence-related markers such as p21, SA-β-gal and cell phenotypes such as nuclear size. BP was removed from cell culture medium and MCF7 was continuously cultured to evaluate whether BP-induced cell senescence is reversible or not. Senescence was induced by BP treatment with upregulation of p21 and SA-β-gal. Furthermore, cells that had experienced senescence began to proliferate when cells were cultured in BP-free medium. And the decreased colony-forming ability tended to be recovered when BP was removed from culture medium. In addition, significant nuclear translocation of AhR and ER, which are BP-binding proteins, was followed by increased expression of senescence markers such as p21 and γH2AX. Since cellular senescence did not occur in the triple-negative breast cancer cell line 4T1 which lacks ER, we hypothesize that AhR and ER might contribute to BP-induced reversible cellular senescence and are currently working on the mechanism of this process.

Fibrinolysis is the process by which plasmin degrades fibrin and the activity appears to depend on the balance between the tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Although irradiation causes hemorrhagic tendency due to a decrease in platelets, it is likely that abnormality of fibrinolysis is involved in the hemorrhage. In this study, we investigated the effects of γ-irradiation on the fibrinolytic system of vascular endothelial cells and its mechanisms. The fibrinolytic activity in the conditioned medium of the human vascular endothelial cell line EA.hy926 increased after γ-irradiation. At that time, accumulation of both t-PA and PAI-1 proteins also increased in the conditioned medium by γ-irradiation with a higher expression of their mRNAs. On the other hand, the expression of t-PA was increased by ATP, and the γ-irradiation-induced expression of t-PA was suppressed by siRNA-mediated knockdown of either P2X4 or P2Y2. The γ-irradiation-increased expression of t-PA mRNA was suppressed by pretreatment with an Akt inhibitor, but not with a PI3K inhibitor. Although the phosphorylation of Akt protein was increased by γ-irradiation, the increase was suppressed by siRNA-mediated knockdown of either P2X4 or P2Y2. These results suggest that γ-irradiation activated fibrinolysis in the liquid phase of vascular endothelial cells by induction of t-PA synthesis, which is mediated by the P2X4/P2Y2-Akt signaling pathways activated by ATP released from damaged cells after exposure to γ-irradiation.

Compound-based models of liver injury are widely used in studies of hepatotoxicity, but long-term continuous administration is a major burden. Models in which compounds are mixed with drinking water are less costly in terms of labor. While the thioacetamide (TAA) drinking water model (drinking water/hepatotoxicity model) is widely used, knowledge of drinking water/hepatotoxicity models for other compounds is limited. Focusing on the changes over time of multiple liver injury models is expected to deepen our understanding of the mechanisms of hepatotoxicity. Therefore, the aim of this study was to establish a drinking/hepatotoxicity model with compounds other than TAA. Through a literature review, compounds that increase ALT, a marker of hepatotoxicity, were extracted. We focused on methylenedianiline (MDA), which shows a different toxicity profile from TAA in oral administration studies and is inexpensive and water soluble. TAA (300 mg/L) and MDA (750 mg/L) were administered to mice drinking water for one week. Peak ALT levels were observed on the second day for the TAA group and on the third day for the MDA group, and both groups showed marked individual differences in peak ALT levels. Flow cytometry revealed a correlation between the accumulation of neutrophils in the liver and ALT levels, and only in the TAA group was there an accumulation of eosinophils in the liver during recovery of ALT. In summary, a new drinking/hepatotoxicity model with MDA was established. RNA-seq analysis of the samples obtained in this study is currently underway.

