PM2.5 refers to atmospheric particulate matter (PM) that have a diameter of less than 2.5 micrometer. Skin is an important target of PM2.5 as well as respiratory system considering that skin is large organ of the human body and always exposed to PM2.5 outdoors. Most of studies on PM2.5 exposure to skin were conducted by the method that PM2.5-diluted suspension were exposed to keratinocytes or other cells inside skin. However, skin is exposed to PM2.5 in the air, a system of air-liquid interface exposure can be fundamental to investigate the effects of PM2.5 on skin. The aim of this study is to develop a novel air-liquid interface exposure system using skin 3D culture model.

We used LabCyte EPI-MODEL model as human 3D skin model. The model was incubated in the chamber box under 37℃ and 5％ CO2 condition. PM2.5 suspended solution was atomized with an ultrasonic nebulizer for 1 hr continuously from the surface side of stratum corneum. PM concentration in the chamber was measured by a particle counter equipped with a diffusion dryer to be kept at the concentration of 10 and 100 μg/m3 in the chamber. Thiobarbituric acid reactive substances, an index of lipid peroxidation, was elevated by PM2.5 exposure. Protein levels of inflammatory mediators also were upregulated by PM2.5. Interestingly, these increases under air-liquid interface exposure were significantly potentiated compared with liquid exposure of PM2.5. Taken together, our air-liquid interface exposure system can reproduce actual situation of PM2.5 exposure and be valuable to evaluate the effects of PM2.5 on skin.

Inorganic arsenic is known to induce cancers in various organs. Our previous study showed that arsenite exposure induces premature senescence in human hepatic stellate cell line LX-2, followed by senescence-associated secretory phenotype (SASP) that could contribute to arsenic-induced carcinogenesis in the liver. However, it is unclear what mechanism by which arsenite induces premature senescence in hepatic stellate cells. In this study, we performed RNA-seq in the human hepatic stellate cell line LX-2 under the conditions in which premature senescence is induced by arsenite exposure and searched for target pathways that induce premature senescence.

We found 981 genes whose expression significantly increased more than 2-fold and 949 genes whose expression reduced to less than half compared to the control group. Enrichment analysis using Metascape was performed on these genes to determine what pathways are altered by arsenite exposure. Analysis using TRRUST revealed that NFKB1 and RELA were involved in the regulation of genes with increased expression. Among the changed genes, FTH1 and FTL, the heavy and light chains of ferritin involved in iron metabolism, were particularly highly expressed. These increased expressions were confirmed by using real-time PCR. We also measured intracellular Fe²⁺ after arsenite exposure using FerroOrrange and observed a significant increase in Fe²⁺ compared to the unexposed cells. These results provide new candidate target pathways for the mechanism by which arsenite exposure induces premature senescence in LX-2 cells.

Recently, air pollution due to volatile organic compounds has increased. It is suggested that these diffused chemical substance cause chemical substances allergies. As a result, many people have gotten the sick building syndrome due to aromatic hydrocarbons. The adsorption characterization of aromatic hydrocarbons onto activated carbon was investigated using the flow method. The removal efficiency of aromatic hydrocarbons in the gaseous phase was estimated based on the adsorption kinetic constants and the saturated amount adsorbed of aromatic hydrocarbons on the activated carbon. The saturated amount of benzene and toluene adsorbed was greater than that of xylene adsorbed because the molecular sizes of benzene and toluene are smaller than that of xylene. This result indicated that the aromatic hydrocarbons adsorbed to the pore in activated carbon, because the activated carbon has the hydrophobic surface and pore. Generally speaking, the adsorption rate in gaseous phase depends upon the surface polarity and porosity of adsorbent. The amount adsorbed of aromatic hydrocarbons onto the activated carbon with the elapsed time was measured. The adsorption kinetic constant increased in the order of xylene, toluene, and benzene. Those of the three xylene isomers were similar. These results indicated that the adsorption rate of benzene by the activated carbon was the fastest and the kinetic constant depended upon the different between the boiling point and the melting point and the molecular size of the aromatic hydrocarbons.

Diphenylarsinic acid (DPAA) was one of non-natural arsenic compounds found in the well water in Kamisu, Ibaraki, Japan in 2003, which induced neurological symptoms in people having used this well water. Our previous studies revealed that DPAA could induce the aberrant astrocyte activation including promotion of transient cell growth and following cell death, increases in phosphorylation of MAP kinases (ERK1/2, p38MAPK, and SAPK/JNK) and activation of downstream signaling pathways such as transcription factors (CREB, c-Jun, and c-Fos) and antioxidant proteins (Nrf2, heme oxygenase-1 (HO-1), and Hsp70) in cultured rat cerebellar astrocytes. Here, we investigated a possible role of calpain, a calcium-dependent cysteine protease, in DPAA-induced aberrant astrocyte activation. A cleavage product ofα-II spectrin, a major calpain substrate, appeared in astrocytes exposed to 10 µM DPAA for 96 h, suggesting calpain activation by DPAA. ALLN, a calpain inhibitor, suppressed DPAA-induced cleavage of α-II spectrin. ALLN inhibited 10 µM DPAA-induced cell growth and 50 µM DPAA-induced cell death. Furthermore, ALLN potentiated DPAA-induced increase in the protein expression of Hsp70, suppressed phosphorylation of ERK1/2 and SAPK/JNK, potentiated DPAA-induced c-Jun protein expression and phosphorylation, and suppressed DPAA-induced c-Fos phosphorylation with few protein expression change. These results suggested that DPAA induced calpain activation, which contributed both inhibitorily and facilitatively to DPAA-induced aberrant astrocyte activation depending on the activation phenotypes.

In Kabwe town in the Republic of Zambia, heavy metal pollution has been widespread due to lead and zinc mining activities since the early 20th, and it continues even after the official cessation of industrial operation in 1994. In addition to soil, high concentrations of lead have been detected in humans, domestic animals, and wild rodents (Yabe et al. 2015, Nakata et al. 2017, Nakayama et al. 2011).

We employed a species of wild lizards (Trachylepis wahlbergii) as a target organism for biomonitoring focusing on land use patterns. This species was considered to be suitable because they live in relatively small home ranges, they only prey on insects, and they are widely distributed from residential areas to grasslands.

An initial field survey was conducted in 2017 to collect the lizards and soil of their habitat. Liver and other organs were collected from 224 lizards. Metal analysis of lizard and soil samples was performed by ICP-MS after acid digestion with a microwave system. The highest concentration in the liver was 200μg/g, which is much higher than the previous result in the same area using other animal species. Also, biological concentrations were relatively high in places near the mine and areas without vegetation. A follow-up survey was conducted in 2021 to further investigate the species-specificity of accumulation pattern, chronological shift, and relationship of these points with changes in land use pattern. The analytical results of 112 lizards are presented together with environmental parameters and geographical analysis.

Introduction: Environmental pollution by microplastics (MPs) is expanding, and there are concerns about the health effects of MPs and the chemicals contained in MPs. This study focuses on intestinal inflammation (Leaky Gut syndrome; LGS) caused by consumption of high-fat diets, and investigates the effects of MP on obesity and diabetes to clarify the toxicity of MP on the metabolic system.

Methods: C57BL6/J mice fed a high-fat diet (HFD) with or without polystyrene MPs were examined for effects on the metabolic disorder and LGS. Fluorescent-labeled MPs standard were orally administered to the particle-exposed groups.

Results: There was no significant weight difference between the HFD and HFD+MPs groups, but glucose intolerance was significantly exacerbated in the HFD+MPs group, and liver enzymes and serum lipids were elevated. Significant worsening of fatty liver was observed. Fluorescence microscopy showed that labeled MPs accumulated in the intestinal mucosa of the small intestine in the HFD+MPs group, and the concentration of short-chain fatty acids in the feces was significantly decreased. In addition, flow cytometry was used to evaluate the dynamics of inflammatory cells in the lamina propria of the small intestinal mucosa, which showed a significant increase in type 1 innate lymphoid cells and a significant decrease in type 3 innate lymphoid cells in the HFD+MPs group, and inflammation-related genes were significantly increased in the HFD+MPs group, while Il22 expression was decreased.

Conclusion: MPs induce intestinal inflammation and exacerbates various metabolic disorders, including impaired glucose tolerance.

