Synaptic loss is the best neuropathological correlate to cognitive decline in Alzheimer’s disease (AD) and related dementias. Contributing factors include not only aggregated proteins such as amyloid-beta and tau, but also environmental factors such as air pollution and pesticides. These agents produce toxic stress from NOx (nitric oxide-related compounds), resulting in disruption of protein function via aberrant redox reactions on proteins, such as S-nitrosylation. Here, we describe distinct enzymes, a ubiquitin protein hydrolase (Uch-L1), a kinase (Cdk5), and a guanosine triphosphatase (Drp1), that act in concert to mediate a series of S-nitrosylation reactions from one to another. This non-canonical transnitrosylation cascade contributes to synaptic damage in AD. We show this series of reactions is kinetically and thermodynamically favored, resulting in mitochondrial fragmentation, bioenergetic compromise, and consequent synapse loss. We also develop a quantitative method based on Nernst equations for thermodynamic assessment of these redox reactions at steady state, as might be expected to occur in a chronic disease. This analysis reveals Gibbs free energies that are quite favorable for forward reaction through the transnitrosylation cascade. We conclude that enzymes with distinct primary reaction mechanisms can form a completely separate network of aberrant transnitrosylation steps. Finally, we will present potential therapies to prevent the resulting loss of synapses and cognitive function due to these aberrant transnitrosylation reactions in AD brain.

Unique bioactive compounds produced by microorganisms always have specific target molecules in the cell. Their identification has been useful information for drug discovery. We have so far identified many gene expression regulators as the targets, including histone deacetylases that control epigenetics, all of which are important targets for new anticancer drugs. However, the identification of target molecules of active compounds requires experience, knowledge, and insight, and systematic analysis has been difficult. We have been trying to establish a bias-free method for the identification of compound targets using chemical genomics with gene disruption and gene overexpression in yeast and phenotypic analysis by systematic gene knockdown in animal cells. In this talk, we will present the history of chemical genetics and the forefront of chemical genomics.

We humans are exposed to a huge amount of chemical substances through our food and the environment. Exposome (exposure to environmental factors) throughout life is expected to be deeply involved in health and longevity. Among these chemical substances, aldehydes are one of the most familiar compounds, and there are many sources inside and outside of living organisms, including not only dietary and environmental factors but also fatty acids and carbohydrate metabolites. Even glucose itself exhibits aldehydic properties. In addition, the peroxidation of polyunsaturated fatty acids produces not only aliphatic aldehydes but also various reactive (toxic) aldehydes, such as α,β-unsaturated aldehydes and ketoaldehydes. Living organisms have defense mechanisms, such as detoxification metabolism against these toxic aldehydes. On the other hand, aldehydes react with nucleophilic amino acid residues on protein molecules, producing unique abnormal structures (adducts). The sum of countless adduct structures (“modification signature”) is memorized as a stimulus for biological responses and is expected to play a role in the biological response mechanism. The most typical response mechanism to adduct structures is the immune system. Recent studies have revealed that certain autoimmune diseases produce antibodies that display unique cross-reactivity to aldehyde modifications. For example, in systemic lupus erythematosus (SLE), which is known to cause overproduction of autoantibodies against DNA, the presence of bispecific IgG antibodies has been shown have affinity for not only DNA but also for modified proteins with toxic aldehydes. Moreover, IgM antibodies, which are involved in biological defense as innate immune proteins, show more diversity in terms of cross-reactivity than IgG antibodies. In particular, with regard to a carcinogenic acrolein, an interesting cross-reactivity of both IgM antibodies and B cells (B-1a cells) that recognize acrolein-modified proteins have been revealed. The existence of innate immune B cells against acrolein-modified proteins suggests that life had to deal with this toxic compound at the beginning of its life and had acquired the ability to use innate immunity as a means to do so. The fact that toxic aldehydes exist somewhere in the immunological memory, which plays an important role in biological defense, is extremely interesting when considering the relationship with health and disease.

Our body possesses the ability to sense environmental stresses and activate cellular defense response. We have identified the KEAP1-NRF2 system. NRF2 is the transcription factor that plays a crucial role in the coordinated expression of cellular defense enzymes against oxidative and electrophilic stresses. KEAP1 acts as a sensor for these stresses and as a subunit of ubiquitin-E3 ligase that constantly degrades NRF2. Nrf2 gene knockout animals are sensitive to a broad array of toxic electrophiles and reactive oxygen species. In contrast, Keap1 gene knock-down animals exhibit a gain-of-function phenotype of NRF2. Modifications to the cysteine residues in KEAP1 negate its ubiquitin ligase activity, which stabilizes NRF2. Genetic as well as pharmacological inductions of NRF2 protect our body from the oxidative injury. We have demonstrated this activity in various disease models. The field continuously evolves with new topics emerging, including inflammation, metabolism, ageing, and neuroprotection by NRF2. Studies of the KEAP1-NRF2 system is now expanding toward human biology utilizing the cohort study of the Tohoku Medical Megabank and space mouse biology utilizing the International Space Station. Both historical contexts and recent advancements in the KEAP1-NRF2 system will be discussed.

We do not fully understand the effects and mechanisms of drugs clinically used in humans. This is because it is impossible to comprehensively measure the affinity for various receptors during drug development, and efficacy and safety studies in humans are also insufficient. In fact, it is not uncommon for a drug to find unknown harmful or beneficial effects on humans. In this talk, I will introduce the following results that aim to identify unexpected beneficial effects of drugs through the analysis of actual clinical real-world data, especially cases of adverse events, and to reevaluate drugs and discover novel drug targets.

(1) Usefulness and the mechanism of a thrombin inhibitor dabigatran for the interstitial lung disease caused by the antiarrhythmic drug amiodarone

(2) Usefulness and the mechanism of lisinopril for bullous pemphigus induced by the antidiabetic DPP4 inhibitors

(3) Usefulness and the mechanism of dexamethasone for tendinopathy caused by fluoroquinolone antibiotics

(4) Neural mechanism of proton pump inhibitors suppressing the risk of obsessive-compulsive disorder increased by dopamine D2 receptor stimulants

In this country, abuse of legally regulated substances such as narcotics, cannabis, cocaine, and psychotropic drugs has been a major social problem since the first, second, and third periods of stimulant abuse after the world war Ⅱ to the present. In addition to the problems of drug dependence associated with these abuses, there were accidents and incidents caused by poisonous chemicals, such as organic solvent thinner which led to a social problem.

Also, there was a period when a social atmosphere called psychedelic due to the abuse of LSD. Thereafter, laws and regulations and public awareness activities are being promoted by the national and prefectural governments. In the 1980s, illicit drugs synthesized by partially modifying the structure and the addition of functional groups of the synthetic drug fentanyl, so-called designer drugs, appeared for personal use and sales, and became a social issue.

The new psychoactive (dangerous) substances (NPS), such as phenethylamines, tryptamines, piperazines, amyl nitrites, and plants containing hallucinogens, which have been known as illicit, legal, and evasive drugs since around the year 2000, and have caused accidents due to their use evading legal regulations. After public comment, the name has finally settled to be NPS. This country, which had traditionally implemented legal regulations based on substance names, have introduced the regulatory concept of comprehensive designation based on chemical structure in 2008, and at once a large number of substances have designated to be designated drugs, and prohibited to use, possess　and sale. However, synthetic cannabinoid-containing plant products and cathinone derivatives have then emerged to avoid the comprehensive designation, and various substances with acting on central nervous system have been used both domestically and internationally. In particular, with the spread of the Internet and the progress of globalization, it has become relatively easy to obtain substances that have central nervous effects from sales sites. Under these circumstances, the government takes measures such as on-site inspection of stores selling drugs, measures against the internet site, quick evaluation for specifying designated drugs, border control measures for NPS, and exposing criminals. Many local governments, including Tokyo and Osaka Prefectures, are collecting information as quickly as possible on the actual use of chemical substances like NPS in Japan and overseas, independently verifying their pharmacological effects, and analyzing their usage status and toxicity both domestically and internationally. Based on the results of the evaluation by the expert committee, it is designated as a governor-designated drug, and then regulated as a minister-designated drug. To date, 41 cathinone-based drugs, 62 cannabinoid-based drugs, 5, LSD-based drugs, and 10 ketamine-based drugs have been designated by the governor. While NPS have become a social issue, we should also be aware of the current situation where the enormous amount of legally regulated narcotics, stimulants, cannabis, etc. have been seized at Japan customs. So, we should think that the use of centrally acting drugs is widespread in this country. As mentioned above, the abuse of NPS and other legally regulated substances may be quelled by strict regulations and educational activities by national and local governments, but it has not been overcome due to globalization. All members of this society involved in pharmaceuticals and other chemical substances will learn about the reality of drug abuse in this country and take an interest in social measures.

