Based on significant advances in stem cells and culture systems and the social demand for alternatives to animal testing, development and application of MPS is growing worldwide are strongly pursued; European Organ on a Chip Society (EUROoCS) and the international society iMPSS have been established. According to the new definition of MPS by the U.S. FDA, MPS is a very broad term that includes all advanced cell culture systems, and perfusion-based organ-on-a-chip is now positioned as part of MPS. Now, the only non-MPS system is two-dimensional static pure culture. The demand for MPS from various industries is increasing day by day. Especially in the field of drug discovery, where new modalities are pursued, human immune responses must be included in the evaluation, and the use of human cells is desirable. In Japan, four MPS prototypes were developed through the AMED project (2017-2021). In the second phase (2022-2026), the prototypes will be improved, and the protocols will be developed in collaboration with pharmaceutical companies to achieve the required Context of Use (CoU), aiming for international standardization. In principle, in vivo responses should be reproduced in vitro by fully utilizing a variety of organ-constituting cells and MPS technologies, but it is not clear what combinations are necessary and sufficient for physiological functions. Systematic research is required to break through the insurmountable barrier of the gap between vitro and vivo responses, and only with this background can MPS meet the demands of various applications.

Perfluorooctane sulfonic acid (PFOS) is thermally and chemically stable and repellent to water and oil. Thus, it has been used in various materials such as coating and fire extinguishing agents. Its production and import have been banned due to its toxicity. However, its chemical stability has allowed them to accumulate in the environment. Not only hepatotoxicity and immunotoxicity, but also developmental neurotoxicity of PFOS has been considered as a major problem. We have analyzed the developmental neurotoxicity of PFOS exposure in rodent hippocampus and cerebellum using behavioral and cell-physiological methods. Regarding hippocampal function, maternal PFOS exposure (1 mg/kg/day) from 1 to 14 days after birth decreased the scores of the object recognition and location tests, and visual discrimination test. In addition, hippocampal glutamate and GABA concentrations were decreased. However, there was no change in glutamate and GABA receptor levels. Regarding the cerebellar function, similar exposures caused decreased motor coordination and learning, and electrophysiological studies showed decreased long-term depression in Purkinje cells. However, as in the hippocampus, the molecular mechanisms causing the behavioral changes are still not clear. On the other hand, a decrease in thyroid hormone (TH)-mediated Purkinje cell dendrite arborization and type 2 deiodinase activity were observed in vitro, suggesting the involvement of the TH system. In this presentation, we would like to introduce our data and discuss future prospective to study the PFOS toxicity.

Increase of developmental disorders, such as autism spectrum disorders, (ASD), attention deficit/hyperactive disorders (ADHD) is serious problem in Japan. The cause of increase is mainly environmental for those with a genetic predisposition. Environmental factors include nurture, parental age, nutrition, infection, and so forth, and environmental chemicals are the most likely risk factors, because of their rapid increase in the past 70 years and the evidence-based various epidemiological and animal model studies. Especially, those data have shown that perinatal exposure to organophosphate pesticides (OP) have more risk for developmental disorders, through inhibition of cholinesterase. Cholinergic system is important for mammalian brain development. In addition to OP, our data showed that neonicotinoid insecticides (NEO) disrupt mammalian cholinergic synaptic transmission, through nicotinic acetylcholine receptor, and disturbed neurite extension. (Plos One,7,2012, IJERPH, 13,2016). Various reports have suggested neurodevelopmental toxicity of NEO (Environ Res, 187, 2020). In Japanese children and infant, OP and NEO have been highly detected in their urine (Environ Res,147,2016, Environ Int, 134,2020, Sci Total Environ, 750, 2021). Both of signal toxicity (J Toxicol Sci, 41, 2016) of OP and NEO may be one of causal factors of developmental disorders.

The developing brain is susceptible to the effect of environmental factors, including exogenous neuroactive chemicals, which can hinder the formation of the normal neural circuit and potentially have adverse effects on higher brain functions in adulthood. Therefore, it is important to consider the characteristics of the developing brain, which is highly sensitive to neuroactive chemicals, and carefully examine late effects on higher brain functions, which are difficult to detect by conventional neurotoxicity tests. To analyze the effects of neuroactive chemicals on higher brain functions in mice, we developed a battery of behavioral tests as a system to objectively and quantitatively, along with methods to collect neuroscientific evidence such as gene expression analysis.

