Introduction: Reactive sulfur species (RSS), such as cysteine persulfide (CysSSH) and glutathione persulfide (GSSH), exhibit high antioxidative property, playing a crucial role in suppressing oxidative stress. The gastrointestinal tract serves as a major site for sulfur metabolism, with gut bacteria contributing to this process. However, it is unclear if commensal gut bacteria produce RSS. We hypothesized that RSS produced by gut bacteria influence the host's antioxidant capacity by contributing to the systemic RSS pool and also serve to protect the gut microbiota from oxidative stress.

Results: The systemic levels of RSS were lower in germ-free mice compared to SPF mice. Gut bacteria enzymatically produced CysSSH from cystine. The plasma RSS level was increased in mice given cystine, which was strongly suppressed by gut microbiota depletion. Single bacterial cultures showed high CysSSH production capacity in Lachnospiraceae and Ruminococcaceae families. Administration of cystine protected mice from concanavalin-A-induced oxidative liver injury in a gut-microbiota-dependent manner. Furthermore, RSS-donor increased RSS levels in the bacteria with low CysSSH production.

Discussion and Conclusion: We identified gut microbes that produce CysSSH from cystine, thereby influencing the systemic RSS level in the host. Our study proposes new mechanisms by which gut-microbiota-derived metabolites contribute to enhancing host antioxidant capacity. Additionally, RSS may enhance antioxidative capacity in bacteria, suggesting a novel gut bacterial interaction facilitated by RSS.

Photodynamic therapy (PDT) is a treatment that uses a laser to irradiate tumor-affinity photosensitizer that have accumulated in tumor tissue. We have previously demonstrated that PDT using a photosensitizer sodium talaporfin (TS-PDT) has an antitumor effect on glioblastoma multiforme (GBM), the most common and aggressive malignant brain tumor. Recently, we found that retinol, one of the retinoids, enhances the cytotoxic effect of anticancer drugs against GBM. In this study, we investigated the antitumor effect of retinol in combination with TS-PDT on GBM, with the aim of developing a more effective treatment for GBM. We found that retinol at a non-cytotoxic concentration enhanced the cytotoxic effect of TS-PDT in U251 cells. In addition, retinoic acid, the active metabolite of retinol, enhanced the cytotoxic effect of TS-PDT. Therefore, retinoids may potentiate the cytotoxic effect of TS-PDT on GBM. Furthermore, retinol and retinoic acid also increased the cytotoxic effects of TS-PDT in T98G cells, which are more drug-resistant GBM cells than U251 cells. These results suggest that retinoids may be potential candidates for combination drugs to enhance the antitumor effect of TS-PDT on human GBM.

Reactive oxygen species (ROS) can react with highly unsaturated fatty acids (HUFAs) of membrane phospholipids to induce lipid peroxidation. Thus, HUFA-containing phospholipids levels might be correlated with sensitivity to ROS-induced cell death. Acyl-CoA synthetase long-chain family members (ACSLs) catalyze the synthesis of long-chain acyl-CoA from long-chain fatty acids. Among five ACSL isozymes, ACSL4 prefers HUFAs as its substrate and thus plays an important role in maintaining HUFA-containing phospholipids levels. We recently reported that the levels of HUFA-containing phospholipids decreased in lungs from ACSL4-deficient (KO) mice and that ACSL4 deficiency attenuated both paraquat- and methotrexate-induced lung injury via the suppression of lipid peroxidation. In this study, we investigated the role of ACSL4 in bleomycin-induced pulmonary fibrosis. We revealed that after administration of bleomycin, the level of phosphatidylcholine (16:0/20:4)-derived peroxide was increased in wild type (WT) mice, the increase was significantly suppressed in KO mice. Similarly, the levels of fibrosis markers such as hydroxyproline and α-SMA, and the gene expression of E-cadherin and IGF-1 were increased in WT mice, but these increases were suppressed in KO mice. Furthermore, the oxygen saturation was decreased in WT mice from day 14 after administration, but maintained at a high level in KO mice. These results suggested that bleomycin-induced inflammatory and pulmonary fibrosis were also attenuated by ACSL4 deficiency.

The purpose of this study was to determine the effect of hydrogen water intake on exercise endurance in mice, with a focus on the antioxidant properties of hydrogen and its potential to improve metabolism.

In the study, 8-week-old male C57BL/6J wild-type mice were divided into two groups: one given distilled water and the other given hydrogen water. We used the treadmill all-out test to examine the physical endurance capacity of the mice. We also measured muscle damage indicators, oxidative stress markers, and innate immune response（NF-κB） in the tibialis anterior muscle using blood biochemical analysis and Western blotting techniques. In addition, AMPK activation, levels of the lipase ATGL, and expression of CPT1b, a key enzyme in β-oxidation, were analyzed.

The results showed that after 4 weeks, mice consuming hydrogen water had significantly improved endurance capacity compared to the control group. These mice also showed a reduction in markers of muscle damage, a decrease in oxidative stress, and a suppression of NF-κB. Importantly, the hydrogen group also showed reduced body fat percentage, activated AMPK, and lower levels of blood-free fatty acids with increases in CPT1b and ATGL levels.

These results suggest that long-term intake of hydrogen water could potentially improve exercise endurance by attenuating oxidative damage and inflammation in skeletal muscle through the antioxidant effects of hydrogen. In addition, the study highlights the role of hydrogen water in enhancing fatty acid utilization, which could contribute to improved lipid metabolism in exercise.

Cardiac remodeling involves compensatory alterations in heart mass, geometry, and function in response to hemodynamic stress or cardiac injury. Previous studies have attributed myocardial variations to the size of cardiomyocytes. Recently, supersulfide, a sulfur-catenated molecule, has emerged as a crucial regulator of cardiac robustness. Our earlier findings revealed a heightened presence of supersulfide in healthy mouse hearts, which undergoes catabolism to hydrogen sulfide (H2S) following myocardial infarction. Despite these observations, the precise role of supersulfide metabolism in governing cardiac cellular functions remains elusive. In this study, we use cardiomyocytes isolated from ventricular of neonatal rat cardiomyocytes (NRCMs). We found that supersulfide, but not H2S, positively regulates the size of cardiomyocytes. qPCR results elucidated supersulfide anabolism related gene, Solute carrier family 7 member 11 (Slc7A11), may have involved in regulating size of cardiomyocytes. Slc7A11 gene knockdown efficiently induced cardiomyocyte atrophy. These findings suggest that supersulfide plays a key role in regulating cardiac cell remodeling induced by receptor stimulation. The anabolism and catabolism of supersulfide in cardiac cells could provide a new strategy for the treatment of pathological cardiac remodeling.

Calcium ions (Ca2+) and reactive oxygen species (ROS) serve as pivotal signaling molecules in diverse vascular processes, including contraction and proliferation. However, the precise mechanism through which ROS modulate Ca2+ signaling in vascular smooth muscle cells (VSMCs) remains largely unexplored. This study delved into the contribution of mitochondrial ROS to Ca2+ transients in the context of angiotensin II (Ang II) signaling. Ang II was observed to induce a predictable elevation in both Ca2+ levels and ROS generation in rat VSMCs. Notably, Ang II rapidly triggered mitochondrial fission, a process inhibited by both a Ca2+ chelator and intracellular Ca2+ depletion, indicating its dependency on Ca2+ signal. Ang II prompted the formation of mitochondrial ROS detected by mitoSOX and HyPer7-MLS. Treatment with mitochondrial ROS scavengers, such as mitoTEMPO and mitoQ, attenuated Ca2+ transients and mitochondrial fission, implicating mitochondrial ROS in these processes. Further exploration revealed that Ang II activated dynamin-related protein 1 (Drp1), leading to its translocation from the cytosol to the mitochondria, and inhibition of Drp1 with Mdivi-1 mitigated mitochondrial ROS production and Ca2+ elevation. Indeed, interventions targeting Drp1 or mitochondrial ROS attenuated vascular contraction and acute elevation of blood pressure induced by Ang II. In conclusion, mitochondrial ROS and Drp1 play crucial roles in Ang II-induced Ca2+ signaling in VSMCs, thereby modulating vascular contraction and blood pressure regulation.