Dogs and pigs, together with cynomolgus macaques, are important animal species in drug metabolism and toxicity studies. However, some differences in drug metabolism patterns are occasionally noted due to species differences in cytochromes P450 (P450, CYP), important drug-metabolizing enzymes. To understand the species differences, we have identified and characterized P450s in a systematical manner in these animal species. In dogs, pigs, and cynomolgus macaques, a 1-to-1 relationship to human P450s were found for most P450s (including CYP1A, CYP2D, and CYP2E), but not for some P450s, including CYP2Cs partly due to species-specific P450s such as cynomolgus CYP2C76. Moreover, some P450s show substrate specificity and hepatic expression profile different from humans in dogs, pigs, and cynomolgus macaques. These results partly explain species differences in drug metabolism. Finally, we also have identified and characterized P450s in tree shrews, the species once categorized as primates and currently used mainly for virus research, to assess their usefulness in drug metabolism studies, and will present some of the results. This presentation will be mainly on CYP1A, CYP2A, CYP2B, CYP2D, and CYP2E.

Flavin-containing monooxygenases (FMOs) are a family of heteroatom-containing drug oxygenation enzymes that consist of functional enzymes (FMO1–5) in humans. Human FMO3 and FMO1 are the major hepatic and renal forms, respectively. Monkey, dog, and pig FMO1 and FMO3 have been identified. Expression of FMO1 have been detected in dog and pig livers. Pig liver microsomes had high oxygenation activities toward typical substrate benzydamine than human, monkey, and dog liver microsomes. The pharmacokinetic profiles of FMO-mediated benzydamine N-oxide were different for control and humanized-liver mice. In terms of the interactions of FMO substrates, limited interactions with FMO substrates, such as itopride and trimethylamine, were detected in humanized-liver mice. These results suggest that selection of suitable animal models is important for understanding human pharmacokinetics of substrates mediated by FMO enzymes.

Humanized-liver mice are attractive experimental models for drug metabolism and toxicological studies. Although chimeric mouse livers express multiple drug-metabolizing enzymes, including cytochrome P450 (P450), UDP-glucuronosyltransferase, and aldehyde oxidase, their drug-metabolizing activities have not been comprehensively analyzed. In this study, we conducted in vitro metabolic assays to characterize hepatic drug-metabolizing enzyme activity in humanized-liver mice. Human and humanized-liver mice exhibited similar hepatic microsomal coumarin 7-hydroxylation and flurbiprofen 4'-hydroxylation activities. In contrast, the regioselectivity of propafenone hydroxylation catalyzed by P450 2D enzymes differed between humans and mice, with chimeric mouse liver microsomes preferentially catalyzing propafenone 4'-hydroxylation similar to mouse liver microsomes. Unique human olanzapine N10-glucuronidation activity was detected in chimeric mouse liver microsomes. In addition, SGX523 is metabolized by chimeric mouse liver cytosol to the less soluble 2-quinolinone metabolite that is likely involved in the clinically observed obstructive nephropathy. Interestingly, in the kidneys of humanized-liver mice receiving repeated orally administered SGX523, we observed amorphous material accumulation in the renal tubules and inflammatory cell infiltration. Over all, the hepatic drug-metabolizing enzyme activities are similar in humans and humanized-liver mice. Understanding the hepatic drug-metabolizing potential of humanized-liver mice can enhance their effective use in drug development.

Evaluation of hepatic cytochrome P450 (P450)-mediated fractions metabolized of drugs is important for evaluating drug interactions and effect modifiers in pharmacogenetics. In an in vitro system, ~80% inhibition of single-on-target P450 activity in human liver microsomes pretreated with time-dependent inhibitors was observed under limited off-target effects. Sustained experimental human hepatocytes (HepaSH cells) harvested from quality-controlled transplanted mice are available. R- and S-Omeprazole 5-hydroxylation by human P450 2C19 in HepaSH cells was slightly (~2-fold) autoinduced by R,S-omeprazole. Modeled hepatic/plasma exposure to omeprazole in subjects with P450 2C19 poor metabolizers is likely associated with hepatic toxicity. Humanized-liver mouse systems pretreated with furafylline and tienilic acid would be useful, creating a standard in vivo animal model to investigate the contributions of specific P450 1A2 and 2C9, and some apparent contradictions were observed for P450 2D6 and 3A4 (the in vivo fraction metabolized in hepatocytes treated with paroxetine and azamulin and the in vitro fraction metabolized). Similarly, unexpectedly high hepatic exposure modeled with data from the paroxetine pretreatment-humanized liver mouse model, possibly in intermediate-metabolizer patients harboring P450 2D6*10, may contribute to the adverse effects recorded for atomoxetine prescribed alone.

NADPH oxidase 2 (NOX2) represents a potential therapeutic target for inflammatory disorders. However, clinically applicable NOX2 inhibitors have yet to be available despite extensive research efforts. In our quest for NOX2 inhibitors, we focused on the regulatory subunit p40^phox, which exclusively functions with NOX2, rather than the catalytic subunit gp91^phox, aiming to impede the complete assembly of the NOX2 complex. Chemical compounds were designed to interfere with the interaction between the PX domain in p40^phox and its ligand phosphatidylinositol 3-phosphate (PI3P), and screened for their inhibitory potential, leading to the finding of a hit DG401. DG401 exhibited inhibitory activity against NOX2 in cells within the range of 0.1–10 μM, with high selectivity over other NOX isoforms. Indeed, it disrupted the interaction between p40^phox and PI3P, and prevented the recruitment of p40^phox to the phagosomal membrane during zymosan phagocytosis. In animal model, it displayed oral efficacy in attenuating complete Freund's adjuvant (CFA)-induced inflammation and reactive oxygen species generation. This study provides substantial support for the conceptual rationale that the pharmacological inhibition of NOX2 serves as a viable strategy for treating immuno-inflammatory disorders. In addition, it underscores the potential of regulatory subunits such as p40^phox as promising targets for selectively inhibiting NOX isoforms.

The robustness of cardiomyocytes is supported by their superior redox homeostasis, and disruption of redox homeostasis leads to the onset and progression of cardiac disease. Supersulfides, which include catenated sulfur atoms (R-S_nSH) such as cysteine persulfide (Cys-SSH), have been recently identified as highly reactive sulfur metabolites and recognized as a key molecule to regulate redox homeostasis. This study aimed to elucidate the role of sulfur metabolism in maintaining cardiac robustness, and the impact of abnormal sulfur metabolism on ischemic and electrophilic stress-mediated cardiac dysfunction.

Supersulfides are reduced to hydrogen sulfides in cardiomyocytes after ischemic and electrophilic stress. This supersulfide catabolism decreased the contractile function of cardiomyocytes through mitochondrial hyperfission. We also found that the depletion of supersulfides promotes mitochondrial fission by decreasing polysulfidation of mitochondrial fission factor Drp1 at Cys644.

Our results suggest that the redox modification of Drp1 Cys644 has a pivotal role in the ischemic and electrophilic tolerance of cardiomyocytes. We found that this polysulfidated Cys644 is modified by S-glutathionylation. Drp1 was glutathionylated by oxidized GSSG but not reduced GSH. GSSG-mediated Drp1 glutathionylation protected cardiac function against ischemic and electrophilic stress in vitro and in vivo. These results suggest that sulfur metabolism has a pivotal role in ischemic and electrophilic stress-mediated cardiac dysfunction through Drp1 Cys644 redox modification.