Long-term exposure to arsenic may lead to either acute or chronic toxicity, mainly through drinking-water and food. Anemia is one of the major symptoms of chronic arsenic toxicity. Erythropoietin (EPO) is an essential hormone for hematopoiesis and its suppression causes anemia. Previously we reported that 24-hour treatment with arsenate promotes EPO production in HepG2 cells, which are EPO-producing cells. On the other hand, the long-term effects of arsenate on EPO production are unknown. Therefore, in this study we analyzed EPO production in HepG2 cells exposed to a long-term and low concentrations of arsenate. HepG2 cells to which 10μM arsenate was added and passaged for 3 weeks were designated as adapted HepG2 cells. Cells were harvested 24-hours after the addition of the substance promoting EPO production and the level of EPO mRNA was measured using Real-Time RT-PCR. The amount of reactive oxygen species (ROS) was evaluated by the fluorescence microscopy using the ROS indicator. The level of EPO mRNA in adapted cells was significantly lower than that in non-adapted HepG2 cells. Tempol, a ROS scavenger, suppressed the increase in levels of ROS and EPO mRNA by 100μM arsenate in HepG2 cells. These results suggested that EPO production was suppressed by not increasing ROS production by arsenate addition in adapted cells and it was considered that adaptation to arsenic promoted ROS degradation. Adapted cells are expected to attenuate physiological responses with ROS signaling.

Sodium dichloroisocyanurate-96% (NaDCC) is commonly used to treat drinking water, wastewater, and industrial water. However, exposure to NaDCC by inhalation can cause toxic effects in humans such as respiratory diseases. In this study, we evaluated the potential toxicity of NaDCC following a 13-week subchronic inhalation exposure in Sprague-Dawley rats. The animals were exposed to NaDCC at concentrations of 0.4 (low), 2.0 (middle), and 10.0 (high) mg/m3. In addition, male and female rats from the high (10.0 mg/m3) group were set up as the recovery group for 2 weeks. In bronchoalveolar lavage fluid, a concentration-dependent increase in the total number of cells was observed, with a significant increase in neutrophils occurring in both sexes exposed to 10.0 mg/m3 compared to the negative control group. In the 10.0 mg/m3 NaDCC treated group, lung organ weight was significantly increased in the female animals. Furthermore, histopathological examination showed eosinophilic droplets in the olfactory/respiratory epithelium, mucous cell hyperplasia, atrophy/degeneration of the tracheal branches, and wall thickening of the alveolar ducts in the nasal cavity of both sexes from the 10.0 mg/m3 group. Adverse effects of NaDCC exposure were observed in both sexes during a 2-week recovery period. Based on our findings, the no adverse effect concentration observed level following NaDCC inhalation exposure for 13 weeks was considered to be 2.0 mg/m3. These results are expected to be effective in providing a scientific basis for inhalation toxicity data of NaDCC.

[Introduction]

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are widely used type of brominated flame retardant, which is employed as additives in the manufacturing of office and home electronic equipment. Although endocrine-disrupting effects of TBBPA and HBCD have been reported, information about neurotoxicity is limited. In this study, we investigated the effects of TBBPA and HBCD on necroptosis signaling in PC12 cells.

[Methods]

PC12 cells were treated with TBBPA and HBCD for 24 hours, and then cell viability was measured by CCK8 and LDH assay. The relative mRNA expression levels of RIPK1, RIPK3, and MLKL were determined by real-time RT-PCR. Protein levels of RIPK1, RIPK3, and MLKL were determined by Western blot analysis.

[Results and Discussion]

Cell viability was decreased after treatment with TBBPA and HBCD for 24 hours in a concentration-dependent manner. RIPK3 and MLKL mRNA and protein levels were significantly increased after treatment with TBBPA and HBCD, whereas changes in RIPK1 levels were minimal. Necrostatin-1, a necroptosis inhibitor, significantly inhibited the cytotoxicity induced by TBBPA and HBCD.

[Conclusions]

These results suggest that TBBPA and HBCD activate necroptosis signaling in PC12 cells.

Thyroid hormones are a critical role in cardiac function and brain development, and the act of thyroid hormones is mediated through the activation of nuclear thyroid hormone receptors (TRs). Although in vitro reporter gene assays are widely used to detect endocrine disruptors, a TR-responsive reporter gene assay has not been established except for a few human cell-based bioassays. In the previous study, we developed yeast-based TR responsive reporter gene assays. In this study, we investigated whether thyroid hormones and anti-thyroid hormone compounds were effectively detectable using four yeast-based reporter gene assays for TRα and TRβ in human and Xenopus tropicalis.

In 96-wells plates, yeasts were incubated with the various concentrations of test substances for 16 hours. The antagonistic activities of chemicals were examined by competition between the chemical and thyroid hormone simultaneously added at the following concentrations. To distinguish antagonist activity from the cytotoxicity, the CYC yeast strains established in W303a and corresponding mutants for each receptor that constitutively expressed the lacZ reporter were used for comparisons.

In both of TRα and TRβ assays in human and Xenopus tropicalis, β-galactosidase activity was induced by thyroid hormones in a dose-dependent manner. All anti-thyroid hormone compounds tested in this study inhibited thyroid hormone-induced in human TR assays, but some compounds had no antagonistic effects in the assays for Xenopus tropicalis. Further studies are needed to determine whether other compounds can be evaluated in our assays.

The present study aimed to establish in vivo and in silico assay systems to evaluate anti-androgenic potentials of environmental chemicals using zebrafish. We assessed transcript expression of sulfotransferase 2st3 (sult2st3) in embryos exposed to androgen 17α-methyltestosterone (TES) alone or in combination with anti-androgens, such as flutamide (FLU), p,p’-DDE (DDE), vinclozolin (VIN), linuron (LIN), and fenitrothion (FEN). Developmental toxicity of these anti-androgens was also monitored to see if the toxicity is associated with anti-androgenic potency. The expression of sult2st3 was significantly induced by TES at later stages of embryonic development. The TES-induced expression of sult2st3 was inhibited by FLU to the level of DMSO control (IC₅₀ = 5.7μM), suggesting the role of androgen receptor (AR) in the sult2st3 induction. Similarly, DDE, VIN and LIN repressed the TES-induced expression of sult2st3 in a concentration dependent manner (IC₅₀ = 0.35, 3.9, and 52μM, respectively). FEN suppressed sult2st3 expression almost completely at the highest concentration tested. FEN and LIN induced broader toxicities to zebrafish embryos, suggesting no clear relationship between developmental toxicities and anti-androgenic potency. In silico docking simulation also showed that all five chemicals were interacted with zebrafish AR, being Arg702 as a key amino acid for the ligand binding. Our findings suggest that the combination of in vivo and in silico assessments using zebrafish becomes a promising tool to predict anti-androgenic potentials of environmental chemicals.

Bisphenol A diglycidyl ether (BADGE) is an epoxy resin and synthesized by reacting bisphenol A (BPA) and epichlorohydrin. BADGE has been used for the inner coating of canned food and beverages. BADGE can easily migrate from the containers and become a contaminant. In the previous studies, we examined the effects of BADGE exposure to the dams on the behavioral, structural, and developmental abnormalities in the offspring. We reported that maternal BADGE exposure (1.5 mg/kg/day) during gestation and lactation periods could accelerate neuronal differentiation in the fetuses and induce anxiety-like behavior in juvenile mice. In addition, a direct BADGE (1-100 pM) exposure promoted neurite outgrowth and neuronal connection in the primary cultured cortical neurons. In this study, we examined involvement of estrogen receptor β (ERβ) in the abnormal brain development induced by BADGE exposure. The histological analysis demonstrated the increase in the ERβ-positive signals in the cortex of offspring from BADGE-exposed dams at postnatal day 1. In primary cultured cortical neurons from SD rat embryos at 15-day gestation, the elongation of neurites induced by BADGE (100 pM) treatment was significantly inhibited by ERβ antagonist ICI182,780 and GPER antagonist G15. These data suggest that BADGE exposure can accelerate neuronal differentiation via ERβ-mediated signal pathways.

Hydroxylated fullerene [C60(OH)n], a type of nanomaterial used in cosmetics and pharmaceuticals, can be introduced into the aquatic environment by human activity. C60(OH)n tends to bind with organic pollutants including perfluorooctane sulfonic acid (PFOS), which is of concern for its effect on aquatic organisms. Here, we investigated the combined effects of C60(OH)n and PFOS on larval zebrafish. Zebrafish eggs (about 30 eggs in each experiment, n=3) were cultured at 26℃ with 16 h light/8 h dark photoperiod in six different solutions; DMSO (15mg/L), C60(OH)n (10mg/L), PFOS (5 mg/L), PFOS (10 mg/L), PFOS (5 mg/L)+C60(OH)n (10 mg/L), and PFOS (10 mg/L)+C60(OH)n (10mg/L). After exposure for 7 days, the viabilities of larvae treated with solutions of PFOS+C60(OH)n were almost the same as that with PFOS alone. To the contrary, larvae treated with solutions of PFOS+C60(OH)n had higher malformation rates (length, spine curvature, and eye width) than those treated with PFOS alone. Metabolomic analysis detected an increase in metabolites related to purine metabolism in the larvae treated with C60(OH)n and PFOS together. Our results may suggest that the coexistence of C60(OH)n and PFOS has a greater effect on larval zebrafish than PFOS alone.