Long-term exposure to inorganic arsenic causes not only skin symptoms but also various toxicities, such as anemia. However, the impacts of low levels of inorganic arsenic have not yet been elucidated. We reported that exposure to low levels of arsenate, a pentavalent arsenic, affected the production of erythropoietin (EPO) in HepG2 cells. This study investigates the impact of low-level long-term arsenate exposure on EPO production both in vitro and in vivo. In vitro experiments involved subculturing HepG2 cells for three weeks and treating them with arsenate at levels ranging from 1–10 µM. Compared to untreated cells, EPO production and responsiveness to EPO production stimuli were reduced. In the in vivo experiments, mice were exposed to arsenate at a dose of 60 nmol of arsenate per day for two months. Compared to untreated mice, treated mice showed decreased EPO mRNA levels, although there were no differences in body weight or hematocrit. The stimulating impact of EPO production on liver and kidney cortical cells recovered from mice was significantly reduced in arsenate-treated mice. In HepG2 cells treated with arsenate, while ROS production increased, the response to remove oxidative stress was also enhanced, suggesting that the promotion of EPO production by ROS production was attenuated. There is a possibility that long-term low-level arsenate exposure may not generally be harmful, but it might cause anemia and other health problems by preventing the body from producing enough EPO.

【Background】

Linezolid (LZD) can cause thrombocytopenia associated with high LZD plasma concentrations due to renal dysfunction, etc. This study aimed to clarify whether the rate of achieving LZD concentrations that avoid the toxicodynamic target, that is, the safety target achievement rate, can predict the onset of thrombocytopenia.

【Methods】

Subjects were adult patients hospitalized at Shimane University Hospital, who received LZD treatment. Based on predicted two PK parameters (Parameters A and B) for each patient, the safety target achievement rate (LZD trough concentration of less than 8 μg/mL, at which the risk of thrombocytopenia is low) was calculated. Univariate, multivariate and receiver operating characteristic (ROC) analyses were performed to assess the relationship between patient characteristics including the safety target achievement rate and the onset of thrombocytopenia.

【Results and Discussion】

Patients (n = 77) aged 72 ± 11 years old and weighing 56.7 ± 10.9 kg, with a creatinine clearance of 60.5 ± 47.2 mL/min, were analyzed. A multivariate analysis of several factors stratified with the cutoff values obtained by ROC analysis revealed that the duration of LZD therapy and the safety target achievement rate were significant factors (Parameter A and B: odds ratios for the duration of LZD therapy were 7.436 and 4.712; odds ratios for the safety target achievement rate were 0.060 and 0.167, respectively).

【Conclusion】

The risk of LZD-induced thrombocytopenia, which increased with the duration of LZD therapy, may be predicted using the safety target achievement rate.

【Purpose】

Vancomycin(VCM) is a trump for the infection in a hospital. However, VCM has a huge clinical problem in that it causes acute kidney injury (AKI) frequently though, the detailed mechanism of developing VCM-induced AKI has not yet been elucidated. Circadian rhythms are observed in biological function, so administration focused on administration time suppresses side effects. Therefore, we carried out an analysis of whether circadian machinery influences VCM-induced AKI.

【Methods】

Each analysis uses the Uni-nephrectomized (UNx) mice and RAW264.7 cell, mice macrophage-like cells.

【Results and Conclusion】

The kidney function of UNx mice VCM-administered at 9 A.M. only decreased in intravenous administration at 9 A.M. or 9 P.M. To investigate the cause, an omics analysis of the kidney after VCM administration was conducted, as a result, the involvement of myeloid leukocytes was suggested. Then, when the cell population in the kidney was measured using flow cytometry, there was an administration time-dependent difference in the number of macrophages. Besides, depleting macrophages using liposomes including clodronate improved kidney function in administration at 9 A.M. After synchronizing clock gene expression by dexamethasone (DEX shock) in RAW264.7 cells, the cells were stimulated by VCM, and then inflammatory cytokines mRNA level was different depending on the elapsed time after DEX shock. Our study is expected to lead to the optimization of the therapy by VCM focused on circadian rhythms and the therapy for VCM-induced AKI targeting monocytes/macrophages.

[Backgrounds] Benzimidazoles are a synthetic opioid subclass recently emerging on the illicit drug market. Although we have found opioid-related behavioral toxicities of some benzimidazoles in mice, their detailed mechanism of action and pharmacokinetics, especially in the brain, are still unknown. This study aimed to reveal the distribution of a benzimidazole analog, metonitazene (MNZ), in mouse brains. [Methods] Eight-week-old male C57BL/6J mice were i.p. injected with MNZ at 10 mg/kg and sequentially sacrificed at 0, 5, 15, 60, 90, 240, and 480 min after the injection. While the sera and portions of the brains were analyzed by LC-MS/MS, the remaining brains were dissected and prepared for sagittal cryosections, followed by image analysis using DESI-MSI. [Results & Discussion] MNZ concentrations in the sera and brains were highest at 5 and 15 min after injection, respectively, with peak values of 1.13 µg/mL and 2.63 µg/g brain, and then rapidly decreased. In the DESI-MSI analysis, MNZ signals visualized with a product ion at m/z 100.11 were observed throughout the brain, but strong signals were detected in the brainstem (midbrain, pons, and medulla), thalamus, and hypothalamus, which are the regions where μ–opioid receptors localize and regulate analgesia, respiratory depression, and the formation of drug dependence. Combined with our previous studies with behavioral observation tests and a conditioned place preference test, these results strongly suggest that the induction of neurological toxicities of MNZ in mice is indeed mediated by μ–opioid receptors.

The kidney is involved in drug excretion, where characteristic secretion and reabsorption of drugs occur in the proximal tubules. However, drug concentrations increase easily inside tubular cells, leading to much toxicity. Sex differences are known to exist in cisplatin-induced nephrotoxicity and pioglitazone-induced edema, but the detailed mechanisms remain unclear. Many drugs are excreted or reabsorbed by transporters, and membrane proteins such as transporters are difficult to comprehensively analyze for protein expression levels. Therefore, we aimed to elucidate the mechanisms underlying sex differences in side effects in the kidney by utilizing membrane proteomics, a method specialized in membrane proteins established by our collaborators.

Because sex differences are influenced by sex hormones and sex chromosomes, we used sex reversed (FCG) mice that can distinguish between two effects. Brush border membrane vesicles from the kidneys of FCG mice were purified, and underwent membrane proteomics, obtaining expression profiles. Next, total RNA was purified from the kidneys, and gene transcription profiles were obtained through microarray analysis, identifying molecules showing sex differences derived from sex gonads and sex chromosomes. Two sex-differentiated transporters involved in cisplatin transport were found. These two transporters have sex chromosome- and gonadal-derived sex differences, which could explain the clinical studied reporting higher mortality from cisplatin-induced nephrotoxicity in males and a reversal of mortality in older patients.

【Background/Purpose】

Drug-induced liver injury (DILI) is a major cause of discontinuation of clinical trials and withdrawal after marketing; it is necessary to evaluate drugs with DILI risk in the preclinical stage. CDM-Y, newly synthesized as a cccDNA modulator, is a novel orally administrable hepatitis B drug candidate with IFNα-like activity. CDM-Y-induced cell death was observed when its compound was tested using BAtox, detecting a bile acid-dependent toxicity developed by our laboratory. Therefore, this study aimed to analyzed the cell death mechanism of CDM-Y-induced BAtox.

【Method/Results】

We used HepaSH cells isolated from human liver chimeric mice, in which HepaSH cells have similar hepatocyte function compared to primary human hepatocytes. We confirmed that CDM-Y did not inhibit BSEP function and bile acid amidation process. When we examined various CDM compounds, CDM-Y had the highest pharmacological activity and induced BAtox more than other CDM compounds. So, we focused on its pharmacological effects. After knocking down Tyrosine kinase 2 (TYK2) and Janus kinase 1 (JAK1) with siRNA system in HepaSH cells, CDM-Y-induced BAtox was increased under siTYK2 condition, while it was attenuated under siJAK1 condition. Furthermore, when we evaluated the phosphorylation level of TYK2 and JAK1, JAK1 was only phosphorylated.

【Conclusion】

Ther present study suggested that the CDM-Y-induced BAtox was caused by JAK1 phosphorylation. Based on these results, it is necessary to create CDM-Y derivatives by considering the signal balance between JAK1 and TYK2.