Reactive sulfur species (e.g., cysteine hydropersulfide; CysSSH, and glutathione hydropersulfide; GSSH) exhibit high nucleophilicity and thus is thought to play an important role in repressing oxidative stress. However, adaptive cell responses to excess reactive sulfur stress are not well understood. Using cystathionine gamma-lyase as a CysSSH-producing enzyme overexpression to induce sulfur stress, we showed in vivo and in vitro that levels of CysSSH are strictly regulated in mice via CysSSH export from tissues and cells, suggesting an adaptive response to reactive persulfides. Interestingly, among all amino acids, cystine (CysSSCys) was found to be essential for CysSSH export from primary mouse hepatocytes, HepG2 cells, and HEK293 cells during excess reactive sulfur stress, that the cystine/glutamate transporter (SLC7A11) contributes, at least partially, to CysSSH export. We established HepG2 cell lines with knockout of SLC7A11 and used them to confirm SLC7A11 as the predominant antiporter of CysSSCys and CysSSH. We observed that the poor efflux of excess CysSSH from the cell enhanced cellular stresses induced by excess reactive sulfur exposure, such as polysulfidation of intracellular proteins, mitochondrial damage, and cytotoxicity.

These results suggest that a cellular mechanism for maintaining intracellular reactive sulfur homeostasis via extracellular efflux of excess reactive sulfur could prevent the adverse effects of excess reactive sulfur. Our findings suggested the existence of a safeguard against excess reactive sulfur.

Echinochrome A (Ech A), a marine bio-product isolated from sea urchin eggs, is known to have cardioprotective effects through its strong antioxidant and ATP-sparing capabilities. However its biological effect and underlying mechanisms were not clear. Our recent studies investigated the novel effect of Ech A on mitochondria function and biology, cardiac protection against cardio toxins and cardiac contractility and Ca2+ handling. The studies figure out (1) Ech A enhances the mitochondrial biogenesis and oxidative phosphorylation (2) Ech A protect cardiac cells from different kinds of cardio toxins including tert-Butyl hydroperoxide (tBHP, organic reactive oxygen species (ROS) inducer), sodium nitroprusside (SNP; anti-hypertension drug), and doxorubicin (anti-cancer drug) (3) Ech A negatively regulates cardiac contractility by inhibiting SERCA2A activity, which leads to a reduction in internal Ca2+ stores (4) Ech A promotes the differentiation of stem cells into cardiac cells through regulation of mitochondrial function (5) Ech A is effective in treating diabetic nephropathy by protecting mitochondrial function. These novel effects of Ech A suggest possible clinical application and drug repositioning of Ech A to various cardiovascular diseases.

In non-clinical toxicity evaluations for drug development, the primary objective is to elucidate the target organs of toxicity induced by the test-article and the relationship between the dosage and exposure. Moreover, it is important to determine whether the observed changes are reversible or not, for risk assessment. In the general toxicity studies, the reversibility of changes may be scientifically discussed based on the characteristics, extent, and severity of the lesion, and regenerative capacity of the organ which showed the toxicity. However, when it is difficult to predict reversibility, it should be evaluated by conducting a study that includes a drug-free period after the administration period. Although it is not necessary to observe the complete reversibility, drug-free period will be carefully set to examine reversibility, as the half-life of some drugs, such as antibody drugs, is long.

In this presentation, it will be introduced the overview of common concept of reversibility evaluation and the settings of recovery animals and drug-free period in the general toxicity studies.

The adrenal glands are the most sensitive target organ among the endocrine organs for compounds that have high fat solubility or that affect steroid hormone metabolism, since the adrenal cortex is particularly involved in the synthesis and secretion of various steroid hormones using cholesterol as a substrate. In the toxicity studies of these compounds, vacuolation and necrosis in the adrenal cortex are frequently observed. In our study, during a 4-week repeated-dose toxicity study in dogs, Compound X induced diffuse cortical vacuolation (lipidosis) accompanied by macroscopic adrenal enlargement and weight gain at the end of the administration period. After a 4-week recovery period, these lesions transitioned into cluster lesions consisting of cells with larger vacuoles in the middle cortical region of the adrenal. This made it difficult for us to determine the reversibility of vacuolation lesions in the adrenal cortex, although macroscopic enlargement and weight gain of the adrenal recovered. Therefore, in the subsequent 13-Week toxicity study in dogs, we set the recovery period to 12 weeks to confirm the reversibility of the vacuolation lesions mentioned above, and as a result, we were able to confirm the reduction in frequency and severity of the lesions after the recovery period. Prolonging the recovery period is useful for reliably confirming the reversibility of a lesion, but it is a trade-off with accelerating the drug development process. In this presentation, we would like to discuss how to interpret reversibility of vacuolation lesions in the adrenal cortex and the appropriate recovery period, considering the well-known information about adrenal gland’s physiological turnover and lipid metabolism and our experimental data about the above Compound X and Aminoglutethimide, which induce vacuolation in the adrenal cortex in animals.

Respiratory exposure is one of the most recognized routes of chemical exposure in the workplace. We have been done the hazard assessment of chemicals using a rodent and a whole-body inhalation exposure apparatus. Notably, only a limited number of chemicals exhibit organ-targeted toxicity specific to the respiratory system. In this presentation, chemicals that have been found to induce toxicity in the bronchial and alveolar regions will be selected and their recoverability will be discussed from a histopathological perspective.

To commence, male and female p53KO1 and rasH22 mice were exposed to chroloetane for 6 h/day, 5 days/week for up to 26 weeks using a whole-body inhalation exposure system. Outcomes revealed a concentration-dependent manifestation of vacuolar degeneration within the bronchial epithelium of mice lung.

Secondly, F344 rats were exposed of cross-linked water-soluble acrylic acid polymer (CWAAP)3,4 utilizing diverse protocols for systemic inhalation exposure and repeated intratracheal administration. Noteworthy lesions were discerned within the alveolar region in a manner contingent upon dosage and concentration.

Within this presentation, we aim to delve into the toxicologic pathological alterations induced by these chemicals, encompassing considerations of their recuperative potential.

[references]

1.https://anzeninfo.mhlw.go.jp/user/anzen/kag/pdf/chukigan/75-00-3_p53ko_MAIN.pdf

2.https://anzeninfo.mhlw.go.jp/user/anzen/kag/pdf/chukigan/75-00-3_rasH2_MAIN.pdf

3.Takeda T, Yamano S, Goto Y, et al. Dose-response relationship of pulmonary disorders by inhalation exposure to cross-linked water-soluble acrylic acid polymers in F344 rats. Part Fibre Toxicol. 2022 Apr 8;19(1):27. doi: 10.1186/s12989-022-00468-9.

4.Yamano S, Takeda T, Goto Y, et al. Mechanisms of pulmonary disease in F344 rats after workplace-relevant inhalation exposure to cross-linked water-soluble acrylic acid polymers. Respir Res. 2023 Feb 13;24(1):47. doi: 10.1186/s12931-023-02355-z.

Gastric mucosal injuries induced by toxic insults generally show recovery after the withdrawal of the causative agent. One such agent is fusarenon-X (FX), a type B trichothecene mycotoxin and a potent gastric chief cell toxin. There are only a few reports regarding the recovery process after injury to chief cells. Therefore, we proposed an FX-induced mucosal injury to determine chief cell recovery after injury. Crl:CD(SD) rats were treated with a single gavage dose of FX (1.5 mg/kg or 3.0 mg/kg) or vehicle, and the stomach was examined histopathologically for 31 days post- treatment including immunohistochemical staining for gastric and pancreatic differential markers. After 48 hours post-treatment, a single cell necrosis and a decreased number of chief cells was noted. However, the single cell necrosis and decreased number of chief cells began to recover after day 2 and day 10, respectively. Eosinophilic changes in chief cells, which was positive for trypsinogen, suggested incomplete pancreatic acinar differentiation, was observed after day 2 and disappeared at the end of study. Doublecortin and calcium/calmodulin-dependent kinase-like-1 positive gastric progenitor cells and Trefoil factor 2 -positive mucus neck cells were ectopically observed at the base of the gastric glands within 10 days and within 3 days post-treatment, respectively. Additionally, pancreatic acinar metaplasia, which was positive for both trypsinogen and amylase, was observed 5 days post-treatment in the higher dose group. All histopathological findings described above showed the complete or tendency of recovery 31 days post- treatment. In conclusion, FX-induced gastric mucosal damage composed of chief cells injuries followed by promoted ectopic reproduction of gastric progenitor cells and abnormal differentiation of chief cells during its recovery process.

