Heart failure may be caused by prolonged exposure of drugs such as anti-cancer drugs, and/or there may be species differences in the occurrence of side effects. It is difficult to accurately predict the heart failure risk of drugs in the current evaluation systems including animal echocardiography and contractile assessment using animal hearts. Therefore, development of new in vitro assay system using human cells, such as human iPS cardiomyocytes, which are well understood for predicting the risk of drug-induced proarrhythmia is strongly expected.

In recent years, the development of cell culture platforms called microphysiological system (MPS) has been became active, thus we have been working on the development of cardiac MPS which can detect drug-induced contractile dysfunction. In this study, we constructed a contraction measurement system using three-dimensional myocardial tissue generated from human iPS cardiomyocytes (h3D heart tissue) on a polydimethylsiloxane micro device.

We confirmed that isoproterenol, a β-adrenoceptor agonist, showed positive inotropic and chronotropic action. Then, we examined the effects of 72-hour exposure to doxorubicin, an anthracycline anti-cancer drug, on contraction in h3D heart tissue. As a result, we observed a concentration- and exposure time-dependent impairment of contraction. Furthermore, we investigated the difference in contractile dysfunction depending on the method of exposure to doxorubicin. In the future, it is necessary to compare and verify the validity of this evaluation system with human clinical data.

Due to environmental factors such as ionizing radiation and genotoxic chemicals, cells are exposed to extensive genomic stresses every day. These genomic stresses trigger recovery from the injury, cell death, and cellular senescence. Thus, it is important to understand and predict the ability of tissues to maintain the steady-state homeostasis in response to the injury.

DNA double-strand break (DSB) is the most severe genomic stress, and if they are not repaired correctly, genetic mutations and/or loss of function might be induced. In a challenge for establishing real-time and quantitative analysis of cell fate triggered by DSBs, we cloned the foci-forming element of mouse 53BP1, one of the DSB repair proteins, and generated a fusion gene connected with a fluorescent protein. We further generated fluorescent fusion genes with two other fluorescent genes expressing two different cell-cycle indicators (a part of hGmnn and hCdt1) and finally constructed a tricistronic vector named “Focicle”. Next, we knocked in the Focicle gene into the mouse ROSA26 region by CRISPR/Cas9, then established cell lines enable us quantitative live-cell imaging of cell cycle-dependent genomic stress. By time-lapse imaging of a cultured cell line transfected with the Focicle gene, we observed dose-dependent induction and extinction of DSB foci upon irradiation.

Furthermore, to achieve imaging of genomic stress in whole-tissue in mouse, we established a new mouse strain, and organoids from the mouse. We consider that our model is useful to connect the knowledge obtained from organoids to whole body.

The time course of pathological remodeling occurring in the cynomolgus monkey hearts against atrioventricular block condition was studied (n=10). The atrioventricular block induced the ventricular and atrial dilation followed by the ventricular hypertrophy. Interstitial fibrosis in the ventricle was also observed, which was associated with gradual increases in the plasma angiotensin II and aldosterone concentrations. These anatomical and histological adaptations were in accordance with the changes in gene expression profiling reflecting fibrosis and hypertrophy. Systolic/diastolic blood pressure after the atrioventricular block was kept equal to or lower than that before the block, which was partly explained by lack of increase in the plasma catecholamine levels. While no marked difference was observed in pharmacokinetic variables of dl-sotalol between 1 month and 7 month after the atrioventricular block, 10 mg/kg of dl-sotalol hydrochloride induced torsade de pointes (TdP) in 6 out of 10 animals with time during the experimental period of ≦15 months. Animals showing greater QT-interval prolongation after the atrioventricular block developed dl-sotalol-induced TdP earlier. Each TdP spontaneously terminated, which would be partly explained by relatively small "effective size of the left ventricle" in the atrioventricular block monkey heart. These findings would provide a fundamental knowledge for better utilizing the chronic atrioventricular block monkeys as a proarrhythmia model for detecting drug-induced TdP.

Benzo[a]pyrene (BaP), is a known genotoxic and carcinogenic agent. This study aims to investigate the underlying mechanisms and histopathological changes within mouse brain in relation to BaP exposure. Sixteen wild type male mice (10 weeks age) were allocated into 4 groups and exposed to BaP at doses 0, 2.88, 8.67, 26.00 µg/mice (that are relevant to occupational exposure) respectively, by pharyngeal aspiration once/week for 4 weeks. The effects of BaP on motor function were assessed by landing foot spread test, short-term memory and anxiety like behavior were evaluated by using Y-maze and elevated plus maze test. Noradrenergic axon density was evaluated by IHC in hippocampal CA1 and CA3 area. Result showed no alteration on body weight gain through the treatment, while motor function decline in all treated groups in third and fourth weeks. In addition, both acute and chronic exposure to BaP with different doses revealed a decrease percentage of spontaneous alterations compared to the control mice. For anxiety like behavior, the mice receiving middle dose showed reduced open arm exploration behaviour as indicated by lowering open total ratio for entries than the other groups, implying increase in anxiety like behavior. The density of noradrenergic axons significantly decreased in CA3 area of hippocampus (low and middle groups) and CA1 area for all treated groups. These finding data suggesting that BaP might be induced neurobehavioral alteration and neurotoxicity. Further study is needed to elucidate the underlying mechanism of neuro-morphological changes induced by BaP.

In Japan, cerebrovascular disease is the third leading cause of death, and ischemic stroke accounts for more than half of all cerebrovascular disease deaths. In the chronic phase of ischemic stroke, treatment is limited to prevention of recurrence and reduction of complications and sequelae, therefore it is important to be able to provide appropriate treatment in the hyperacute and acute phases. The cells in the penumbra region, where blood flow is reduced, are damaged after the infarction but can be saved from cell death with appropriate treatment. The control of tissue inflammation is worth to improve pathological progression at the penumbra, and microglia and related other immune cells are major cellular targets for that purpose. Minocycline, a tetracycline antibiotics, is used in the treatment of infections caused by unconventional bacteria such as mycoplasma pneumonia and chlamydial infections. On the other hand, minocycline is known to inhibit the activation of microglia. In this study, we investigated the effect of minocycline on the pathological progression of ischemic stroke. We used a photothrombotic ischemic stroke model mouse and examined cellular dynamics of microglia and astrocytes. The results indicated that the astrocytes in the minocycline-administered brain have bigger cell body and thicker astrocytic processes, compared with those in the control saline-administered brain. This result indicates a functional relationship between microglia and astrocytes. I would like to discuss the effects and benefits/risks of minocycline on the pathological progression of ischemic stroke.

Fetal alcohol spectrum disorders (FASD) are diseases occurring on children by an excess alcohol intake by pregnant women. FASD is prevalent worldwide: the average rate of FASD is about 15 per 10,000 newborns. FASD with mild symptoms are not properly diagnosed and can result in intellectual disability, attention-deficit hyperactivity disorder (ADHD), and alcoholism in adulthood, therefore FASD is a socially important disease.

