Organotins such as tributyltin (TBT) and triphenyltin (TPT) were previously utilized in antifouling paints to deter fouling organisms. However, their usage has since been banned due to concerns over marine pollution. Previously we have indicated that organotins exert various effects on different species via retinoid X receptors (RXRs), with Mytilus galloprovincialis expressing RXRs sensitive to organotins. While it is plausible that organotins influence Mytilus galloprovincialis through RXRs, the specific mechanisms remain elusive. We hypothesized that organotins may hinder mussel adhesion via their RXR (mgRXR) and sought to elucidate the transcriptional regulation of the adhesion-associated molecule (argX). Initially, we amplified the putative argX promoter region from the Mytilus galloprovincialis genome by PCR and inserted it into a firefly luciferase reporter vector. Cells were co-transfected with the reporter vector and the mgRXR-expression vector, followed by a reporter assay. The argX promoter activity exhibited a concentration-dependent increase upon exposure to 9-cis retinoic acid, TBT, and TPT. Subsequently, we divided the argX promoter region into several fragments and analyzed their respective promoter activities. Notably, increased promoter activity was observed in the divided promoter regions located 30 to 50 base pairs upstream of the transcription start site following exposure to the test substances in a concentration-dependent manner. These findings suggest that organotins may modulate the transcriptional regulation of the argX gene via mgRXR.

Epidemiological studies showed that ambient and subway particles cause vascular dysfunction. However, vascular function implications of co-exposure of these particles with LPS are unknown. In this study, HUVECs were exposed to α-Fe2O3, Fe3O4 (main components of subway particles), vehicle exhaust particles (VEP) and urban aerosols (UA) and ICR mice received α-Fe2O3 and Fe3O4 intratracheally with or without LPS. Without LPS, iron oxides reduced endothelial cell viability, increased LDH release, altered production of pro-inflammatory cytokines like IL-6 and IL-8 and decreased Plasminogen Activator Inhibitor 1 (PAI-1), a fibrinolysis regulator and Endothelin 1 (ET-1), a vasoconstrictor more than VEP and UA. At non-cytotoxic concentrations, iron oxides and LPS impaired ET-1 and PAI-1 more than VEP and UA. Although, low production of ET-1 and PAI-1 occurred with or without LPS, cells exposed to all particles and LPS had increased production of IL-6 and IL-8. In vivo, both types of iron oxides with LPS heightened the production of cytokines (KC, TNF-α, IL-6 and IL-12) in BALF. Immune profile and histological evaluation showed that LPS increased neutrophils, lymphocytes and oedema around murine lung blood vessels. Iron oxides and LPS were more toxic than iron oxides alone to murine lung cells. These results suggest that iron oxide-rich particles impact vasomotor and fibrinolytic function more than other ambient particles on endothelial cells. Although iron oxide alone may be less toxic, LPS presence exacerbates inflammation and immune response, which may impair vascular function.

Dysfunction of the circadian clock has been implicated in the pathogenesis of cardiovascular disease. Particularly, the CLOCK protein is a core molecular component of the circadian oscillator, so that mice with a mutated Clock gene (Clk/Clk) exhibit abnormal rhythms in numerous physiological processes. In this study, we analyzed the relationship between heart failure and Clock in 5/6 nephrectomy (5/6Nx) mice, which induce heart failure by chronic kidney disease (CKD). Surprisingly, cardiac inflammation and fibrosis were attenuated in Clock mutant (Clk/Clk) 5/6Nx mice even though they had high blood pressure and increased serum angiotensin II levels. A search for the underlying cause of the attenuation of heart disorder led to identification of the monocytic expression of G protein-coupled receptor 68 (GPR68) as a risk factor of CKD-induced inflammation and fibrosis of heart. 5/6Nx induced the expression of GPR68 in circulating monocytes via altered CLOCK activation by increasing serum levels of retinol and its binding protein (RBP4). The high-GPR68-expressing monocytes had increased potential for producing inflammatory cytokines, and their cardiac infiltration under CKD conditions exacerbated inflammation and fibrosis of heart. Our present study reveals an uncovered role of monocytic clock genes in CKD-induced heart failure¹. Furthermore, we used a high-throughput screening approach to identify an inhibitor of GPR68 and found that this compound has a preventive effect on CKD-induced heart failure².

¹Yoshida Y et al. Nat Commun., 2021.

²Yoshida Y et al. Trans Res., 2024.

【Background】We have developed the chronotoxicology and previously demonstrated that metal-induced chrono-toxic effects were different depending on metal, respectively. Therefore, individual investigation for metal-induced diurnal toxic effect is important. Cisplatin is a platinum-based medicine that is widely prescribed to treat various types of cancers. However, cisplatin is well known to cause severe adverse effects such as nephrotoxicity and ototoxicity. Several literatures have shown that clock gene regulates cisplatin-related homeostasis genes such as Oct2 and Mate1. Although some part of clock gene involved with cisplatin-induced nephrotoxicity, association with other clock genes was still unknown. The aim of the present study is to investigate whether seven kinds of clock gene (Ciart, Cry1, Cry2, Npas2, Per1, Per2, and Per3) regulate CDDP-induced renal toxicity or not using renal cortex tubule cell lines (MuRTE61 cells).

【Results & Discussion】Cisplatin treatment decreased MuRTE61 cell viability in a dose-dependent manner. It increased the Cry2 expression levels after 24-h. Notably, Cry2 overexpression alleviated the Cisplatin-induced cell proliferation suppression, apoptosis, and platinum contents in MuRTE61 cell. Moreover, Cry2 overexpression upregulated the efflux-related transporter (Atp7a and Mrp2). These results suggest that Cry2 involved with cisplatin-induced nephrotoxicity and cisplatin-induced renal toxicity is associated with Cry2 expression via the regulation of Atp7a and Mrp2.

The Ames test is widely used to assess the mutagenic effects of chemical substances in pharmaceutical and industrial products on DNA. Positive results occur when the colony count exceeds double that of the negative control. However, the strength of mutagenicity can vary even with the same positive result. One indicator commonly used to measure mutagenicity strength in the Ames test is the specific activity value, which is the ratio of the colony count difference between the test and control divided by the dose. Higher values indicate higher mutagenicity. Chemicals with specific activity values above 1,000 are classified as having "strong mutagenicity" according to the Industrial Safety and Health Act. Recently, in silico technology that predicts mutagenicity from compound structures has been developed, enabling cost-effective and rapid evaluation. However, there is currently no software that provides information on mutagenicity strength. This makes it challenging to assess the potential for avoiding mutagenicity by modifying chemical structures after obtaining a positive result. Additionally, early access to mutagenicity severity information would facilitate compliance with legally required measures under the Industrial Safety and Health Act. To address this, we have developed a machine learning model that predicts mutagenicity strength using both in-house and publicly available data on specific activity values. We have also expanded our system to provide information on mutagenicity strength, in addition to binary determinations of negative or positive, by incorporating the model into a mutagenicity prediction system.

【Purposes】

The blood-brain barrier (BBB) is an important mechanism in central nervous system (CNS) toxicity because it controls the transport of substances between the blood and brain. Therefore, in vitro models of BBB function similar to those in humans are important for the evaluation of CNS toxicity. However, the rarity and weak tight junctions in existing BBB models are problematic. To solve these problems, human iPS cell-derived brain microvascular endothelial cells (iBMECs) with robust tight junction have been developed. But, other functions of iBMECs have not been evaluated, and very few reports have compared drug permeability in iBMECs with that in in vivo models. This study aimed to evaluate iBMECs by comparing the drug permeability between iBMECs and mices or humans.

【Methods】

Expression of genes and proteins related to tight junctions and transporters was analyzed in iBMECs using RT-qPCR and immunofluorescence staining. Drug permeability P_app values were determined through permeability assays. The obtained P_app values were correlated with literature values in in vivo models.

【Results】

iBMECs have robust tight junctions, and the expression of the excretory transporter BCRP was higher compared to human BMECs. P_app values in iBMECs correlated well with those in mices, and correlated well with drug translocation in humans.

【Conclusions】

This study showed a high correlation between drug permeability in iBMECs and in vivo models. Therefore, drug permeation in iBMECs reflects in vivo conditions, which may be applicable to drug permeation prediction and safety assessment.