Nonclinical toxicity studies of anticancer agents are conducted to understand their profiles such as target organs, exposure-response relationships, and reversibility, same as other pharmaceuticals. However, unlike drugs for non-oncology indication, anticancer drugs have been known often to have narrow or negative safety margin expressed as exposure multiple between systemic exposure at nonclinical efficacious dose/predicted efficacious dose in humans and that at toxic dose in the toxicity study. This means that it is important to evaluate the reversibility of the anticancer agents’ toxicity from the perspective of clinical development and risk assessment.

For the correct judgement of the reversibility in histopathological examination, it is essentially critical to understand lesions including their severity and pathological stage accurately and to express them using appropriate terminology and grades at the end of the dosing period. Then, the characteristics of the findings at the end of the recovery period are to be carefully evaluated to examine whether they recovered rapidly after the end of the administration period, were in the middle of recovery, or did not recover at all. When the lesions are determined to be in the process of recovery, the length of time required for complete recovery is sometimes estimated considering the turnover of affected cells. This process requires the knowledge of histology and physiology as well as the knowledge of pathology.

In this symposium, we will introduce the lesions and reversibility observed in organs such as the gastrointestinal tract, lymphohematopoietic system, and testes, which are often injured by anticancer agents, focusing on histopathological images.

Titanate nanosheet (TiNS) is 2D material with thickness of about 1 nm, and it is expected to be applied for various kinds of industrial use. TiNS is composed of titanium and oxygen as well as titanium dioxide (TiO₂) particles, but it has a unique feature of lepidocrocite-type crystal structure, which suggests different toxic effects. Therefore, we proceeded with the analyses for toxicity by cell culture experiments. TiNS caused caspase-dependent apoptosis of human monocytes with giant vacuoles, in which titanium was identified. TiNS induced the expression of autophagy-related genes, but the results suggested a toxic mechanism through abnormally increased lysosomal function. Recently, the significance of lysosome-derived Ca²⁺ signals has been pointed out. Therefore, we focused on the lysosomal Ca²⁺ release channel of transient receptor potential mucolipin (TRPML). It was found that the monocytes exposed to TiNS showed increases in intracellular Ca²⁺ concentration and mRNA levels of v-ATPase and TRPML1 genes following incorporation of TiNS. Additionally, it was also confirmed that Ca²⁺ binds on TiNS. ML-SA1, which works on release of Ca²⁺ from lysosomes as TRPML agonist, increased apoptosis of monocytes in the culture with TiNS. The intracellular Ca²⁺ chelating agent BAPTA-AM suppressed the increases in the mRNA levels of lysosomal genes. It is known that lysosomal cell death is dependent on catepsin (CTS), but CTS inhibitor exacerbated apoptosis caused by TiNS exposure. Those findings indicate that the increase in TRPML1 channel augmented lysosome-derived Ca²⁺ signals, which induced the expression of the lysosomal genes, leading to a positive feedback loop that amplify the Ca²⁺ signal and then causes apoptosis in monocytes. The toxic effects of TiNS due to its unique crystalline structure and three-dimensional feature illustrate a new mechanism of immunotoxicity by metal nanomaterials.

Particle pollution is known to induce allergic inflammation in the lungs. These particles function as adjuvant and trigger allergic immune responses characterized by the induction of IgE and activation of eosinophils. However, the underlying mechanisms by which inorganic particles induce allergic immune responses are still unclear. In this study, we are focusing on the role of alveolar macrophages and are investigating the effect of inorganic particles on the activation. So far, our research has shown that inflammatory particles such as aluminum salt (alum) and silica stimulate alveolar macrophages to release IL-1alpha.The release of IL-1alpha is mediated by cell death, specifically through a dead cell factor known as a damage-associated molecular pattern (DAMP). Interestingly, despite both being aluminum, aluminum oxide particles did not induce cell death and IL-1alpha, indicating that the chemical properties of particles play a role in the function of alveolar macrophages. In this symposium, we will discuss the impact of particle chemical properties on immune responses.

Carbon nanotubes (CNTs) have attracted great interest for use in multiple fields including electronics, material science, biology and medicine. However, concerns have been raised regarding their potential risks. When multi-walled CNTs (MWCNTs) enter bodies, they are recognized by macrophages, which trigger inflammatory responses involving the activation of inflammasomes and the secretion of interleukin-1beta (IL-1beta) [1]. In animal models, this inflammatory response can progress to chronic inflammation, fibrosis, and even mesothelioma. However, it is poorly understood how macrophages recognize MWCNTs on their cell surface.

Recently, we found that murine T cell mucin immunoglobulin 1 (Tim1) and Tim4 contain a unique cluster of aromatic residues in their extracellular domain that facilitate direct recognition of MWCNTs [2,3]. Given the negligible expression level of Tim1/4 in human lung macrophages, we used molecular dynamics modelling to uncover alternative cell surface human receptor harboring a similar aromatic residue cluster, identifying a human macrophage receptor sialic acid immunoglobulin-like binding lectin (Siglec)-14 [4]. Here I focus on the molecular interactions between CNTs and biological systems and discuss therapeutic approaches to combat the toxicity of MWCNTs.

[1] M. Nakayama, Front. Immunol., 9, 103 (2018).

[2] S. Omori et al., Cell Rep., 34, 108734 (2021).

[3] M. Kuroiwa et al., Sci. Total Environ., 875, 162586 (2023).

[4] S-I. Yamaguchi et al., Nat. Nanotechnol., 18, 628 (2023).

Chronic arsenic exposure due to the contamination of drinking water with high concentrations of arsenic compounds has become a serious problem throughout the world, particularly in Asian regions such as Bangladesh and Cambodia. Chronic exposure to arsenic compounds has serious health effects, with carcinogenesis being the most serious. According to a WHO study, tens of millions of people in Bangladesh are exposed to arsenic and rore than 200,000 arsenic-exposed patients die from cancer each year.

Some studies have been reported aimed at clarifying the carcinogenic mechanism caused by arsenic exposure, however, the detailed mechanism is not well understood. The basis for this is that although arsenic compounds are carcinogens that are recognized to cause cancer in humans, this cannot be reproduced in animal models. Based on these investigations, arsenic compounds are considered to be "non-genotoxic carcinogens."

Our laboratory is currently investigating the possibility that the immune system might be impaired in the carcinogenic mechanism caused by arsenic compounds. Through a series of research studies, we discovered that arsenic compounds weaken the function of natural killer (NK) cells, which are involved in killing cancer cells in innate immunity. In this symposium, we will introduce the mechanism by which the cancer cell-killing function of NK cells is impaired by arsenic compounds.

Drug-induced liver injury (DILI) is a major cause of withdrawal after marketing, so it is necessary to evaluate DILI risk. Mitochondrial toxicity is one of the crucial factors for predicting DILI, and several methods have been established. Although these methods have been widely used due to their high throughput, this does not deny the importance of another mitochondrial toxicity mechanism. Among them, we have focused on mitochondrial membrane permeability transition (MPT), common to drugs that cause severe DILI. While our research has only focused on hepatocytes, we try to evaluate the effects of MPT on inflammatory cells. When acetaminophen was administered to mice that lack a component of MPT in bone marrow-derived cells, we confirmed that acetaminophen-induced liver injury could be involved in the MPT in platelets. MPT has not been actively evaluated because its contribution to liver injury is unclear, and isolated mitochondria from the organs of experimental animals are required for evaluation. Regarding this point, when MPT is induced in platelets, different activation occurs compared to the general activation process; we try to construct an alternative method to conventional MPT evaluation by using platelet activation. Furthermore, there are species differences in the difficulty of predicting liver injury. Species differences mean not only differences between humans and animals but also between experimental animals, so selecting an appropriate animal species is crucial depending on the purpose of the experiments. We have also investigated species differences between animals using lipopolysaccharide, which can induce MPT in rats and mice.