Indium tin oxide (ITO) is one of the most widely manufactured materials with broad applications, such as flat panel displays, touch panels, and solar panels. Whereas cases of indium-related lung disease, such as pulmonary emphysema and fibrosis, have been reported worldwide, the effects of ITO on the progression of epithelial-mesenchymal transition (EMT) are completely unknown. EMT is a crucial process whereby fully differentiated cells undergo morphological changes from the epithelial phenotype to the mesenchymal phenotype and is associated with pulmonary fibrosis. The purpose of this study was to understand the inductive mechanism of indium-related lung diseases, focusing on the fibrotic potential of ITO nanoparticles (NPs) through EMT induction. We found that a conditioned medium obtained from THP-1-derived macrophages stimulated with ITO NPs induced morphological changes, high motility, and EMT progression in A549 cells. Furthermore, we identified that interleukin-1β (IL-1β) is an ITO NP-mediated EMT inducer based on the results of cytokine array as well as cellular physiological and biochemical analysis. In addition, we found that IL-1β-exposed cells co-expressed epithelial and mesenchymal markers and abolished the proper localization of cell-cell junction-related proteins, which is similar to the partial EMT phenotype. Taken together, these findings suggest that IL-1β is released from macrophages stimulated with ITO NPs and is able to induce EMT progression in alveolar epithelial cells, thereby potentially triggering the genesis and development of pulmonary fibrosis.

Titanium (IV) oxide (TiO₂) is generally considered to be a safe compound, however, there has been long-lasting discussion about the safety of TiO₂ as a food additive because it may contain nanosized particles (particles with at least one dimension less than 100 nm). Since we have found that the toxicity of nanosized silver particles greatly varies depending on the particle size, we examined the toxicological effect of nanosized TiO₂ with a very small crystallite size of 6 nm for repeated oral administration. For dispersion, nanosized anatase TiO₂ with a crystallite size of 6 nm was suspended in 0.2% disodium hydrogen phosphate (D50=Ca. 200 nm in solution), and then gavaged for 28 days to 6-week old male and female F344/DuCrj rats at doses of 0, 10, 100, and 1000 mg/kg bw/day. During the treatment period, no mortality was observed, and no toxicologically significant changes were observed in body weight, general condition, hematology, organ weights, and histopathology. Serum biochemistry showed a significant increase in TG in the 1000 mg/kg bw/day females. In ICP-MS analysis, trace amounts of titanium were detected in the liver of all rats including controls. In histopathological examination, small particles that are appeared to be agglomerates of the TiO₂ nanoparticles were observed without foreign-body reaction in the mucosal area of the nasal cavity in rats given TiO₂. However, these particles were not noticed in the intestinal mucosa. Because the significant increase in TG in the 1000 mg/kg bw/day females was not associated with other toxicological change, the NOAEL in this study was considered to be 1000 mg/kg bw/day.

[Introduction]

To develop artificial autologous bone, novel ferromagnetic nano-ferrite particles (NFPs) are taken up by cells under control of a magnetic field to produce 3D cell-derived artificial bone and calcification. This study evaluated the safety and cellular response of NFPs engineered by our group in osteoblasts (OBs).

[Methods]

NFPs of approximately 20 nm were prepared with iron oxide and measured by scanning electron microscopy. Cell viability was evaluated by the Alamar Blue method after exposure to NFPs at 0.04-0.57 mg/mL for 24 h. NFP uptake was evaluated by flow cytometry (FCM), and NFP uptake images were visualized by transmission electron microscopy (TEM). OB calcification function was quantitatively assessed after Alizarin Red staining.

[Results]

TEM showed that NFPs accumulated in large amounts in OBs, which exhibited high cell viability. FCM revealed maximum NFP uptake at concentrations of 0.14 mg/mL or higher. Uptake peaked at 4 days of exposure. The calcification ability of OBs changed with NFP concentration and became affected above 0.57 mg/mL.

[Discussion]

Considerable NFP accumulation in OBs was seen, with limited effect on survival. The NFP uptake peak at 4 days of exposure suggested long-term safety. Since calcification was inhibited at higher NFP concentrations, the non-internalized extracellular NFPs might have disrupted the formation of calcified nodules.

[Prospects]

NFPs appear well-tolerated and safe in OBs. Future studies will evaluate 3D modeling and calcification of NFP-encapsulated OBs to develop tailor-made artificial autogenous bone.

Neurotoxicity is a major side effect of many chemotherapeutic treatment regimens with an estimated 60% of patients enduring neurotoxicity events. This neurotoxicity can form significant dose-limiting barrier in clinical therapies. Current cellular models primarily rely on 2D monocultures of animal or iPSC derived neuronal cells. Animal cell systems suffer from the species differences. Simple, iPSC-based monocultures are insufficient to model the full pathology behind induced neurotoxicity.

Human dermal fibroblasts and Schwann cells were used to populate a 96-well format polyHIPE scaffold. These cells secrete extracellular matrix proteins (ECM) proteins to create a humanised dermal equivalent structure and protein microenvironment. Neuronal cells are cultured in the dermal equivalent to produce an innervated dermis upon which an epidermis is formed. TUJ1 (neuron specific microtubule) and substance P (Neurotransmitter) staining demonstrates neuroactive neurites throughout the model. Neurostimulants Capsaicin (TRPV1 agonist), Menthol (TRPM8 agonist) and KCL induced increased Substance P release compared to controls and sub-threshold stimulation.

Histologically, our model demonstrates structural similarities to human tissue. TUJ1 staining highlights a robust neuronal population within the model with clear neurite extension. Functional assessment of the model shows high sensitivity, concentration specificity to a range of neurostimulatory compounds. As such this model demonstrates a novel high-throughput 96 well platform for the screening of neuro-irritants in vitro.

Cannabis is the most popular illicit drug among young people. While its effect is likely to disrupt normal brain functions in the developmental stage, the mechanism is still unclear. In this study, we have investigated hazard risks of early-life exposure to cannabinoids immunocytochemically using cultured rat hippocampal neurons. Dendritic spines are postsynaptic structures, and drebrin plays a key role in the dendritic spine formation of neurons and is responsible for the morphological plasticity of dendritic spines. In this study, the effects of CP55940, a synthetic cannabinoid, have been investigated on the distribution of drebrin in developing neurons. Hippocampal neurons prepared from frozen embryonic rat hippocampal neurons (SKY neuron, AlzMed, Inc., Tokyo) were incubated in 96-well microplates. After 7, 14 or 21days (DIV7, 14, 21), the cultured neurons were treated with CP55940 (from 100 nM to 10 µM). After the process of immunocytochemistry to visualize drebrin, MAP2 and cell nucleus; neuron number, dendrite length, and drebrin cluster number were automatically quantified using our algorithms. Neuronal death was observed when 10 µM CP55940 was applied from DIV7 or 14, but not from DIV21. The lower concentration of CP55940 significantly changed the ratio of high-intensity drebrin clusters as well as increased the number of drebrin clusters per dendrite length. Identification of drebrin clusters and analysis of their numbers and distribution are promising to detect the toxicity of cannabis.

Neurotoxicity of acrylamide (ACR) has been reported in human cases and experimental animals. Our previous study demonstrated that ACR induced dose-dependent degeneration of noradrenergic axons, which has been reported extensively related with various neurodegenerative diseases. Recently we reported activation of microglia in murine brain after exposure to environmental electrophiles, although the underlying mechanism remains largely unclear. To investigate the mechanism of ACR-induced degeneration of noradrenergic neurons, in this study noradrenergic neuron 1C11^NE model, as well as BV2 microglia cell line were used. Differentiated 1C11^NE neurons were exposed to ACR or to conditioned medium (CM) from ACR-treated BV2 microglial cells (ACR-CM), and various analyses were carried out. The results showed that treatment with ACR did not decrease cell viability of 1C11^NE , nor increase LDH release. Exposure to ACR-CM also did not affect the viability of 1C11^NE cells, while the LDH release was increased. Quantification of neurite length showed that direct treatment of 1C11^NE cells with ACR did not decrease the neurite length, while treatment with ACR-CM significantly decreased the neurite length. Transcriptomic RNA-seq analyses for neurons and microglia showed linkage between ACR-CM treatment and various neurodegenerative diseases. Moreover, RNA-seq results also revealed a predominant relationship between proteasome pathway and neurodegeneration induced by ACR. The above results suggest that microglia and proteasome pathway play critical roles in environmental electrophile ACR induced degeneration of noradrenergic neurons.