The conventional read-across method for the in silico evaluation of toxicity of chemical substances uses the Tanimoto coefficient and the degree of agreement between the results of in vitro tests as similarity indices. These indices are not weighted for each variable and are not optimized for the toxicity. On the other hand, in the QSAR method, the predicted value is optimized by weighting each variable using machine learning technology, but it is difficult to show the process and basis of the prediction. Therefore, in this study, we developed a new read-across method that uses machine learning technology to define toxicity-specific similarities to optimize similarity indices and clarify the basis for prediction. We predicted the LLNA Category 4 class, which stands for the skin sensitization potency of chemicals. Using molecular descriptors and data from each alternative method of skin sensitization as explanatory variables, a LightGBM model that predicts LLNA EC3 values that express the intensity of skin sensitization was constructed. Because this model has a function that converts the properties of chemicals into skin sensitization intensity, the difference in predicted values can be treated as the distance between chemicals in the optimized space with respect to skin sensitization. For each validation data, 10 analogues in order of the similar predicted value were collected among the training data. The median LLNA Category (in vivo) of the analogues was determined as the predicted value. The accuracy for the 4-class potency categorization was 0.60, and the accuracy was 0.95 when the error of one class was allowed.

A chemical language model, analogous to natural language processing, is employed in machine learning to handle compound structures, with SMILES representation as its principal input format. However, disparities in processing methods across databases lead to notational inconsistencies, even in Canonical SMILES. This study explores the influence of these 'dialects' in the applications of chemical language models for QSAR tasks.

An initial investigation revealed discrepancies in SMILES representations regarding stereochemistry across databases, which could impact the application of chemical language models. To assess the influence of these inconsistencies, three pre-processing methods were implemented: standard procedures, explicit stereoisomer assignment via 3D structure calculation, and exclusion of stereoisomeric data. Three corresponding models were constructed and evaluated for their performance on the Ames test dataset, focusing on translation and classification accuracy. Findings indicated notable enhancements in translation accuracy with the proposed pre-processing methods, while classification accuracy showed marginal improvement.

From the above, we found that (1) compound databases have many notational inconsistencies, and (2) taking stereoisomerism into account may contribute to improving the accuracy when applying chemical language models. It is expected that the operation of an appropriate chemical language model will contribute to the improvement of QSAR tasks, such as Ames mutagenicity prediction.

Norovirus is the leading cause of acute gastroenteritis worldwide. There is currently no effective vaccine against norovirus. The recent development of virus-like-particle (VLP), which have the same antigenic properties as noroviruses but lack genes for self-replication, has made it possible to develop a vaccine using VLP as antigens. This study aimed to compare the efficacy and adverse reactions of the antibody-producing capacity of VLPs with various adjuvants.

Mice were vaccinated intramuscularly with 2 injections of norovirus VLP with 8 different adjuvants, and plasma was collected dynamically for 7-10 weeks after vaccination to measure antibody titres by ELISA. Compared with the control VLP-treated group (without adjuvant), the adjuvant-added groups significantly increased the maximal antibody production and the duration of antibody production. However, some unfavorable effects were also observed in some of the adjuvant-added groups.

These results suggest that some adjuvants have the power to increase the antibody production efficiency of norovirus VLPs, albeit with some degree of side-effects. This study provides us with evidence to determine the optimized condition of adjuvant administration in combination with VLP based on efficacy and safety evaluations.

Since its emergence in late 2019, COVID-19 has become a major global health challenge, and COVID-19 vaccines has raised concerns regarding their safety profile, particularly in the pediatric population. Although rare, studies have reported myocarditis and pericarditis following COVID-19 vaccination. In this study, using a robust database, we investigated the risk of myocarditis in children who received COVID-19 vaccines. We used the VAERS, a comprehensive database to track adverse events after mRNA COVID-19 vaccination. Data were extracted from 2022; we focused on the occurrence of myocarditis among vaccine recipients. All children received one of the following vaccines approved in the United States: Moderna, Pfizer/BioNTech, or Johnson & Johnson (Janssen). Vaccination rates were calculated using the 2022 CDC and Prevention data from the United States. Analysis of the 2022 VAERS data revealed the highest myocarditis reporting frequency in the 18–64 years group, ten cases in the 5–11 years group, and no case in the 0–4 years group. The reported cases of myocarditis per million were the highest among adolescents (12–17 years) at 8.1 cases, followed by 0.84 cases in the 5–11 years group, and no case in the 0–4 years group. This large-scale study highlights the risk of myocarditis in children who received COVID-19 vaccination. The highest incidence rate was observed in adolescents (12–17 years), with notably low rates observed in children aged ≤11 years. These findings are important for public health messaging and can significantly affect vaccine uptake decisions based on informed risk assessment.

[Introduction]Gapmer antisense oligonucleotides (Gapmer) are chemically modified with artificial nucleic acid at both ends. Some Gapmers have hepatotoxicity, and it has been reported a relation between base sequence and chemical modification. Therefore, in this study, we focused on the increase in blood alanine aminotransferase (ALT), an indicator of hepatotoxicity, in animal tests, and aimed to develop a machine learning model to predict ALT elevations from base sequences and chemical modification information.[Method]Data on Gapmers’ sequences, modifications, ALT, and doses were collected from papers on single-dose studies in mice. The objective variable was the presence or absence of ALT elevation. A positive substance was defined as a substance that showed an increase of 10-fold or more of ALT value compared to the control group, and a negative was defined as an increase of less than 10-fold. As explanatory variables, each sequence was converted to the frequency of 2 or 3-base subsequences. The sugar modification structure information was calculated from alvaDesc. A random forest classification model was constructed.[Result/Discussion]We obtained data on 138 Gapmers from 16 studies. Of these, 86 were positive substances and 52 were negative. The model performance was ROC-AUC: 0.84, sensitivity: 0.88, and specificity: 0.55. In this study, we collected data on ALT in single-dose studies of Gapmers and developed a high-performance prediction model using only the substance information. This model is expected to support the efficient design of Gapmers to avoid ALT elevation.

We have previously reported an Artificial Neural Network (ANN) model for predicting LLNA EC3 to assess the risk of skin sensitization using in vitro tests. In this study, we report on the ANN model developed using Amino acid Derivative Reactivity Assay (ADRA), which can be performed at lower concentrations and is Known for low frequency of co-elution, as opposed to Direct Peptide Reactivity Assay (DPRA), which is a known method for evaluating the covalent binding of test substance and skin proteins.

We defined LLNA EC3 as the objective variable, and used characteristic values obtained from ADRA, KeratinoSens™, h-CLAT, and toxicity alerts from Toxtree as descriptors. We developed two models based on data of ADRA at molar and gravimetric concentrations, and used Python and scikit-learn, a machine learning library, to develop the ANN models. In addition, we evaluated the robustness of the models by cross-validation and compared their prediction accuracy with our previously reported ANN model¹⁾ using characteristic values of DPRA.

We compared the two models with the DPRA-based model using R² and RMSE, the results were comparable in both models. In addition, no substances of external data were underestimated for the LLNA Category. Furthermore, the predictive value between the two models developed using ADRA showed a high correlation (R²=0.865). Finally, we consider that the results suggest the usefulness of ANN models using ADRA for risk assessment of skin sensitization.

Reference

1) Hirota et al., J Appl Toxicol. 2018, 38, 514-526

Gastrointestinal toxicity (GI-tox) is a common clinical adverse event. It is very important to clarify GI-tox concerns in the nonclinical stage, because GI-tox significantly reduces patients' QOL.

Up to now, the main in vitro evaluation system for GI-tox has been monolayer culture of single cell line. Intestinal organoids contain undifferentiated cells and have a three-dimensional structure, thus GI-tox can be evaluated in a closer environment to the body. Previous studies revealed that GI-tox inducible compounds caused a decrease in cell viability. In addition, since the size of organoids is also reduced, it was considered that GI-tox could be evaluated by organoid size quantification.