In pharmaceutical development, developmental toxicity tests using animals to assess impacts on embryos and fetuses are essential, as clinical trials present significant challenges. These tests require substantial investments of time, effort, and resources due to species differences. Some drugs, like thalidomide, do not affect rodents but cause severe malformations in humans. Moreover, the specificity of new modalities, such as nucleic acid therapeutics, toward human complicates safety assessment through traditional animal testing. Addressing this urgent issue necessitates the development of high-throughput, accurate in vitro methods capable of precisely predicting developmental impacts. Our research has developed a developmental toxicity evaluation method using human iPSCs, focusing on the disruption of signal transduction by chemical substances (DynaLux/c). This method has demonstrated high accuracy and comprehensive coverage in detecting developmental toxicants, particularly through analyzing dynamic changes in FGF-SRF signal disruption by a 24-hour live-cell luciferase assay.

Currently, we have enabled continuous detection of dynamic signal disruptions by incorporating a real-time luminescence measurement device with cell culturing capabilities into DynaLux/c. This integration significantly reduces testing time to one week and allows for detailed analysis. Additionally, we've also established the Wnt-TCF/LEF signal disruption reporter system, like the FGF signal. Now, we're attempting to elucidate the molecular mechanisms of DynaLux/c detecting developmental toxicity.

Thyroid hormones (THs) are essential for brain development. Environmental chemicals with TH disruption are of regulatory concern for potential adverse effects on the developing brain. Although quantitative relationships between chemical-induced perinatal TH insufficiency and abnormal brain development have not been fully understood, several adverse outcome pathways (AOPs) associated with TH disruption have been identified, highlighting the need for efficient chemical safety screening and a reduction in animal testing. When using in vitro assays for regulatory purposes, there remains a challenge in application due to insufficient knowledge of TH signaling mechanisms during critical periods in target tissues, such as the developing brain. Indeed, many factors, including TH supply and chemical exposure, affect brain TH. We focused on a weight-of-evidence approach using an in vivo study, the Comparative Thyroid Assay (CTA) in rats, and began verifying the feasibility of a modified CTA by adding parameters (brain TH concentrations and brain heterotopia) but reducing the number of rats. The results indicate that the modified CTA has the potential to serve as an in vivo assay for identifying even mild perinatal TH disruptors. The modified CTA could also be instrumental in gathering data to construct a physiologically based pharmacokinetic model for the perinatal period, with the aim of employing New Approach Methodologies. This model could significantly contribute to our understanding of TH disruptions during critical stages of development and improve safeguards for human health.

Developing alternative methods for carcinogenicity tests remains challenging. We have been working to develop a read-across method to predict non-genotoxic carcinogenicity by using chemical structural information and in vitro test results related to carcinogenic mechanisms. In read-across, the toxicity of the target substance is predicted from the toxicity of tested substances (i.e., source substances) with similar chemical and/or biological properties. To this end, we collected the results of two-year rat carcinogenicity tests of pesticides, and the presence or absence of tumors is set as the objective variable. The outline of the method is as follows: 1) Calculate the Euclidean distance between substances based on molecular descriptors calculated by using commercial software. 2) Select substances within a certain distance from each test substance as source substance candidates. 3) Conduct in vitro experiments related to carcinogenicity for test substances and primary source substances. 4) Select only substances whose experimental results match the test substance as the final source substance. 5) Perform read-across with the final source substances with the incidence rate of each tumor in the dataset as the criterion for positive judgment. We mainly work on tumors associated with epithelial cell damage, such as the nasal cavity, stomach, and bladder tumors, and those associated with nuclear receptor activation and drug-metabolizing enzyme induction, such as liver and thyroid tumors. In this session, I will share our recent progress of this study for discussion.

The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential to maintain cellular functions. However, the relationship between the disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. To continuously analyze the metabolite fluctuations that occur during IR injury in the living state, a tubular semipermeable membrane (microdialysis) was implanted in the heart wall. The glutathione is released from the cell during ischemia-reperfusion and is unable to remove intracellular reactive oxygen species. The depletion of intracellular glutathione increases reactive oxygen species and causes ferroptosis due to lipid peroxidation and excessive oxidation of lipids in the cell membrane. Additionally, this extracellular release of glutathione was found to be primarily mediated by multidrug resistance protein 1 (MRP1), a specialized transporter. When MRP1 function was inhibited by drugs, glutathione not only scavenged reactive oxygen species but also reduced oxidized lipids, thereby supporting cardiomyocyte survival. Furthermore, this treatment targeting ferroptosis was found to have some efficacy even when initiated several hours after ischemia-reperfusion injury. Targeting the increase in oxidized lipids observed after 6 hours of myocardial infarction is expected to lead to effective treatment by creating a time advantage before the administration of therapeutic agents.

Recently, Tetrodotoxin (TTX), a well-known pufferfish poison, and several analogues were detected in gastropods and bivalves from European waters. For this reason, the European Food Safety Authority (EFSA) has conducted a risk assessment of TTX in marine bivalves. Based on the results of the acute oral study in mice, “apathy” was selected as the most sensitive endpoint, and 0.25 μg/kg bw as the group Acute Reference Dose (ARfD) of TTX and its analogs was derived. Thus, in mice, oral administration of TTX is most sensitive to inducing “apathy”, a little-known toxic symptom in humans, suggesting an effect on the central nervous system.

For seeking the molecular mechanisms of acute poisoning due to TTX, we applied our Percellome Toxicogenomics that had been developed for the mechanism-based predictive toxicology using time- and dose-dependent transcriptomic responses induced by a chemical. The 24-hour Non-Observed Effect Level was selected as the high dose. Murine liver and hippocampal mRNA were collected after a single oral administration of TTX at each concentration of 0, 30, 100 and 300 μg/kg bw (4 doses x 4 time points, triplicate), and absolute gene expression levels of approximately 45,000 probe sets were obtained by the Percellome method using GeneChip MOE430v2 (Affymetrix) for comprehensive analysis. Signal networks related to stress responses and cytokines were extracted from both liver and hippocampus. Neuronal signatures in hippocampus were less pronounced than expected. The inter-organ relationship of acute TTX toxicity will be discussed based on comprehensive analysis on its molecular mechanism.

When treating a patient with poisoning, the patient may or may not be known to be " poisoning" of some kind. Therefore, it is important to always consider the possibility of "symptoms due to poisoning" when treating patients. In particular, "unexplained symptoms" is a key word to suspect poisoning. There are also situations in which a patient is known to be poisoned by something but does not know "what" he or she is poisoned by. In such cases, the toxidrome is used to identify the substance that is poisoning the patient. The term "toxidrome" was coined from "toxic syndrome" and was first used around 1970. It is used as a concept for emergency response by classifying the causative agent of poisoning based on symptoms and signs, and there are various ways of classification. In the ACLS (Advanced Cardiovascular Life Support) EP (experienced provider) course, there are five classifications: sympathomimetic, cholinergic, anticholinergic, opioid, and sedative/hypnotic, which are commonly used in clinical practice. On the other hand, the Advanced Hazmat Life Support (AHLS) course, which is an educational course on responding to toxic gases and chemicals, has five classifications: irritant gas, asphyxiant, cholinergic, corrosive substance, and halogenated hydrocarbon. The classification should be made according to the symptoms and signs of the situation. In any case, the significance of classification by toxidrome is that it can lead to necessary emergency treatment even if the causative drug or toxicant is not identified. The "Standard Medical Guide to Acute Poisoning," supervised by the Japanese Society for Clinical Toxicology, also emphasizes this point, allocating a chapter to the toxidrome and explanations of various clinical tests, including analyses to complement it, as the standard approach in the treatment of acute poisoning.

According to the Global Terrorism Database, the total number of terrorism attacks has increased since 2010, with more than 10,000 attacks occurring annually worldwide. While the overwhelming majority of these attacks are by explosives, chemical terrorism (C-terrorism) using chemical agents is common. Another characteristic of terrorism in recent years is that chemical weapons, originally developed for the battlefield, are now being used against civilians in cities and conflict areas.