Previous studies have revealed the importance of microglia and astrocytes in FASD onset and progression. However, the relationship between microglia and astrocytes is not well determined. We utilized mouse FASD model to clarify the relationship between microglia and astrocytes. The administration of ethanol from postnatal days 4 to 6 reduced the activity of microglia and increased astrocyte reactivity. In addition, the ethanol exposure changed the localization of astrocytes in the cerebral cortex. Forced reduction of microglial activity by minocycline similarly triggered astrocyte reactivity, suggesting the suppression of astrocyte reactivity by microglia in the developing cerebral cortex. We also found that C-C chemokine ligand (CCL) 26 was upregulated in the ethanol-exposed cerebral cortex. An antagonist of CCR3, the main receptor for CCL26, enhanced the ethanol-induced astrocyte reactivity and change of the localization of astrocytes, suggesting that CCL26-CCR3 signaling attenuates the ethanol-induced phenotypic transformation of astrocytes. These results indicate novel mechanisms underlying the regulation of astrocyte reactivity in the cerebral cortex exposed to FASD.

We previously established an animal model of Parkinson’s disease (PD) induced by chronic low-dose rotenone as an environmental neurotoxic pesticide that reproducible behavioral and central and enteric neurodegenerative features of PD, and revealed that rotenone-induced enteric neurodegeneration is caused by dysfunction of enteric glia using primary cultured enteric cells. However, the mechanism of enteric neurodegeneration and inflammation are still obscure. In this study, we examined changes in enteric cellular environment in the enteric epithelium and myenteric plexus of the rotenone-induced PD model mice. Chronic subcutaneous administration with low-dose rotenone (2.5 mg/kg/day) for 4 weeks using an osmotic mini pump reduced the number of dopamine neurons in the substantia nigra and the intestinal myenteric neurons and glial cells of mice. Furthermore, it produced translocation of HMGB1 to cytosol towards the bowel lumen. These results suggest that the epithelial barrier disruption, inflammatory reactions and dysfunction of enteric glia would be involved in the rotenone-induced enteric neurodegeneration.

Female F344 rats were intratracheally administered crystalline silica (Min-U-Sil5; 20 mg/rat) or sintered indium tin oxide (ITO, 10 mg/rat), and sacrificed at 3 days, 4 weeks and 26 weeks after the administration. At all dissection periods, both silica and ITO increased neutrophils and LDH activity in the bronchoalveolar lavage fluid (BALF). Histopathological examination revealed that ITO caused alveolar proteinosis via potent macrophage injury, while silica increased perivascular lymphocytic infiltration and Min-U-Sil5-phagocytosed macrophages in the alveolar interstitium during the acute phase. In the chronic phase, Min-U-Sil5-treated rats were suffered from multifocal lesions including fibrosis and hyperplasia. In agreement with this, metabolomics analyses showed that ITO accumulated a large amount of phospholipids in the alveolar region. Furthermore, sphingomyelins and ceramides levels in BALF are also suggested to be increased by ITO and Min-U-sil5. In the plasma 4 weeks after intratracheal instillation of ITO and/or Min-U-Sil5, an increase in fatty acid amides, such as oleamide, and lysophosphatidylcholines were suggested. These changes may be candidate markers of lung damage caused by toxic particulate matters.

【Introduction】Exposure of pregnant rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces prolactin expression levels during lactation. The suppression of prolactin levels is then inherited by the next generation of female rats. As prolactin promotes milk secretion, this study aimed to analyze milk secretion and the milk nutrient content of first and second generation (F1/F2) female offspring with the same low prolactin constitution as their F0 mothers and grandmothers during lactation, to explore the significance of low blood prolactin levels in female offspring.

【Methods】Wistar rats were orally administered TCDD (1 µg/kg) or corn oil at gestational day 15. At 7 weeks of age, pregnancy was induced in the F1 and F2 females. Their breast milk was collected on postpartum day 5. Total protein and relative whey acidic protein (WAP) concentrations were determined.

【Results】The results showed that maternal exposure to TCDD in F0 dams significantly reduced total milk production in the F2 offspring, while milk production in F1 offspring tended to be reduced during lactation. An analysis of the milk protein content of the F1 offspring during early lactation showed that the total protein concentration did not change, but the WAP concentration significantly increased in F0 dams maternally exposed to TCDD. These findings indicate that the whey protein composition of milk in lactating F1 offspring differed substantially, and the total amount of protein (except WAP) was significantly reduced in rats with, compared to those without, F0 maternal exposure to TCDD.

The Ogasawara Islands, which have a unique ecosystem, are facing the problem of ecosystem disturbance caused by non-native species such as rats, and have taken countermeasures by dusting with an anticoagulant rodenticide (ARs). ARs act on VKOR to stop the vitamin K cycle of the blood coagulation system, causing systemic bleeding and death of the target. Although ARs are widely used, primary exposure by direct ingestion of the ARs by non-target animals and secondary exposure by ingestion of rodents killed by the ARs are major problems. Then, when using ARs, it is necessary to consider whether they threaten other species inhabiting the target area. In particular, the Ogasawara Islands are home to endangered endemic species, such as the Ogasawara Greenfinch (Chloris kittlitzi) and the Bonin flying fox (Pteropus pselaphon), and thus primary exposure to ARs may be a concern. Then, evaluating their susceptibility is important.

In this study, we aimed to evaluate the effects of ARs application on endemic species in these islands. We used microsomes extracted from the livers of the Greenfinch (C. sinica), a sister species of the Ogasawara Greenfinch, and the Bonin flying fox and conducted in vitro susceptibility testing. As a result of the metabolism test, in addition to warfarin (WF), its hydroxylated forms, 4’-, 6-, and 8-OH-WF, were identified in the Greenfinch, and 4’-, 6-OH-WF in the Bonin flying fox, suggesting that metabolism for these substances take place. In addition to this study, we are now conducting a VKOR inhibition test and plan to conduct a more detailed evaluation.

Neonicotinoid insecticides (NNIs) are one of the most abundantly used insecticides all over the world these days. For the present risk assessment of NNIs-induced toxicities, experimental animals such as mice, rats, and rabbits have been used. However, these experimental animals are taxonomically distant from many wild mammals, and not appropriate as animal model. Thus, we focused on musk shrew (Suncus murinus) as a novel animal model. Musk shrew is classified within Euarchontoglires as other many wild mammalian species. Because of high carnivorousness, musk shrew is possible to have different metabolic characteristics from experimental animals. The objective of this study is to reveal the characteristics of metabolism towards acetamiprid (ACP), one of NNIs on musk shrew. Pharmacokinetics analysis, in vitro metabolic assay, and genetic analysis of cytochrome P450 (CYP) were performed using musk shrews, mice, and rats. As a results of pharmacokinetic analysis, concentration of ACP in blood decreased more quickly in musk shrews than mice. By in vitro metabolic assay, it was suggested that musk shrews have higher metabolic abilities towards ACP compared to mice and rats. By genetic analysis, some of CYP2A and CYP3A isoforms of musk shrews were thought to be particularly important in ACP metabolism. From the results above, unique metabolic characteristics of musk shrews were indicated. To elucidate the characteristics of CYP-dependent metabolism of xenobiotics in musk shrews, it is expected to reveal the function of each CYP isoform using recombinant CYP enzymes in the future.

【Background & Aim】

There is an increasing concern about the effects of environmental chemical substances on the endocrine system and reproductive function. Some chemical substances such as ethylene oxide have been suspected to affect reproductive function and sex hormones in animal studies. However, there are limited evidences in the effect of commonly used chemicals on human sex hormone levels. The purpose of this study is to analyze the association between blood levels of frequently used chemical substances and sex hormone levels in human.

【Methods】

Of the participants in NHANES (2013-2016), 816 adult males were included in the analysis. Using random forest regression model, we analyzed the relationship between blood levels of 40 common chemical substances and blood testosterone (TST) and estradiol (EST) by variable importance. Ethylene oxide (EtO), which had the highest variable importance, was investigated for dose dependence on TST and EST levels by multivariate regression analysis.