Objective: Circadian rhythm is endogenous 24-hr oscillations usually entrained to the daily environmental cycle of 12/12 h light/dark (LD). It has been reported that circadian rhythm disruption (CRD) induces sleep disorders and delirium. However, there are few reports about CRD caused by advanced cancer, although it is well known that patients with advanced cancer often develop delirium. This study investigates the CRD induced by advanced cancer using cancer-induced cachexia model mice.

Methods: 85As2 cells, derived from human stomach cancer, were implanted into 8 week-old male BALB/c nude mice (2x10⁵ cells/mouse). Home-cage activity was measured after 2 weeks from implantation under LD. Mice were then placed in constant darkness (DD) or light (LL), and finally returned to LD cycle. The day-night variation was analyzed using Actogram.

Results: The active phase of 85As2-implanted mice gradually shifted from dark to light between 2 to 4 weeks after implantation, and was almost reversed after 4 weeks. The free-running period under DD or LL condition was significantly shorter compared to the control. Even after returning to LD, the active phase remained reversed in 85As2-implanted mice.

Summary: 85As2-implanted mice demonstrated the reverse of day-night variation without synchronization of photoperiod, and shortened free-running period, suggesting that 85As2-implantation may directly affect the circadian rhythm formation. The neural activity and clock gene expression in the suprachiasmatic nucleus (the center of circadian clock), need to be investigated further.

Astrocyte and neural network activities are closely related. It has been reported that in evoking convulsions, astrocyte oscillation precedes neural activity. If astrocyte responses to seizurogenic compounds 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 MEA system, and whether responses to seizurogenic compounds could be detected based on astrocyte oscillations. To detect the astrocyte response, seizurogenic compounds were administered to human astrocytes cultured on MEA plate and the evoked response was measured. Oscillation by seizurogenic compounds was detected in the cultured astrocyte-only sample. In addition, the potential in the low frequency range was increased dose-dependently with pilocarpine. Simultaneous measurement of Ca2+ imaging and MEA showed that pilocarpine increased the frequency of extracellular potential synchronized with Ca2+ oscillation in astrocyte. In co-culture system of human iPS cell-derived astrocytes and neurons, pilocarpine also dose-dependently increased the potential below 1Hz, which reflects astrocyte oscillation. The response of cultured astrocytes was detected for the first time using MEA. In addition, the response of astrocytes to seizurogenic compounds was detected in a dose-dependent manner. Also, extracellular potential measurement by MEA might be able to more accurately assess the astrocyte activity. The present study demonstrated the efficacy of a method for predicting seizure liability of drugs based on astrocyte responses.

Background

Human induced pluripotent stem cell derived-neuronal cells (hiPSC-NCs) can be a powerful in vitro model for evaluating neuronal toxicity. We aimed to construct a stable architecture of hiPSC-NC networks using gelatin hydrogel fibrous nonwoven (GHFN) because nonwoven structure of GHFN is expected to confine the position of hiPSC-NCs in the scaffold and provide a stable neuronal network. Using this model, drug effects on spontaneous firing and conduction were examined by calcium imaging.

Methods

iCell DopaNeurons were seeded to laminin-coated GHFN. The hiPSC-NCs attached on cell culture plate without polycation coating. After 14 days in culture, calcium fluorescence images were captured with confocal imaging system CQ1 (Yokogawa Electric Corp.) and drug effects were analyzed.

Results

Many small clusters were formed and captured at contact point between the bottom of the GHFN and culture plate. After 2 days culture, neuronal network was formed by extending neurites from each spheroid and connected with each other. Over 7 days culture, these neurites were bundled and became thicker. Over 14 days of culture, the neuronal network was stable and showed synchronized firing. The firing pattern was changed from single peak to prolonged multiple peaks (burst-like peaks) by 4-aminopyridine. Synchronous firing was disturbed by tetrodotoxin without delay of conduction.

Conclusion

We succeeded in construction of spontaneous firing hiPSC-NC network using GHFN and analysis of network firing pattern between remote clusters.

[Background]

Cardiotoxicity in anti- cancer drugs treatment is a serious and complex issue. To predict these toxicities in vitro, multi-parameter toxicity tests by hiPSC-CM has been needed. The gelatin hydrogel fibrous nonwoven (GHFN) is a nice scaffold for the evaluation of various cardiac toxicity parameters, as hiPSC-CMs culture on GHFN show dynamic contractile motion and matured-like structure. We evaluated contractile impairment, change of nucleus and sarcomere structure, proarrhythmic risk and mitochondrial toxicity by anti-cancer drugs　treatment with hiPSC-CM cultured on GHFN.

[Methods]

iCell cardiomyocytes² were cultured on GHFN for 7-14 days, and anti-cancer drugs (doxorubicin, nilotinib, belinostat) were exposed for 72 hours. Time-lapse imaging was recorded by confocal imaging system (CQ1, Yokogawa Electric Corp.). Contractile response was analyzed from bright field images. Proarrhythmic effects were evaluated by calcium imaging. Mitochondrial toxicity was assessed by JC-10 fluorescence. Shape of nucleus and sarcomere structure were observed by Hoechst staining and a-actinin immunostaining.

[Results]

Doxorubicin (1μM) stopped the contraction and caused cell death after 72 hours. Nilotinib (10μM) decreased the contraction to 50 % after 72 hours. Arrhythmia, mitochondrial toxicity, and irregular sarcomere structure were observed after 72 hours. Belinostat (1μM) caused dysrhythmia after 72 hours.

[Conclusion]

By using GHFN culture system multiple aspects of cardiotoxicities by anti-cancer agents could be analyzed in hiPSC-CM.

Acceleration or inhibition of the actin-myosin complex with myosin ATPase modulators (myotropes) can lead to increased or decreased myocardial force production, respectively. Thus, identifying the human effect of myotropes during preclinical development can aid in the determination of drug-induced contractility risk. We used adult human primary cardiomyocytes from organ donors to evaluate the effects of 7 myotropes on contractility and Ca2+ transients at 1Hz. Treatment effects on contractility and Ca2+ amplitude were expressed relative to the myocyte’s specific baseline control. We report that negative myotropes exerted concentration-dependent decreases in contractility (Mavacamten; IC50=0.2µM), N-benzyl-p-toluene sulphonamide (IC50=16µM), Blebbistatin (BBS, IC50=2.6µM)). Unlike BBS, derivatives of BBS, HydroxyBBS, (S)-3'-aminoBBS and Para-aminoBBS, caused biphasic effects on contractility. Omecamtiv Mecarbil, myosin ATPase activator, increased contractility with EC50 value of 0.6µM. All 7 myotropes showed no potential to alter Ca2+ amplitude even at concentrations that had maximal effect on contractility. For example, 0.3µM Mavacamten and 2µM Omecamtiv Mecarbil changed the Ca2+ amplitude by -3% and -10%, respectively. In contrast to myotropes, a β-adrenoceptor agonist (Isoproterenol 0.3µM) and a Ca2+ channel blocker (Verapamil 10µM) increased (122%) and decreased (60%) Ca2+ transient amplitude, respectively. Thus, adult human cardiomyocytes provide a useful translational strategy for the early assessment of new myotrope candidates with a novel mechanism of action.

Plasma cytokines and/or histamine are often measured in the safety assessment of biopharmaceuticals, due to concerns about hypersensitivity reactions such as cytokine release syndrome and anaphylaxis. The present study aims to clarify the effects of infused histamine on the cardiovascular (CV) system and their mechanism in anesthetized (Ane) and conscious (Con) cynomolgus monkeys.

Histamine was intravenously infused in 4 telemetered female cynomolgus monkeys (3-5 kg, 4 years old) at consecutive dose levels of 2.5 to 10 µg/kg (Ane) or 2.5 to 20 µg/kg (Con). CV parameters were analyzed using a telemetry system, and plasma histamine and catecholamine (epinephrine and norepinephrine) concentrations were measured. The animals received saline or histamine receptor antagonists (either 0.3 mg/kg diphenhydramine [H1] or 1 mg/kg ranitidine [H2] or both H1 and H2) before administration of histamine.