Therefore, we aimed to establish an image analysis method for evaluating GI-tox using images of organoids. Then, we developed an application that can recognize organoids from bright-field images and extract feature values such as area and color. Next, the application was used to evaluate the GI-tox of clinical compounds. The result showed that the combination of the IC50 value for organoids area reduction and the maximum clinical blood concentration of each compound allowed the development of an evaluation system to identify compounds with potential GI-tox. Based on the above, this evaluation system was useful for distinguishing GI-tox at the nonclinical stage and for safety evaluation strategies.

Background:

Myocarditis induced by immune checkpoint inhibitors (ICIs) is a rare yet fatal side effect, posing challenges in predicting its onset amid the increasing use of ICIs in cancer treatment. Despite a mortality rate over 40%, understanding the mechanisms and risk factors remains a research focus. This study aims to predict ICIs-induced myocarditis onset using machine learning with data from a Japanese hospital database.

Methods:

Dataset Creation: We extracted ICI-prescribed patient data from the MDV oncology database, using the occurrence of myocarditis within a year post-first ICI prescription as the dependent variable.

Model Construction: We developed models using LightGBM and Random Forest algorithms, including two bagging models trained on undersampled non-event data.

Model Evaluation: We assessed model performance through 5-fold cross-validation and predictive accuracy on evaluation data, using F1 and G-means scores.

Model Interpretation: SHAP was used for visualizing predictions on evaluation data.

Results & Discussion:

The incidence of ICI-induced myocarditis was around 0.4%. Models with undersampling and bagging exhibited enhanced predictive accuracy. The significance of known risk factors as predictors implies the potential existence of unexplored risk factors. This study's model aims to enhance prediction and, consequently, the safety of cancer care.

【Background and Objectives】Drug-induced kidney injury (DIKI) is one of the factors in the failure of drug discovery and development. Because the prediction ability for DIKI by conventional animal testing is low, construction of in vitro system is desirable. We reported three-dimensional cultured proximal tubular epithelial cells (3D-RPTEC) highly expressing drug transporters including OAT1¹⁾, and utility of the systems in evaluating DIKI. Recently, various modalities including nucleic acids and middle-molecules have been developing as medical agents, and the establishment of evaluation methods for kidney injury caused by these modalities is required. This study investigated whether 3D-RPTEC can evaluate the uptake and DIKI involving the megalin-mediated endocytic pathway.

【Methods】Uptake of FITC-albumin in 3D-RPTEC was evaluated. After exposing DIKI compounds to 3D-RPTEC, the intracellular ATP levels were measured. Furthermore, organelle markers were observed using the confocal image cytometer CQ1.

【Results and Discussion】Uptake of FITC-albumin in 3D-RPTEC was increased linearly up to 180 minutes and showed concentration dependence. Moreover, the uptake at 4 °C was decreased by approximately 75 % compared to 37 °C, indicating receptor-mediated uptake. Gentamicin exposure led to a concentration-dependent decrease in intracellular ATP levels and altered intensity of Lyso Tracker and Mito Tracker, suggesting that gentamicin affects the functions of mitochondria and lysosomes. These results indicated that 3D-RPTEC can evaluate endocytosis-mediated DIKI.

Reference

1) Ishiguro N et al, Drug Metab Dispos, 51:1177-87 (2023).

In the development of drugs, foods, and cosmetics, it is important to properly evaluate not only the efficacy of compounds but also their safety. However, the mechanism of toxicity is complex, and the safety evaluation of compounds is difficult. Drug-induced liver injury (DILI) is an adverse event that causes hepatic decompensation and liver failure. Computational methods for predicting DILI are desired in order to reduce development costs and animal experiments. In this study, we propose a machine learning method to predict DILI from pharmaceutical big data such as chemical structure fingerprints of compounds, cell morphology phenotypes, gene expression profiles, and cell morphology image profiles.

We tested our proposed method on its ability to predict DILI for compounds for which toxicity information on DILI is available from the U.S. Food and Drug Administration (FDA). When the structures of the test compounds were similar to the structures of the training compounds, the prediction accuracy with chemical structures was high. In contrast, when the structures of the test compound were dissimilar to the structures of the training compounds, the prediction accuracy with gene expression profiles and cell morphology phenotypes was high, and the prediction accuracy with chemical structures was low. The results indicate that DILI prediction by cell morphology and gene expression information is independent of the structures of the compounds in the training data. The proposed method is expected to be useful in practical applications.

Read-across (RAx) as a promising alternative method for repeated dose toxicity (RDT) is a technique that infers the toxicity of target chemicals from the toxicological data of structurally similar chemicals. Although the consideration between structural similarities (SS) and toxicological similarities (TS) is need for RAx, there are few cases in which the relationship between SS and TS has been analyzed. Therefore, we analyze the relationship between the SS and the NOEL differences, which is one of the points of departure.

This analysis uses RDT data in 621 chemicals for about 210 thousand combinations in the Hazard Evaluation Support System Integrated Platform (HESS) database. In addition, we extracted features of chemical pairs that had big NOEL differences despite having high SS (high SS calculated by Tanimoto coefficient with Toxprint : > 70 %).

As a result, it was confirmed that SS and NOEL differences are inversely correlated. In addition, halogens were extracted as a feature of chemical pairs that had big NOEL differences despite having high SS. By excluding chemical pairs with halogens, the proportion of chemical pairs with small NOEL differences increased in high structural similar chemical pairs. Furthermore, the NOEL differences among high structural similar chemical pairs were 43.9-fold at 5% tile value and 20-fold at 10% tile value.

Therefore, this study shows SS and NOEL differences are inversely correlated in RAx and clarified the structure alerts lead to big NOEL differences and quantitative NOEL differences in high structural similar chemical pairs.

[Background & Objectives] Potential adverse effects of drug candidates on the central nervous system (CNS) have been assessed in Safety Pharmacology studies with behavior analysis which performed in a relatively subjective manner. Since heart rate vary according to autonomic nervous activities which reflect CNS activities, we hypothesized that drugs effect on CNS can affect heart rate variability (HRV). The purpose of this study was to evaluate whether some frequency-domain parameters of HRV can be an objective index for detecting CNS undesirable effects.

[Materials & Methods] Rats implanted ECG telemetry system were used and their ECG data were obtained after administration of drugs 1) targeting CNS, 2) causing CNS side effects, and 3) having no obvious effect on CNS. R-R intervals were dissected to perform frequency-domain analysis obtaining the spectral power in the low frequency range (LF, 0.03 to 1.0 Hz) and the high frequency range (HF, 1.0 to 3.0 Hz), and LF/HF ratio. Data were analyzed for at least one hour before as baseline and 6 hours after drug administration every one hour.

[Results & Discussion] Drugs used in this study caused no obvious changes in heart rate. Drugs having no obvious effect on CNS hardly changed any HRV parameters, whereas drugs targeting CNS or causing CNS side effects significantly increased LF/HF from baseline in a dose-dependent manner, with changes in the latter drugs being rather evident. This study suggests that LF/HF, which represents autonomic nervous system balance, could be an indicator of drug-induced undesirable effects on the CNS.

Objective: Dasatinib, a second-generation BCR-ABL inhibitor for the treatment of chronic myeloid leukemia (CML), outperforms the first-generation imatinib in efficacy and onset. Its therapeutic and adverse effects are closely tied to plasma concentrations, emphasizing the need for pharmacokinetic analysis. High gastric pH can reduce dasatinib's solubility and absorption, thus lowering its concentration in the body. This study evaluates the impact of gastric acid suppressants (H2 receptor antagonists or H2RAs and proton pump inhibitors or PPIs) on dasatinib’s effectiveness and safety, leveraging long-term, comprehensive prescription data.

Methods: This analysis involved patients diagnosed with CML and treated with dasatinib, using data from the Japan Medical Data Center (JMDC). We compared outcomes between patients who were prescribed gastric acid suppressants after starting dasatinib and those who were not, focusing on therapeutic efficacy and side effects. Efficacy was measured by the transition to another BCR-ABL inhibitor or death due to CML, analyzed with Kaplan-Meier curves and log-rank tests. Major side effect were scrutinized across groups.

Results & Discussion: There were 743 patients in this study. The difference in event times between groups was not significant. Although co-treated patients exhibited more side effects, notably agranulocytosis showed a significant difference.