【Results&Discussion】

In random forest regression, EtO had the greatest importance on TST and EST levels among the 40 evaluated chemicals. In addition, the importance of EtO on the levels of TST and EST was greater than age and smoking habits, whose effects on sex hormones are known. The results of multivariate analysis by smoothing spline regression and linear regression analysis revealed that higher level of EtO significantly contributed to the increase in TST level. There was no statistically significant relationship between EtO and EST levels. These results suggest that EtO is associated with increasing level of TST in men.

Neuronal electrical activity with high-spatio-temporal resolution is useful for understanding brain functions and elucidating neurological disorders, and for drug screening and safety assessment of drugs. CMOS-MEA is excellent in detecting detailed electrical activity patterns of neural networks due to the large number of electrodes. In this study, we investigated the electrical activity characteristics of brain slices, brain organoids, and cultured neural networks using CMOS-MEA with 236,880 electrodes, which have the highest specifications in the world. In the measurement of brain slices, we succeeded in measuring the interregional propagation of the hippocampus and cerebral cortex, ventral tegmental area in detail, and detected changes of propagation patterns due to drug administration. In sensory neuron measurement, calculation of axon conduction velocity in single neuron and drug responses based on firing pattern of each neuron were detected. In human iPS cell-derived central nervous system networks and human cerebral organoids, network activity was detected on a cell-by-cell basis, and changes in propagation patterns due to drug administration were detected. It was found that CMOS-MEA with 236,880 electrodes and a large measurement area can measure the electrical activity characteristics of ex vivo and in vitro neural networks and single neuron in detail. It was suggested that big data with high temporal resolution is effective for elucidation of neural circuit function and drug evaluation based on new neural activity information.

Although many cell lines have been developed as ideal in vitro models, there are many reports that imprinted differentially methylated regions (DMRs) are not properly maintained in such cell lines. Aberrant DNA methylation in imprinted DMRs could cause loss of imprinting (LOI) in imprinted gene clusters. LOIs have often been observed in cancer cells, cultured cell lines, and aged tissues.Recently, a three-dimentional (3D) culture system, the organoid, has been developed and used as a model that reproduces an environment closer to that in vivo; however, whether epigenetic status is maintained in organoids is not clearly established.

In this study, whole-genome bisulfite sequencing of colon and colon-derived orgnaoids from F1 mouse crosses (JF1 x C57Bl6/J) was performed to compare the DNA methylation status between the original tissue and tissue-derived orgnaoids. Global DNA methylation was decreased by 10.38% in the colon-derived organoids, however, most of the decreased methylation locus were not near promoter-associated CpG islands (CGIs), suggesting that most of the DNA methylation changes might not alter gene expressions. On the other hand, imprinted DMRs were maintained. Finally, by screening CGIs with methylation levels of approximately 50% and using SNPs between JF1 and C57BL6/J to distinguish paternal and maternal allels, at least three CGIs were isolated as a novel imprinted candidates. Organoids appear to be an excellent model system for paving the way for drug screening and toxicity testing, including the identification of epigenetic changes.

Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are involved in drug resistance in cancer, and share some molecular characteristics. To reveal the molecular networks responsible for cancer malignancy and responsiveness to therapeutics, gene expression and molecular networks in diffuse-type gastric cancer (GC), which is resistant to anti-cancer drugs, and intestinal-type GC were analyzed with Ingenuity Pathway Analysis (IPA). The injury or sustained reactive oxygen species (ROS) causes resistance in human GC. ROS has multiple roles such as development and progression of cancer, or apoptotic induction causing anti-tumor effects. EMT, which is cellular phenotypic change from epithelial to mesenchymal-like feature, demonstrates CSC-like characteristics in human gastric cancer. Adverse Outcome Pathway (AOP) 298 “Chronic ROS leading to human treatment-resistant GC” has been developed in OECD AOP project. This AOP298 consists of Molecular Initiating Event (MIE) (KE1753) as chronic ROS, followed by Key Event (KE) 1 (KE1754) as sustained tissue damage / macrophage activation / porcupine-induced Wnt secretion, KE2 (KE1755) as proliferation / beta-catenin activation, KE3 (KE1650) as EMT, and Adverse Outcome (AO) (KE1651) as human treatment-resistant GC. In this AOP, we focus on the role of chronic ROS with sustained level to induce the therapy-resistance in human gastric cancer. EMT is induced by Wnt/beta-catenin signaling, which confers rationale to have Wnt secretion and beta-catenin activation as KE1 and KE2 on the AOP, respectively.

[Purpose] UDP-glucuronosyltransferases (UGT) are pivotal xenobiotic-conjugating enzymes for drugs, environmental pollutants and dietary compounds such as polyphenols. UGT isoforms of mammal are encoded in several gene family and show various substrate specificity and regio-specificity towards structurally different compounds. In this study, orthologus enzymes of mammalian UGT1A1 and 1A6 were functionally analyzed in comparison with those of human.

[Methods] Mammalian UGT1A1 and 1A6 (human, cynomolgus monkey, marmoset, rat, and mouse) were expressed in budding yeast cells, Saccharomyces cerevisiae AH22, using a multicopy plasmid (pGYR). Microsomal fractions were prepared from each transformat of yeast cells. Protein expression of UGT1A was confirmed by Western blot analysis.

[Results and Discussion] In the identity of amino acid sequence, UGT1A1 and 1A6 show 93-95% of identity in each isoform of primates or rodents. All UGT1A1 isoform have a glucuronidating activity toward bilirubin as degradation product from heme. UGT1A1 and 1A6 showed the ability of glucuronidation towards several substrates such as scopoletin, bisphenol A, and 1-hydroxypyrene with isoform- and species-dependent difference of activity. In addition, several polyphenols as dietary functional compounds were regio-specific glucuronidated by UGT1A1 and 1A6. These results indicated that some differences in enzyme properties of UGT1A1 and 1A6 need to be considered when extrapolating the glucuronidation of xenobiotics in model animals to human.

In vivo cardiomyocytes produce most of their energy from mitochondrial oxidation of free fatty acids. However, it is difficult for iPSC-derived cardiomyocytes in vitro culture to mimic the energy metabolism of cardiomyocytes in vivo due to insufficient oxygen supply. In this study, we evaluated the effect of oxygen permeability on human iPS cell-derived cardiomyocytes using a high oxygen permeable culture plate, named as InnoCellTM, newly developed by MITSUI CHEMICALS, Inc. First, iPSC-derived cardiomyocytes were seeded on the highly oxygen permeable culture plates and cultured for several days, and we detected cardiomyocyte markers and mitochondria-regulated genes was enhanced. Next, the effect on pharmacological response was verified by measuring calcium transients. The arrhythmogenic effects of the multichannel blocker Bepridil were clearly detected on oxygen-permeable plates compared to polystyrene plates. We would like to discuss the effect of using the highly oxygen-permeable plates in an in vitro assay using iPSC-derived cardiomyocytes.