To elucidate the mechanism of renal injury induced by the cisplatin (CDDP), we used immortalized cells (S1, S2, S3 cells) derived from mouse proximal tubule S1, S2, and S3 segments. We found that S3 cells are sensitive to CDDP than S1 and S2 cells. We established CDDP-resistance cells (Cis-R cells) by gradually exposing S3 cells to CDDP from low concentrations, and these Cis-R cells acquired an approximately 14-fold resistance to CDDP. In this study, we aimed to elucidate the mechanism involved in the vulnerability of S3 cells to CDDP by comparing between S3 cells and Cis-R cells. Comparison of intracellular Pt concentrations in S3 and Cis-R cells after addition of CDDP showed higher concentrations in Cis-R cells, whereas the amounts of Pt in DNA after addition of CDDP was lower in Cis-R cells. CDDP is known to bind proteins, therefore we hypothesized that decoy proteins, which capture CDDP in the cytoplasm and thus mitigate its toxicity, might be highly expressed in Cis-R cells. Using a microarray, we extracted a group of genes that were highly expressed in mRNA levels in Cis-R cells compared to control cells, and found that selenoprotein P (SeP), a selenocysteine-containing protein that has been reported to bind to CDDP. was highly expressed in Cis-R cells. We detected that addition of selenite to S3 cells induced intracellular SeP and suppressed CDDP-induced cytotoxicity. Furthermore, inhibitory effects of senite on CDDP cytotoxicity was abolished by knockdown of SeP. These results suggested the involvement of SeP as one of the factors contributing to resistance in Cis-R cells.

[Background] From the point of view of sustainability, economic efficiency, and throughput, in vitro toxicity tests are growing in importance. In most cases, test substances are dissolved in water. However, safety test of some substances is difficult due to their insolubility. We therefore explored novel solvents with high biocompatibility and found that Zwitterionic Ionic Liquids (ZIL) enhanced the solubility for certain food-related substances such as ellagic acid and showed lower toxicity compared to DMSO. This study aimed to find the benefits and limitations using ZIL in safety testing.

[Methods] Ellagic acid was dissolved in 1% DMSO, 1% or 2% ZIL, then treated for 24 hours on HepG2 cells at 0, 30, 100, 300, and 600 μM to measure cell viability. Drug interactions were surveyed using human liver-derived microsomes and human hepatocytes for Cytochrome P450 (CYP) inhibitory tests. Additionally, CYP induction tests on human hepatocytes and P-glycoprotein (P-gp) inhibitory tests on Caco-2 cells were conducted.

[Results] Co-treatment with ZIL alleviated ellagic acid-induced cytotoxicity and improved hazard predictivity for hepatotoxicity. ZIL expressed inhibitory effects on all CYP isozymes tested and exhibited inducing effects on all CYP isozymes tested. No P-gp inhibitory activity was observed.

[Conclusion] As a novel solvent for food-related substances, ZIL was found to be beneficial in terms of improved safety and solubility, with enhanced safety testing performance for low solubility substances. Attention to impact on metabolic activity is, however, necessary when using ZIL in tests with CYP metabolic activities.

[Purpose] Vascular irritation is one of the safety risks in the development of injectable medicine. There are several methods to evaluate vascular tolerance in non-clinical settings. However, there is no standardized method as an in vitro testing system. In this study, we constructed a testing system for vascular irritation more quickly and conveniently in vitro, using the trans-endothelial electrical resistance (TEER) value in human umbilical vein endothelial cells (HUVEC) as an index, and examined its usefulness.

[Method] After seeding HUVEC in a Transwell, we continuously measured TEER value and incubated it until the TEER value reached a plateau. After removing the culture medium, we added a solution mixed with tested solvent and the culture medium and measured the TEER value. As tested solvents, we used solvents that were irritant or no-irritant in in-house general toxicity tests or local tolerance tests by the rabbit's posterior auricular vein. In addition, Pasil and Acyclovir were used as standard formulations. The evaluation of the TEER value was performed one hour after adding the solution because the assumed administration time of the tested solvent is one hour.

[Results/Discussion] A correlation was observed between in vivo vascular irritation and decreases in TEER value, indicating that this evaluation system may be useful as a system for simply evaluating vascular tolerance in vivo. This in vitro system is expected to contribute to the 3Rs by evaluating vascular tolerance in place of in vivo tests.

Information obtained from biodistribution tests conducted in non-clinical studies is important in the development of regenerative medicine products.

Some evaluation methods have been reported for detecting administered human cells, including quantitative PCR (qPCR), flow cytometry (FCM), and immunohistochemical staining (IHC). Since each product has its strengths and weaknesses, it is necessary to develop an optimal evaluation method for each product.

In a previous study, we measured human mesenchymal stem cells (hMSCs) in the blood of mice using qPCR and FCM methods. As a result, differences in hemodynamics were observed depending on the time elapsed after administration, suggesting that differences in measurement methods may have an effect.

In this study, we developed qPCR and IHC analysis methods to evaluate the distribution in mouse tissues, and compared the bio-distribution of hMSCs 30 minutes after administration.

In the qPCR method, the number of hMSC cells was calculated using the standard curve method and compared between each tissue, and 3931 to 4530 hMSC cells were detected in the lungs.

In the IHC method, immunostaining using anti-HLA (class 1 A, B, C) antibodies was performed on the lungs, liver, spleen, cerebrum and cerebellum, kidneys, and heart, and HLA-positive cells were found in the blood vessels of the lungs. It was seen here and there.

There is little knowledge of evaluating the same sample using different measurement methods, and it is hoped that the results of this study will help in selecting evaluation systems when developing regenerative medicine products.

[Background] Gut microbiome (GM) is closely related to the host in many aspects. In addition, GM is sensitive to changes in the gut environment and rapidly changes their composition depending on what host animals ingest. By focusing on this sensitivity and analyzing changes in the GM, this study aims to evaluate the effects of environmental toxicants (ETs) on the host. We administered each ET to chickens (Julia, 6-weeks-old) and analyzed changes in the GM.

[Methods] Chickens were divided into 6 groups (DW, corn oil, lead, imidacloprid, warfarin, and nanoplastic) and each group was administered orally one time. The animals were kept in isolation for one day prior to administration, and fresh feces were collected daily until three days after administration. Bacterial DNA was extracted from each feces and analyzed using a next generation sequencer targeting the V3-V4 region of 16S rRNA.

[Results and Discussion] Significant reductions in alpha diversity were observed in the lead, imidacloprid, and warfarin groups. The relative abundance of Lachnospirales and Oscillospirales increased in the lead group. In the imidacloprid group, the composition of the main flora did not differ from that of the control group, but the number of bacterial species decreased from the day of administration and was comparable to that before administration 3 days later. The warfarin group also showed a decrease in the number of bacterial species, which did not recover even after 3 days. As described above, characteristic fluctuations in GM were observed for each ET even after a single administration.

Introduction: Chronic kidney disease (CKD) poses a significant risk to cardiovascular health and life expectancy. Uremic substances such as indoxyl sulfate (IS) are implicated in this complication, being elevated in CKD patients due to intestinal dysbiosis and resistant to hemodialysis¹. Previous research has highlighted the role of vitamin A (VA) accumulation in increased uremic substance production, yet the precise mechanism remains elusive. This study aims to explore role of VA in immune maturation and investigate the mechanism behind enhanced production of intestinal bacteria-derived uremic substances.

Method: CKD mice (5/6 Nephrectomy: 5/6Nx) were established by removing 5/6ths of the kidneys from ICR mice and raised for 8 weeks. A sham group served as a control for comparison.

Results: VA-free feeding prevented abnormal immunoglobulin A (IgA) secretion and accumulation of uremic substances, including IS, in 5/6Nx mice, indicating VA accumulation disrupts intestinal immunity. Analysis of intestinal IgA-producing cells revealed increased retinoic acid secretion by dendritic cells in Peyer's patches of 5/6Nx mice, promoting germinal center reactions and IgA-producing cell differentiation. Inhibitors targeting this pathway reduced IgA secretion in the intestinal tract and accumulation of IS and other substances in 5/6Nx mice. This study unveils a novel mechanism of the kidney-gut axis mediated by VA and IgA, with implications for the development of therapeutic interventions.

Reference: 1. Fukuoka K, et al., Biochem Biophys Res Commun. 2023.

[Objective] In this study, we fed potassium tripolyphosphate (K5P3O10) to uninephrectomized mice and investigated phosphorus homeostasis during renal injury.

[Materials and Methods] Six-week-old C57BL/6J mice underwent right uninephrectomy and were fed a diet containing 1.5% K5P3O10 for 4 or 8 weeks (high-P diet group). The control group (low-P diet group) and sham treatment group were fed a diet containing 0.3% K₅P₃O₁₀. After the feeding period, blood Ca and P concentrations were measured, and gene expression analysis of phosphate transporter-related genes (slc34a2a, slc34a2b, slc34a2c) in the kidney and small intestine and vitamin D hydroxylase (CYP24A1, CYP27B1) in the kidney was performed.

[Results] P intake was proportional to the feeding concentration. Blood P concentration increased in the high-P diet group, but no change was observed in blood Ca concentration. The expression of slc34a2b in the jejunum and CYP24A1 in the kidney tended to increase in the high-P diet group. There were no differences between groups in the ileum. Kidney CYP27B1 was significantly increased in the high-P diet group compared to the low-P diet group.