The co-administration of H2RAs or PPIs appears to have a negligible effect on the efficacy and side effect profile of dasatinib in CML patients.

Cardiac safety evaluation is one of the important factors in new drug development, and it requires good correlation between preclinical study and clinical trial. However, animal models are used in the preclinical studies of drug development have limitations in predicting human responses due to interspecies differences. In addition, the hERG assay concerns persist regarding its accuracy, as hERG-blockade does not consistently correlate with QT prolongation in humans.

With recent advance in the field of stem cells research, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have well developed to mimic human cardiac electrophysiology and broadly used in the evaluation of cardiac safety. For this reason, the ICH guideline was updated to be use hiPSC-derived CMs for cardiac safety evaluation in 2022.

NEXEL produces highly functional hiPSC-derived CMs (Cardiosight®-S) that validated for the new test method in the updated guideline. The hiPSC-derived CMs not only provide accurate prediction of cardiac toxicity through multi-ion channel analysis, but also provides the great accessibility by high throughput capability. Thus, the use of hiPSC-derived CMs in the drug development could a new paradigm under the updated ICH guideline.

Mice topically treated with MC903, a Vitamin D3 analog, develop type 2 inflammation, scratching, and skin barrier dysfunction, resembling atopic dermatitis (AD), making them a suitable model for AD. Previous studies suggest that variations in MC903 application conditions, including dosage, frequency, and duration, influence the onset, progression, and severity of dermatitis. It is essential to understand the time-course of disease components common with AD. However, under the same application conditions of MC903, the time-course of inflammation, scratching, and skin barrier dysfunction remains unclear. In this study, we aimed to clarify this issue by characterizing the time-course of pathological conditions with MC903 being applied to the mouse ear every other day for four weeks. Compared to the control mice treated with ethanol, ear thickness in MC903-treated mice significantly increased from Day 8 post-application. Scratching behavior increased from Day 3, and transepidermal water loss, a marker of skin barrier dysfunction, increased from Day 14. Histological analysis revealed granulocyte infiltration in the dermis from Day 4, and increased mast cells and CD3-positive T cell infiltration from Day 8. ELISA and/or real-time PCR analysis showed increased TSLP, IL-4 and IL-13 involved in type 2 inflammation from Day 8 to Day 28. In conclusion, in MC903-treated mice, the onset and progression patterns of inflammation, scratching, and skin barrier dysfunction varied, and pathological condition encompassing all these phenotypes was established from Day 14.

Currently, carcinogenesis studies in rodents require a long period of time. Even using genetically modified animals, the administration period is about 26 weeks. Therefore, the problems of labor and cost have been pointed out. Organoids are widely used in human and animal cancer research because they can reproduce in vivo epithelial tissue structures and gene expression patterns in culture dishes. Although the usefulness of carcinogenesis models using organoids derived from normal mouse tissues (lung, liver and mammary gland) has been reported in previous studies, there are no reports of carcinogenesis models using organoids derived from normal bladder tissues. In this study, we attempted to create a carcinogenic organoid model using normal rat bladder tissue. Organoids were prepared using bladder tissue from 6-week-old F344/DuCrlCrlj rats, treated with 2-Acetylaminofluorene (2-AAF) for 6 days, and then transplanted into immunodeficient mice, resulting in tumor formation 7 days after implantation. On 21 days after transplantation, the tumor tissue removed and confiemed the expression of bladder epithelial cell markers (CK5 and CK7) and histopathological features. Subsequently, organoids were prepared from tumor tissue and transplanted into immunocompromised mice, resulting in tumor re-formation. Tumor tissue removed after the second organoid implantation was histopathologically confirmed to be squamous cell carcinoma.

In this study, for the purpose of evaluate of anti-cancer drug sensitivity and side effects in an environment closer to the living body, mammary tumor organoids produced from mammary tumor affected cats and normal intestinal organoids from mice were perfused with the anticancer drug toceranib after being mounted on a microfluidic device, and comparisons with the non-perfusion condition were made. Analysis was performed.　Cell viability was analyzed after perfusion of cat mammary tumor organoids with culture medium treated with 10 µM toceranib at 9.5 µl / min for 48 hours, and survival was significantly reduced in organoids perfused with the anticancer drug. In addition, the expression of apoptosis-related genes such as p53 and caspase9 was elevated in mammary tumor organoids of cats perfused with toceranib. Next, mammary tumor organoids and mouse normal intestinal organoids were mounted on the same device and perfused with toceranib under the same conditions. Similar to mammary tumor organoids, the survival rate of normal intestinal organoids was also decreased by perfusion of toceranib. However, apoptosis-related genes were not upregulated; instead, the expression of necrosis-related genes such as RIPK3 was increased. Furthermore, intestinal organoids perfused simultaneously with the necrosis inhibitor HS-1371 and toceranib showed a trend toward inhibition of necrosis and increased survival. These results suggest that perfusion with toceranib induces apoptosis in mammary tumor cells and necrosis in normal intestinal cells. They also revealed the usefulness of organoid-loaded microfluidic devices.

Diabetic retinopathy (DR) is one of the most common complications of diabetes. The current gold standard for treating DR involves using anti-VEGF antibodies to inhibit angiogenesis in late-stage proliferative diabetic retinopathy. However, this approach often results in recurrence and numerous complications, emphasizing the need for new treatment options. Recent findings suggest that VEGF inhibitors might inadvertently cause vascular damage, diminishing efficacy and potentially accelerating disease progression. In contrast, vascular endothelial growth factor receptor inhibitors (VEGFRi) can simultaneously block the activation of both embedded and soluble VEGFR, offering more significant therapeutic potential. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in the cellular response to DNA damage, is activated in the early stages of DR. Inhibiting PARP can effectively alleviate DR progression, making it a viable therapeutic target. These insights underpin the potential of a combined VEGFRi and PARPi approach for DR treatment. We used bEnd3 cells, exposing them to media with varying glucose concentrations under normoxic and hypoxic conditions. The expressions of PARP, VEGFR were evaluated under these conditions. Our research demonstrated significant upregulation of PARP and VEGFR3 in the endothelial cells of individuals with DR, underscoring their importance in disease progression. In conclusion, the dual inhibition of PARP and VEGFR3 presents as a novel and promising therapeutic approach for DR. However, further studies are required to determine the efficacy of this strategy conclusively.

Exacerbating the severity of diabetic retinopathy (DR) might result in the loss of vision. Histone deacetylases (HDACs) have a role in several processes and are responsible for controlling gene expression during the epithelial-mesenchymal transition (EMT). However, the precise effects of AZP, as HDAC inhibitors on EMT and their role in the progression of diabetic retinopathy (DR) are still not well understood. A comprehensive in silico analysis was conducted to discover the hallmarks of DR, possible molecular targets, and to analyze interactions between AZP and salient potential targets using molecular docking. The protein levels associated with EMT and HDACs were determined using Western blot. The db/db mice as a DR model, were administrated with and without AZP, while WT mice were used as controls. Ophthalmology examination was conducted using optical coherence tomography (OCT) and fundus fluorescein angiography (FFA).

HDAC6 had a negative effect size and was found to be elevated in PDR, and EMT was a pathological characteristic of DR. High glucose generates elevated protein levels of HDAC1, 3, 4, 6, and mesenchymal characteristics in HG-induced ARPE-19 cells and the DR animal model. HDACtr3qqq324,6 interacts with AZP. AZP treatment reduced the upregulation of HDAC1, 3, 4, 6, and reversed EMT protein levels. Furthermore, treatment of AZP enhances retinal vasculature and thickness in a DR animal model. By blocking EMT, AZP as an HDAC inhibitor restores retinal thickness and vasculature in DR patients and satisfies the necessary safety-good medication criteria. AZP may have a role in the management and prevention of DR.

Background: The dry form AMD (dAMD) has been proven that many could observe a high amount of lipofuscin accumulated between the retinal pigment epithelium cells (RPE) and Bruch’s membrane in the retinal layer. Bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E), the best-characterized lipofuscin fluorophore, has also been reported to lead RPE to autophagy dysfunction. Here, we provided an effective histone deacetylase (HDAC) inhibitor drug, Aza-PBHA (Azp). Due to its potential, we hypothesized that Azp can suppress the blue light-induced accumulation of A2E in dAMD model by regulating the autophagy pathway and accordingly represents the candidate of potential development drug of dAMD.