Paxlovid, a combination of nirmatrelvir (NTV) and ritonavir (RTV), has been granted emergency use authorization by the US FDA for the treatment of COVID-19. The objective of this study was to compare the effects of Paxlovid and RTV on the activities of cytochrome P450 (CYP) isoforms and to predict drug-drug interaction potential using pooled human liver microsomes and LC-MS/MS system. In the direct inhibition assay, the activities of nine CYP isoforms including CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 were determined. NTV inhibited CYP2E1, 2C19, and 3A4 with an IC₅₀ value of 18.0, 61.5, and 77.8 µM, respectively. RTV exhibited inhibitory potential against CYP2B6, 2C8, 2C9, 2C19, 2D6, and 2E1, with IC₅₀ values ranging from 4.60 to 11.6 µM, and exhibited very strong inhibition against 3A4 with IC₅₀ values of 0.302 µM. In NTV/RTV, the IC₅₀ values for CYP2B6, 2C8, 2C9, 2C19, 2D6, and 2E1 ranged from 4.77 to 11.7 µM, and 3A4 was 0.230 µM, showing a similar tendency to that of RTV alone. In the time-dependent inhibition (TDI) assay, as ritonavir was rapidly depleted during the microsomal incubation, the IC₅₀ values were calculated based on the remaining amount of ritonavir after the 30 min preincubation. The IC₅₀ values of RTV and NTV/RTV in CYP3A4 using midazolam as a substrate were 0.014 and 0.034 µM, respectively, and IC₅₀ shifts were 3.52- and 1.58-fold, respectively, after preincubation. In conclusion, these results suggest that NTV interferes with the metabolism of RTV by acting as a substrate of CYP3A4, showing attenuation of TDI potential of RTV.

Background and Objective:

The need for safe and effective gapmer antisense oligonucleotide (ASO) targeting central nervous system (CNS) diseases remains unmet because of drug-induced neurotoxicity. Introduction of chemical modification in ASO such as 2'-O-methylation (2'-OMe) and 5'-methyl have been reported to mitigate hepatotoxicity of systemically administered ASO by modulating ASO–protein binding (Shen, W. et al., Nat Biotechnol. 2019, Vasquez, G. et al., Nucleic Acids Res. 2021). Based on this finding, this study aims to reduce late-onset neurotoxicity of intrathecally administered ASO by introducing chemical modification into ASO.

Method:

Chemical modifications such as 2'-OMe and 5'-cyclopropane (5'-CP) were introduced in one deoxynucleotide monomer in gap regions of three ASO sequences. For in vitro assay, Neuro-2a or BE(2)-M17 cells were transfected with ASO for 72 hours, followed by cytotoxicity assessment: cell count and lactate dehydrogenase release measurement. For in vivo assay, mice were intracerebroventricularly injected with ASO, followed by chronological measurement of change in body weight and motor function (open-field test).

Result:

Both 2'-OMe and 5'-CP at specific gap positions mitigate late-onset neurotoxicity of one ASO sequence without major reduction in efficacy (knock down activity). For another ASO sequence, on the other hand, 2'-OMe increased the late-onset neurotoxicity of ASO. Even for this sequence, 5'-CP at specific positions reduced the late-onset neurotoxicity.

Conclusion:

Our study demonstrates introduction of optimal chemical modification into ASO can mitigate late-onset neurotoxicity. By enhancing therapeutic index, this drug design will enable clinical application of ASO targeting CNS diseases.

[Purpose] G-quadruplex (G4), a higher-order structure of DNA or RNA, is formed in guanine-rich nucleic acid sequences. Such a G4-DNA in genome affects gene transcription via the modulation of chromatin structure and accessibility of transcriptional factors. We noticed that there are three sequences that can potentially form G4 structure on the CYP3A4 upstream region. The purpose of this study is to examine whether G4 modulates CYP3A4 transcription.

[Results and Discussion] The treatment with pyridostatin, a G4 stabilizer, increased CYP3A4 mRNA level in HepG2 cells and primary human hepatocytes. In addition, CYP3A4 induction by rifampicin, a ligand of preganane X receptor (PXR), was enhanced by pyridostatin. With a qPCR stop assay to assess the G4 formation, it was demonstrated that three guanine-rich sequences on the CYP3A4 upstream region can form G4. Formaldehyde-assisted isolation of regulatory elements assay and chromatin immunoprecipitation showed that the stabilization of G4 on the CYP3A4 upstream region relaxed chromatin structure to enhance PXR binding. By screening from FDA-approved drug library (765 drugs) to identify small molecules that stabilize G4 on CYP3A4 using qPCR stop assay, digoxin was identified. The treatment with digoxin (not a ligand of PXR) increased CYP3A4 expression. Collectively, we uncovered a novel regulation mechanism of drug-metabolizing enzymes by stabilizing G4. It should be noted that small molecules, which affect G4 formation on the CYP3A4 upstream region, potentially cause drug-drug interaction via the modulation of CYP3A4 expression.