At the scene of a chemical terrorism disaster, it is not easy to identify the myriad of substances that can cause poisoning, and it can take time to diagnose the problem. Furthermore, the complex nature of the information and the uncertainty of specimen collection also make identification difficult. On the other hand, treatment of lethal and urgent chemical agents must be initiated immediately. Supportive care is needed for respiratory arrest and cyanosis, and antidote therapy may be considered. Furthermore, “Aging" is said to diminish the effects of oximes, the antagonists of nerve agents, in as little as 5 hours for sarin and 2 minutes for soman. It may be too late to begin treatment once the causative agent has been identified, so the initial treatment of C-terrorism injuries requires an approach in which the toxic agent is estimated while treatment is also initiated. In addition to the five toxidromes used in clinical practice, different toxidromes have been devised for chemical terrorism.

In this session, we will review the toxidromes of chemical agents (nerve agents, asphyxiants, and opioids) that have properties suitable for terrorist attacks, i.e., high volatility and rapid onset of incapacitating or lethal effects, as well as the actual models prepared for the Tokyo 2020 Olympic and Paralympic Games and other events to counter C-terrorism.

The eukaryotic genome is divided into two domains: euchromatin and heterochromatin. Furthermore, heterochromatin is broadly classified into two types, constitutive and facultative heterochromatin, which are epigenetically regulated by the H3K9 methylation and H3K27 methylation (Polycomb repressive complex：PRC) regulatory systems, respectively and separately. Interestingly, these two heterochromatin states are maintained by the other regulatory system to some extent from each other if the respective responsible epigenomic machinery fails to work.

Recently, various epigenome changes and plasticity have been observed in response to environmental stimuli during developmental stages and long-term life activities including aging, suggesting that these epigenome changes are associated with pathological or physiological biological dysfunction.

In this context, I would like to discuss with participants how the heterochromatin-regulated plasticity we are finding contributes to human health and disease.

The epigenome defines cellular identity. When environmental exposures induce epigenomic alterations, these alterations can be linked to the development of various diseases, including cancer, neurological disorder, and type 2 diabetes, as they are precisely inherited after cell division. In the context of cancer, epigenomic alterations occur in epithelial cells in normal-appearing tissues, which can produce the “field for cancerization.” We previously demonstrated that the severity of DNA methylation alterations was correlated with cancer risk [Gut, 64:388, 2015; Gut, 66:1751, 2017], and identified chronic inflammation as a trigger of DNA methylation alterations in epithelial cells [J Clin Invest, 130:5370, 2020]. Recently, we explored that intrinsic factor, such as chronic inflammation, aging, and hypoxia, changed DNA methylation and H3K27 acetylation in fibroblasts and endothelial cells, key components of the tissue microenvironment. Furthermore, in normal gastric fibroblasts, upregulation of H3K27 acetylation was involved in the formation of “field for cancerization” and influenced gastric cancer cell identity. These findings indicate that biological responses to the intrinsic factors induce epigenomic alterations within the normal tissue ecosystem. We are now investigating the role of extrinsic factors, chronic phycological stress, in epigenomic alterations and their impact on gastric cancer development. This presentation will introduce how epigenomic alterations driven by both intrinsic and extrinsic factors contributes to carcinogenesis.

Since the developmental mechanisms during fetal stages are strictly regulated, fetus is susceptible to the influence of environmental stresses such as maternal nutritional status and chemical exposures. Experimental studies using animals have revealed that exposure of pregnant animal to agricultural fungicides (such as vinclozolin) can affect the phenotype of descendants for several generations. On the other hand, recent research has also demonstrated that paternal environmental stress can also impact the phenotype of future generations. For instance, rearing of male mice by either normal or low-protein diets results in increased expression of cholesterol metabolism genes in the livers of offspring from the paternal low-protein diet group. These phenomena suggest the existence of inheritance pattern as non-genetic traits, indicating the epigenetic elements in spermatozoa to be altered by environmental stress and inherited by descendants.

In order to comprehensively capture sperm epigenetic alterations induced by paternal environmental stress, we have independently established novel methods for the high-purity fractionation of mature sperm and mapping of sperm histones. Utilizing these techniques, we have detected sperm epigenetic changes induced by low-protein diets in mice and by rearing in space environments, thereby elucidating some of the molecular mechanisms underlying phenotypic changes in the next generation. In this presentation, we will focus on the molecular mechanisms of paternal stress inheritance via stress-responsive transcription factor ATF7.

Although the molecular mechanisms of intergenerational transmission of effects by environmental factors are still largely unclear, the sperm epigenome will be involved in the transmission of effects mediated through sperm.

We are studying the mechanisms of the effects of arsenic, an environmental chemical, on the next generation. We found that gestational arsenic exposure of C3H mice, whose males are prone to developing liver tumors after growth, increases liver tumors in the F2 through their F1 males. We also found that gestational arsenic exposure induces genome-wide DNA hypomethylation in F1 sperm, and that the number of hypomethylated CpG are increased, especially in the LINE and LTR. Since hypomethylation of retrotransposons leads to adverse transposition activity and has been reported to be involved in carcinogenesis in somatic cells, it may contribute in part to the mechanism of liver tumor increase in arsenic F2 groups. Furthermore, the methylation state of arsenic F1 sperm was maintained in arsenic F2 embryos, whereas that was cancelled in arsenic F2 sperm. Our experimental system of gestational arsenic exposure in C3H mice has shown no increase in liver tumors in the F3, and the results of F2 sperm support the above discussion.

Recently, we have shown that genome-wide DNA hypomethylation occurs in type A spermatogonia of arsenic F1, but there is no increase in hypomethylated CpG in LINE. Arsenic may affect the expression of Dnmt and Tet during spermatogenesis after type A spermatogonia and induce an increase in hypomethylated CpG in F1 sperm.

Epidemiological evidence from analyses of millions of individuals has shown that environmental factors early in life, including the intrauterine environment, are associated with the development of disease in early childhood or adulthood. The involvement of epigenetic changes by environmental factors has been suggested as a mechanism for future disease development; this concept was proposed by Dr Gluckman and Dr Hanson as the Developmental Origins of Health and Disease (DOHaD) hypothesis and is widely accepted. Studies demonstrating the DOHaD hypothesis are indeed ongoing in humans: DNA methylation is a useful molecular biological indicator within the epigenome to assess past and future effects simultaneously, and many prospective cohort studies have reported associations between various environmental factors and DNA methylation changes. We have also analysed the effects of the intrauterine environment, including pregnancy complications, on offspring in Japanese subjects, focusing on DNA methylation changes. We would like to present these results and discuss the mechanisms by which the early environment can lead to later disease development, and link this to the discussion at this symposium.

Navigating the protein targets of drugs (hereafter, targetome) and deciphering the specific mechanisms of action at the molecular level of these interactions are critical steps in the development of drugs to treat human diseases. We have developed target protein identification methods including conventional affinity chromatography using labeled small molecules as well as recent methods using label-free small molecules such as Drug Affinity Responsive Target Stability (DARTS) and Cellular Thermal Shift Assay (CESTA) in combination with LC-MS/MS analysis to identify the targetome of drugs. The direct interaction between drug and the target protein is validated using biophysical, and bioinformatics tools. In addition, the biological relevance of this ‘drug-targetome-phenotype’ interaction is verified by genetic modulation, facilitating structure-based better drug design. In this presentation, our studies on target identification of ‘drug-targetome-phenotype’ interaction for navigating new mechanism of small molecules, target proteins, and their translational impact will be presented by introducing our case studies of protein target identification and validation of small molecules perturbing autophagy.