[Discussion] Changes in transporters related to phosphorus absorption were confirmed in the jejunum, suggesting that phosphorus absorption in the intestine is mainly carried out in the jejunum. During early kidney injury, phosphorus homeostasis was regulated in the jejunum rather than the kidney. Furthermore, an increase in the expression level of CYP27B1 in the kidney was observed, suggesting an increase in the amount of calcitriol synthesis.

Recently, it has been suggested that there are more than 3,000 proteins are acetylated in human cells, which are thought to regulate diverse biological processes. Recently, it has been shown that beside acetylation, lysine residues undergo various types of acylations. Some of these lysine acylations occur depending on acyl-CoA derived from endogenous carboxylic acids such as fatty acids. We are exposed daily to a variety of compounds with food, including carboxylic acids. These living environment-derived carboxylic acids may modify lysine residues of proteins in vivo through chemical reactions. Indeed, we found that L-theanine, a well-known amino acid unique to green tea, has the potential to modify to proteins in cells. This study aimed to explore novel lysine acyl modifications induced by L-theanine using click chemistry, a selective reaction between alkyne and azide. Cells were treated with alkyne-labeled L-theanine, and L-theanine alkynes incorporated into proteins as acylation were detected by click chemistry with fluorescent azide. These analyses suggest that many proteins are modified by L-theanine. Furthermore, chase experiments revealed that L-theanine modification is reversible. Currently, we are conducting to comprehensively search for proteins modified by L-theanine using click chemistry with biotin azide, and would like to introduce this attempt in this presentation.

trans-Fatty acids (TFAs) are unsaturated fatty acids with one or more trans double bonds in the carbon-carbon bond, and are classified into industrial TFAs (iTFAs), which are rich in processed foods, and ruminant TFAs (rTFAs), which are abundant in milk and meat. Previous studies have associated iTFAs, but not rTFAs, with various diseases including cardiovascular diseases. However, the underlying mechanism has long remained unclear. We previously showed that TFAs promote apoptosis induced by extracellular ATP (eATP) and DNA damage, important risk factors for TFA-related diseases. We further demonstrated that iTFAs, but not rTFAs, are specific promoters of eATP-induced apoptosis. However, it is still unknown whether iTFAs also specifically promote DNA damage-induced apoptosis.

Here, we showed that in multiple cell lines such as U2OS cells (osteosarcoma), iTFAs including elaidic acid (EA), but not rTFAs including trans-vaccenic acid, promoted cell death accompanied by elevated activation of c-jun N-terminal kinase (JNK), a stress-responsive MAP kinase that had been previously identified as an effector of TFAs. Furthermore, pro-apoptotic effect of iTFAs was strongly suppressed in the presence of oleic acid (OA), the geometrical isomer of EA. Taken together, these results suggest that iTFAs are potent promoters of DNA-damage induced apoptosis, which is effectively mitigated by OA. We are now conducting a cell-based screen for identification of compounds that can effectively ameliorate TFA toxicity as well as OA.

Various lysine acyl modifications are related to epigenetic gene expression and are responsible for regulation of diverse biological phenomena. Lysine acylations occur depending on acyl-CoA derived from endogenous carboxylic acids. We are daily exposed to various carboxylic acids in the environments. We hypothesized that when these compounds are taken into the body, they may modify histones in a similar way to endogenous fatty acids, thereby altering gene expression and affecting health. Therefore, we developed a simple and efficient assay system to explore for histone modifiers among the myriad of carboxylic acids in our living environment, utilizing the interaction between acylated histones and reader proteins. Using this assay system, we searched for carboxylic acids with high consumption and found that several compounds have histone-modifying ability. Among them, we focused on aspartame, which is widely used as a sweetener, and examined changes in gene expression induced by aspartame. However, RNA-seq analysis revealed that aspartame had little effect on gene expression. These results suggest that histones may not be the primary target of aspartame modification. Indeed, analyses using alkyne-labeled aspartame derivatives suggest the presence of numerous proteins in cells that can be modified by aspartame. In this presentation, we would like to introduce our efforts to comprehensively search for proteins modified by aspartame using chemical biology approaches.

Introduction: We previously reported disruptive hippocampal neurogenesis by maternal exposure to neonicotinoid pesticide imidacloprid (IMI) in rats. In this study, we investigated the protective effect of amorphous formula of curcumin (CUR) against IMI-induced disruptive neurogenesis.

Methods: Maternal rats were administered 750 ppm IMI in diet and 120 ppm CUR in drinking water from gestation day 6 to day 21 post-delivery at weaning. After weaning, offspring continued to receive CUR until postnatal day 77 in adulthood.

Results and Discussion: IMI persistently suppressed hippocampal neurogenesis and synaptic plasticity of granule cells until adulthood. IMI decreased the expression of antioxidant enzyme genes on weaning, suggesting increased susceptibility to oxidative stress during the weaning period, whereas no oxidative stress effect was evident in adulthood. These results suggest involvement of suppression of GABAergic interneuron regulation on neurogenesis and synaptic plasticity by increased sensitivity to oxidative stress at weaning. CUR showed sustained interneuron-mediated neuroprotection; however, because CUR did not exert antioxidant effects, CUR may have restored neurogenesis and synaptic plasticity through direct protective effects on interneurons.

Conclusion: IMI persistently suppressed hippocampal neurogenesis and synaptic plasticity of granule cells, involving suppressed interneuron regulation through oxidative stress toxicity at weaning. CUR sustained neuroprotection on neurogenesis and synaptic plasticity through direct protective effects on interneurons.

Imidacloprid (IMI) is a widely used neonicotinoid insecticide; the developing cerebellum is known for its high sensitivity to neurotoxicants. However, neurotoxic potential of IMI on cerebellar development remains unknown. This study examined the maternal exposure effect of IMI on cerebellar development in rat offspring. Dams were exposed to IMI (83, 250, and 750 ppm in diet) from gestation day 6 until day 21 post-delivery on weaning, and offspring were maintained without IMI exposure until postnatal day (PND) 77 in adulthood. Behavioral tests revealed that the grip time in forepaw grip test and inclined plate degree in inclined plane test were decreased at ≥ 250 ppm both in the adolescent and adult stages. In the cerebellum, number of CALB1 ^＋ Purkinje cells decreased at 750 ppm on PND 21, and sustainedly decreased until PND 77, appearing at ≥ 83 ppm. IMI at 750 ppm increased Iba1 ^＋ microglia and CD163 ^＋ M2-type microglia in the molecular layer (ML) and internal granular layer of the cerebellar cortex, accompanying upregulation of Il6, Il10, and Tnfα in the examination at 750 ppm. Cerebellar malondialdehyde level was elevated at 750 ppm, accompanied by Sod2 downregulation. TUNEL^＋ apoptotic cells were also increased in the ML at 750 ppm, accompanied by Casp3 upregulation. Obtained results suggest that maternal IMI exposure induced oxidative stress damages and neuroinflammation in the developing cerebellum causing apoptosis of migrating granule cells followed by progressive loss of Purkinje cells and long-term deficits in motor activity and coordination functions in offspring.

In nonclinical safety studies, animals may exhibit abnormal behaviors due to the deterioration of their condition or due to central nervous system (CNS) toxicity. Sensitive continuous monitoring methods are necessary to detect the acute onset or recurrence of such symptoms. In this research, we have developed four new AI-based models to analyze abnormal behaviors of cynomolgus monkeys from video data.

The summaries of each model are as follows:

Reduced activity level: By tracking the center of the animal’s pose skeleton, the extent and speed of movement are calculated, enabling quantitative detection of drug-induced decrease in activity.

Stereotypic behavior: Wavelet transformations of body part trajectories can be used to detect stereotypic locomotor behaviors (e.g., pacing and somersaulting), allowing quantification of these behaviors induced by excitement and stress.

Vomiting: Recognizing vomit through the use of vomitus object detection models provides information on the exact timing and frequency of vomiting.

Convulsion: Classification algorithms established from video data of pentetrazole-induced seizures can detect occurrences in real-time, which not only improves toxicity assessment but also enables the provision of veterinary care promptly.

These four models improve upon previous observation methods by providing quantitative, automated detection with enhanced sensitivity. By utilizing these new indicators, it is expected to improve the accuracy of toxicity evaluation and optimize animal care.