Recent studies have reported that environmental factors are related to the onset of chronic diseases such as cancer and diabetes. The attractive concept of "exposome" was proposed as a total of lifetime exposure by C. Wild. However, the various measurement items make the exposome research extremely challenging. Additionally, most studies focused on lifetime exposure, but molecular mechanisms remain unclear. Therefore, in this study, we focused on DNA methylation that regulates gene expression epigenetically and analyzed the effects of the environmental electrophiles that easily bind to proteins as a model for the exposome. First, the effect of 45 electrophiles on DNMT3B enzyme activity, which is related to DNA methylation, was examined, and we identified 4 electrophiles that reduce its enzyme activity. From here, we focused on 1,2-naphthoquinone (1,2-NQ), an air pollutant. The binding sites of 1,2-NQ in DNMT3B were analyzed using the LC-MS/MS analysis, and specific lysine and histidine residues were identified. Next, we examined transcriptome changes for 1,2-NQ using SAGE. We revealed that the expression of chemokines that regulates inflammatory response was increased. Additionally, we showed that 1,2-NQ and the universal DNMT inhibitor, 5-aza-2’-deoxycytidine induced chemokines in a time-dependent manner. Moreover, we found that 1,2-NQ-induced chemokines promote lung cancer A549 cell growth.Our results indicate that 1,2-NQ covalently modifies DNMT3B and induces epigenetic disorders to alter gene expression such as chemokines that promote lung cancer proliferation.

Currently, tissue transcriptome data in toxicology are abundantly accumulated in public databases. Deconvolution is a method for estimating the immune cell proportions from transcriptome data and extracts immune responses from the database. On the other hand, the impact of modeling considering tissue specificity and species differences on the estimation performance of this method was not clear. In this study, we evaluated these considerations in deconvolution for application to large-scale toxicological databases. We obtained our own datasets with corresponding RNA-seq data and immune cell proportions in diverse liver injury models induced by small molecule compounds and evaluated the impact of considering tissue specificity. As a result, we found that modeling with liver-specific cell types showed higher estimation performance. The proposed model also outperformed the conventional model in analyzing time series data after acetaminophen administration obtained from a public database. Deconvolution is mainly applied to humans and mice, and there is no dataset or method suitable for rats, which are frequently used in toxicological studies. Therefore, we obtained our own RNA-Seq of rat immune cells and constructed a rat-specific deconvolution model. We showed the superiority of the rat-specific model, and application to a large toxicological database provided novel findings, such as similar immune cell behavior induced by cycloheximide and LPS. These results indicate that consideration of tissue specificity and species differences is important for the precise estimation of immune response by deconvolution. The application of the model with these considerations to a large-scale toxicological database is expected to obtain aggregated knowledge on immune responses and contribute to the understanding of the mechanisms of toxicity.

Several chemicals have been found to be toxic by inducing epigenetic alteration in cells. It is required to develop an effective assay to evaluate epigenetic toxicity of such substances quantitatively in human cells. In this study, we have developed the "epi-TK assay" based on the principle of the thymidine kinase (TK) gene mutation assay using human lymphoblastoid TK6 cells, which enables the quantitative evaluation of the DNA methylation status at the endogenous TK gene locus. Using CRISPR/dCas9-DNA methyltransferase (DNMT3A) fusion system, we established the mTK6 cell line, in which TK gene expression is epigenetically regulated by DNA methylation at the TK promoter region of TK6 cells. The RT-qPCR revealed a marked reduction in the expression of TK gene in mTK6 cells. In addition, bisulfite sequencing analysis revealed highly methylated CpG loci within 250 bp upstream of exon 1 of the TK gene. To assess the ability of epi-TK assay to evaluate the epi-genotoxicity, mTK6 cells were treated with DNA methyltransferase inhibitors 5-aza-2'-deoxycytidine (5-AZ), GSK-3484862, and RG108. Upon treatment with 5-AZ and GSK-3484862 for 24 hours at 37℃, the TK revertant frequency was dramatically increased (230-fold and 1,900-fold, respectively) compared to the solvent control. In contrast, no significant increase of TK revertant frequency was observed after 24 or 48 hours exposure to RG108. Notably, analysis of some substances, e.g., dimethyl sulfoxide, displayed the decreased TK revertant frequency. These results suggest that the epi-TK assay can be utilized to assess both DNA demethylation and methylation states on the TK reporter gene. Using the assay, the evaluation of epi-genotoxic chemicals and their mechanisms of action are currently in progress.