Here, we mainly focus on the results of analyses using acetamiprid, a neonicotinoid insecticide. Neonicotinoids are a new class of insecticides that bind to insect nicotinic acetylcholine receptors selectively and exhibit insecticidal activity. Though knowledge of the potential neurological effects of neonicotinoids is beginning to accumulate, there is still limited information available to discuss the behavioral toxicity of neonicotinoids for mammals including humans. In this symposium, we would like to discuss the potential neurobehavioral toxicity resulting from exposure to neonicotinoids during the prenatal and postnatal lactation periods, and its relationship with the factors contributing to central nervous system psychiatric disorders associated with behavioral abnormalities.

While the development of the central nervous system is regulated by a strict genetic program, it is also known to be affected by various environmental factors. For example, it has been suggested that exogenous chemicals may disrupt proper neural circuit formation and sometimes be a cause of developmental and psychiatric disorders. However, how the genetic program of neurodevelopment is affected remains largely unexplored. Autism Susceptibility Candidate 2 (AUTS2) gene has been associated with a variety of disorders, including autism spectrum disorders, intellectual disabilities, ADHD, mood disorders, schizophrenia, and language disorders Prof.Tanemura’s group at Tohoku University found that administration of the pesticide acephate to juvenile and adult mice decreased the expression of the AUTS2 gene in the hippocampus and cerebrum, and that the mice exhibited different behavioral abnormalities upon administration at different time points. This suggests that exogenous chemicals can alter the expression of genes important for neurogenesis and affect the development of the cranial nervous system. By utilizing mouse animal models, we have shown that the AUTS2 gene/protein plays an important role in the development of the cerebral cortex, hippocampus, and cerebellum. In this symposium, I will present the role of AUTS2 in the development of the brain nervous system and detail the mechanisms involved in the development of upper layer neurons in the cerebral cortex. We will also assess how the dysfunction of AUTS2 impairs brain functions and leads to various psychiatric disoders. The relationship between AUTS2 and exogenous chemicals is also discussed.

In order to detect all odors perceived by the human sense of smell (according to one theory, there are about 400,000 types of simple odors, and an enormous number of types of complex odors), conventional semiconductor sensors using metal oxide or organic polymers as sensing molecules have limitations in terms of specificity and selectivity of the sensing molecules. In particular, it is extremely difficult for artificial sensing molecules to discriminate optical isomers of odor molecules that can be identified by the human sense of smell. Therefore, we have developed a human olfactory receptor cell array sensor that uses approximately 400 types of olfactory receptors that support the human sense of smell as sensing molecules. Using this sensor, in principle, all odors perceived by the human sense of smell can be recorded and stored as digital data, which is a completely new method of odor management. In the near future, it will also be possible to transmit this odor information to remote locations and reproduce it through "human olfactory DX (digital transformation). As a result, we believe that a completely new smell/fragrance industry will emerge. In this symposium, we would like to present an overview of this sensor, introduce examples of social implementations that have already been realized, and discuss future developments and technical issues.

Magnetic resonance imaging (MRI) with a high-frequency magnetic field is a powerful, non-invasive tool to detect and monitor addicted human brain. MRI can also be applied to small animals in non-clinical toxicological studies. This approach enabled us to evaluate time-course changes in pathological and anatomical findings and their reversibility in the same animals. It can also detect the presence and location of induced lesions and/or deposition of abnormal proteins, assisting with the selection of sections for subsequent histopathological examination. In fact, MRI has been used to detect amyloid plaques by using amyloid-beta precursor protein transgenic mice; this demonstrates the usefulness of MRI as a complementary tool for conventional histopathology. Thus, MRI widens the range of potential non-invasive imaging modalities, expanding the scope of non-clinical studies. However, the widespread use of MRI systems in non-clinical studies are hindered by obstacles. With progress in design technology, a novel compact MRI system with a high-field permanent magnet (~1.0 tesla) has been developed. The new system is portable and self-shielded; it can be placed in most institutions. Compared with conventional MRI systems, the new system has been utilized in several non-clinical studies, including those on hepato- and neuro-toxicities. However, no evidence has been found on the evaluation of addiction and drug abuse, and reproductive toxicity in vivo by MRI. I discuss the usefulness of a ready-to-use, novel compact MRI platform in evaluating their toxicity.

Acutely intoxicated patients can suddenly show abnormal changes with ABCDE (Airway/Breathing/Circulation/Dysfunction of CNS/Body temperature) until they are out of toxicity range. Unfortunately, some symptoms, such as lethal arrhythmias, lead to fatal outcomes. Therefore, rigorous patient monitoring is needed. This presentation will introduce the standard or expected monitoring devices to promote awareness of what to look for when using them.