Purpose: This study aims to evaluate the protective effects of Azp against blue light-induced retinal degeneration and its underlying mechanisms.

Material and Methods: A2E-laden retinal pigment epithelial-19 (ARPE-19) cell damage was used to evaluate the protective effects of Azp on retinal degeneration. MTT assay was used to ensure the impact of Azp in A2E-laden ARPE-19 cells, while the western blot was performed to observe the expressions of autophagy-related marker and investigate the mechanisms of action of Azp. Immunostaining was used to locate the accumulation in the retinal then further evaluate the in vitro and in vivo efficacy.

Results: Azp alleviated the blue light-induced A2E-laden ARPE-19 cell damage, further suppressed the induction of autophagy pathway.

Conclusion: Azp protects against blue light-induced retinal degeneration by modulating A2E-related autophagy pathway.

In the era of advancing technology, our lifestyles are closely linked to electronic devices, particularly the ubiquitous 3C products. An area of concern is the exposure to blue light (BL), known for its higher energy despite a shorter wavelength. Prolonged BL exposure can potentially cause retinal pathology, with geographic atrophy (an advanced form of dry age-related macular degeneration) being a recent focus. This study investigates the mechanism and evaluates the effectiveness of Azatyrosine-Phenylbutyric Hydroxamides (AzP) treatment, assessing its impact on C3 and the membrane attack complex (MAC) expression to understand its regulatory effects on the complement system.

In our in vitro study, AzP demonstrated protective effects on cell growth under BL exposure. Protein imprinting analysis revealed increased C3 expression with prolonged BL exposure, indicating activation of the complement system. However, the addition of AzP slightly reduced the expression of C3, suggesting its regulatory role. Polymerase chain reaction experiments confirmed the AzP-induced decrease in C3 expression, emphasizing its regulatory impact. In animal experiments, AzP treatment alleviated the reduction observed in electroretinography waveforms due to exposure to BL, indicating a restoration of visual function. Retinal histological analysis confirmed a thickness recovery and a structural damage mitigation. These findings underscore the potential of AzP in preventing light-induced retinal damage, indicating its involvement in the complement system.

The widespread use of 3C electronic devices has led to a significant increase in retinopathy cases, particularly in younger populations. Age-related macular degeneration (AMD) is exacerbated by 3C device usage and lacks effective pharmaceutical treatments. Medicinal fungi, with their ease of fermentation, present a promising avenue for pharmaceutical development. This study investigates the potential of Cordyceps cicadae and Hericium erinaceus in preventing or treating AMD.

We examined the oxidative stress markers, reactive oxygen species (ROS), and Nrf2/Keap1 pathway in blue light (BL)-exposed retinal pigment epithelial cells (RPE) and photoreceptor cells (661W). Additionally, C57BL/6 mice, exposed to BL for 12 hours daily over 28 days, served as an AMD model to evaluate the protective effects of Cordyceps cicadae and Hericium erinaceus through oral treatment.

In vitro analyses revealed that extracts from these medicinal fungi significantly attenuated BL-induced ROS production and altered Nrf2/Keap1 signaling pathway in RPE and 661W cells. In vivo studies showed that the extracts from these medical fungi effectively alleviated both functional and structure damage in the retinas of C57BL/6 mice induced by BL.

The findings suggest that the components derived from Medicinal fungi, specifically Cordyceps cicadae and Hericium erinaceus, may serve as potential preventive or therapeutic agents for AMD, particularly as it relates to damage induced by BL. Further research is warranted to fully establish their potential clinical applications.

Soluble epoxide hydrolase (sEH) is an enzyme responsible for metabolizing lipid epoxides into diols, with epoxyeicosatrienoic acids (EETs) among its most well-characterized substrates. Given the antiplatelet activity of EETs, it was hypothesized that inhibiting sEH would decrease platelet reactivity by building up EETs in platelets. Treatment with 14,15-EETs successfully inhibited the aggregatory response of rat and mouse platelets to collagen. However, contrary to expectations, the sEH inhibitor AUDA did not affect aggregation, and there was no discernible difference in reactivity between platelets from sEH-null (Ephx2^-/-) and wild-type (WT) mice. Furthermore, in an arterial thrombosis model, the thrombogenic potential in Ephx2^-/- mice was comparable to that in WT controls. Notably, sEH activity in mouse platelets was relatively low, comprising only 26 or 39% of that in rat or human platelets, respectively, and the ratio of sEH activity in platelets to liver was substantially lower in mice (0.011) compared to rats (0.845) or humans (0.045), indicating significant species differences. Although concurrent treatment with 14,15-EETs and AUDA reduced the response of rat platelets to collagen, such inhibition was not observed in AUDA-treated platelets from Ephx2^-/- mice. In conclusion, sEH inhibition may not suffice to reduce aggregation but could desensitize platelets to aggregatory stimuli. However, this effect is not evident in mouse platelets, likely due to the inherently low activity of sEH. Therefore, the mouse model may not be suitable for studying platelet function in the context of sEH.

Drug repositioning is a strategic approach aimed at discovering new use for approved drugs by exploring their potential for expanded indications. NADPH oxidase (NOX) is an enzyme complex that generates superoxide by transferring one electron from NADPH to molecular oxygen. Among its isoforms, NOX2 stands out as a potential target for inflammatory and thrombotic disorders. In this study, a drug library screening was conducted to identify existing drugs with NOX inhibitory activity. Cell-based assays, specific to NOX1–5, were performed using three different ROS probes such as WST-1, Amplex Red, and coumarin boronic acid. This screening identified LS14 as a potential NOX inhibitor with selectivity for NOX2 over other isoforms. Counter assays confirmed that LS14 is not a non-selective ROS scavenger, and its activity was not attributed to cytotoxicity. In vivo efficacy was assessed using a complete Freund’s adjuvant-induced paw inflammation model and a FeCl3-induced arterial thrombosis model in mice. Oral administration of LS14 effectively reduced paw thickening and ROS formation at a dose of 1 mg/kg, demonstrating comparable efficacy to NOX2 knockout. Additionally, LS14 prolonged vascular occlusion time at the same dose. Moreover, LS14 inhibited collagen-induced platelet aggregation and P-selectin exposure in a concentration-dependent manner. In conclusion, LS14 emerges as a selective NOX2 inhibitor with in vivo efficacy. Further studies hold promise for uncovering additional therapeutic indications for LS14.

Animal-based traditional Chinese medicine (TCM) has a long history of use in treating various health conditions throughout Asia. Despite its widespread use, there is a lack of research on the genotoxic and carcinogenic effects associated with prolonged consumption of these animal-based TCM substances, raising safety concerns. In this study, we aimed to evaluate the genotoxicity and carcinogenicity of animal-based TCM using in vitro assays such as comet assay, micronucleus test, and transformation assay on selected samples. Our results indicate that within the tested experimental concentrations (< 800 μg/mL), the animal-based TCM did not exhibit significant cellular genetic damage in the in vitro CHO-K1 cell micronucleus test and HepG2 cell comet assay. Furthermore, no significant changes in cell proliferation of HepG2 cells and transformation of Bhas 42 cells were observed during the carcinogenicity assessment. Our findings suggest that the animal-based TCM showed no genotoxic or carcinogenic effects in in vitro assays within the tested concentration range (< 800 μg/mL). This provides a toxicological foundation for future clinical applications of animal-based TCM. In conclusion, our study highlights the importance of evaluating the safety profile of animal-based TCMs before they are used for medicinal purposes. Further research is needed to identify specific compounds responsible for genotoxic effects and develop safer alternatives or purification methods for these medicines.

Key words: Animal-Based Traditional Chinese Medicine, Genotoxicity, Carcinogenicity.