We have reported an approach for evaluating developmental toxicity through an FGF-SRF signal reporter assay utilizing human iPS cells (S. Kanno et al., iScience, 2022). Signal interactions are vital for the regulation of fetal development, and certain developmental toxicants are known to disrupt signaling pathways. Considering these facts, we hypothesized that developmental toxicity eventually relates to signal disruption, and thus established a signal reporter assay. In this assay, chemiluminescence associated with FGF signaling activity was continuously monitored, demonstrating that signal disruption occurred at different time points depending on developmental toxicants. By integrating signal disruption over time, it was possible to accurately distinguish known developmental toxicants. In this study, we further improved the approach for more comprehensive analysis of signal disruption. In our previous approach, chemiluminescence was manually measured at 2, 4, 6, 8, 10, and 24 hours of culture. Because continuous measurements over several days were difficult to perform, disruption between the time points might be missed and longer-term disruption tests were not possible. To address these issues, we conducted the assay using a real-time luminescence measurement system. The continuous monitoring of chemiluminescence reveals that there were two peaks in for 72 hours, unlike the single peak in 24 hours. Furthermore, we found that some developmental toxicants, such as valproic acid, enhanced signal disruption after more than 24 hours and these were readily detected by continuous and long-term luminescence monitoring. Our approach holds great potential as a screening tool for developmental toxicants.

Drug-induced cholestasis accounts for half of drug-induced liver injury (DILI). In terms of that, we have already established bile acid-dependent toxicity system (BAtox) using primary human hepatocytes (PHHs). PHHs are the gold standard for BAtox, but it has the following drawbacks: i)lot-to-lot differences, and ii) Same lot cannot be stably supplied. We focused on Hu-liver cells prepared from human liver chimeric mice to solve these problems. In the present study, we evaluated the usefulness of Hu-liver cells for BAtox compared to PHHs’s data. Biliary excretion index (BEI) was evaluated on day 5 Hu-liver cells derived from 3 lots of donor hepatocytes. BEI values were 52-87%, equivalent to or higher than PHHs. After that, Bile acids and 22 drugs were co-exposed on day 5 Hu-liver cells, and BAtox was evaluated after 24 hrs. We confirmed that one of the three lots had low susceptibility, which could be derived from the lot-to-lot difference of donor PHHs. In addition, using the BAtox’s data of Hu-liver cells, high DILI risk and low DILI risk drugs were separated with 63-83% sensitivity and 63-83% specificity, which showed similar prediction accuracy as PHHs. Compared to PHHs, some drugs (flutamide, leflunomide, ticlopidine) did not induce BAtox in Hu-liver cells. We thought that altering CYP activity between two cells could affect BAtox susceptibility. Present study suggested some differences between Hu-liver cells and PHHs, but Hu-liver cells can adapt to BAtox.