Methylmercury (MeHg) is the causative substrate for Minamata disease, and it is known to induce sensory impairment. The neurotoxicological mechanism of MeHg has been widely studied in the central nervous system, but not as much in the peripheral nervous system. It has been reported that the number of neurons in the rat dorsal root ganglion (DRG) decreased 14 days after MeHg exposure (Day14). In addition, from behavioral analysis, only hypoalgesia was observed during Day11 to 48 and then, recovered to control level. However, the histological background for hypoalgesia-specificity remain unclear. In this study, we investigated the possibility that hypoalgesia originates in the peripheral nervous system. MeHgCl (6.7 mg/kg/day) was orally administered into 9-week male Wistar rats for 5 days, followed by 2 days without administration, and this cycle was repeated once. Seven, 14, 28, 42, 56, and 70 days after the beginning of MeHg exposure, frozen sections from L4-5 DRG were prepared and immunostained by nociceptive and mechanoreceptive cell markers. DNA microarray analysis for Day14 was also performed. Histological analysis showed that the number of mechanoreceptive and nociceptive cells have both decreased around Day14 and recovered afterwards. Microarray data also did not show specific cell death of nociceptor cells. These data suggest that the hypoalgesia-specificity may be caused by the impairment of central but not peripheral nervous system.

Methylmercury (MeHg) is recognized as a factor in Minamata disease, leading to neuronal degeneration in the CNS. While MeHg exposure is known to impact sensory neurons in the PNS, its toxicological effects remain inadequately explored. Recent studies indicate an accumulation of macrophages within the dorsal root ganglion (DRG) in MeHg-exposed rats. However, the role of macrophage behavior in MeHg-exposed rat DRG, whether contributing to neurodegeneration or neuroprotection, remains unclear. To address this, we conducted immunohistochemistry to elucidate the role of macrophage in MeHg-exposed rat DRG. Male Wistar rats (9 w) received oral MeHgCl (6.7 mg/kg/day) for 5 days, with a subsequent 2-day break, repeated once. Concurrently, macrophage activation inhibitor, minocycline (30 and 100 mg/kg/day), was administered by i.p. Sections from L4-5 DRG were immunostained for Iba1, CD68, and NeuN at intervals of 7 to 70 days post-MeHg exposure. The number of Iba1+ macrophages increased significantly from day 28 until day 70. CD68+ macrophages showed a transient increase at days 14 and 28. Interestingly, neuron counts remained unchanged throughout the study period. Minocycline administration failed to suppress macrophage activation under our experimental conditions. While our data demonstrate fluctuations in the numbers of Iba1 and CD68+ macrophages, the relationship between accumulated and activated macrophages and neurons remains elusive. We aim to discuss potential associations between macrophages and neurons in MeHg-exposed rat DRG during the poster session.

In vitro assessment of drug responses within the central nervous system (CNS) is critical for evaluating drug toxicity in neurological disorders. Microelectrode array (MEA)-based evaluation systems commonly employ co-cultures of neurons and glial cells, leading to enhanced neural network maturation. Given the distinctive roles of astrocytes and neurons in CNS drug response, there arises a necessity to distinguish between neuronal and astrocytic functions in co-culture setups. This study aims to address this challenge by employing independent component analysis (ICA) to segregate neural signals originating from neuron and astrocyte co-culture systems on MEA. Rat primary neurons from 17-day pregnant rats and human iPS cell-derived neurons were cultured on MEA plates. Both co-culture and monoculture were established independently for each plate. Following data acquisition, ICA was employed to disentangle the neural signals acquired from the co-culture conditions into their independent components. Each component was categorized based on its source and subsequently separated and reconstructed. Notably, each cell-derived component exhibited characteristics in the low-frequency band. The application of ICA enabled the extraction of individual cell-derived components from co-culture data. Moreover, the extracted data post-ICA exhibited similar trends to monoculture data concerning drug response. This study successfully extracted neuronal and astrocytic signals from co-culture conditions using ICA, offering potential for evaluating drug responses of distinct neuronal cell types.

Assessment of histopathological changes in the brain is essential for in vivo neurotoxicity testing. However, whole brain histological analysis is labor-intensive and costly. In this study, we have developed an efficient method for three-dimensional imaging of larval zebrafish brain exposed to neurotoxic compounds, enabling quantitative analysis of cell death and microglia dynamics. Zebrafish embryos were exposed to neurotoxic agents at multiple concentrations and durations up to 24 hours. For labeling, fluorescent dyes that can quickly stain structures such as white matter, cell membrane and dead cell throughout the body, and transgenic zebrafish that specifically express fluorescent proteins in microglia were used. The brains of the samples mounted on observation plate were scanned using a two-photon excitation microscope. Data such as the position and shapes of dead cells and microglia were automatically acquired from all images using an ImageJ macro. Additionally, qualitative evaluation of histological properties and the coordinates of specific brain region were obtained manually. Statistical analysis of the data was performed using R, adjusting for individual variations in body orientation and size based on coordinate data, which facilitated sample comparison. Variations in metrics representing cell count and morphology in each region of the brain result from differences in test compound, concentration, and exposure time. Our method streamlines the analysis of histological changes in the brain caused by neurotoxic substances.

Proteoglycans (PGs) are one of the extracellular matrix components, which are composed of a core protein and one or more glycosaminoglycan side chain(s). Vascular endothelial cells mainly synthesize perlecan, biglycan, and syndecans, which are involved in the regulation of cell proliferation and the blood coagulation-fibrinolytic system. Atherosclerosis is a common vascular lesion in advanced countries, resulting in a local hypoxia caused by intimal thickening of the vessel wall. Under hypoxic conditions, vascular endothelial cells induce hypoxic responses and transmit signals toward cell survival; however, the effects of hypoxia on PGs have not been elucidated. In this study, we found that biglycan and perlecan mRNA expressions increased in a time-dependent manner, while syndecan-1 mRNA expression was unaffected by hypoxia; the increase in biglycan and perlecan mRNAs was mediated by HIF-1α. On the other hand, hypoxia decreased core protein expression of PGs tested. There was no significant difference in whole protein amount in the cell layer and conditioned medium between normoxic and hypoxic cultures, indicating that hypoxia reduced PG core protein synthesis selectively. Since decrease in PGs promotes thrombus formation and vascular permeability, a reduced synthesis of PGs core protein in vascular endothelial cells under hypoxic condition would lead to worsening atherosclerotic lesions. HIF-1α-mediated induction of PG mRNA expressions may reflect a protective response against vascular vulnerability caused by the decrease in PGs core protein under hypoxic conditions.

Atherosclerosis is a major lifestyle-related disease in advanced countries. In the lesion, intimal thickening causes narrowing of the vessel wall and forms a local hypoxic environment that exacerbates the progression of the disease. Proteoglycans (PGs) are macromolecules that consist of glycosaminoglycan chains attached to a core protein. PGs play an important role in the regulation of vascular functions. We have found that the core protein expression of biglycan, perlecan, and syndecan-1, the major PGs synthesized by bovine aortic endothelial cells, are reduced under hypoxic culture condition, but the extrapolation to human cells remains unclear. In this study, we used a human vascular endothelial cell line EA.hy926 cells and analyzed the expression of PGs under hypoxic conditions. In hypoxic condition, biglycan mRNA increased after 24 h, perlecan mRNA decreased in a time-dependent manner, and syndecan-1 mRNA showed no significant change. On the other hand, biglycan, perlecan, and syndecan-1 core protein expression were all reduced under hypoxic condition. These results suggest that the decrease in PGs core protein expression under hypoxic conditions is a common phenomenon independent of species. PGs bind to antithrombin III and various growth factors/cytokines, each of which has an important role in vascular antithrombogenicity and repair from injury by transmitting signals to cells via core proteins and glycosaminoglycans. Therefore, the decrease in PGs caused by hypoxia contribute to the development of vascular disorders in atherosclerosis.

　We are exposed to environmental chemicals such as pesticides and PM2.5, in daily life. Long-term exposure to these chemicals has been associated with the development of chronic diseases such as cancer. Although epigenome dysregulation has been suggested as the underlying mechanism, the toxic factors and molecular mechanisms remain unknown. In contrast, environmental electrophiles have been reported to form protein adducts and regulate enzymatic activity.

　We identified four environmental electrophiles that inhibited the enzymatic activity of DNA methyltransferase DNMT3B, an epigenomic regulator, using chemical screening. We found that one of the environmental electrophiles inhibited DNMT3B enzymatic activity through direct modification, induced DNA hypomethylation, and upregulated inflammatory cytokines. In this study, we focused on dithianon, which is currently used worldwide as a pesticide. We analyzed the effect of dithianon on STAT1, which regulates the inflammatory response.

　First, we evaluated the ability of dithianon to inhibit DNMT3A/B enzymatic activity and found that both enzymatic activities were suppressed in a concentration-dependent manner. Moreover, dithianon increased the phosphorylation of STAT1 after 48 h of treatment in A549 cells.