Several kinds of nanomaterials have been versatile due to their usefulness, and their application will expand in the future. Therefore, we can be exposed to nanomaterials. However, nanomaterials may have unexpected biological effects due to their small size. Thus, the collection of safety information on nanomaterials is needed. In this regard, the effects of chemicals on the male parents and next generation have been attracting attention recently. Here, we selected silver nanoparticles, one of the most common nanomaterials, as a model nanomaterial to evaluate effects on reproductive tissue and feeding behavior. ICR male mice were orally treated with silver nanoparticles with 10 nm in diameter (nAg10) for two consecutive weeks. During experiment, there was no significant changes of body weight and food intake in nAg10-treated group compared to control group. After the last treatment, silver in blood and testis was analyzed by ICP-MS, and we found that silver was significantly elevated in the group treated with high concentrations of nAg10 compared to the control group. Moreover, the male mice were mated with normal female mice, and the number, weight, and height of offspring were evaluated. As a result, there was no significant differences between the groups. These results suggest that nAg10 can be absorbed intestinally and distributed to the testes, and that nAg10 may have no effect on pregnancy outcome under this administration condition. In the future, we will examine the effects of long-term treatment with nAg10 on pregnancy outcome.

Recently, microplastics (MPs) and nanoplastics (NPs) have been gained attention as environmental problems. MPs are plastic particles with a diameter of 5 mm or less and are present in the environment. They have been detected not only in table salt and drinking water, but also in human blood and placenta. NPs, with a diameter of less than 1 µm, raise concerns about unknown effects on human health due to their tiny size, and knowledge about their biological effects is limited. To understand the toxicity of MPs and NPs, it is necessary to conduct kinetic and hazard analyses using particles with diverse physical properties. In this study, we focused on polyethylene (PE), which is difficult to obtain nano-sized particles and tried to prepared fluorescent labeled PE (nPE) for dynamics analysis. First, green fluorescent nPE was prepared using Qdot, which enables not only fluorescence observation but also quantitative evaluation by measuring the contained zinc. Following a previous study, PE was dissolved in xylene and dropped into DMSO containing Qdots and warmed to 110-115°C to form particles, and then nPE were collected by filtering. The prepared nPE particles smaller than 1 µm were observed by SEM. Fluorescence microscopy also revealed fluorescence signals from nPE. In the future, we will obtain information on the physical properties and attempt to evaluate their kinetics in vitro and in vivo. Furthermore, we aim to clarify the biological effects of NPs in the environment by preparing NPs made of different polymer types and degraded NPs that mimic NPs in the environment.

It is pointed out that the circadian clock has been involved in the resistance to the treatment of cancer immunotherapies. One of the factors is that the circadian clock is closely related to the function of macrophages(Mφ), the starting point of tumor immunity, but no effective countermeasures are currently available. On the other hand, we have established a method to regulate the biological clock by noninvasive microcurrent stimulation (MCS)¹. In this study, we analyzed the effects of MCS on Mφ.

Firstly, we observed cellular changes induced by MCS on Mφ using RAW264.7, suggesting that MCS does not affect Mφ differentiation into M1 or the immune checkpoint, but acts on actin polymerization and increases motility. To evaluate the effect of MCS on the phagocytosis of Mφ, we examined the effect of MCS using fluorescently labeled beads and found that MCS increased bead phagocytosis of RAW264.7.

Secondly, we investigated the detailed mechanism by which MCS increases phagocytosis of Mφ. Using NGS analysis as a starting point, we found that MCS alters the circadian clock system, which decreases the expression rhythm of MCS-related kinase (MRK) and that the decreased expression of MRK leads to an increase in phagocytosis, revealing a series of mechanisms by which the decreased expression of MRK causes the increased phagocytosis.

Finally, the results of MCS on tumor-bearing mouse revealed that MCS exerts its anti-tumor effects by increasing Mφ infiltration into tumors and increasing the tumor phagocytosis of Mφ. The results of this study are expected to lead to the application of MCS as a safe and effective cancer therapy.

The association of specific human leukocyte antigen (HLA) types with the onset of Stevens-Johnson syndrome and toxic epidermal necrolysis has been demonstrated for several drugs. Allopurinol (ALP) and its active metabolite oxyprinol (OXP) have been reported to be associated with HLA-B*58:01 and reactivation of human herpesvirus type 6 (HHV-6) at onset. We have previously found a peptide from 601-780 bp of HHV-6 U54 antigen protein that specifically activates cytotoxic T cells with HLA-B*58:01 upon addition of ALP/OXP. In this study, we investigated experimental conditions for the MAPPs (MHC-associated peptide proteomics) method, which identifies antigenic peptides that bind specifically to HLA.

HEK293 cells expressing HLA-B*58:01 and the co-stimulatory molecule CD80 were transfected with a plasmid containing the 601-780 bp region of HHV-6 U54 for expression and cultured in the presence of OXP. From the lysates of these cells, complexes of HLA molecules and HLA-binding peptides were purified by immunoprecipitation using anti-HLA antibodies. We eluted the complexes from the magnetic beads by acid or thermal elution method. Samples eluted by each method were analyzed and compared by SDS-PAGE and silver staining. In both elution methods, a band around 45 kDa was confirmed, suggesting that the HLA molecules bound to the antibody beads were separated. In addition, only in the thermal elution method, elution of anti-HLA antibodies was observed. These results suggest that acid elution method is appropriate for elution of HLA-peptide complexes by the MAPPs method.

【Introduction】Skin sensitization is important toxicity for the safety assessment of chemicals. Currently, it is need to develop new safety evaluation methods that don’t use animals. We aimed to develop a regression model to predict skin sensitization intensity (EC3) by using toxicity database and machine learning. Moreover, to improve interpretability, we interpreted prediction results using SHAP values. 【Method】We used 154 substances from Guideline (OECD No. 497, 2021) as model building and internal validation, and 72 substances from document (Hoffmann, 2022) as external validation. The regression model was built using XGBoost with EC3 as the objective variable. In vitro tests on adverse outcome pathway (AOP), chemicals information were used as explanatory variables. The applicability domain was set based on the k-nearest neighbor method and in vitro test results. 【Result and Discussion】We calculated the percentage of substances whose predicted values were within 1/5 to 5 times true values within the applicability domain to evaluate the performance of the model. As a result, we found 80% for internal validation and 65% for external validation were within the range. Based on SHAP values, it was confirmed that in vitro test results contributed significantly and positively to the prediction, suggesting that this model takes AOP into account. Using toxicity information and machine learning, we were able to construct a model to predict EC3. In addition, SHAP values allowed us to check the contribution to prediction and to build a model with interpretability.

Macrophages are polarized by various stimuli and produce inflammatory cytokines and reactive oxygen species (ROS). ROS are messengers that propagate inflammation. Conversely, selenium supplied by selenoprotein P (SeP) induces the expression of glutathione peroxidase (GPx), an antioxidant enzyme, and is involved in the termination of inflammation by ROS regulation. We aimed to elucidate the variations of selenium metabolism and its regulation in inflammatory macrophages.

We utilized SeP purified from human plasma and sodium selenite as selenium sources. Mouse macrophage RAW264.7 cells were polarized into an inflammatory state by lipopolysaccharide (LPS) stimuli.

Polarization to inflammatory macrophages suppressed SeP-induced GPx1/4 expression, and the levels of ApoER2 were also low. SeP accumulation was observed due to the inhibition of its degradation. The induction of GPx1/4 expression via sodium selenite treatment was also suppressed, suggesting that the intracellular selenium utilization is inhibited.

We focused on the role of nitric oxide (NO), which is induced in inflammatory macrophages. NO donor (DETA NONOate) stimuli of unpolarized RAW264.7 cells reproduced the reduction of GPx1/4. Through the aBPML assay, developed by our research group to detect selenocysteine (Sec)-specific modifications, we revealed that Sec within SeP is modified by NO. Furthermore, NO inhibited Sec lyase activity, as determined by using the lead complexation method.

These results suggest that selenium utilization is suppressed in inflammatory macrophages, with NO playing a crucial role as the mediator of selenium metabolic remodeling.

Recent advancements in immune checkpoint inhibitors (ICIs) underscore the importance of effectively managing immune-related adverse events (irAEs). This study aimed to systematically monitor the real-world occurrence of irAEs and assess their prognostic implications, including the influence of subsequent steroid therapy.