It is well known that activated microglia is involved in the pathology of central nervous system injury caused by toxic substances. In these circumstances, microglia are thought to change their shape, which may be an indicator of toxicity of toxic substances to the central nervous system. Therefore, in this study, we attempted to establish a method to quantitatively evaluate the morphology of microglia and examine neurotoxicity and neurodevelopmental toxicity. To efficiently evaluate the change of microglia shape in the whole brain in vivo, transgenic zebrafish larvae expressing fluorescent proteins in neurons and microglia were exposed to ethanol, which is well known for its neurotoxicity. Whole-brain live imaging was performed with multiphoton laser scanning microscope. The obtained images were processed and analyzed using Image J. Three-dimensional data of microglia present in specific regions of the zebrafish brain was obtained. For quantitative assessment of microglia shape, projections of each microglia were evaluated using Image J shape descriptors. As a result, it was found that circularity or solidity of microglia in the optic tectum of zebrafish larvae treated with 2% ethanol for 24 hours increased and approached 1.0 compared to the control group. Circularity approaches 1.0 when the shape is nearly circular, and solidity approaches 1.0 when the outer contour is nearly flat; therefore, it was thought that acute exposure to ethanol caused the increase in the proportion of ameboid microglia, which is near-circular and less-branched in the optic tectum. This result indicates that microglial morphological changes caused by neurotoxic substances could be evaluated quantitatively. We are currently testing various neurotoxic substances by similar experiments.

Background: To determine glutathione (reduced form: GSH, oxidized form: GSSG), which is known as an important biological antioxidant, the colorimetrical method utilizing DTNB is widely used. However, the method does not give us sufficient sensitivity, especially for the determination of GSSG, and the simultaneous detection of GSH and GSSG. Although LC-MS can analyze GSH and GSSG simultaneously with high-sensitivity, the access to LC-MS is limited due to an economical reason. We recently found that glutathione can be detected fluorometrically by using the König reaction, which is usually utilized for the detection of cyanide. In this study, we evaluated whether the technique can be applicable to the simultaneous determination of intracellular GSH and GSSG.

Methods: GSH and GSSG were separated by our HPLC system, and reacted with 0.03% chloramine T and pyridine-barbituric acid reagents to form fluorescent compounds on-line. The fluorescent compounds were detected at 583 nm (excitation) and 607 nm (emission). As an application of the technique to a biological sample, the change of GSH/GSSG ratios in rat pheochromocytoma cells, PC12, exposed to 250 or 500 μM paraquat for 24 h were evaluated.

Results & Discussion: Limits of quantification of the technique were 18.3 nM for GSH and 33.9 nM for GSSG. This indicated that our developing method was more sensitive than some LC-MS methods. Good linearity (R²>0.999) was obtained in ranges of 0.05-50 and 0.05-10 μM for GSH and GSSG, respectively. Precision and accuracy were 2.39-10.9% and 84.8-96.5%, respectively. The decrease in the GSH/GSSG ratio in PC12 cells was observed with the increase of paraquat concentration.

(Introduction)

The large effort to switch the in vivo evaluation assay of skin sensitization of chemicals to the alternative non-animal assays has been paid because EU banned animal tests for cosmetics. However, an alternative method of evaluating intensity has not been developed, although it is important for risk assessment. We have developed a machine learning (ML) based-model to predict EC3 values in Local Lymph Node Assay (LLNA EC3), which is used as an indicator of the intensity of skin sensitization, but the model had not been established for the practical use, such as explanatory, interpretability and conducting external validation. In this research, we developed ML model to predict LLNA EC3 values for the practical use.

(Method)

We used safety data of 143 chemicals, which were involved in the Defined Approach Guideline at OECD (No.497). We randomly divided them into training and external validation data with a 4:1 ratio and built a regression model with XGBoost. The objective variable was LLNA EC3 value, and the explanatory variables were in vitro test data (DPRA, KeratinoSens^TM, h-CLAT), relating to the adverse outcome pathway of skin sensitization, chemical properties, and molecular descriptors and alert information obtained from QSAR ToolBox.

(Result and Discussion)

Using data of 29 chemicals as external validation data, we calculated predicted LLNA EC3 values using our model and compared with values from experiments. The coefficient of determination R² value was 0.68. The results of three in vitro tests ranked high in variable importance in our model, suggesting a significant contribution to the predicted results. These results suggested that our model was useful for a practical use to predict the intensity of skin sensitization because our model had an explanatory and interpretability, and its performance was evaluated by external validation.