Exogenous histamine increased plasma histamine and catecholamine levels in both Ane and Con monkeys. Marked increases in LV dP/dt max and heart rate (HR) were observed from 2.5µg/kg in Ane monkeys and from 10µg/kg in Con monkeys. These responses were drastically inhibited by pretreatment with H1 and H2 receptor antagonists. No changes were observed in other CV parameters at any dose in either Ane or Con monkeys.

In conclusion, an increase in plasma histamine causes increases in cardiac contractility and HR, which are mediated by H1 and H2 receptors and catecholamines released by histamine stimulation and might be a little more sensitive in Ane monkeys compared with Con ones.

【Aims】Recently, the interdisciplinary field of cardio-oncology has emerged to study the mechanism and prevention of cardiac dysfunction associated with cancer treatment. In this study, we investigated possible therapeutic effects of voluntary wheel running (VWR) on cardiac dysfunction observed in cancer-cachexia model mice established in our laboratory, since exercise therapy is known to be effective for patients with chronic heart failure.

【Methods】Mice were individually housed with free access to a wheel from 2 to 6 wks after implantation, to determine the effects of VWR on cancer cachexia-induced cardiac dysfunction.

【Results】VWR significantly suppressed the loss of heart and skeletal muscle weight as well as general symptoms of cachexia. Moreover, left ventricular ejection fraction significantly improved in cachexia group with VWR, compared to those without VWR. Microarray analysis revealed that the gene expression of “X”, which is an enzyme belonging to E3 ubiquitin ligase family but has not been reported to be related to heart failure, increased in the myocardium of cachexia mice, and that this increase was suppressed by VWR.

【Conclusions】These results suggest that the myocardial impairment associated with cancer cachexia may be caused by a different mechanism than that caused by heart failure, and that VWR may improve not only cachexia symptoms but also cachexia-induced cardiac dysfunction. In addition, the enzyme “X” may be one of key factors which are associated with myocardial atrophy and cardiac dysfunction on cancer cachexia. The pathway mediated by the enzyme “X” is currently being further analyzed.

[Background] In vitro hERG channel inhibition assays, in vivo electrocardiogram assays using conscious animals, and various types of studies are currently performed for predicting drug-induced torsade de pointes (TdP) in drug safety evaluation based on ICH S7B guidelines. In addition, best practice of in vitro hERG assay was presented in ICH S7B Q & A Section 2.1 in Feb. 2022 to support an integrated risk assessment of the potential of drugs that induce Tdp and/or other types of arrhythmias. The best practice recommends that in vitro hERG assays are performed at physiological temperature instead of room temperature, which has been widely used.

Therefore, we performed in vitro hERG assays using a protocol that has been conducted in safety pharmacology studies and using a protocol that refers to the best practice to evaluate differences in drug reactivity between these methods.

[Methods and results] In the best practice, we conducted experiments at room or physiological temperature using the ramp down voltage protocol in reference to the CiPA protocol (Jul. 30, 2021). In the conventional method, we conducted experiments at room temperature using the step voltage protocol.

We will report the results of comparison between various experimental conditions regarding the IC₅₀ and hill coefficients of compounds reported to have temperature dependence on drug reactivity and those reported to have Tdp risk.

Drug-induced liver injury is one of the leading causes of market withdrawal in the pharmaceutical industry and poses a serious health risk to affected patients. Here, we developed a 3D in vitro model of the human liver, the OrganoPlate LiverTox compatible with automated liquid handling and validated for hepatotoxicity screening. To build the model, up to 96 independent 3D perfused cultures were established on MIMETAS’ OrganoPlate 2-lane. Induced pluripotent stem cell-derived hepatocytes (iHep) in extracellular matrix were added to a microfluidic channel, following which endothelial and Kupffer cells were added to an adjacent channel. Characterization of the model revealed hepatocyte function including CYP3A4 activity and albumin production for up to 14 days. Fetal hepatocyte marker alpha-fetoprotein (AFP) declined over the 14 day culture, supporting iHep maturation. Assay validation studies using troglitazone as a positive hepatotoxic control revealed robust Z-factors ≥ 0.2 for albumin, urea, iHep viability (propidium iodide staining), and iHep nuclear size (Hoechst 33342 staining) assay readouts. Using these assays, 159 compounds of known hepatotoxicity were screened in the OrganoPlate LiverTox (50 µM, 72 h) and ranked by a composite score by combining the assay readouts. A follow-up dose response evaluation of select hits suggested the albumin assay to be the most sensitive readout in calculating TC50 values. Together, the OrganoPlate LiverTox is a promising platform for hepatotoxicity detection and has the potential to be used in a high throughput screening capacity.

Since the liver plays an important role in drug metabolism, in vitro tests using primary human hepatocytes (PHH) are one of the most important experimental tools of drug screening. However, in Japan, collecting PHH from humans raises ethical concerns, resulting that there is always a shortage of mature and normal PHH for research use. To solve this problem, hepatocytes derived from iPSCs (iPS-HEP) are promising as the alternative cell sources of PHH. Therefore, we focused on the culture medium for hepatocytes. In the first place, we evaluated the performance of the developed medium by measuring the amount of albumin secreted by PHH into the culture supernatant with ELISA. In the second place, we treated PHH with the drug-metabolizing enzyme inducer and the gene expression levels of the metabolizing enzyme were evaluated by RT-PCR. In the third place, we evaluated the function of iPS-HEP by ELISA and RT-PCR using this medium in the late stage of iPS-HEP differentiation induction. As a result, it was confirmed that there is no significant difference in the albumin secretion from PHH and gene expression levels of the metabolizing enzyme in both the commercially available media and the developed medium. However, when the drug-metabolizing enzyme inducer was added to iPS-HEP, a similar tendency of the drug response was observed even though the gene expression levels were significantly lower than PHH. In conclusion, we report that our developed medium can be used for PHH and iPS-HEP and could be a promising medium for various hepatocyte studies.

[Background]

Lorlatinib (LOR) is a potent small-molecule anaplastic lymphoma kinase inhibitor approved for the treatment of patients with non-small cell lung cancer. In a drug-drug interaction (DDI) study in healthy human participants, liver enzyme elevations were observed when a single 100 mg dose of LOR was administered after multiple doses of rifampin, a strong cytochrome P450 (CYP) 3A inducer and a pregnane X receptor (PXR) agonist. A series of in vitro and in vivo studies were conducted to evaluate potential mechanisms for the observed clinical toxicity.

[Methods]

To investigate the involvement of CYP3A and/or PXR in the observed liver toxicity, studies were conducted in cynomolgus monkeys administered LOR alone or with coadministration of multiple doses of known CYP3A inducers that are predominantly PXR agonists (rifampin, St. John’s wort) or predominantly constitutive androstane receptor agonists (carbamazepine, phenytoin) and a net CYP3A inhibitory PXR agonist (ritonavir).

[Results and Discussion]

Results from these studies identified cynomolgus monkeys as a pharmacologically relevant nonclinical model, which recapitulated the elevated liver function test results observed in humans. Furthermore, liver toxicity was only observed in this model when LOR was coadministered with strong CYP3A inducers, and the effects were not restricted to, or exclusively dependent upon, a PXR activation mechanism. These results generated mechanistic insights on the liver enzyme elevations observed in the clinical DDI study and provided guidance on appropriate product safety label for LOR.

Bioluminescence imaging has become an essential part of life science research and has routinely used to detect target cells in various tissues and to monitor disease processes. By introducing genes of luminescent enzymes, luciferases, into the target sites, it is possible to detect light emission from there. Although the firefly bioluminescent system is the most widely used, the yellow-green light is not able to penetrate deeper through living organisms and has significant limitations for its use in bioluminescence imaging. To overcome the problem, we have developed three longer wavelength luciferin analogues, "AkaLumine", "TokeOni" and "SeMpai" based on the substrate of the firefly bioluminescence system, which are extremely transmissive to living organisms. Unexpectedly, without introduction of a luciferase gene, we observed the luminescence from the liver of mice in vivo when AkaLumine and TokeOni were administered. This luminescence was reduced by the treatment of CYP inhibitors, and was affected by liver diseases. In order to study the luminescence in other organisms, these analogues were added to the extracts of pill bugs and blow flies, and the luminescence was observed. This suggests that the new luminescence system is conserved in animals.