　These results indicate that dithianon could be an epigenomic regulator that inhibits the enzymatic activity of DNMT3 and induces STAT1 phosphorylation. We will investigate the mechanism of STAT1 activation by dithianon.

Oxidative stress injury to vascular endothelial cells covering the lumen of blood vessels is important in the early development of atherosclerosis. It has been reported that ATP causes the progression of atherosclerosis, but the detailed mechanism has not been understood. In this study, we investigated the effects of ATP on oxidative stress on vascular endothelial cells, the initial process of atherosclerosis, and the involvement of ATP receptors, P2X and P2Y receptors, as molecular mechanisms. We found that ATP enhanced the injury effects of hydrogen peroxide on vascular endothelial cells and that ATP is a potent mediator of the oxidative stress induced by hydrogen peroxide. We found the following results,

(1) ATP suppressed the expression of heme oxygenase-1 (HO-1), a protective factor against oxidative stress.

(2) ATP suppressed the expression of HIF-1α, a regulator of HO-1 expression.

(3) ATP suppressed the induction of HO-1 mRNA expression by hydrogen peroxide treatment.

(4) The P2Y receptor antagonist Suramin abrogated the suppression of HO-1 mRNA expression by ATP and the potentiation of its injury effects by hydrogen peroxide, but not by the P2X receptor antagonist PPADS.

These results suggest that ATP may enhance cytotoxicity by inducing an abnormal response to oxidative stress via P2Y receptors in vascular endothelial cells.

Perlecan is a high-molecular-weight molecule of heparan sulfate proteoglycan (HSPG) that regulates proliferation and anticoagulant activity by activation of FGF-2 and antithrombin III in vascular endothelial cells. Although the changes in the expression of perlecan are crucial for the exacerbation of inflammatory vascular lesions such as atherosclerosis, the detailed mechanisms remain unclear. In inflammatory vascular lesions, ATP are secreted from cells and platelets, regulating cellular functions through purinergic receptors. In this study, we investigated the effects of ATP on the expression of perlecan and its mechanisms in vascular endothelial cells. When treated with ATP, the expression of extracellular perlecan core protein was decreased in vascular endothelial cells. The decrease was not observed with ADP or adenosine. Transmembrane HSPGs, syndean-1 and syndecan-4 were also decreased by ATP. When purinergic receptors (P2X4R, P2X7R, P2Y1R and P2Y2R) constitutively expressed in vascular endothelial cells were nocked down, ATP-decreased perlecan expression was recovered, especially P2Y2R. The typical Akt inhibitor, Akt inhibitor Ⅷ, exhibited such similar inhibitory effect on perlecan expression as ATP, whereas no change occurred with an Akt activator, SC79. These results suggested that ATP suppressed the expression of perlecan core protein in vascular endothelial cells and the suppression was mediated through the inhibition of Akt signaling by P2Y2R. In inflammatory lesions, the suppression of perlecan expression by released ATP is implicated in inducing thrombus formation by the inhibition of anticoagulant activity.

Proteoglycans are composed of glycosaminoglycan(s) such as chondroitin/dermatan sulfate and heparan sulfate, a repeating structure of uronic acid and amino sugars, bound to a core protein. In vascular, proteoglycans are involved in the regulation of cell proliferation and the blood coagulation/fibrinolytic system. Vascular endothelial cells covering of the inner surface of blood vessels are locally exposed to a hypoxic environment when the vessel wall thickens in atherosclerosis. We have shown that the core protein expression of proteoglycans produced by vascular endothelial cells is altered under hypoxic conditions. The present study aimed to elucidate the effects of hypoxia on the gene expression of glycosaminoglycan synthase. Under hypoxic conditions, the expression of CHSY1, an elongating enzyme of chondroitin/dermatan sulfate, was decreased in sparse culture. Similarly, the expression of CHPF2, an elongating enzyme of chondroitin/dermatan sulfate chains, was downregulated regardless of cell density. On the other hand, the expression of EXT1, EXT2, and EXTL3, enzymes that elongate heparan sulfate, was suppressed in the dense culture at 8 h after hypoxia, while it turned to increase at 24 h. In the sparse culture, EXT1 expression increased at 8 h and decreased at 24 h after hypoxia, and EXT2 expression was continuously suppressed until 24 h after hypoxia. Future studies are needed to clarify the effects of these changes on protein expression and chain length of glycosaminoglycans.

Cardiotoxicity significantly contributes to drug attrition during the new drug development process and the withdrawal of drugs already in the market. Although the hERG assay under the ICH S7B has been a key tool for assessing hERG-related arrhythmic risks since 2005, concerns persist regarding its accuracy, as hERG-blockade does not consistently correlate with QT prolongation in humans. Recent advances in the stem cell field now enable the generation of highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that faithfully replicate native behavior of human ventricular myocytes, offering a more precise assessment of the proarrhythmic potentials of candidate drugs.

Currently, these cells are under active investigation, particularly within the ICH guideline revision (CiPA initiative), showing their potential as novel assays for preclinical cardiotoxicity testing. NEXEL’s highly pure human iPSC-derived cardiomyocyte, Cardiosight®-S, is also a part of many cell products that are being tested for the purpose and has been validated for its effectiveness.

The outcomes generated by NEXEL’s NeXST service have undergone validation across numerous cases, encompassing the evaluation of both reference drugs and unknown drugs. Through optimization tailored to Cardiosight®-S in various platforms, NeXST service is highly versatile for various purposes within cardiac toxicity screening.

In conclusion, Cardiosight®-S, along with the NeXST service, emerges as a promising alternative source for the early determination of cardiac risk in both drug discovery and preclinical studies.

[Background] γ-H2AX is a useful marker for detecting bladder and renal carcinogens, and combinations of γ-H2AX and stem cell markers increase the sensitivity. Here, we investigated the possible application of γ-H2AX and stem cell markers in rats for early detection of hepatocarcinogens (HCs).

[Methods] Immunostaining for γ-H2AX and liver stem cell markers (ALDH3A1, APN/CD13, and EpCAM) was performed using liver samples of male F344 or Crl:CD(SD) rats treated with HCs or non-HCs for 28 days. Significant increases in positive-cell ratios for γ-H2AX, hepatocytic expressions for ALDH3A1, and semi-quantitative scores for APN and EpCAM, respectively, were used as criteria.

[Results] Including previous reports, immunohistochemical analysis of γ-H2AX, ALDH3A1, APN, and EpCAM were performed for 49, 84, 40, and 56 chemicals, respectively. The sensitivity and specificity of each marker alone were highest for ALDH3A1 at 71.1%/78.3%, and others were γ-H2AX at 48.5%/75.0%, APN at 48.1%/23.1%, EpCAM at 58.8%/68.2%. The combination of γ-H2AX and ALDH3A1 improved the sensitivity to 78.9%. In addition, ALDH3A1 showed different expression patterns depending on the chemicals; PPAR agonists tended to show characteristic canalicular pattern and biliary carcinogens tended to induce expression in cholangiocytes.

[Discussion] As ALDH3A1 was the most sensitive alone or in combination with γ-H2AX, it was suggested that ALDH3A1 can be a useful marker for early detection of HCs. Our results also suggest that ALDH3A1 expression pattern may be related to the mechanism and target cells of HCs.

In a reproductive and developmental toxicity study using cynomolgus monkeys at our facility, females were mated by housing them with males for 3 days. We hypothesized that cynomolgus monkeys, which exhibit periodic menstruation (25 to 35 days), ovulate 11 to 16 days after the onset of menstruation, and 3 days of this period were set as the mating period. However, the pregnancy rate with this method was approximately 20%. To improve pregnancy rates, a simple and non-invasive method of predicting ovulation time and optimizing the mating period is necessary. Therefore, we conducted ultrasound examinations (using a 38 mm, 5 to 18 MHz linear transducer) of the ovaries and uterus of 23 cynomolgus monkeys over time to search for indicators of ovulation. The primary follicle diameter and endometrial thickness were measured on Menstruation Days (MDs) 2, 10, 12, 14, 16, 18, and 20 (until the corpus luteum was confirmed), with the start of menstruation as MD 1. The results suggested that the period when the primary follicle diameter was at least 4.5 mm coincided with the period immediately before ovulation, and therefore primary follicle diameter was considered to be a good indicator of the ovulation. Additionally, the number of days on which primary follicle diameter was at the maximum differed for each animal. In contrast, there was no apparent change in endometrial thickness over time. These results suggest that monitoring primary follicular diameter by ultrasound is an effective tool for predicting the timing of ovulation and optimizing the mating period in cynomolgus monkeys.