As thyroid hormones (THs) are essential for brain development, concern has been raised that mild THs disrupting chemicals may have potential to interfere with the developing brain. Since guideline studies to evaluate developmental neurotoxicity require significant resources, a simple screening assay would be valuable. The Comparative Thyroid Assay (CTA) is a candidate screening test, but it requires several animals and advanced techniques of sampling and THs measurements. We are currently attempting to develop a downsized CTA that reduces the animal number, while adding extra parameters in offspring (brain THs levels and histology). We had shown that 6-propylthiouracil (10 ppm) induced severe (> 50%) reduction of THs and offspring brain abnormalities, while phenobarbital (PB, 1000 ppm) induced mild (approx. 30%) reduction of THs but did not induce brain abnormalities. Here we investigated suitability of doses tested and reproducibility of the findings with PB. 1000 and 1500 ppm PB were fed to 10 pregnant rats/group from (1) gestational days (GD)6 to 20, and (2) from GD6 to lactation day 21. THs levels and brain histology were examined. The previous findings at 1000 ppm were reproduced. Compared to 1000 ppm, 1500 ppm revealed similar or slightly more potent THs suppression but was an excessive dose as evidenced by neurological signs in all dams and reduced pup survival. These findings suggest that the modified CTA is feasible and reliable and mild suppression of THs may have little impact on offspring brain development. Further studies should be investigated.

Stress is one of the environmental factors affecting development. It has been reported to be associated with the risk of developing developmental disorders. The social-defeat stress (SDS), the one type of psychosocial stress, model has been established as an animal model of this stress. In this study, we evaluated developmental stage-dependent abnormalities in spontaneous activity and social interaction in pups (prenatal SDS exposure model mice) of mice exposed to SDS by aggressive mice during developmental stages. Preganant C57BL/6J mice were brought into contact with aggressor male mice between embryonic days 10 and 15 and after that, reared together for 24 hours through a perforated clear acrylic plate. The pups were then analyzed for spontaneous activity, anxiety-like behavior, social interactions, and light/dark rhythms during development and mature stages. In the open field analysis, distance and duration were significantly increased during the developmental stages. Home cage monitoring analysis showed a significant increase in distance during the light period and frequency of contact with another individual during the dark period. In the social interaction test, behavioral characteristics against Stranger mice (mice meeting for the first time) were altered by prenatal SDS exposure. These behavioral abnormalities showed the similar phenotypes with those identified in mouse models of autism. These data indicate that prenatal SDS exposure might induce developmental disorder-like behavioral abnormalities.

Reactive sulfur species (RSS) exhibit high nucleophilicity/antioxidative property and thus is thought to play an important role in repressing electrophilic/oxidative stress. On the other hand, excess amount of RSS (sulfur stress) may disrupt redox homeostasis and increase health risk. Here, we examined adaptive response against intracellular sulfur stress. The present study suggests that there are cystine-dependent antiporters including SLC7A11 to excrete excess intracellular CysSSH.

Nikon Corporation, in collaboration with research group of Professor Masayuki Murata*, Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, has developed a new signal pathway analysis method that estimates and visualizes the activated signal pathway from immunostaining images of dozens of proteins utilizing intracellular localization information of proteins.

In this method, at first the proteins are quantified where, when, and how the protein behaves in the cell.

Next, by calculating the correlations of those quantified data, the relationship between proteins including intracellular localization information is estimates and visualized.

This make it possible to capture changes in the signal pathway due to drugs.

In this presentation, we will introduce an example of applying this method to insulin response research.

*Currently, “Multimodal Cell Analysis Collaborative Research Cluster, Institute of Innovative Research, Tokyo Institute of Technology”, “Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology”, “International Research Center for Neurointelligence, Institutes for Advanced Study, The University of Tokyo”.

Cell free DNA (cfDNA) circulates in the blood. Since cfDNA is derived from the dead blood cells, it is also present in healthy person, however, ctDNA (circulating tumor DNA) released from cancer cells has a mutation in a cancer-related gene in the blood of cancer patients. In recent years, since ctDNA having a mutation in a cancer-related gene released from a tumor exists in the blood of a cancer patient, the development of liquid biopsy using cfDNA as a biomarker for carcinogenesis has been extensively promoted.

Since there are DNA methylation sites specific to various organs in the body, cfDNA with DNA methylation specific to the target organ would leak into the blood stream when the target organ is damaged.

Therefore, in this study, cfDNA with liver-specific DNA methylation was used as a liver injury-specific biomarker in the blood of male C57BL/6J mice orally administered with carbon tetrachloride (70 mg/kg) which could induce liver injury.

The liquid biopsy by using DNA methylation of cfDNA as biomarkers would accelerate a rapid evaluation of chemical substances and drugs in Nonclinical Safety Evaluation.

Introduction

Fructose is known to be one of the most important risk factors for metabolic syndrome, and there are many reports on its toxic effects on the body. In this study, we investigated the effects of lighting environment during blood sampling on the blood biochemical parameters of rats fed a high-fructose diet.

Methods

Six-week-old male SD rats were divided into two groups: normal diet (CD) group and high fructose diet (HFD) group, and fed ad libitum for 4 weeks. Blood samples were collected before grouping and at 1, 2, 3, and 4 weeks after grouping between 9:00 and 11:00 a.m., and blood biochemical parameters were measured. After the final blood collection, livers were collected from euthanized animals between 9:00 and 11:00 a.m. under non-fasting conditions for histopathological examination.

Results and conclusions

Compared with the CD group, the HFD group showed an increase in liver weight and lobular central fat droplets in both the light and dark periods, and the degree of increase was similar in both periods. In blood biochemical tests, alanine aminotransferase (ALT) and glutamate dehydrogenase activity (GLDH) were elevated in the HFD group, but ALT and GLDH appeared one week earlier in the light and dark periods, respectively. The increase in leucine aminopeptidase activity was observed only in the light period, and the decrease in aspartate aminotransferase activity was observed only in the dark period.

Although tests in animal experiments are generally conducted under light conditions, the present study revealed that there was a difference in the response of rats to fructose in the light and dark periods.

We have previously shown that the effects of small molecules numerized by omics analysis and then fed into a latent variable model, which contributes to understanding the toxicity of the compounds. In this study, we tested whether numeric information extracted from pathological images by representation learning, a kind of deep learning method, contributes to the prediction of toxicity.

By using Open TGGATEs, we constructed a model that numerizes pathological images (chemical-leveraged model, CL model). We then compared the descriptive power of the conventional pathological findings, the baseline model without learning, and the CL model on images that were not used in the model construction. We evaluated whether the numerical information reflects the characteristics of the compounds by clustering, and found that the numeric information derived from the CL model formed clear clusters for each compound. Since the differences among compounds may be confounded by the differences between experiments due to treatment, we used the numeric information as input to predict the mode of action of the treated compounds. The results showed that the accuracy of the CL model (0.72) was higher than that of the baseline model (0.67). The pathological findings were hardly predictive (0.22).

Our representation learning model could extract numeric information that reflect the characteristics of pathological images. Since the numeric information is considered to be sensitive to toxicity, these could reduce the burden on pathologists and to reduce the number of animal studies in nonclinical studies.

[in Japanese]

We generated STA551, a Switch-Ig^TM antibody that binds to CD137 in an extracellular ATP concentration dependent manner; this is intended to reduce the effect on normal tissues and induce tumor-selective activity. Although STA551 is assumed to have higher target binding in tumors than in normal tissues, it is technically challenging to detect the binding of the switch antibody in vivo without significantly interfering with the ATP concentration. In this study, we investigated the target binding of STA551 in vivo using intravital imaging with two-photon microscopy and attempted to verify the concept that it reduces systemic immune activation.

Human CD137 knock-in mice were inoculated with the mouse cancer cell line LLC1/OVA/hGPC3 and intravenously administered fluorescent-labeled anti-CD137 antibodies with enhanced binding to the mouse Fcγ receptor. Using flow cytometry, we found that CD137 expression was higher in tumor than in spleen, and that T cells and NK cells were major CD137 expressing cells. According to intravital imaging, both the conventional and switch anti-CD137 antibody showed binding in tumor; however, in spleen, the fluorescence of the switch antibody was much weaker than the conventional anti-CD137 antibody and comparable with the control antibody.