Cytochrome P450 (P450, CYP) and UDP-glucuronosyltransferase (UGT) are drug-metabolizing enzymes that play crucial roles in phases I and II of drug metabolism, respectively. The expression levels of P450 and UGT in the liver are well known to be upregulated up to several tens of folds by exposure to ligands of aryl hydrocarbon receptor and nuclear receptors. However, the effect of such changes in expression levels on enzyme function remains unclear. On the other hand, the authors have focused on the functional interaction between P450 and UGT. It is evident that P450 and UGT form a complex on the ER membrane and modulate their function each other [1, 2]. To address these issues, we focused on a doxycycline-inducible expression system in this study. The ORFs of CYP3A4, CYP2E1, UGT1A1, and UGT2B7, which are important P450 and UGT members in both biodefense and toxicology, were subcloned into the pTetOne vector, which enables the regulation of gene expression by doxycycline in a single plasmid. The sequence of the target product was confirmed by Sanger sequencing analysis. Currently, we are confirming the induction by doxycycline and creating a stable expression system using HEK293 cells. In the future, we will use the obtained stable cells to analyze the effects of P450 and UGT expression levels on their enzyme functions and P450-UGT interaction.

[1] Ishii, Y., et al., Drug Metab. Rev., (2010).

[2] Miyauchi, Y., et al., Biol. Pharm. Bull., (2021).

[Aim] Our previous study shown that the concentration of acetamide (AA) in the blood and liver of F344 rats was higher than in Wistar rats after AA administration for 4 weeks, as was the incidence of micronucleated hepatocytes. To investigate the carcinogenic mechanism of AA from the viewpoint of rat strain differences, we compared toxicokinetics and histopathological changes in the liver of rats after a single dose of AA.

[Methods] Male F344 and Wistar rats were given a single oral or intravenous administration of AA at 100 or 2000 mg/kg bw. Blood and liver samples were used for AA analysis and histopathological examination.

[Results] In both strains, plasma AA concentration reached C_max at 4 hr after oral administration and at 0.5 hr after intravenous administration, and then decreased. No strain differences were observed in C_max and T_1/2. AA concentrations in the liver correspond to that in the plasma, and had no differences between strains. Histopathologically, the number of binuclear hepatocytes with irregularly shaped smaller nuclei increased in the liver of F344 rats at 24 hr rather than that of Wistar rats.

[Discussion] Since there were no differences in AA concentrations in plasma and liver after a single-dose administration, the strain differences in AA concentrations observed in the 4-weeks feeding study were considered to be due to repeated administration. On the other hand, strain differences were observed in the morphological changes of nuclei at 24 hr, suggesting that AA metabolites contribute to the formation of micronucleated hepatocytes.

Nonalcoholic steatohepatitis is a lifestyle-related disease and an increasing threat worldwide. Hepatic fibrosis caused by chronic liver diseases including nonalcoholic steatohepatitis (NASH) is closely correlated with mortality among liver lesions. Thus, it is important to identify factors that can serve as diagnostic and therapeutic targets for hepatic fibrosis. In the present study, we investigated the function of CD44 in the development of hepatic fibrosis, especially the proliferation of bile duct epithelia, in choline-deficient, methionine-lowered, L-amino-acid (CDAA) diet-fed rats. Male Fischer 344 rats were fed CDAA for 2, 4, 13, or 26 weeks. This diet increased the levels of serum parameters indicating liver injury, such as aspartate (AST) and alanine (ALT) aminotransferase; upregulated inflammation- and fibrosis-related gene expression in the liver. The expression of both CD44 mRNA and protein was significantly increased throughout the experimental period. CD44 protein was observed in some of the bile duct epithelium, around which hyaluronic acid was deposited, and these bile duct lesions were concordant with the area of hepatic fibrosis. Furthermore, the CD44-positive bile ducts were higher in the cell proliferation activity than that of CD44-negative bile ducts. These results suggested that CD44 contributed to the promotion of NASH-related hepatic fibrosis through hyaluronan deposition and ductular reaction. CD44-positive bile duct epithelium could be a therapeutic target for hepatic fibrosis.

The glomerulus is the structure in the kidney that filters waste products and small molecules from the blood into the urine, Essentially, the glomerulus is formed from an endothelial layer, a basement membrane and a size and charge selective sieve formed by podocytes. Damage to podocytes cells effectively destroys the size and charge selective filter, permitting the passage of larger and larger proteins from the blood into the urine, resulting in proteinuria. Currently there are no in vitro model of the glomerulus,

We have developed an in vitro 3D model of the glomerular filtration barrier (GFB), using human primary podocytes and endothelial cells derived from kidneys surplus to transplant requirements. We have developed a method for isolation of podocytes which yields a ~90% pure population. The podocytes express all known podocyte markers, including synaptopodin, WT1, nephrin, NEPH1, podocin, CD2AP, and podocalyxin. The podocytes also have central cilia, shown by pericentrin and acetylated tubulin staining. Cells are grown in cell culture inserts to generate an in vitro proteinuria assay. We have used this to demonstrate that our model retains the size selectivity of the in vivo GFB using a range of FITC-labelled dextran molecules of differing molecular weights and charges. We have shown that this size selectivity is disrupted using known podocyte toxins, adriamycin and puromycin. Our model is constructed in a 96-well plate format, making it amenable to high throughput analysis of GFB function and drug screening of compounds that affect GFB permeability and health.

Renal toxicity remains a major issue in clinical trials and stresses the need for more predictive models fit for implementation in early drug development. Here, we describe the use of a high throughput, microfluidic platform for the detection of drug-induced nephrotoxicity. A microfluidic platform (Mimetas’ OrganoPlate) was combined with renal proximal tubule epithelial cell lines (PTEC) and exposed to fluid shear stress. A 12-compound nephrotoxicity screen across multiple laboratories was performed in collaboration with sponsors and the NC3Rs. ciPTEC-OAT1 or RPTECs (Sigma) seeded against an ECM gel under perfusion flow were used to establish a proximal tubule-on-a-chip. Tubules with polarized epithelium containing functional transporter expression were obtained. Drug-induced toxicity was assessed by exposing the tubules to 4 benchmark compounds with known clinical effect and 8 blinded compounds supplied by the sponsors for 24 and 48h. Epithelial barrier tightness and drug-transporter interactions were evaluated. Parallel to this, cellular damage and stress were assessed using various read-outs. Finally, gene expression analysis was performed to assess AKI markers. The Nephroscreen revealed that a combination of cell viability, LDH and miRNA release were the most predictive readouts in determining nephrotoxicity. Most of the blinded compounds resulted in toxicity detected by at least one of the functional read-outs. Nephroscreen provides a reliable standardized and automatable system for efficacious identifying nephrotoxicants and revealing their mode of action.

[Background] In toxicology and pharmacology studies, glomerular filtration rate (GFR) is considered a gold standard in assessing renal function. The renal clearance of labeled inulin measured by photometers has been known as a filtration marker for the determination of GFR. Preclinically, a non-invasive GFR measurement method was recently developed, in which near-infrared fluorescent-labeled form of inulin is scanned with fluorescence molecular tomography (FMT). [Methods] In this study, an IVIS Spectrum based method for determining GFR ex vivo using fluorescence-labeled inulin (GFR-Vivo 680) was evaluated in conscious male C57BL/6 mice orally administered with vehicle or immunosuppressive agent - cyclosporine A (CsA, 80 mg/kg) for 14 days. [Results] Based on a two-compartment model fitting, estimated GFR (eGFR) was 235.40 ± 52.46 and 188.94 ± 18.97 μL/min; p<0.01 in vehicle-treated and CsA-treated mice, respectively. The results corresponded well to FMT imaging showing a reduction in GFR in the CsA treated mice, which yielded comparable GFR values (229.21 ± 60.96 and 150.80 ± 35.06 μL/min, p<0.01), together with previously reported values in literature. [Conclusion] The results suggested that this way to measure GFR ex vivo using an IVIS system and inulin probe could be applied to renal function testing in nonclinical studies.

[Aim] Maladaptive repair of renal tubule after renal tubular injury leads to irreversible renal fibrosis. We have shown CD44 expression in renal tubules in maladaptive repair using several renal fibrosis model rats. Here, we investigated the potential of CD44 as a biomarker to evaluate renal fibrosis by examination of CD44 expression in the kidney and urine of allopurinol (APL)-induced renal fibrosis model rats.

[Methods] Six-week-old male SD rats were treated with APL by gavage at a dose of 0, 100, and 150 mg/kg for 28 days, and fresh urine were collected at day 28. Serum biochemistry and histopathology of the kidney were examined. Immunohistochemistry for CD44 was performed in the kidney, and CD44 protein expressions in the kidney and urine were analyzed by western blotting.