Development of alternative methods to rat carcinogenicity tests is highly demanded, but it remains very difficult. This study aimed to establish a method for predicting non-genotoxic carcinogenicity by read-across using molecular descriptors and in vitro assay data. From the results of 2-year rat carcinogenicity tests of agrochemicals published by the Food Safety Commission of Japan, we selected 80 carcinogens that cause benign or malignant tumors in liver, thyroid, testis, uterus, ovary, breast, nasal cavity, stomach, or bladder/urethra and 46 non-carcinogens that showed no carcinogenicity in these organs, and subjected these chemicals to various cytotoxicity tests in HepG2 cells and reporter assays for several nuclear receptors. Analyses using Fisher's exact test demonstrated significant associations between thyroid tumor and PXR activation, testicular tumor and AHR activation, stomach tumor and changes in GSH levels, and bladder/urethra tumor and AHR activation. Then, read-across prediction for these 4 types of tumors were performed using neighboring substances selected based on Euclidean distance between substances calculated using alvaDesc molecular descriptors, with or without stratification of the test substances by in vitro assay results. The results indicated that the stratification improved prediction accuracy (concordance rate: 0.571-0.921) compared to the accuracy based on descriptors alone (concordance rate: 0.421-0.556), suggesting that in vitro assay results are useful for read-across prediction of non-genotoxic carcinogenicity.

Repeated-dose toxicity (RDT) test is important for the safety assessment of chemical substances. Meanwhile, the development of non-animal RDT evaluation methods is highly demanded. In this study, we aimed to improve read-across accuracy for RDT using in vitro test data in addition to chemical structure information. Male rat 28-42-day RDT test data of 326 substances were obtained from Hazard Evaluation Support System Integrated Platform (NITE, Japan). By grouping similar endpoints (EPs), 6 hepatotoxicity-related and 2 hematology-related group EPs (gEPs) were defined, and if a substance was positive for any EP in a gEP, the substance was considered positive for the gEP. We used 2649 descriptors from alvaDesc (Alvascience), and the results (positive/negative) of rat cytochrome P450 inhibition tests and HepG2 cell-based cytotoxicity tests and high-content analyses. Thirty-two substances were selected as test substances, and their neighbor (i.e., similar) substances were selected from the rest of 294 substances based on Euclidean distance between substances. Sensitivity (0.5 - 1.0), specificity (0.1 - 0.7), and balanced accuracy (0.6 - 0.8) differed significantly among gEPs. Next, we selected an in vitro test parameter most associated with each gEP by Fisher's exact test, and only the neighbors whose results matched those of test substances were used for read-across. This selection improved prediction accuracy for some gEPs (hepatomegaly, dyslipidemia, and anemia). These results suggest that using gEP-associated in vitro test data can improve read-across accuracy for some gEPs.

We have reported that the cell proliferation, osteoblast-related gene expression status, alkaline phosphatase activity, and calcification response of preosteoblast MC3T3-E1 cells (MC) differed according to culture medium. This comprehensive study extracted candidate genes as in vitro indicators of osteogenic function under different culture conditions towards ultimately replacing in vivo experiments. MC cultured in αMEM without vitamin C (VC) or DMEM (non-treated media) were seeded onto culture micro plates. After 1 day, we replaced each medium with either non-treated medium or calcification induction medium containing VC and β-glycerophosphate. Detailed gene expression status under the 4 conditions at 3 days after induction was analyzed by microarray. Genes displaying a large change in induction or difference in expression were verified by qPCR. Microarray analysis revealed remarkable differences in the types of genes upregulated by the calcification induction between αMEM and DMEM. The upregulation of known osteoblast differentiation-related genes (Ibsp, Alpl) was observed only in αMEM. Associated with bone repair in vivo, Gpnmb expression was markedly higher in DMEM and became upregulated by calcification-inducing stimuli. qPCR validation assays confirmed the microarray results. Specific MC gene expression varied with culture medium. Thus, certain gene and medium conditions may accurately reflect the in vivo response to bone formation processes and serve as functional indicators. Future studies will aim to clarify such conditions towards an international standard method.