The xenobiotic-responsive nuclear receptor CAR is highly expressed in the liver, while its activation associated hepatocarcinogenesis in rodents. We found that CAR induces hepatocyte proliferation through interacting with YAP, a cell growth-associated transcriptional cofactor, in mouse liver, but our recent results suggest that CAR also induces hepatocyte proliferation through a pathway other than YAP. Recently, it was reported that the CAR-mediated induction of Gadd45b, is involved in cell proliferation and apoptosis, is important for CAR-dependent hepatocyte proliferation in mice. In this study, we investigated the mechanism for Gadd45b transcription by CAR to fully elucidate the mechanism for CAR-mediated hepatocyte proliferation. Mice were intraperitoneally injected with phenobarbital (PB), a CAR activator, twice at 24-hour intervals, and livers were harvested 3 hours after the last injection. qRT-PCR revealed that Gadd45b mRNA levels were increased by PB. We investigated the methylation levels of the CpG sites on the Gadd45b by PCR in combination with HpaII, cleaves non-methylated CpG sites. The results showed that methylation levels decreased with PB. Finally, we performed reporter assays using the mouse Gadd45b promoter. When CAR was exogenously expressed and chemically activated, reporter activity was increased, while pre-treatment of the reporter plasmid with the CpG site-methyltransferase attenuated the basal reporter activity regardless of CAR activation. These results suggest that CAR promotes Gadd45b transcription in mouse livers by inducing the demethylation of the CpG sites of the Gadd45b promoter.

Nitric Oxide (NO), which is produced in vivo and exists as an air pollutant, has various effects on the body. We have previously shown that NO inhibits the activity of DNA methyltransferases (DNMTs) by modifying the cysteine thiol group of proteins via S-nitrosylation. DNA methylation plays an important role in regulating gene expression and maintaining genomic stability. Thus, the inhibition of DNMT activity reduces the level of DNA methylation and causes aberrant gene expression.

In this study, we analyzed the effects of exposure to exogenous NO on human nasal septal epithelial carcinoma cells, RPMI2650 cells, at 48 h after treatment with GSNO, an NO donor. First, we performed transcriptome analysis and found that GSNO exposure increased the expression of various genes. Gene ontology of the upregulated genes included DNA replication, DNA damage response, and cell cycle arrest. RT-qPCR analysis revealed that connective tissue growth factor (CCN2/CTGF) and early growth response factor (EGR1) levels increased 48 h after GSNO treatment. Furthermore, treatment with 5-azacytidine, a DNMTs inhibitor, increased the expression of CCN2 and EGR1 in a time-dependent manner, suggesting that the upregulation of these genes could be mediated by DNA demethylation.

Our results indicate that NO exposure increased the expression of tumor suppressor genes in RPMI2650 cells. We will focus on the DNA methylation status of the target genes to clarify the effects of NO-induced epigenomic changes on gene expression.

Recently, “Exposome” was proposed as a total lifetime exposure by C. Wild. Most studies have focused on lifetime exposure; however, the molecular mechanisms remain unclear. Therefore, in this study, we focused on DNA methylation, which epigenetically regulates gene expression, and analyzed the effects of environmental electrophiles that easily bind to proteins as a model for the exposome study.

First, we examined the effects of 45 electrophiles, including air pollutants and pesticides, on DNMT3B enzyme activity, which is one of the major DNA methylation enzymes. We identified four electrophiles that reduced DNMT3B enzyme activity. Among these four candidates, we focused on 1,2-naphthoquinone (1,2-NQ), an air pollutant. The binding sites of 1,2-NQ on DNMT3B were analyzed using LC-MS/MS, and specific lysine and histidine residues were identified. Next, we examined transcriptome changes in 1,2-NQ by RNA-Seq. We found that the expression of chemokines that regulate inflammatory responses was increased. Additionally, we showed that 1,2-NQ and the DNMT inhibitor induced the expression of chemokines, including CXCL8, in a time-dependent manner. In addition, we analyzed the DNA methylation levels of CXCL8 enhancer sites using bisulfite sequencing and found that 1,2-NQ decreased its methylation levels. Moreover, we revealed that 1,2-NQ-induced chemokines promoted A549 lung cancer cell growth. Our results indicate that 1,2-NQ covalently modifies DNMT3B and induces epigenetic disorders that alter gene expression, such as chemokines that promote lung cancer proliferation.

[Introduction]

17β-Estradiol (E2) is a major risk factor for breast cancer. E2 is metabolized by the CYP1 family enzyme to 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2), which are highly genotoxic. However, the mechanisms underlying their genotoxicity are not fully understood. This study aims to evaluate the genotoxicity of E2 metabolites using mouse Bhas42 cells and human MCF-7 cells.

[Methods]

The E2 metabolites were treated with Bhas42 and MCF-7 cells for 24 hours. The levels of indicator genes for genotoxicity were determined by quantitative RT-PCR in Bhas42 (12 genes) and MCF-7 (6 genes). In addition, the genotoxicity of E2 metabolites was evaluated in Bhas42 cells using the malignant transformation assay and in MCF-7 cells using the comet assay method.

[Results and Discussion]

The expression of all 12 oncogenic indicator genes was induced by 2-OHE2 in Bhas42 cells, with higher levels of induction than by 4-OHE2. However, treatment with E2 metabolites did not accelerate the oncogenic transformation of Bhas42 cells. In MCF-7 cells, 2-OHE2 induced the expression of three out of the six oncogenic indicator genes, which is consistent with the results observed in Bhas42 cells, suggesting that 2-OHE2 is genotoxic. However, the comet assay results showed that 4-OHE2 was slightly more genotoxic than 2-OHE2. The genotoxicity of E2 metabolites was observed prominently in Bhas42 cells, and its strength varied among the test systems. The differences in genotoxicity of E2 metabolites may depend on the expression status of estrogen receptors.

Aim:

Acetamide (AA) found in various foods is a potent carcinogen in rat liver. We have shown that AA induces characteristic large micronuclei in rat hepatocytes which show chromothripsis-like molecular pathological features. To clarify the involvement of chromothripsis in AA hepatocarcinogenesis, we performed mutation analysis using a next-generation sequencing (NGS) and optical genome mapping (OGM) on AA-induced rat liver tumors.

Method:

6-week-old male F344 rats were given a diet containing 2.5% AA for 26～30 weeks. Normal livers in the control group, hepatocellular adenoma and carcinoma in the AA-treated group were subjected to NGS and OGM analysis.

Results:

In NGS analysis, large and random copy number gains were observed in AA-induced liver tumors on various chromosomes, and copy number gains of Chr7 including the oncogenes c-Myc and MDM2 were commonly observed. In OGS analysis, drastic interchromosomal translocations were observed in AA-induced liver tumors.

Discussion:

Chromothripsis is known to cause complex copy number and structural alterations through chromosome shattering and rearrangement. The large and random copy number gains and drastic interchromosomal translocations observed in AA-induced liver tumors supported the involvement of chromothripsis in the hepatocarcinogenesis of AA. The detailed locus of chromosomal translocations and their relationship with copy number gains of oncogenes is currently under investigation.

SLCO2A1 is a prostaglandin transporter in the body. Our laboratory’s previous research implied its role in inflammation regulation via PGE2 transport. We also found that cigarette smoke extracts (CSE) reduced SLCO2A1 function and expression in alveolar epithelial cells derived from mouse lungs. This study aimed to explore the effect of CSE on human SLCO2A1 mRNA expression and promoter activity across cell lines from organs susceptible to smoking-induced inflammation (lung, gut, and liver). We investigated the effect of CSE alongside AhR inhibition using perrialdehyde (PAH) and PD98059 on SLCO2A1 mRNA expression in different cell lines (such as LoVo, HepG2, NCI-H292). Most cell lines exhibited reduced SLCO2A1 mRNA expression after CSE treatment, except HepG2. Treatment with PAH and PD98059 restored SLCO2A1 mRNA levels after CSE exposure, indicating that AhR signaling is involved in CSE-mediated SLCO2A1 downregulation. Luciferase reporter assays using various SLCO2A1 promoter constructs (from 0.2 to 3.7kb upstream of transcription starting site) revealed significantly decreased activity with CSE in pGL3/3.7k promoter construct, and only PD98059 restored activity after CSE treatment, implying that CSE reduces SLCO2A1 transcription via AhR. Mutations in two AhR consensus sites within the promoter region restored luciferase activity after CSE treatment, affirming that there is an AhR and DNA interaction for SLCO2A1 transcription. In summary, CSE negatively modulates SLCO2A1 expression through AhR/DNA interaction.