[Results] Blood urea nitrogen and serum creatinine were increased with dose dependency in rats treated with APL. In histopathological analysis, interstitial collagen fibers were increased with dose dependency in the kidney of rats treated with APL. Renal tubules in fibrotic lesions were dilated or atrophied, which clearly expressed CD44 in immunohistochemistry. Western blotting analysis revealed an increased expression of CD44 in the kidney and urine of rats treated with APL.

[Discussion] The results of the present study demonstrated that CD44 was expressed in renal tubules in maladaptive repair. Urinary CD44 level was considered to increase following CD44 expression of the renal tubules in fibrotic lesions, suggesting potential of urinary CD44 as a non-invasive biomarker to evaluate renal fibrosis.

In treatment of hepatitis B virus (HBV), it is usually difficult for us to control with emergence of drug resistance. As HBV often reactive after treatment was stopped, patients must keep it for long term. Recently, we have developed E-CFCP, as a candidate drug of HBV for patients with drug-resistant HBV. As it has high antiviral activity and the half-life also is longer, patients can take it in a once-weekly dosing. We expect that E-CFCP can greatly improve the quality of life of patients. However, effects of E-CFCP are unclear in renal. The aim of this study is to clarify the effects of E-CFCP in the kidney, especially organic acid transporter（Organic anion transporters：OATs, Organic cation transporter：OCT）. We conducted cell viability studies using mouse-derived renal cortical cells (S2, CCD, cTAL) and uptake studies using radioisotopes to determine the effects of E-CFCP on the kidneys. In cell viability studies, E-CFCP has no cytotoxicity in all cell lines. We also examined the effect of drugs at high concentration using S2 cells. E-CFCP has no cytotoxicity even at high concentrations. In the substrate uptake assay, there was no inhibition of substrate uptake by E-CFCP, the transporter is not involved in the intracellular transport of E-CFCP and is unlikely to cause cytotoxicity. In conclusion, E-CFCP, a novel HBV antiviral drug, is unlikely to cause renal damage. It may be a novel great candidate drug of HBV for patients with drug-resistant HBV.

Purpose:

In ophthalmology examinations in preclinical safety studies using cynomolgus monkeys, full-field electroretinography (ERG) is used to evaluate the function of various cell types in the retina, and recently, optical coherence tomography (OCT) is increasingly used to investigate retinal morphology. By quantifying retinal thickness, retinal toxicity can be objectively evaluated; however, quantitative data on retinal thickness remain largely unreported.

We collected retinal thickness data from 10 cynomolgus monkeys using OCT and conducted ERG in order to investigate the relationship between retinal thickness and ERG waveforms.

Materials and Methods:

Using OCT images of the horizontal and sagittal planes from cynomolgus monkeys (10 males, ages 5 to 6), the thickness of the whole retina and of each retinal layers was measured every 500 μm up to 2500 μm from the fovea. ERG was performed in accordance with the International Society for Clinical Electrophysiology of Vision protocol.

Results and Discussion:

In OCT, thickness was highest in regions 1000μm from the fovea, particularly from the fovea to the nasal and ventral regions, where the retina was 70% thicker than at the fovea. In ERG, individual decreases in oscillatory potential were noted, but there was no correlation between potential and retinal thickness. We hope that the retinal thickness data and basic quantitative ERG data obtained from the present research will provide a useful index for evaluation of retinal toxicity.

Colorectal cancer is a malignant tumor that develops in the large intestine and ranks high in the number of morbidity and mortality. Chemotherapy FOLFOX or FOLFIRI is combined with anti-EGFR antibody drugs such as cetuximab (C-mab) to treat patients with unresectable/advanced recurrent colorectal cancer. This therapy cannot be expected to be effective for RAS gene variants. In addition, the efficacy and safety of RAF gene polymorphisms have been established by antibody drugs, and they will become further important in the chemotherapy. Therefore, this study analyzed RAS/RAF gene polymorphisms in patients with stage III-IV colorectal cancer who underwent FOLFOX± C-mab combination therapy. In addition, based on the literature, the efficacy and safety of C-mab and discussed. Three samples, blood, non-cancerous site, and cancerous site, were provided by the same patient for the gene analysis. Genomic variant was analyzed by the direct sequence method. As a result, the number of KRAS mutants was found from 5 patients (38.5%) and no BRAF (V600E) variant was found. Since large-scale clinical trials have shown that RAS mutant patients are less effective with anti-EGFR antibodies, it is possible to perform genetic analysis before the introduction of chemotherapy to determine the appropriateness of the introduction of anti-EGFR antibody drugs. It is also important from the viewpoint of preventing side effects such as peripheral neurotoxicity to improve quality of life.

Although blood thyroid hormone levels are useful for detection of antithyroid chemicals in rodent toxicity studies, they are highly variable depending on conditions of blood sampling, so the development of more appropriate method is still expected. Our recent study showed that in rats treated with thyroid peroxidase inhibitors for 28 days, histopathological and immunohistochemical analyses can be more efficient indicator than blood hormone levels. Here, we administered promoters of thyroid hormone metabolism, phenobarbital sodium salt (PB) and nicardipine hydrochloride (NCD), to six-week-old male and female SD rats (5 rats/group) at doses of 10, 30, 100 mg/kg and 15, 50, 150 mg/kg, respectively, for 28 days. Decreased serum T4 and increased TSH were observed in males of 100 mg/kg PB and 150 mg/kg NCD and in both sexes of 150 mg/kg NCD, respectively, and thyroid weights were increased in these groups. Histopathological analysis revealed that hypertrophy of thyroid follicular cells was found in both sexes of >30 mg/kg PB and >50 mg/kg NCD groups. In immunohistochemistry, TSH-positive area in the anterior pituitary gland was significantly increased in the groups with increased serum TSH levels, and was considered useful for detection of antithyroid chemicals. Histopathological findings in the thyroid were observed from lower doses without significant changes in serum T4 and TSH, which was consistent with that obtained using thyroid peroxidase inhibitors, suggesting that they can be more sensitive parameters for detecting antithyroid effects by promoting hormone metabolism.

Alkaline phosphatase (ALP) is distributed in various tissues including the liver, bone, and small intestine, and blood ALP is widely used as the marker of possible tissues damages. A treatment-related decrease in food intake is often observed in repeated-dose toxicity studies, and this decrease can exert secondary effects on several toxicological parameters, including blood ALP. However, it remains unknown which tissues ALP are related to the changes in blood ALP by decreased food intake. We measured ALP isozymes (ALP1, 2, 3 and 5) in serum, liver, bone, and small intestine in male and female rats fed with 65 and 45% of ad libitum food consumption daily for 4 weeks and conducted ALP staining in the tissues. As the results of isozymes analyses, the percentages of ALP3 and 5 were high in serum of normal rats, while those of ALP1 and 2 were low. In food-restricted rats, the serum ALP was increased, which was associated with the increased ALP5 and 2. The increased ALP5 was considered due to the increased ALP5 in the liver and small intestine. It was unclear which tissue contributed to the increased ALP2, but it might be related to an increase in females showing ALP positive staining in the bile canaliculi of the liver. Although the liver ALP1 was increased by food restriction, there were no leakages of the ALP1 into serum, which were consistent with no findings suggestive of liver damage. These results are considered to provide useful information to distinguish between treatment-related effects and secondary effects by malnutrition when serum ALP changes in toxicity studies.

Corn oil was subcutaneously administered to Crl:CD(SD) rats for 4 weeks at a volume of 0 (negative control; saline), 1, 2, or 5 mL/kg/day and evaluated the local and systemic toxicity for the purpose of setting the volume for repeated subcutaneous dosing toxicity studies using corn oil as a vehicle.

High body weight and decreases in some organ weights relative to body weight were observed at 2 and 5 mL/kg/day. Subcutaneous swelling at dosing sites was observed by clinical observation in all corn oil-treated groups and necropsy revealed the subcutaneous retention of corn oil at the sites.

In the histopathology, accumulation of administered substance and granulation tissue were observed at the dosing site. Accumulation of administered substance was also observed in inguinal skin, axillary lymph node, alveolus of lung and bronchus regardless of sexes and dosing corn oil volumes. In addition, multiple white nodules in abdominal cavity and liver were observed in a female at 5 mL/kg/day at necropsy, which were considered to be related to lipogranuloma at peritoneum and hepatic capsule.

In the hematology, slight decrease in RBC was observed in females at 5 mL/kg/day.

There were no test substance-related toxic changes in the blood biochemistry, urinalysis and ophthalmology.