Toxicant-induced liver injury necessitates the identification of targets and therapeutic agents. Gα12 signaling axis has been implicated in cell viability. This study aims to explore the role of the Gα12 signaling axis in endoplasmic reticulum (ER) stress-induced ferroptosis by toxicant, and the effect of NEMO as a cell-survival component in this process. Gα12 overexpression in hepatocytes increased toxicity, promoting lipid peroxidation, inflammation, and ferroptosis. IRE1α-dependent Xbp1 transactivated Gna12, facilitating Gα12 overerexpression. In this event, the level of miR-15a, identified as an ALOX12 inhibitor, was decreased. Thus, Gα12 overexpression by ER stress contributes to hepatocyte ferroptosis through ROCK1-mediated dysregulation of ALOX12. In an effort to find a cell-survival component that acts against ferroptosis in association with Gα12, we next focused on NF-κB essential modulator (NEMO), a protein known as a regulator of inflammation and cell death. Nrf2 transcriptionally induced NEMO. Hepatocyte-specific overexpression of Gα12 inhibited NEMO in mice. Conversely, Gα12 deficiency prevented toxicant from inhibiting NEMO, suggesting post-transcriptional control. Moreover, decrease of Gα12 lowered miR-125a, a novel inhibitor of NEMO. The significance of Gα12 in NEMO-dependent hepatocyte survival was confirmed via ROCK1. These results were validated in human specimens. Together, our results show that Gα12 overexpression by toxicant-induced ER stress causes ferroptosis in the liver through ALOX12, which can be overcome by Nrf2-dependent NEMO induction. Our findings may provide ways to ameliorate toxicant-induced hepatic injury.

Type 2 Diabetes Mellitus (T2DM) is characterized by insulin dysfunction, brought about by chronic hyperglycemia. The endoplasmic reticulum (ER) has been implicated in diabetic cardiomyopathy (DCM) progression. This study aimed to determine a novel target for new therapeutic modality to address T2DM-induced cardiomyopathy. Cereblon (CRBN) has been found to function in cardiovascular disease and so, this study further elucidated how CRBN can induce cardioprotection via attenuation of ER stress response in T2DM. 11-week-old C57BL/6 and CRBN knockout mice were fed with high fat diet for 4 weeks and administered with streptozotocin to induce T2DM in vivo after which samples were collected when mice reached 20 weeks of age. Mouse cardiac fibroblasts treated with high glucose and palmitic acid were used to induce ER stress in vitro. Pharmacologic and genetic CRBN knockdown was also done on in vitro T2DM mode using TD-165, a PROTAC-based CRBN degrader, and shCRBN adenovirus, respectively. Western blot and RT-PCR analyses were used to assess ER stress protein and RNA expression under T2DM conditions. Higher CRBN levels were observed in the T2DM model in vivo and in vitro. ER stress proteins were modulated upon knockdown of CRBN, both genetic and pharmacological, suggesting the possible interaction of CRBN with ER stress proteins. Additionally, fibrosis was also alleviated upon knockdown of CRBN. Knockdown of CRBN regulated the quantitative expression of ER stress proteins and RNA indicating that CRBN may be used as a novel target in alleviating ER stress in T2DM.

Diabetes is a chronic metabolic disorder that affects nearly 10% of adult people worldwide. It is characterized by high levels of blood glucose, which can lead to a range of complications, such as cardiovascular disease, neuropathy, and retinopathy. Recently, mitochondrial quality control has been highlighted as a potential therapeutic target for treating diabetes and its complications. We previously reported that mitochondrial hyperfission by forming a protein complex between dynamin-related protein (Drp) 1 and filamin A (FLNa), mediates chronic heart failure and cilnidipine, developed as an L/N-type Ca²⁺ channel blocker, improves heart failure by inhibiting Drp1-FLNa protein complex. Therefore, we investigated whether cilnidipine improves hyperglycemia of various diabetic model mice.

Cilnidipine treatment improved systemic hyperglycemia and mitochondrial morphological abnormalities in STZ-exposed mice, without lowering blood pressure. In contrast, cilnidipine failed to improve hyperglycemia of ob/ob mice, by suppressing insulin secretion. Therefore, we have identified a Ca²⁺ channel-insensitive cilnidipine derivative (1,4-DHP) that does not inhibit insulin release. 1,4-DHP improved hyperglycemia and mitochondria morphology abnormality in ob/ob mice fed high-fat diet. These results suggested that maintaining mitochondrial quality by inhibition of Drp1-FLNa becomes a new strategy for diabetes treatment to treat diabetes and diabetic complications.

Per- and polyfluoroalkyl substances (PFAS) are widely used for consumer products. Some of PFAS have been listed as POPs due to their persistent and bioaccumulative natures, as well as toxicity. The present study aimed to evaluate the biological effects of representative PFAS and to understand the possible mode of action using developing zebrafish. Embryos exposed to perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) elicited pericardial and yolk-sac edemas and reduction of blood flow in trunk vessels at 96 hours post fertilization in concentration-dependent manner, with greater potency observed in PFOS than in PFHxS. The behavioral assay revealed that embryos exposed to PFOS increased motility without a major effect on swimbladder inflation, indicating hyperactivity. RNA-seq analysis showed that many differentially expressed genes (DEGs) were upregulated by both compounds, whereas a smaller number were the shared downregulated DEGs. Among DEGs are neuronal and behavioral pathways most highlighted in the enrichment analysis, which supports the behavioral disorder. We found that genes important for calcium metabolism, such as pth1a, pth2, and calca, were largely upregulated following exposure to both PFAS. These results, together with existing reports, suggest that behavioral disorders and possibly circulatory failure caused by PFAS exposure might occur through disruption of calcium signaling in developing zebrafish. We are underway to measure other endpoints to see if there is any association with transcriptomic changes caused by PFAS in zebrafish.

PFAS are some of the most prominent man-made chemicals in history due to their unique properties, such as water and oil repellence, chemical and physical resistance and surfactant nature, which cannot be reproduced by non-fluorinated hydrocarbons. PFAS comprise a wide variety of substances with carbon–fluorine (CF2) groups as a common molecular skeleton but exhibit structural differences including different chain lengths, moieties and branches. Due to their long-term and wide-range use, PFAS are found everywhere on the globe, even in environments far from human activities, such as the Arctic and Antarctic. There are increasing numbers of studies about the environmental fate and transport of PFAS, their toxicity to ecosystems, animals and humans and their effects on human society and economics. These studies have revealed the unique properties of PFAS, especially their long-range transport potential, environmental persistency and human health effects. Many alternatives have been introduced to the market with the aim of overcoming the problems associated with PFAS, but some of them have already been recognised as unsafe, i.e., regrettable substitution. In this presentation, the current evidence about PFAS health risks is summaries and discussed

Perfluoroalkyl substances (PFAS) are synthetic organic compounds that have been produced for over six decades and they are used widely in industries due to their unique physicochemical characteristics like both hydrophobic and hydrophilic properties. Recently, Perfluorooctanoic acid (PFOA), Perfluorooctane sulfonic acid (PFOS), Perfluorohexanesulfonic acid (PFHxS), and their salts are designated as Persistent Organic Pollutants (POPs) under the United Nations Environment Programme (UNEP) Stockholm. In relation to the global regulation of these specific compounds, industries have changed to use of PFAS with lower persistence and bioaccumulation. These PFAS include shorter-chain PFAS like perfluorobutane sulfonic acid (PFBS) and perfluorobutanoic acid (PFBA), as well as Gen-X (hexafluoropropylene oxide dimer acid; HFPO-DA), dodecafluoro-3H-4,8-dioxanonanoate (ADONA), and 6:2 chlorinated polyfluoroalkyl ether sulfonate (F53B). Despite of the effort to reduce PFAS use, PFAS have been ubiquitously detected in water systems due to their wide use and low removal efficiency in water treatment processes. Accordingly, there has been increasing concern about potential human exposure via drinking water consumption. Therefore, this study aimed to evaluate the current PFAS contamination status of Korean environment including human samples. In addition, we estimated the daily intake of these contaminants via various exposure pathways and assessed human exposure to PFAS in South Korea. The detailed results will be presented in conference.