The Japanese Standard Allergens of 2015 (JSAs), the representative allergens in Japan with clinically diagnosed positive patients, have not been evaluated for in vitro sensitization tests. At the last JSOT meeting, we demonstrated that the 11 kinds of JSA can activate nuclear factor erythroid2-related factor 2 (Nrf2), an indicator of skin sensitization, in human epidermal keratinocytes HaCaT cells. In this study, we investigated the contribution of metabolic activation by cytochrome P450 (CYP) enzymes to the expression of sensitization of JSAs. Nine kinds of JSA extracts showing significant Nrf2 activation ability and benzo[a]pyrene (BaP), a carcinogenic compound with strong skin inflammation, were used as test chemicals. The reporter cells (HaCaT-Nrf2-Luc) were treated with the test chemicals in the presence/absence of SKF525A, a non-specific P450 inhibitor, and the effects on luciferase induction were assessed. In addition, the effect of pretreatment with siRNA targeting CYP1A1 or CYP1B1 on BaP-induced luciferase induction was examined. Since Nrf2 activation by JSA extracts and BaP was significantly reduced by the pre-treatment with SKF525A, P450 enzymes expressed in HaCaT cells have an important role in the expression of sensitization by these compounds. Furthermore, BaP-induced Nrf2 activation was decreased by CYP1A1 siRNA treatment and increased by CYP1B1 siRNA treatment, suggesting a contribution to metabolic activation and detoxification, respectively. We are currently perfoming similar studies using JSA extracts.

[Objective] To investigate the pathogenesis of islet fibrosis in SDT (Spontaneously Diabetic Torii) rats, an animal model of non-obese type 2 diabetes mellitus. We investigated the pathogenetic mechanism of islet fibrosis in female SDT rats.[Materials and Methods] Rats were fed a basal diet (CRF-1), and blood and organs were collected for various analyses on the day of necropsy at 8, 16, 24, 32, and 40 weeks of age.[Results] Body weight increased with age in weeks. In blood biochemistry tests, blood glucose levels were not elevated, however, blood insulin levels increased markedly with age. These results indicated that female SDT rats were a pathological model of severe insulin resistance. Histopathologically, islet irregularity, enlargement and hemorrhage were observed from 24 weeks of age. Islet fibrosis was also observed from 24 weeks of age and was frequently observed in all animals at 32 and 40 weeks of age. Furthermore, CD44-positive cells, which are thought to be involved in fibrosis, were observed at 24 and 32 weeks of age, with more positive cells at 32 weeks of age. However, CD44-positive cells decreased at 40 weeks of age.[Discussion] CD44-positive cells have been implicated in the pathogenesis of islet fibrosis in SDT rats, a pathological model of diabetes mellitus. Although CD44-positive cells decreased, islet fibrosis was more pronounced at that time. This phenomenon suggests that CD44-positive cells are involved in the early and progressive stages of islet fibrosis.

In toxicity studies, one of the core endpoints is observation of clinical symptoms including vital signs (VSs). Measurement of VSs has already been realized in safety pharmacology in drug development, but not in toxicity studies on chemical substances. We believe that VSs monitoring in easy-to-use approach can achieve both refinement and reduction in toxicity studies. In the past, we have developed a wearable device for rats (WDr) and achieved measurement of VSs (pulse rate, SpO2, respiratory rate, and body temperature) from a conscious and unrestrained rat over time at low cost (JSOT 2022). For further improvement of the accuracy of toxicity assessment, we attempted to reduce the weight of the WDr, build a program to cope with body motion noise, and evaluate the activity. By redesigning the sensor arrangement and circuitry from the conventional WDr, the weight was reduced by 47% while mounting an additional 6-axis sensor. In addition, we developed an analysis program that recognized abnormal changes in pulse waveforms due to body motion and artifacts and excluded them from the analysis. We orally administered chemicals to rats wearing the WDr and confirmed their pharmacological effects and the recovery process. The results showed that changes in VSs induced by these chemicals could be detected more accurately. Furthermore, we succeeded in obtaining the amount of activity of the animals from the acceleration data. In the future, we aim to achieve both further refinement of toxicity evaluation and reduction in the number of test animals and costs.