In conclusion, the in vivo biodistribution of switch antibody was successfully assessed by intravital imaging with two-photon microscopy. These results reveal that STA551 showed tumor-selective binding, demonstrating the concept that STA551 can avoid systemic immune activation while exerting an anti-tumor effect.

One of the challenges in the current risk assessment for chemicals is that conventional toxicity assessment systems for assessing individual chemicals cannot evaluate the risk of combined exposure to chemical mixtures. Based on the concept of adverse outcome pathway (AOP), common key events in toxic mechanisms could be used as indexes to assess the combined effects of multiple chemicals. At the last conference, we reported a novel neurotoxic mechanism in which a pyrethroid deltamethrin caused an imbalance in the proteolytic system via the activation of mitophagy and inhibition of proteasome activity. In this study, we examined the neurotoxic effects of multiple pesticides by evaluating these endpoints as key events. We exposed 10 pesticides to mouse neuroblastoma Neuro-2a cells and measured mitochondrial membrane potential and proteolytic activities. The combined effects of pesticides on cell viability were measured by wst-8 assay and analyzed by CompuSyn software. Our data showed that a pyrethroid pesticide, permethrin or deltamethrin, and a phenylpyrazole pesticide, fipronil, shared common key events, such as mitochondrial dysfunction and accumulation of p62, an autophagy substrate marker. Combined exposure to these pesticides induced a further decrease in cell viability compared to single exposure, which showed synergistic effects with a combination index < 1. These data constitute the first analysis of synergistic neurotoxicity caused by combined exposure to different pesticides relation to the common key events of AOP for neurotoxicity.

Cadmium (Cd) is a toxic metal that is widely distributed in the environment. It was reported that maternal blood Cd levels during pregnancy are positively associated with the risk of early preterm birth. Hypertensive disorders of pregnancy (HDP) are major causes of premature births. Since there is an association between Cd concentration and the risk of HDP development in the human placenta and Cd causes HDP in animal studies, we thought that Cd-induced HDP development leads to premature births. The onset of HDP involves two steps: placental hypoplasia and associated vascular endothelial damage. The placenta is formed through the proliferation and differentiation of cytotrophoblast cells into extravillous trophoblast (EVT) and syncytiotrophoblast (ST) cells. EVT infiltrates the uterus and remodeling of the spiral arteries. Previous work showed that long-term exposure to low concentrations of Cd (100 – 400 nM) inhibited EVT differentiation in human trophoblast stem cell model CT27. It was reported that a high concentration of Cd about 1 µM inhibited the migration of HTR-8/SVneo, EVT-like cells, but not clear about the effect of low Cd exposure on EVT function. Here, we showed that low concentrations of Cd also inhibit HTR-8/SVneo migration with long-term Cd exposure. These results suggest that Cd causes HDP through the disorder of the placental formation process by causing EVT dysfunction in addition to inhibiting differentiation into EVT. Further research is needed to verify which pathway Cd affects EVT. We are also analyzing changes in gene expression involved in migration.

Selenoprotein P (SeP) is a plasma protein containing 10 residues of selenocysteine (Sec) in one molecule. SeP transports selenium (Se) to several tissues and is degraded to induce the expression of other selenium-containing proteins (selenoproteins e.g., glutathione peroxidases GPx) having antioxidant capacity. We have previously shown that methylmercury covalently binds to Sec in SeP (Se-mercuration) and disrupts the selenium supply function of this protein, which result in the enhancement of oxidative stresses. In this study, we revealed that Se-mercuration of SeP does not affect its endocytosis and lysosomal degradation but further metabolization via selenocysteine lyase (Scly). Not only that, but we also found that super sulfide species, which contain sulfur chain in a molecular and recently known as a new type of post-translational modification of the protein, canceled Se-mercuration of Sec and recovered selenium supply function of SeP. These results suggest that 1. Se-mercuration of SeP causes disruption of selenium metabolism by substrate inhibition of Scly, and 2. Super sulfide can recover the disruption via supporting the release of Se-mercuration.

[in Japanese]

Our previous study demonstrated that Cd inhibited the total activities of PPAR transcription factors in several kidney cell lines. In this study, we investigated the involvement of PPAR in the Cd renal toxicity using the human proximal tubular cells (HK-2 cells). PPAR consists of three isoforms. Among the isoforms, only the PPARD knockdown significantly conferred the resistance to Cd. The transcriptional activity of PPARδ was decreased by not only PPARD knockdown but also Cd treatment. Using the DNA microarray analysis, it was identified PPARD knockdown downregulated the gene expressions of apoptosis related genes. Therefore, we examined the involvement of PPARδ in the Cd-induced apoptosis. PPARD knockdown decreased the apoptosis signal as wells as caspase-3 activity. These results suggest that PPARδ may play an essential role in Cd renal toxicity and apoptosis pathway may be involved in the PPARδ-related Cd toxicity.

　Methylmercury (MeHg), a worldwide environmental pollutant, seriously damages the central nervous system. We previously reported that exposure to 2.5 µM MeHg for 96 h induced cell growth, activation of MAPKs and transcription factors, and expression of HO-1 and Hsp70 in normal rat cerebellar astrocytes (NRA). In the present study, we investigated effects of antioxidants (Trolox, N-acetyl-cysteine (NAC), glutathione (GSH)) on MeHg toxicity in NRA. Cell viability and intracellular ROS were evaluated in NRA exposed to 2 or 4 µM MeHg and 0–200 µM Trolox, 0–4 mM NAC, or 0–4 mM GSH for 96 h. Protein expression was evaluated in NRA exposed to 2 µM MeHg and 25, 50 µM Trolox or 2, 4 mM NAC for 96 h. Cell viability was increased by exposure to 2 µM MeHg, which was suppressed by Trolox or NAC. However, co-exposure to 2 µM MeHg and GSH induced remarkable cell death. Exposure to 4 µM MeHg caused cell death, which was suppressed by NAC. Exposure to 2 µM MeHg increased intracellular ROS, which was suppressed by Trolox or NAC. However, co-exposure to 2 µM MeHg and GSH inversely induced a further ROS increase. Exposure to 4 µM MeHg decreased ROS, which was suppressed by NAC. Exposure to 2 µM MeHg increased phosphorylation and/or expression of MAPKs and transcription factors and expression of HO-1 and Hsp70, while it decreased expression of Nrf2 and SOD-1, which was attenuated by Trolox or NAC. These results suggested that ROS was crucially involved in the MeHg toxicity in NRA, and that there were qualitative differences in the mechanism of toxicity induced by low (2 µM) and high (4 µM) MeHg.

The chronic arsenic exposure is known to be related to the progression of atherosclerosis, although the pathogenic mechanisms are not fully elucidated. One of the risk factors for atherosclerosis is disruption of the blood coagulation-fibrinolysis system regulated by vascular component cells, such as vascular endothelial cells and smooth muscle cells. We previously reported that arsenite suppress the fibrinolytic activity of the vascular endothelial cells through selective inhibition of the expression of t-PA via activation of Nrf2. On the other hand, effect of arsenite on expression of genes involved in blood coagulation system was not fully elucidated. In this study, we investigated the effect of arsenite on expression of genes involved in blood coagulation and anticoagulation system in vascular endothelial cells and smooth muscle cells. We obtained the following results.