Perfluorinated compounds, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), are persistent organic pollutants that can cause severe toxicity in mammals. However, the underlying molecular mechanisms are not clearly understood. The aim of our study is to investigate mode of actions for PFOA- or PFOS-induced nephrotoxicity in male rats. PFOA (20 mg/kg b.w.) or PFOS (20 mg/kg b.w.) were administered by oral gavage for consecutive 20 days. Additionally, we compared the apoptotic cell death in normal rat kidney epithelial (NRK52E) cells. Data showed that PFOA and PFOS significantly increased the blood urea nitrogen (BUN) and creatinine levels in serum of rats. PFOA and PFOS significantly increased malondialdehyde (MDA) levels, decreased GSH peroxidase (GSH-Px) activity in kidney tissues. PFOA and PFOS exposure significantly elevated urinary protein biomarkers including pyruvate kinase M2 (PKM2), kidney injury molecules-1 (Kim-1), and selenium-binding protein 1 (SBP1) in urine of rats. Histological analysis revealed epithelial degeneration and necrotic cell death were exhibited in the proximal tubules of the kidney. For further investigation of the potential mechanism of PFOA- or PFOS-induced renal cell apoptosis, the expression of Bcl-2/Bax ratios were reduced in NRK52E cells. PFOA- or PFOS-mediated ROS production was increased in a dose-dependent manner in NRK-52E cells. These data indicated that PFOS-induced nephrotoxicity was closely related with the renal tubular cells apoptosis via the JNK signaling pathway.

PRL is highly expressed in malignant cancer tissues, such as metastases of colorectal cancers. We found an evolutionarily conserved magnesium ion efflux transporter, CNNM, as a direct binding molecule of PRL. PRL inhibits the transporter function of CNNM and increases intracellular magnesium levels. Through analyses using CNNM gene-deficient mice and cultured cancer cells, we also found that intracellular magnesium accumulation due to inhibition of CNNM function alters cellular functions and causes malignant progression of cancer. In cells expressing high levels of PRL, ATP levels increased along with magnesium levels, and lysosomal exocytosis, in which lysosomes fuse with the plasma membrane and expel protons and other substances accumulated in the lumen, was found to be activated. Malignant cancer tissues are known to be acidic, and the active proton efflux allowed them to grow actively in an acidic environment. Deletion of CNNM in the nematode C. elegans resulted in a shorter lifespan, which was attributed to an increase in reactive oxygen species (ROS) in the intestinal cells, in conjunction with an increase in magnesium and ATP. We found that such ROS production also occurs in mammalian cells and is important for lysosomal exocytosis. In this symposium, we will introduce the intracellular magnesium regulation and ROS production, which was revealed by the studies on PRL/CNNM, and its role in the progression of cancer malignancy.

Ferroptosis, a form of programmed cell death triggered by iron-dependent lipid peroxidation, has been associated with various diseases. Organisms possess defense mechanisms against oxidative stress, including antioxidant enzymes like glutathione peroxidase (GPx) and thioredoxin reductase, which contain selenocysteine in their active sites. GPx4 plays a crucial role in reducing lipid peroxides and is directly involved in ferroptosis.

Selenoprotein P (SeP) is essential for delivering selenium to selenium-containing proteins. It is primarily mainly produced in the liver, secreted into the bloodstream, and facilitates selenium transport throughout the body. Recently we found SeP regulates cellular selenium levels, impacting ferroptosis sensitivity via GPx4 expression. In liver cells, inhibiting SeP secretion increases intracellular selenium and GPx4 expression, thus protecting against ferroptosis. Conversely, glioblastoma cell lines experience reduced intracellular GPx4 and heightened ferroptosis susceptibility with SeP inhibition. These findings indicated that SeP not only transports selenium to other organs but also plays a crucial role in selenium circulation within tissues through autocrine/paracrine pathways.

In this presentation, we outline the selenium cycle system managed by SeP, correlating it with intracellular antioxidant capacity and ferroptosis resistance. We also explore cancer progression due to selenium imbalance, incorporating patient tissue analyses and computational insights, and suggest SeP-focused treatment strategies.

Programmed cell death is an essential mechanism to eliminate irreparably damaged cells, and requires strict regulation via signaling molecules. Recently, it has been revealed that various types of stress induce diverse forms of programmed cell death, including apoptosis, ferroptosis, and parthanatos, depending on the situation. “Parthanatos” is defined as a novel type of programmed cell death dependent on the activation of PARP-1, a signaling molecule involved in stress responses during cellular injury induced by various types of stress, such as oxidative stress, and has recently attracted much attention due to its close association with various diseases, such as neurodegenerative disorders and cancer. However, the regulatory mechanisms of parthanatos are still unknown in detail. In this study, we found that parthanatos depends on the formation and fluidity (stiffness) of ALIS (aggresome-like induced structures) triggered by the multifunctional protein p62. ALIS are droplet-like structures formed by LLPS (liquid-liquid phase separation) and probably serve as signaling hubs. p62 recognizes the ubiquitinated proteins indicating intracellular damage, and is also found to function as a sensor to various types of stress, including oxidative stress, through the oxidative modification of cysteine residues in its molecule. Moreover, we recently found that poly ADP-ribosylation by PARP-1 and phosphorylation of p62 enhance the ALIS formation and parthanatos, indicating that parthanatos is tightly regulated by the balance of several post-translational modifications. These results in our research provide a novel insight into the pathological roles of parthanatos.

In recent years, ferric iron divalent iron-mediated lipid oxidation-dependent cell death ferroptosis induced by anticancer drugs such as erastin, that decrease intracellular glutathione and RSL3, that inhibit GPx4 activity, has attracted much attention, and its regulating factors have been identified in rapid succession. On the other hand, we found that disruption of the GPx4 genome gene by addition of Tam causes slowly progressive cell death, lipoxytosis mediated by lipid oxidation without iron, and that the Lipo-1 gene, which is involved in lipid oxidation, and the Lipo-2-6 gene, which functions downstream of lipid oxidation, were identified as the execution factors for the slowly progressive cell death, lipoxytosis by disruption of the GPx4 gene by Tam, using whole genome-wide shRNA library screening. Knockdown cells of these Lipo genes inhibit lipoxytosis but not ferroptosis. We screened the Omura Memorial Natural Products Library for lipoxytosis inducers that inhibit cell death in Lipo knockdown cells and induce cell death in WT cells. In addition, compounds that could be inhibited by lipoxytosis inhibitors were selected, yielding six compounds. Cell death by these compounds was inhibited by the vitamin E derivative Trolox, suggesting that cell death is induced via lipid oxidation. Therefore, we investigated lipid oxidation using NBD-PEN, which can detect lipid radicals. We found that lipid oxidation induced by 2D3, a lipoxytosis inducer, could not be inhibited by iron chelators, but could be inhibited by Trolox. We also found that 2D3-induced lipid oxidation was inhibited by knockdown of the lipoxytosis execution factor Lipo-1, and cell death was also suppressed by Trolox. Thus, cell death by the lipoxytosis inducer 2D3 is an iron-independent cell death mediated by Lipo-1 and different from ferroptosis.

The topic of adverse reactions following immunization has become very familiar to the public in Japan over the past few years. The trigger was, of course, the domestic approval of mRNA COVID-19 vaccines. Similar to the results from pre-licensure clinical trials, post-marketing surveys showed high frequency of fever and fatigue, which has been reported in the media every day, along with the unprecedented speed of promoting the national immunization program. Although the higher frequency of adverse reactions of mRNA COVID-19 vaccines compared to vaccines with conventional modality was initially accepted in balance with the high vaccine efficacy/effectiveness, it gradually became clear that the vaccine efficacy/effectiveness decreases over time. As a result, skepticism about vaccines has come into focus as well as serious adverse events following immunization. These phenomena were inevitable as a public reaction to the initial high expectations for COVID-19 vaccines. However, amidst the abundance of information, confusion arose probably because of insufficient sharing of fundamental knowledge from the perspective of vaccinology or epidemiology, such as the difference between adverse reactions and adverse events, and what can be said from the fact that adverse events occurred following immunization.

In my presentation, I will summarize the status and issues of epidemiological research on adverse reactions and adverse events following immunization, which will serve as the basis for discussing the theme of this session, "Vaccine Science from the Perspective of Immunotoxicology".

Messenger RNA vaccines based on lipid nanoparticles (mRNA-LNPs) are promising vaccine modalities. However, some mRNA-LNP vaccines frequently cause adverse reactions, including local symptoms such as swelling and pain, and general symptoms such as fever. Therefore, developing mRNA-LNP vaccines with fewer adverse reactions is required. In this presentation, I will introduce our findings on the mechanism of mRNA-LNP vaccine induction of adverse reactions and the mRNA-LNP vaccine project to reduce adverse reactions.