Cardiotoxicity is a major concern in drug discovery. Blockade of hERG by small-molecule compounds induces QT interval prolongation due to repolarization inhibition and may cause cardiotoxicity such as severe heart failure, torsade de pointes (TdP) and fatal arrhythmia. When applying for drug approval, verification of hERG inhibition is required for all drug candidate compounds. In response to this requirement, many research institutes are developing hERG inhibition prediction models using in silico assays. However, most existing models only classify the presence of hERG inhibition and don't indicate the strength of inhibition. In this study, a dataset of approximately 15,700 compounds from public databases of small molecule compounds was used to construct quantitative pIC₅₀ prediction models. This model used molecular descriptors generated from each compound as explanatory variables. AutoML was used to search for the best algorithm among a total of 19 machine learning algorithms. Consequently, the prediction accuracy of the ET model on the test data showed an R2 of approximately 0.69. The predicted pIC₅₀ was visualized and compared with the actual pIC₅₀. When threshold for pIC₅₀ was set to 5, the prediction accuracy of discrimination was about 0.85. From SHAP values, the ET model revealed that the n-octanol/water partition coefficient and number of N-basic groups were significant explanatory variables for pIC₅₀ prediction. The model is expected to be used as a screening tool to evaluate the effects of drug candidate compounds on hERG channels.

It is important to clarify the mechanism of carcinogenesis by chemical substances in order to explore methods for cancer prevention and treatment. However, the number of chemical substances is enormous, and searching for them in conventional long-term carcinogenicity tests is time-consuming and labor-intensive. Therefore, we focused on a 3D organoid culture method that can reproduce in vivo epithelial tissue structure and gene expression patterns on a culture dish. Our laboratory has established a method for culturing cancer stem cells or canine bladder cancer organoids in the urine of dogs with bladder cancer. Furthermore, we have succeeded in producing canine normal bladder organoids from bladder mucosa cells noninvasively collected from healthy dogs. In this study, we treated canine normal bladder organoids with bladder carcinogens (2-Acetylaminofluorene and N-Butyl-N-(4-hydroxybutyl)nitrosamine) for 6 days, respectively, and then examined cancer stem cell markers (CD 44) and a DNA damage marker (γ-H2AX), which has been focused on in the early detection of bladder cancer. In addition, we evaluated the tumorigenic potential of the chemical-treated organoids by transplanting them into immunocompromised mice, and verified the usefulness of canine normal bladder organoids as a novel carcinogenesis model. The results suggest that 2-AAF-treated canine normal bladder organoids may have elicited the initiation of carcinogenesis. When 2-AAF-treated bladder organoids were transplanted into immunodeficient mice, tumor formation was observed at 2 weeks after transplantation. However, tumor regression was observed at 4 weeks after transplantation, and the conditions of chemical exposure to organoids are being reexamined.

[Purpose] In the early stages of drug discovery, it is necessary to accurately estimate the safety of candidate compounds by hepatotoxicity assays. Human liver organoids are expected to be a novel hepatocyte model for pharmaceutical research. However, the most widely used extracellular matrix for organoid culture, Matrigel, has concerns about batch-to-batch variation and reproducibility since it is derived from animal. We previously developed HYDROX, a synthetic polymer-derived 3D nanofiber, and reported on its applicability to human iPS cell-derived hepatocyte culture. In this study, we applied HYDROX to human liver organoids culture and examined its applicability to hepatotoxicity assays.

[Methods] Human liver organoids were established by embedding primary human hepatocytes (PHH) in Matrigel. Human liver organoids extracted from Matrigel were seeded on HYDROX-coat plates. HYDROX-culture human liver organoids were observed by transmission electron microscopic. The gene expression levels and drug metabolic activities were examined. Finally, to evaluate the applicability to hepatotoxicity assays, the cell viability when treated with hepatotoxic drugs were measured.

[Results and Discussion] Intracellular organelles such as mitochondria were observed in HYDROX-culture group, which were not observed in Matrigel-culture group. The gene expression level of a hepatocyte marker CYP3A4 and the CYP3A4 metabolic activity in HYDROX-culture group were significantly higher than those in Matrigel-culture group, comparable to those of PHH. In addition, cell viability of HYDROX-culture group when treated with hepatotoxic drugs such as acetaminophen was similar to that of PHH. These results indicated that human liver organoids obtained high liver functions by HYDROX culture, and could be applied to hepatotoxicity assays.