We retrospectively analyzed 1008 cancer patients treated with ICIs between 2014 and 2021, gathering comprehensive irAE data spanning their onset, management, and clinical outcomes. Of these patients, 458 (45.4%) experienced a total of 670 irAEs. Skin toxicity emerged as the most prevalent, followed by pneumonitis, hypothyroidism, hepatitis, adrenal insufficiency, and colitis.

Univariate Kaplan-Meier analysis showed that patients developing irAEs exhibited significantly prolonged overall survival (OS) compared to those who did not (22.1 months vs. 13.2 months, p < 0.0001). Patients administered steroids at a dosage of <2 mg/kg showed comparable prognoses to those without steroid treatment. However, individuals undergoing steroid pulse therapy, particularly for severe pneumonitis and hepatitis, displayed shorter OS (7.8 months vs. 23.4 months, p = 0.016). Moreover, steroid pulse therapy emerged as an adverse prognostic factor in multivariate analysis (hazard ratio: 2.19, 95% confidence interval: 1.34–2.86, p < 0.001).

In conclusion, prompt steroid intervention for irAEs does not compromise prognosis and should be promptly initiated to mitigate toxicity. Nevertheless, pulse therapy for severe cases constitutes a negative prognostic indicator, emphasizing the importance of early detection in managing such irAEs.

Environmental particles are a growing threat to public health, and several studies point to their ability to induce and enhance lung inflammation. Research has also indicated certain particle traits related to lung inflammation. However, due to the complexity of environmental particle composition, studies addressing differences in how distinct particles with unique properties induce and exacerbate lung inflammation are inadequate. Thus, we aimed to investigate what particle properties induce and enhance lung inflammation using three different types of particles: diesel exhaust particles (DEPs), which contain elemental carbon, organic chemicals, and heavy metals; TiO2, which consists of pure inorganic crystalline particles; and Asian sand dust particles (ASD), which contain quartz particles, microorganisms, and allergenic components. We analyzed various acute and allergic inflammatory markers and observed histologically stained lung sections after four intratracheal administrations of particle-only or particle + ovalbumin (OVA) suspensions every two weeks in a mouse model. Our findings revealed significant acute inflammation in TiO2, ASD, and all particle + OVA groups 24 hours after the first administration. Besides, we observed that allergy indicators increased in all particle + OVA groups after four administrations, indicating a progressive tendency. Thus, the effects of three types of particles on lung inflammation were successfully shown to differ.

The pathogenesis of atopic dermatitis (AD) is multifactorial with genetic, immunological, and environmental aspects. Titanium dioxide, polystyrene, and other substances contained in personal care products (PCPs) can aggravate AD, but the definitive effects of PCPs themselves remain to be elucidated. Exogenous environmental stimuli can promote adaptive immunity as one underlying mechanism exacerbating AD, but the responses of innate immunity have not been fully understood. The present research aimed to study whether commercially available PCPs can affect the progress of AD following topical application and the response of group 2 innate lymphoid cells (ILC2s) in the pathologic condition. After eight days of continuous administration on dorsal ear skin of C57BL/6J mice, AD-like symptoms were observed in the MC903 (calcipotriol)-treated mice, with or without PCP particulates. However, a high concentration of PCP+MC903 mitigated the pathology, with milder erythema and edema. Cytokine thymic stromal lymphopoietin and interleukin-4 were also lowered concurrently. Such results suggested frequent application of the PCP conferred transient suppression against MC903-induced pathogenesis. Conversely, higher transepidermal water loss by PCP+MC903 indicated a more compromised skin barrier function. Also, the percentage of ILC2s among ear tissue lymphocytes was higher as reported in AD patients. These results suggest that although the PCP may stimulate ILC2s in the innate immune system, generally it has bifunctional effects that suppress and exacerbate skin immune responses.

Introduction: Chronic kidney disease (CKD) is associated with several complications, especially heart failure (HF) which is a major cause of death. However, there is no effective treatment to control it. In contrast, we revealed that G protein coupled receptor 68 (GPR68), which is highly expressed on macrophages, exacerbates cardiac inflammation and fibrosis in CKD. In this study, we explored GPR68 inhibitors to prevent or improve CKD associated HF¹ .

Method: We established an evaluation system in which GPR68 activity can be detected by luciferase activity using MDA-MB-231 cells and used High throughput screening (HTS) to search for compounds with human GPR68 inhibitory activity.

We used the 5/6 nephrectomy (5/6Nx) ICR mouse as CKD mouse model.

Result: We used HTS dependent on nuclear factor of activated T cells response element luciferase activity and toxicity test to screen for GPR68 inhibitors. Then, we identified the GPR68 inhibitory extract which significantly suppressed inflammatory cytokines in RAW264.7 with GPR68 high expression. Afterwards, we analyzed pathology of 5/6Nx mice administrated the extracts water. As result, the extracts significantly not only suppressed upregulation of cytokines, fibrosis and BNP levels, but also improved weight loss and survival rate in 5/6Nx mice. Furthermore, we identified the inhibitory compound among the alkaloids from extracts and this compound had anti-inflammatory effects as well as extracts. Finally, we found out the extracts and compound suppressed inflammatory cytokines and cardiac fibrosis via inhibition of GPR68 in CKD.

1. Sakugawa M, et al., Trans Res., 2024

In drug discovery, it is extremely difficult to identify hit compounds. To do this efficiently, there are structure generators that automatically generate the structural formula of a compound with the desired activity. However, the previous methods can only consider one target molecule or chemical property. Recently, structure generators that utilize gene expression profiles, which can consider exhaustive cellular responses, have attracted much attention. However, none of the existing methods considers the safety of the compound. In this study, we propose a method for generating the structure of drug candidate compounds based on gene expression profiles, considering not only the desired target activity but also toxicity. We focused on hepatotoxicity. First, we extracted hepatotoxicity-specific genes by comparing the compound-responsive gene expression profiles of compounds that exhibit and do not exhibit hepatotoxicity. Next, we designed a gene expression profile that counteracts the expression pattern of genes involved in hepatotoxicity included in the drug target perturbation response gene expression profile. Finally, the designed gene expression profile was input into a structure generator. As a case study, we applied our proposed method to a kinase, a drug target for cancers, and generated the structures of new inhibitor candidate compounds. Compounds generated with consideration of toxicity were structurally similar to approved drugs, and they are structurally dissimilar to known toxic compounds. These results suggest the usefulness of the proposed method.

Results of nonclinical toxicity studies play an important role in risk management for subjects in clinical trials. It is important to verify and improve the predictability of adverse events through toxicity studies to minimize risks to subjects.

Adverse events in the GI tract, such as diarrhea and constipation, are observed frequently in clinical trials. The GI events in clinical area are known to decrease medication adherence. In this presentation, we will report the results of a survey on the predictability of toxicity studies for adverse events in the GI tract, targeting recently approved small molecule drugs (anti-neoplastic drugs, antidiabetic drugs, etc.).

Based on the approval information, we have selected new drugs and we extracted GI adverse events related to the investigational drug from CTD, interview forms, RMP materials, etc. And we investigated whether similar findings had been observed in the toxicity studies.

In the results, diarrhea or constipation were observed as adverse events affecting the GI tract in the clinical. In the toxicity studies, findings related to these adverse events were observed in the many drugs, regardless of indication. The results showed that diarrhea and constipation were highly predictive. And the results were similar to previous reports that toxicity studies for small molecule drugs were highly predictive of diarrhea and constipation. The reasons for this high predictability are thought to be that there are no major species differences in the physiological functions of the GI tract, and that the dose settings for toxicity studies were optimized.

【Objective】

QSAR prediction helps avoid costs and delays associated in Ames tests for new compounds. We developed DeepSnap-DL, a QSAR technique using 3D compound images for deep learning. This study sought to enhance prediction accuracy by integrating DeepSnap-DL with traditional descriptor-based models using ensemble and consensus methods for Ames models.

【Methods】

This study used Ames test results sourced from the mutagen by MHLW and the Hansen data set. For the descriptor method, we developed a lightGBM-based model using molecular descriptors from MOE. The ensemble method built models similar to the approach of the descriptor method, using the prediction probability of DeepSnap-DL and molecular descriptors. The consensus method involved building a model with an applicability domain for compounds in which DeepSnap-DL and descriptor predictions matched. We assessed generalization performance with a 20% external test set, utilizing metrics like balanced accuracy and MCC.