(1) Arsenite suppressed expression of thrombomodulin (TM), an anticoagulation factor, in vascular endothelial cells.

(2) Arsenite upregulated expression of tissue factor (TF), a coagulation initiator, in vascular smooth muscle cells.

(3) Suppression of TM by arsenite was recovered by siRNA-mediated knockdown of Nrf2 in vascular endothelial cells.

(4) Induction of TF by arsenite was suppressed by siRNA-mediated knockdown of Nrf2 in vascular smooth muscle cells.

These results suggest that arsenite induces a procoagulant state of blood to progress atherosclerosis through suppression of TM and induction of TF expression via Nrf2 pathway activation in vascular component cells.

Metallothionein (MT) is a metal-binding protein that maintains essential metal homeostasis, detoxifies toxic metals, and reduces oxidative stress. It is well known that MT expression is induced by exposure to heavy metals such as cadmium, but there is little information on MT induction by arsenic. Chronic arsenic exposure is known to induce vascular diseases such as atherosclerosis, but the defense response mechanisms against arsenic toxicity in vascular tissue remain unclear. In this study, we examined the induction of MT expression in the thoracic aorta and its perivascular adipose tissue (PVAT) of mice treated with a single administration of arsenite. Male C57BL6/J mice aged 7 weeks received a single intraperitoneal administration of saline 10 mL/kg or sodium arsenite (1.5, 3.0, and 5.0 mg/kg as the total dosage of arsenic). Three hours after administration, the thoracic aorta and PVAT were separated and collected. The thoracic aorta was further divided into the intima fraction and the tunica media/adventitia fraction. Expression levels of MT genes were measured by real-time RT-PCR. The results showed that both mRNA levels of MT isoform Mt1 and Mt2 were significantly increased in the thoracic aorta and PVAT. Also, arsenite significantly increased Mt1 and Mt2 mRNA levels in the intimal and the tunica media/adventitia fractions. These results indicate that exposure to arsenite induces MT gene expression in thoracic aorta and PVAT. It is possible that MT in vascular tissue and PVAT induced by arsenite exposure may play a protective role against arsenic vascular toxicity.

Many epidemiological studies demonstrated that chronic arsenic exposure has been linked to hypertension in persons living in arsenic-endemic areas. However, the mechanism by which arsenic induces hypertension remains unknown. Thus, the present study aims to clarify the molecular mechanisms involved in arsenic-induced hypertension by investigating the suppressing effects of arsenic exposure on the vasoprotective axes of the renin-angiotensin system (RAS). Male C57BL/6JJcl adult mice were exposed to arsenic through drinking water at 0, 8, 80, and 800 ppb for 8 weeks. Blood pressure and body weight are monitored and recorded every week. Experimental results showed that arsenic exposure increased both systolic and diastolic blood pressure in arsenic-exposed mice compared to the control group. Arsenic also increased MDA and Ang II levels and decreased Ang (1-7) levels in the plasma of arsenic-exposed groups compared to the control group. The expression of two key components of the vasoprotective axes of RAS, (i) ACE2 and (ii) MasR was significantly downregulated in mice aortas and hearts after arsenic exposure. Further investigation demonstrated that gene expression of the upstream transcriptional activator of ACE2, specificity protein 1 (Sp1) and SIRT1 was also downregulated by arsenic exposure. Obtained results suggest that possibly the suppression of the vasoprotective axes of the RAS is one of the key mechanisms involved in arsenic-induced hypertension. These findings reveal a novel molecular mechanism underlying arsenic-induced hypertension.

Glycidol is known as a mutagen and rodent carcinogen. N(2,3-Dihydroxypropyl)valine (diHOPrVal) is a hemoglobin adduct of glycidol and is used as biomarker for internal exposure to glycidol. It has been suggested that diHOPrVal is also formed from chemicals other than glycidol. In addition, various factors may affect the formation of the Hb adduct. In this study, the chemicals that produce diHOPrVal and the influential factors on Hb-adducts formation were examined.

In the in vitro assays, the formation of diHOPrVal (m/z 563) was detected from 3 chemicals (2-bromoethanol, 2-lodineethanol, and ethylene oxide; EO) except 2-chloroethanol. The EO–Hb adduct (m/z 533) was detected from EO and all haloalcohols. In contrast, in the in vivo assays, diHOPrVal and EO–Hb adduct were detected from only EO. When the interaction with amino acids was examined, glycidol formed amino acids-adducts. The interaction between these chemicals and human serum albumin (HSA) was analyzed using the protein thermal shift assay. EO and all haloalcohols interacted with HSA as well as Hb. These results suggested that diHOPrVal is formed from chemicals other than glycidol. In addition, the presence of other blood components, such as amino acids and HSA, may affect the formation of chemical–Hb adducts. Further research is expected to elucidate the remaining unknown factors that affect the formation of chemical–Hb adducts.

Enniatins are emerging fungal toxins that have attracted increasing concerns in recent years. In this study, a pharmacokinetic study of enniatin B (ENNB) was conducted in mice to examine absorption of ENNB after oral administration. Next, a 28-day repeated oral toxicity study of ENNB was conducted in mice to obtain a toxicological profile for risk assessment of enniatins. In the pharmacokinetic study, ENNB was administered orally (30 mg/kg) or intravenously (1 mg/kg) to 6-week-old male mice, and pharmacokinetic parameters were calculated. Concentrations of ENNB in feces and gene expression in the liver were also analyzed after oral administration. As a result, bioavailability was estimated to be 85.6%. In feces, an average of 5.26% of the oral dose of ENNB was detected. Liver RNA-Seq analysis revealed upregulation of many metabolism-related genes. In the 28-day toxicity study, ENNB was orally administered to 6-week-old mice at doses of 0, 7.5, 15, and 30 mg/kg, and hematology, blood biochemistry and histopathological examinations were performed. In males at 30 mg/kg, low erythrocyte counts, high BUN levels, and high absolute kidney weights were observed; however, histopathology and related parameters revealed no changes suggestive of ENNB effects. In conclusion, although ENNB is absorbed after oral administration, general toxicity is expected to occur at higher doses than those used in this study.

Microplastics (MPs) refers to plastic particles of 5 mm or less that exist in the environment such as the atmosphere and ocean. Therefore, there is concern about human exposure by various routes. However, even today, the effects on the human have not been clarified. It is known that the surface of MPs existing in the various environments is chemically deteriorated by external factors such as UV rays and waves. Therefore, it is essential to evaluate the biological effects in consideration of the deterioration state. Therefore, we first prepared a standard product that imitated the deterioration state of MPs in the environments. Furthermore, using them, we attempted to evaluate the effects of MPs on cells by cytotoxicity tests.

Polyethylene (PE), which has the highest production volume, was selected as the standard product for MPs. By irradiating the PE powder sample with UV rays of 172 nm, degraded PE with a chemically deteriorated surface was prepared. In addition, it was confirmed that the prepared degraded PE could be present in the environment by comparing with the sample collected from the environment. Next, using the obtained standard product, a cytotoxicity test by MTT assay was performed. As cell lines, RAW264.7 and THP-1, which are immune cells, and A549, HaCaT, and Caco-2, which are epithelial cells of lung, skin, and intestine, were selected. As a result, concentration-dependent cytotoxicity was observed only in degraded PE in all cell lines. This indicates that PE has different effects on cells depending on the presence or absence of deterioration.