Introduction: Cadmium (Cd) has been reported to accumulate in paddy fields, and there is concern about its toxicity. Bagasse (BG), which is a by-product of sugar production, has been utilized as an adsorbent for heavy metals due to its large specific surface area (SSA). Therefore, the treatment method of BG suitable for Cd removal and the detailed mechanism of adsorption were investigated.

Methods: BG was calcined in the temperature range of 200‒1000°C. BG was added to a Cd solution and shaken for 24 h under multiple conditions. The equilibrium Cd concentration was measured using inductively coupled plasma–optical emission spectroscopy. The SSA, pore volume, and pH_pzc (pH of the point of zero charge) were measured.

Results and discussion: The pH_pzc of the BGs ranged from 4.27‒6.51, suggesting that the BG surface may be negatively charged at conditions above these pH values. The amount adsorbed was higher at pH 4‒8 than at the pH 2 and 10. These results indicate that the negative charge on the BG surface and the presence of Cd(Ⅱ) cations are important for adsorption. The amount adsorbed and the SSA of BG increased with increasing calcination temperature and were the highest in BG calcined at 800°C. The amount of adsorbed was significantly associated with the SSA (r=0.837, p=0.038), mesopores (r=0.841, p=0.036), and pH_pzc (r=0.818, p=0.046). These results suggest that the Cd adsorption is related to SSA, the negative charge on the BG surface, and the presence of Cd(Ⅱ) cations, and that BG calcined at 800ºC is suitable for Cd adsorption.

Bisphenol A (BPA) have been used to manufacture polycarbonate plastic and reported to exhibit endocrine-disrupting effects via estrogenic action in animals. There are concerns that BPA has low-dose, adverse effects that could be detected at doses lower than the NOAEL. However, the low-dose effects of BPA cannot be detected in guideline-confirming toxicity tests such as the uterotrophic bioassay, which is an in vivo screening method for detecting the estrogenic activity of chemicals. Therefore, these effects, including their authenticity, have not been fully clarified and the debate about them is ongoing. Here, we tried to develop a novel, highly sensitive, screening method for estrogen-like chemicals using in vivo bioluminescence imaging of estrogen-responsive reporter (E-Rep) mice. First, we compared the detection sensitivity between two endpoints (bioluminescence and uterine weight) within the same E-Rep mouse using 17α-ethinylestradiol (a reference estrogen) and bazedoxifene acetate (a selective estrogen receptor modulator), and demonstrated that the method using bioluminescence as an endpoint provides higher detection sensitivity than the uterotrophic bioassay. Moreover, the method using bioluminescence of E-Rep mice could detect the estrogenic effects of BPA at doses below the NOAEL. Our results suggested that in vivo bioluminescence imaging using E-Rep mice was extremely useful for screening estrogen-like chemicals. Our method could greatly facilitate resolution of questions associated with low-dose effects of estrogen-like chemicals, including BPA.

Microplastics (MPs) are plastic particles with a diameter of 5 mm or less, and those with a diameter of 1 µm or less are specifically defined as Nanoplastics (NPs). These plastic particles are ubiquitous in the environment. Recent studies have shown that MPs/NPs were detected in human body, indicating unavoidable exposure for humans. It is also known that MPs/NPs in the environment undergo degradation due to ultraviolet rays and waves, leading to changes in physical properties such as size and surface properties. Although the kinetics of tiny particles vary depending on their physical properties, the effects of these differences in MPs/NPs are not yet clarified. In this study, we tried to elucidate the intracellular dynamics of MPs/NPs focusing on the surface degradation of them.

First, we prepared degraded polyethylene (d-PE) by exposing ultraviolet rays to PE. Next, RAW264.7（mouse macrophage-like cell line）were exposed to PE or d-PE and SSC (side scatter) was measured by Flow Cytometry. As a result, an increasing trend was observed in the d-PE-treated group. In addition, we prepared fluorescent-labeled PE and d-PE using NileRed, a fluorescent dye that emits green fluorescence in response to hydrophobic groups and red fluorescence when hydrophilicity increases. THP-1, (human monocyte cell line）was exposed to them to evaluate cellular uptake. Fluorescence microscopy observation revealed that the fluorescent-labeled d-PE was either adhered to or incorporated into the cells. Now, we are working on establishing a method for quantifying the intracellular uptake of MPs/NPs.

Exposure to high concentrations of methylmercury (MeHg), as in the case of Minamata disease, is no longer a problem in Japan. There is concern about the effects of exposure of low concentrations of MeHg to the fetus via pregnant women due to ingestion of seafood in which MeHg has accumulated through bioaccumulation. However, the toxic mechanism of MeHg on neuronal differentiation is unknown. Therefore, we investigated the effects of MeHg on neuronal function and epigenetic changes in genes related to neuronal function. LUHMES cells were differentiated and exposed to MeHg (0, 0.1, 1 nM) for 68 days starting from day 2 of neuronal differentiation (D2). Gene expression levels, protein expression levels, and DNA methylation were determined on D10. Neural spike activity was also measured by Maestro MEA system on D4, D10, and D16. Exposure to MeHg 1 nM increased DNA methylation, inhibited neurite outgrowth, and suppressed neuronal spiking. DNMT1 protein level was increased by MeHg 1 nM exposure. DNMT1 methylation, which is involved in DNMT1 degradation, was decreased by MeHg. Furthermore, gene and protein expression levels of SET７，SET８，and LSD1, which regulate DNMT1 methylation, were not significantly different after exposure to 1 nM MeHg. These results suggest that exposure to low concentrations of MeHg during neuronal differentiation suppresses neurite outgrowth via DNA methylation by elevating DNMT1, and that MeHg-induced reduction of DNMT1 methylation may be responsible for the elevated DNMT1.

Background: 1,2-Naphthoquinone (1,2-NQ), which exists in atmospheric particulate matter (PM_2.5), is produced by the incomplete combustion of gasoline and photooxidation of naphthalene. 1,2-NQ acts as an electrophilic molecule and forms a covalent bond with target proteins. We previously reported that 1,2-NQ undergoes N-arylation with epidermal growth factor receptor (EGFR) and activates PI3K–AKT signaling. In this study, we attempted to clarify the effect of 1,2-NQ on the EGFR-ERK signaling cascade.

Methods: NSCLC A549 cells were precultured in serum-free medium followed by treatment with 1,2-NQ. To pharmacologically analyze the effects of the EGFR-ERK pathway, cells were treated with the EGFR inhibitors cetuximab and panitumumab, the RAF inhibitor LY3009120, or the MEK inhibitor U0126 before 1,2-NQ treatment. The effects of 1,2-NQ on EGFR and ERK phosphorylation were assessed by western blotting using specific antibodies.

Results and Discussion: 1,2-NQ induced phosphorylation of EGFR (Tyr1068) and ERK1/2 (Thr202/Tyr204) in a dose-dependent manner. Pretreatment with cetuximab, panitumumab, LY3009120, and U0126 suppressed phosphorylation of ERK1/2-Thr202/Tyr204 induced by 1,2-NQ.　These data suggest that 1,2-NQ activates ERK1/2 through EGFR-RAF-MEK signaling. Further studies are needed to identify the effects of ERK downstream signaling regulated by 1,2-NQ, which may lead to potential therapeutic strategies for COPD/cancer treatment.

[Introduction] PXR has attracted attention as a target for the toxic effects of chemicals due to its low ligand specificity and activated with a wide range of chemicals. Recently, we found four phenolic antioxidants (PAs) have potent agonist activity against human PXR (hPXR) compared to rat PXR (rPXR). Here, we performed in vitro and in silico analyses to study in detail the responsiveness of hPXR to PAs and the factors responsible for species difference.

[Methods] The hPXR agonist activity of the four PAs was evaluated using reporter assay in human HepG2 cells. In　addition, the expression of PXR target genes in response to PAs in human LS180 cells was analyzed using RT-PCR. Then, docking analysis was performed to calculate the binding affinity and mode of binding of PAs to the hPXR LBD. Lastly, the PA responsiveness of the rPXR mutant converted to the human form was assessed by reporter analysis.

【Results & Discussion】 In reporter and mRNA expression analyses, all four PAs showed potent hPXR agonist activity, two of which were more potent at lower concentrations than the known agonist RIF. The known antagonist SPA70 strongly inhibited their effect. Subsequent docking analysis predicted that two polar amino acid residues within the LBD contributed to the interaction with PAs. Lastly, replacement of amino acids in the rPXR with the corresponding human residues reduced constitutive and maximal activity but increased the fold activation by PAs. In conclusion, our study provides details of the interaction of PAs with PXRs, which may be useful for the safety assessment of PAs.