【Results and Discussion】

An Ames test data set comprising 6950 compounds (positive/negative ratio: 1.31/1) was assembled. Compared to the descriptor method, no improvement was observed in prediction performance between the DeepSnap-DL and ensemble methods. Yet, the consensus method showcased superior generalization performance among all prediction models. These results suggest that the prediction model’s applicability domain, established by the consensus method (i.e., the group of compounds for which the descriptor and DeepSnap-DL methods predictions coincide), is useful for QSAR prediction of the Ames test.

Pregnant women and fetuses are vulnerable to chemicals, and some reports said that chemicals exposure increases the risk of miscarriage and premature birth. The healthy fetal growth needs the proper development of placenta through syncytialization. Thus, functional and structural abnormalities in the placenta caused by chemicals could lead to adverse pregnancy outcomes, and there is a need for reproductive and developmental toxicity evaluation focusing on the placenta. However, there is still a paucity of research evaluating reproductive and developmental toxicity caused by chemicals from the perspective of placental function. Here, we selected Valproic acid (VPA) and Fluorouracil (5-FU) as model chemicals, which have been reported to induce birth defects in ICH-S5 and evaluated the effects on the syncytialization process. Human choriocarcinoma cell line (BeWo) was treated with forskolin, an inducer of syncytialization, and each chemical for 48 hours, and cell morphology was assessed by immunofluorescence and expression levels of the marker CGB and its upstream factor ERVFRD-1 by real-time RT-PCR. VPA suppressed the elevation of syncytialization rate and CGB and ERVFRD-1 expression in forskolin only treated group, suggesting that VPA inhibited syncytialization in BeWo. Although 5-FU did not change the syncytialization ratio, gene expression was enhanced compared to forskolin only treated group, indicating that 5-FU may cause placental dysfunction. Now, we try to clarify the mechanism of syncytial abnormalities by comprehensively analyzing the expression-variable molecules.

During the process of placental development, syncytialization leads to structural and functional maturation of the placenta, and then placenta can exchange gas and nutrients between fetus and mother and produce placental hormones necessary for pregnancy maintenance. Several amino acid transporters are known to contribute to placental syncytialization, and inhibiting their expression could suppress the progression of syncytialization. In this regard, our previous study showed that valproic acid, which can cause congenital abnormalities, reduced the expression of SLC6A19, encoding the neutral amino acid transporter B0AT1, in the Human choriocarcinoma cell line BeWo. However, the involvement of SLC6A19 in placental syncytialization has not been cleared. Here, we tried to clarify the role of SLC6A19 in placental syncytialization. BeWo, which was knocked down SLC6A19 by shRNA, was treated with forskolin to induce syncytization, and the expression level of human chorionic gonadotropin β (hCGβ, gene; CGB) was analyzed by real-time RT-PCR. CGB expression was significantly reduced in cells knocked down SLC6A19 compared to control cells. Moreover, immunohistochemical staining showed that the ratio of hCGβ-positive cells in SLC6A19 knockdown BeWo cells was decreased relative to control cells. These results suggest that SLC6A19 might be involved in placental syncytialization. Now, to reveal its mechanism, we investigate the relationship between SLC6A19 suppression and the cAMP/PKA signaling pathway, which is important for placental syncytialization.

ADHD is a developmental disorder that manifests as a complex combination of genetic predisposition, neurotransmitter abnormalities, and environmental factors. The prevalence of ADHD in adults is estimated to be about 5%, and the number of patients receiving pharmacologic treatment is increasing. Methylphenidate (MPH) is used worldwide as a first-line treatment for ADHD. Recently, environmental factors such as smoking have been reported to alter the sperm epigenome and affect the phenotype in next generation. We have previously shown that paternal MPH exposure increases ADHD-like impulsivity in offspring of ICR mice, but the underlay mechanism is obscure. Therefore, we used inbred Balb/c mice to examine the effects of MPH on the sperm epigenome and on the behavior and gene expression in the next generation.

Sires (F0) were subcutaneously administered MPH for 28 days and mated with naive dams. Developmental assessments of offspring (F1) were performed at 4-21 days of age. Upon reaching 4.5 weeks of age, F1 were subjected to behavioral tests such as elevated plus-maze (EPM) test and RNA-seq of the brain were performed. Sperm were collected from F0 after mating for epigenomic analysis.

MPH administration to F0 had no significant effect on F1 development. However, EPM test showed that MPH administration to F0 caused F1 to acquire impulsivity. Paternal MPH exposure in male Balb/c mice heightened impulsivity in the F1, as similar to ICR mice. Results from the RNA-seq analysis of F1 brain and epigenome analysis of F0 sperm will be reported along with the findings mentioned above.

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. we hypothesized that developmental toxicity eventually relates to signal disruption, and thus established a signal reporter assay (DynaLux/c). 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. However, chemiluminescence was　measured manually in this method. Thereby, it was difficult to capture detailed temporal changes and to measure during the night. Therefore, we automated luminescence measurements, establishing a method enabling detailed and prolonged luminescence assessments. The continuous monitoring of chemiluminescence reveals that there were two peaks in 72 hours, unlike the single peak in 24 hours. Moreover, substances with developmental toxicity causing expanded disruption after 24 hours, such as valproic acid, were more accurately detected. The automation of luminescence measurements demonstrated the potential for more precise tracking of signal disruption. Future efforts will focus on optimizing measurement conditions and increasing the number of test substances to further elucidate the utility of this method.

【Objective】

The impact of drug treatments on pregnant women and their fetuses is unclear due to ethical constraints in clinical trials. Therefore, VigiBase was used to perform a comprehensive evaluation of possible adverse drug effects on the fetus to determine risk avoidance measures.

【Methods】

Cases with reported routes of administration listed in VigiBase were selected for analysis. Utilizing MedDRAver25.1, we included adverse terms encompassing topics such as “foetal disorders,” which are classified under the pregnancy and newborn topic. To exclude the influence of adverse events resulting from postnatal medication, we defined side effect terms associated with cases in which the administration route was transplacental as developmental toxicity-related side effect terms. To identify trends in developmental toxicity, we calculated the reported odds ratio and the P value using Fisher’s exact test for all listed drugs.

【Results and Discussion】

Our analysis reveals significant indicators of developmental toxicity in drugs like valproic acid, carbamazepine, abacavir, and others. These drugs have the potential for causing developmental toxicity, as documented in their package inserts. According to the ATC classification, antiepileptics, thyroid therapies, and antivirals for systemic use tended to induce toxicity. We also found that various drugs with no reported developmental toxicity in their package inserts were suspected to cause such toxicity. These findings are expected to assist in determining whether or not to administer certain medications to pregnant women.

Exposure to various environmental pollutants during pregnancy commonly results in fetal blood flow abnormalities. It is essential to evaluate the alterations in brain development caused by fetal blood flow insufficiency. GABAergic interneurons are critically implicated, and their disruption is associated with neurodevelopmental diseases. However, no research investigates impacts of fetal blood flow insufficiency on GABAergic interneurons. The present study aimed to investigate the effects of fetal blood flow insufficiency on GABAergic interneurons. On the gestational day 17, constrictors were applied to the uterine and ovarian arteries. Cerebral cortex was collected from pups at postnatal days 7, 14, and 21 and assessed by flow cytometry. Compared with the sham operation group, significant reduction in GABAergic interneurons (GAD1-positive cells) was observed in the blood flow insufficiency group. Further analysis into the subtypes of GABAergic interneurons, parvalbumin (PV), and somatostatin (SST) positive cells, also showed a significant decline in the proportion of PV and SST positive neurons due to blood flow insufficiency. Normally, the count of GABAergic interneurons, including PV and SST positive neurons, increases with brain development, but this increment was lesser in the blood flow insufficiency group compared to the sham operation group. These findings suggest that blood flow insufficiency during perinatal periods disrupts the balance of brain neurons, contributing to the cross-sectional understanding of the developmental neurotoxicity of environmental pollutants that cause fetal blood flow insufficiency.