Clotting factor Ⅸ (FⅨ) activates coagulation by undergoing vitamin K-dependent conversion of a glutamic acid residue to γ-carboxyglutamic acid (Gla). Splicing variant1 (V1) and variants2 (V2) are registered in NCBI as FⅨ. Both are thought to show coagulation activity, but little is known in coagulation inhibition by warfarin in V2. In this study, we analyzed V2, by measuring coagulation ability and warfarin sensitivity in vitro using cultured cells. We also analyzed the complex with factors Ⅶ and Ⅺ by in silico structure estimation using AlphaFold2.

In the coagulation activity test, V1 showed coagulation activity but V2 did not. Next, in the determination of GlaFⅨ by sandwich ELISA, Gla conversion was inhibited by warfarin in both V1 and V2. Furthermore, quantification of GlaFⅨ and totalFⅨ by Western blot suggested that V2 undergoes Gla conversion. Lastly, the result of in silico structure estimation showed that V2 and factorⅦ did not form a complex, but V2 and factorⅪ bound at a specific domain.

This study shows that V2 doesn’t possess blood coagulation ability when activated by factorⅧ and factorⅪ, but undergoes Gla conversion. In addition, AlphaFold2 showed image in which V2 and FactorⅨ were partially combined.

As a conclusion, V2 may act as a competitive inhibitor of V1 and may be a regulator in blood coagulation activity. In fact, the result of RT-PCR in mouse liver showed that V2 was quantified by about 1.5% compared to V1. Further researches are needed to confirm more accurate quantification and expression in tissue samples and to determine whether V2 acts as a competitive inhibitor against V1.

Reactive carbonyl species (RCS) are compounds with highly reactive carbonyl groups produced in the environment and in vivo. RCS covalently binds to proteins via Michael reaction. However, the target proteins and effect on physiological functions remain unknown. We selected 21 types of RCS and analyzed their effects on phosphatidylinositol-3 kinase (PI3K)–Akt signaling, which is an important pathway for maintaining intracellular homeostasis.

We found that several types of RCS suppressed the phosphorylation of Akt by treatment with epidermal growth factor (EGF) in A594 cells. We focused on methyl vinyl ketone (MVK), which showed the most significant suppression, and tried identifying the mechanism by which MVK interferes with Akt phosphorylation. MVK attenuated the phosphorylation of PI3K, but not EGF receptor (EGFR). This result suggests that MVK act in the pathway between EGFR and PI3K. To determine the modification sites in PI3K, we performed LC-MS/MS analysis. We identified that Cys656 residue in p85 subunit of PI3K was modified with MVK. Cys656 residue is in the SH2 domain that binds to receptor tyrosine kinases such as EGFR and recognizes their phosphorylation. Co-immunoprecipitation analysis revealed that MVK inhibited the interaction between EGFR and PI3K.

These results indicate that MVK modifies Cys656 of p85 subunit of PI3K and attenuates PI3K–Akt signaling by inhibiting EGFR–PI3K interaction, and the other RCS also regulates the function of PI3K by the same mechanism as MVK.

Environmental problems related to water pollution are also included in the SDGs as issue to be solved all over the world. Certain regions in India have reported the harmful impacts of arsenic contamination in groundwater and other water supplies on human health. Numerous industries rely on dyes to color our clothing, food, and houses, and even small amounts of dye wastewater can lead to specific types of water pollution. Wastewater regulations are not stringently enforced, thus, resulting in disorganized wastewater treatment, which leads to dye pollution of lakes or rivers. Currently, expensive and highly sophisticated equipment are used to reduce the toxicity of these contaminants, but do not address the problems mentioned above. This study aimed to explore and verify the practical feasibility of a method that involves the use of tea leaves as an adsorbent for easy implementation in terms of treatment costs, application environments, and technologies.

Used tea leaves were prepared from the 21 most consumed varieties of tea worldwide. The leaves were used as an adsorbent. For the adsorbates, 1 anionic dye and 4 cationic dyes were used for dyes and 6 toxic metals. Dye and metal concentrations were analyzed.

Many tea varieties displayed suitability for adsorption, with high adsorption capacities observed for cationic dyes and 3 metals. The physicochemical characterization and adsorption parameters were examined and practical knowledge for predicting adsorption mechanisms was obtained. This presentation will explain the results of detailed data analyses and describe further experiments for practical application.

Methylmercury (MeHg) is known to cause the severe neural degeneration in the central and the peripheral nervous system, which is called Minamata disease. Recently, we reported that the hypoalgesia was selectively induced in MeHg-exposed rats, which was recovered time-dependently. In addition, the number of neurons in the dorsal root ganglion (DRG) of MeHg-exposed rats decreased during hypoalgesia, while it also recovered to control levels after behavioral recovery. In this study, we performed immunohistochemistry in order to clarify whether neurogenesis occurred overtime in MeHg-exposed DRGs. MeHg (6.7 mg/kg/day) was orally administered to Wistar rats for 5 days and discontinued for 2 days, and this cycle was done once again. BrdU (100 mg/kg/day) was administered intraperitoneally for 5 days from a week before the fixation. Twenty-eight, 42, 56 and 70 days after the beginning of MeHg exposure, rats were fixed by paraformaldehyde and their DRGs were cryosectioned and processed for immunohistochemistry. The sections were immunostained for neuronal, nuclear markers and BrdU antibodies. Now we are exploring the optimal experimental condition to see the neurogenesis, and hope to show the results in the conference.

Particulate matters (PMs), represented by PM2.5, are known to aggravate various respiratory diseases such as chronic obstructive pulmonary disease (COPD) and bronchial asthma. Several epidemiological reports have shown that PMs increase the incidence of the onset of coronavirus disease 2019 (COVID-19), which has recently been raging around the world. However, the detailed mechanisms by which PM affects biological systems and triggers the onset of COVID-19 are not clear. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades host cells using angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) expressed on the host cell membrane. Thus, variations in their expression and function are expected to be associated with the pathogenesis of COVID-19. In this study, we aimed to elucidate molecular mechanisms by which PMs affect the onset of COVID-19 by evaluating the expression of ACE2 and TMPRSS2 upon exposure to various PMs. Here we focused on different types of PM such as PM2.5 collected from ambient atmospheres, diesel exhaust particles, titanium dioxide nanoparticles, and Asian sand dust. We introduce the results of the evaluation of the expression level of ACE2 and TMPRSS2, the identification of their expressing cell types in the lung, and the variation in their functions when these PMs were exposed to mouse and human alveolar epithelial cell lines.

Neonicotinoids and fipronil are globally used insecticides for agriculture including rice farms. To reduce the cost of production, most laying duck farms in Thailand are free grazing systems and these ducks are commonly raised on paddy fields. Free grazing ducks are extensively related to paddy field environments. However, the information on levels of these insecticides in paddy fields linked to residues in ducks and eggs are still lacking in Thailand. Therefore, the objectives and methods in this preliminary study are as follows; (1) to evaluate the accumulation levels of neonicotinoids, fipronil, and their metabolites in paddy fields and residues in ducks and egg ducks, these substances and their metabolites in soil, rice, eggs and blood of ducks will be extracted and analyzed using a liquid chromatography with tandem mass spectrometry (LC-MS/MS). (2) To estimate the exposure and potential toxicity of these pesticides for egg duck consumption in humans, the estimated daily intake of these chemicals will be calculated and compared to the international reference levels. In addition, (3) to investigate the metabolism capacity of neonicotinoids and fipronils in ducks, the in vitro biotransformation will be conducted using duck liver microsome containing cytochrome P450 enzyme. The results of this study will effectively provide the information on environmental levels and metabolic capacity of these insecticides in ducks that might relate to our understanding of the potential toxicological effects in humans and other avian.