In conclusion, as a result of repeated subcutaneous administration of corn oil for 4 weeks, high body weight was observed at 2 mL/kg/day or higher, and accumulation of corn oil was observed in the treated site and/or some organs. In repeated subcutaneous dosing studies using corn oil as a vehicle, the dosing volume should be set in consideration of these effects.

Adeno-associated viruses (AAVs) have been one of the promising modalities as a vector for in vivo delivery of gene therapies. A gene delivered with the AAV vector could be expressed in non-dividing cells as well as dividing cells intracellularly for a long time. Additionally, AAV vectors are low in virulence and show tissue-tropism for each serotype. Therefore, AAV vectors are highly effective and safe for gene delivery to targeted cells. AAV serotype 9 (AAV9) is often used in drug development because of its excellent infectivity in the central nervous system, heart, and muscles by intravenous administration. On the other hand, AAV products contain some impurities derived from the manufacturing process, such as plasmid DNA and empty capsids. Empty capsids are outer shells without the gene, and it is known to cause a decrease in delivered gene efficiency due to a competitive inhibition of the infection and to induce immune responses leading to adverse effects. Although the chromatography and ultracentrifugation methods have been used to remove empty capsids, its contamination cannot be eliminated completely so far.

We believe that understanding the toxicity potential of empty capsids will help us to understand the cause of toxicity induced by AAV products and to estimate the acceptable amount of empty capsids to human administration. In this study, the empty capsids of AAV9 were administered to C57BL/6J mice at doses of up to 3×10¹⁵ capsid/kg, and general toxicity including cytokine measurement was evaluated at 24 hours, 4 days, and 1 month after the administration.

Purpose

Toxicity studies using juvenile animals are becoming increasingly important in the development of pediatric drugs. However, there are few data on dogs. In this study, we investigated the changes of biochemical values in juvenile beagles.

Materials and Methods

Pups of TOYO beagle (Kitayama Labes Co., Ltd.) were examined by having blood drawn from the jugular vein immediately and 24, 48 hours after birth and at 1, 3, 5, and 7 weeks of age. Each blood collection point's data were calculated as mean±SD for each age and trends were examined.

Results

No abnormalities were observed in the mothers and pups. Newborns received abundant nutrients and immunoglobulins from colostrum, and by 2 weeks of age, opening of external ear canals and eyelids were observed. Pups that received only breast milk until 3 weeks of age showed eruption of milk teeth between 3 and 7 weeks of age, ingested feed, and walked by themselves. During first 24 hours after birth, ALP, ChE, γ-GTP, AST, ALT, LDH, LAP, CK, BUN, CRE, UA, and BIL were markedly elevated. These decreased with growth and remained at low levels after one week of age. TG increased until 48 hours after birth and then gradually decreased until 7 weeks of age. T-CHO, HDL-C, F-CHO, and PL were elevated until 3 weeks of age, and then began to decline.

Conclusion

Many blood biochemical values were elevated immediately after birth and gradually stabilized after one weeks old. In the study of juvenile animals, it was considered extremely important to understand the basic blood data.

Purpose

Because of the increasing importance of safety studies using juvenile animals, background data has been accumulated by various research institutes in the pediatric drug development. Therefore, we investigated the developmental changes in hematological and biochemical values in juvenile beagles less than 4 months old.

Materials and Methods

TOYO beagle's puppies were examined by drawing blood from the external jugular vein at 2, 4, 6, 8, 12 and 16 weeks of age. The mean ± SD for each items at each age were calculated to test for differences between the sexes. In addition, the results were compared with those of 12 months old.

Result

In hematological tests at juvenile, RBC, HGB, and HCT increased from low values, while MCV, MCH, and Ret decreased from high values. WBC showed a decreasing trend with age, although there were large individual differences at juvenile. The lymphocyte ratio decreased and the pseudo eosinophil ratio increased after 8 weeks old. Blood biochemical values followed three patterns: increase (TP, ALB, CRE, NEFA, ALT, LAP), decrease (CK, GLU, Ca, IP, γ-GTP, LDH, BUN), and increase/decrease (T-CHO, HDL-CHO, PL, F-CHO, ALP). The other items showed little variation with age. From the results of the significance test, it was judged that there was no difference between sexes. At 16 weeks, there were many test items that did not reach the values of 12 months old.

Conclusion

Variations with age were more observed in juveniles. The results of this study will provide useful basic data for blood tests in juvenile beagles.

[Purpose] Zinc (Zn) is trace element which is essential for life, and Zn deficiency causes adverse effects like dysgeusia. To deal with this, Zn supplement with Zn complex is commonly used. Zinc complex is also reported to have pharmacological effects such as antioxidant activity and liver protection. On the other hand, it is known that excessive Zn intake causes toxicities like pancreatic toxicity, and thus there is a possibility that excessive Zn supplement by Zn complex induces some toxicities. However, the toxicity information is unclear due to few studies for Zn complex. Therefore, Zinc maltol (ZM), which is a typical Zn complex, was administered orally to male rats for 4 weeks to assess the toxicities in the present study. [Method] ZM was administered orally to male SD rats for 4 weeks at a dose of 0, 200, 600 or 1000 mg/kg/day (5 animals per group) to assess the toxicities. [Results and Discussion] At 1000 mg/kg/day, pancreatitis-like histopathological changes and increases in white blood cell parameters and creatine kinase were observed. Decrease in red blood cell parameters and extramedullary hematopoiesis in spleen were observed. In addition, low plasma iron and copper concentration were observed. Given these results, it is judged that ZM induced pancreatitis and anemia at a dose of 1000 mg/kg/day by 4-week repeated oral administration in male rats. The no-observable-adverse-effect-level (NOAEL) of ZM was judged to be 600 mg/kg/day in male rats under the present study conditions.

Juzen-taiho-to, a traditional Chinese herbal medicine, is used for patients with anorexia and fatigue in humans. In our previous study, a granulated Juzen-taiho-to improved vincristine-induced gastrointestinal adverse effects through increasing gastric motility in dogs. However, the safety and efficacy of Hozen-S, the sweet liquid form of Juzen-taiho-to, in dogs have not been investigated. Therefore, we examined whether the long-term administration of Hozen-S to dogs is safe and affects their gastric motility. First, we administered normal-dose (0.8 ml/kg) or high-dose (4.0 ml/kg) Hozen-S to dogs for 42 days and observed their general status (vigorousness, appetite, vomiting, diarrhea and body weight), as well as changes in blood parameters. Next, we administered normal-dose Hozen-S and assessed gastric motility using ultrasonography. Furthermore, we administered normal-dose or high-dose Hozen-S and assessed dog plasma ghrelin levels. Although administration of normal-dose or high-dose Hozen-S increased body weight significantly, there were no other changes in their general status. Hozen-S changed some blood parameters significantly. However, there might be no important clinical implications because almost all parameters changed within the reference range. Normal-dose Hozen-S significantly promoted gastric motility and high-dose Hozen-S significantly raised plasma ghrelin levels. Hozen-S might promote gastric motility by raising plasma ghrelin levels in dogs. In conclusion, Hozen-S may become a safe and effective traditional Chinese veterinary medicine.

Intrathecal (IT) injection allows drug delivery to the central nervous system circumventing the blood-brain barrier; however, before refining our method in cynomolgus monkeys, we observed marked differences in cerebrospinal fluid (CSF) drug concentrations between animals, despite no difference in plasma drug concentration. Insufficient drug delivery to the cisterna magna (CM) in some animals is also reported in the literature. In light of this, we refined our lumbar IT injection method based on previous researches, and identified conditions that can suppress differences in CM CSF drug concentration between individuals. Lumbar IT injection of 4.2 mg/mL dye (Evans blue) solution at 2.4mL/body and 2 mL/min was performed in 12 anesthetized monkeys [age: 3 to 6 years, body weight (BW): 2.4 to 7.0 kg]. CM CSF was collected 1 hour after injection, and absorbance at 620 nm was measured to obtain CSF dye concentration. In addition, 3 monkeys received a 2nd injection after a 1-week recovery period to verify the reproducibility of the experiment results. CSF dye concentrations showed above 10000 ng/mL in 9 monkeys; 2 monkeys 5000 ng/mL and 1 monkey 150 ng/mL. When classified by BW, dye delivery to the CM was favorable in lower BW monkeys, whereas CSF dye concentration decreased in higher BW monkeys. The reproducibility of the results was confirmed in the 2nd IT injection in 3 monkeys. These results imply that, incorporating the animal BW parameter in our refined method can suppress differences in CM CSF drug concentration.