Estrogenic endocrine disrupting chemicals (E-EDCs) have a grave attraction globally, not only for their toxicological effects in the ecosystem but also for their significant impact on human. These chemicals are more likely to occur as a combination rather than a single stressor. However, whether and how the binary exposure to E-EDCs may induce developmental effects remains largely unknown. Here we investigated the single and binary exposure to distinct xenoestrogens, diethylstilbestrol (DES), zearalenone (ZEN) and equine estrogens (EEs) on zebrafish embryos. Significant effects on cumulative mortality, growth retardation, circulatory failure and spinal curvature were observed in DES-exposed embryos (96 hours post fertilization) at 0.3μM, as well as ZEN at 5μM. None of EEs exhibited apparent developmental toxicity even at high concentration. Developmental toxicity of DES was improved by co-exposure to either ZEN or EEs such as equilin, equilenin, and 17α-/17β-dihydroequilenins in a concentration-dependent manner, whereas ZEN-induced toxicity was not improved by co-exposure to EEs. Additionally, DES- and ZEN-induced toxicity was counteracted by co-exposure to an estrogen receptor (ER) antagonist, indicating that ERs are involved in the developmental toxicity of single exposure. Furthermore, our results showed that co-exposure to either ZEN or EEs suppress the transcriptional expression of cyp19a1b and 17β-hsd in comparison to DES alone. These results indicate that the competitive action against the ERs could be the main mechanism of ameliorative effect posed by ZEN and EEs.

Nanomaterials are fine particles of less than 100 nm in short diameter. Although the field of use is expanding in various industries because of their innovative functions, there are concerns about their unexpected biological effects. From this viewpoint, we are evaluating on reproductive toxicity of nanomaterials focused on placenta. Our previous studies showed that silver nanoparticles with a diameter of 10 nm (nAg10) could suppress the placental syncytialization process, which is important for structural and functional placentation. On the other hands, there are still many unclear points about the suppression mechanism of syncytialization by nAg10. Here, we attempted to elucidate their mechanism by focusing on the involvement of reactive oxygen species (ROS).

To assess the effect of nAg10 on ROS production, BeWo, a human choriocarcinoma cell line, was treated with forskolin (frk), syncytialization-inducing reagent, and nAg10. Immunostaining analysis revealed that nAg10 increased ROS production. Then, we co-treated BeWo with the antioxidant N-acetylcysteine (NAC) and nAg10, and evaluated the mRNA expression of hCGβ, which is a syncytialization marker, and syncytin-2, which is involved in promoting syncytialization. The results showed that NAC alleviated the suppression of these genes expression by nAg10. Currently, we consider that nAg10 might affect mitochondria, which is the main production area of intracellular ROS, and try to clarify the mechanism by investigating mitochondrial structure and function after nAg10 treatment.

[Background] While multi-walled carbon nanotubes (MWCNT) have already been widely used in various fields, there is still concern regarding their carcinogenicity because of the asbestos-like shapes. This study aimed to develop an alternative carcinogenicity test to an inhalation method.

[Methods] Male F344 rats were intratracheally administered MWNT-7, a reference MWCNT compound, for 2 years (26 times with 4-week intervals) at doses of 0, 0.125, and 0.5 mg/kg body weight (control, low-dose, and high-dose groups, respectively) from 9 weeks of age. Thirty animals in each group were histologically examined at year 2. Satellite animals were euthanized at month 6, year 1, and year 2 to monitor pathological conditions and the lung and pleural burdens of MWCNT.

[Results & Discussion] The incidence of lung tumors was 3.0, 10.3, and 39.3%, and that of pleural mesothelioma was 0, 13.8, and 42.9% in the control, low-dose, and high-dose groups, respectively. The number of leukocytes and biochemical indices of bronchoalveolar lavage fluids were significantly increased in the MWCNT-dosed groups. At year 2, the lung burden was 0.9 and 3.6 mg/Lung in the low-dose and high-dose groups, respectively. The lung and pleural burdens increased in a time and dose-dependent manner. Since the incidence of lung tumors and the time-course of the lung burden were similar to those of a previous long-term inhalation test (Kasai et al. 2016), the present intermittent exposure test could become an alternative carcinogenicity test. Intratracheal administration methods tend to cause pleural mesotheliomas.

Acrylamide (ACR) is a soft electrophile which is known as a potent neurotoxicant in human. It is discovered accidentally during processing food at high temperatures through the Maillard reaction. Interleukin-1ß (IL-1ß) is one of the proinflammatory cytokines and act as a critical component of brain reaction to any insults especially neuroinflammation, but its role in electrophile-induced neurotoxicity is not exactly understood. Therefore, this study aims to investigate the important role of IL-1ß as one of the core cytokines in ACR-induced neurotoxicity in mice. Ten-week-old male wild type (C57BL/6JJmsSLC) and IL-1ß knock-out mice were allocated into three groups per each and subjected to ACR as follow 0, 12.5, 25 mg/kg body weight by oral gavage for 28 days. Comparative to wild type mice, the result revealed a significant increase in landing foot spread test in IL-1ß KO mice exposed to high dose and decrease density of noradrenergic axon in somatosensory cortex area. In wild type mice, exposure to ACR induced a significant increase in expression of Nrf2 gene and its downstream antioxidant genes HO-1, NQO1and GST-M as well as IL-6 and increased NF-kB1 expression. RNA sequence analysis of mice cerebral cortex showed that IL-1ß knockdown increased the oxidative phosphorylation, AD, PD and ALS phenotype compared to wild type mice. We suggest that presence of IL-1ß may protect the mice brain against acrylamide-induced neurotoxicity through oxidative stress, NrF2/NF-kB1 pathway and p38MAPK/ p21. This data gives new insight about the role of IL-1ß in electrophile neurotoxicity in vivo.

This study investigated the effects of GBE and AGIQ on behavioral abnormalities and disruptive hippocampal neurogenesis in LPS-exposed adult rats. LPS at 1 mg/kg was i.p. injected on days 8 and 10. GBE and AGIQ were dietary administered at 0.5% from day 1 to day 38. Behavioral tests were conducted during day 31–38, and necropsies were conducted on days 11 and 38. On day 11, immunohistochemical analysis of the dentate gyrus revealed that LPS increased microglia and astrocytes, and both GBE and AGIQ increased CD163+ cells. Gene expression analysis revealed that LPS upregulated genes related to inflammatory and oxidative stress. In the open field test, there was no difference by treatment, but in the contextual fear conditioning test, LPS decreased fear memory acquisition and GBE facilitated extinction response of fear memory. On day 38, LPS decreased DCX+ or TUBB3+ cells; however, GBE restored the number of TUBB3+ cells, and AGIQ increased PCNA+ cells and decreased TUNEL+ cells. Among interneurons, GBE increased RELN+ cells and AGIQ increased CALB2+ cells. LPS upregulated Eomes, Dcx, and Pcna, while AGIQ downregulated Dcx and Pcna. LPS upregulated Reln, while GBE downregulated Calb1 and Reln. In summary, GBE and AGIQ showed a recovery trend from the LPS-induced decreased fear memory acquisition. GBE further enhanced fear extinction by learning, which might involve the enhancement of the late differentiation in neurogenesis through enhancing neural migration by RELN, while AGIQ had a protective effect by controlling the proliferation and apoptosis of neurogenesis.