The Airway/Breathing monitoring is performed to evaluate the airway patency and the appropriate ventilation status, but there are inaccuracies to be aware of. Percutaneous arterial oxygen saturation (SpO₂) is inaccurate under specific conditions, such as low perfusion and abnormal hemoglobin existence. Non-invasive pulse CO oximetry can measure abnormal hemoglobin, but it doesn't have enough accuracy to diagnose.

While there are many circulatory monitoring devices, Electrocardiogram (ECG) is one of the most helpful devices for detecting QT elongation and arrhythmia. Recently, the ideas of "QT nomogram" and "ECG toxidrome" have been proposed to improve the QTc calculation and support the diagnosis.

The purpose of neuromonitoring is 1) to evaluate sedation and 2) to monitor the degree of brain injury and function. In particular, when treating poisonings that can cause seizures, continuous electroencephalography monitoring should be performed to detect Nonconvulsive Status Epilepticus in case of prolonged disturbance of consciousness.

Lastly, poor body temperature control is associated with poor outcomes. Therefore, core temperature monitoring is the most recommended and accurately measured by intravascular, esophageal, or bladder thermistors.

Proper monitoring should be chosen as each monitoring device may not be accurate depending on the types of substances or the patient's condition.

One of the scientific aspects in daily clinical practice is "clinical reasoning", the process of logically and correctly interpreting the information from medical history and physical examination to reach an appropriate diagnosis. However, because patient's complaints are strongly subjective depending on patient's background, it would easily lead to a diagnostic error especially if the doctors could not translate the chief complaints to the appropriate medical term and recognize what the mechanism of the symptom is. In emergency room, even though there is few information when the patients are in state of disturbance of consciousness or shock, vital signs and visual inspection are high degree objectivity. In particular, the concept of toxidrome, which is important in clinical setting of toxicology, is based on appropriate analysis from vital signs and visual inspection, to reveal which normal mechanisms are impaired. Based on assessments from toxidrome, we can easily understand the pathophysiology in the patients, seek additional information, and finally reach the appropriate therapy without relying on tests. At this time, in order to recognize the slight abnormalities, it is essential to understand the normal mechanisms, which play an important role for maintaining homeostasis. On the other hand, it is also important to explain the condition of patient and educate the patient, in the practice of medicine. A good relationship with patients is established through careful explanation of the pathophysiology based on normal mechanism as well as hospitality. However, although the integration of basic and clinical medicine is emphasized in recent medical education, there are few attending doctors who teach based on this concept. In order to provide the best personalized medical care, clinicians must practice based on understanding the basic medicine.

Since 2000, the main cause of mortality in developing countries has shifted from infectious to non-communicable diseases, and in recent years, mortality from non-communicable diseases has exceeded that from infectious diseases in Africa. Environmental pollution in particular has become a major health threat in developing countries. Like the pollution experienced by Japan in the past, Africa and Asia are undergoing rapid development, and in many cases, environmental considerations have taken a backseat in an economy-first society. At the same time, however, what damage has actually been done and what kind of damage is possible in the future is not known. In this symposium, we asked researchers who have conducted surveillance and epidemiological studies of environmental pollution overseas and have been conducting energetic research to give lectures. In addition to so-called classic environmental pollution such as arsenic and lead, the symposium will introduce the latest data on the problem of waste overseas and the plastics problem, which has become a global issue in recent years.

Emerging Asina countries where the economic growth is remarkable have serious problems with increasing wastes, and hence the environmental release and human exposure of harmful chemicals containing in wastes are of great concern. In addition, aquatic environment pollution by chemicals derived from drainage and subsequent risks for aquatic organisms also attract extensive attention due to undeveloped and ineffective sewerage systems in such countries. We have been conducting research on contamination status and risk assessment of halogenated flame retardants (HFRs) such as polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCDs) containing in electric and electronic equipment wastes (e-waste) and end-of-life vehicles (ELV), and of pharmaceuticals and personal care products (PPCPs) in industrial/domestic drainage, in emerging Asian countries. Thus far we found the elevated levels of not only various HFR compounds but also organophosphate flame retardants (PFRs) in settled dust samples collected from informal e-waste and end-of-life vehicle processing sites, implying that workers handling there are exposed to considerable amounts of HFRs and PFRs via dust ingestion. In surface river waters of major cities, high concentrations of parabens, which are almost completely removed in sewerage treatment plants, were detected, suggesting the direct discharge of domestic/industrial wastewater into the rivers. Furthermore, elevated levels of bisphenols and triclosan were observed in some river water samples and the concentrations exceeded ecotoxicologically predicted no-effect concentration (PNEC) values that estimated from the NOEC or LOEC values reported for aquatic organisms.

Many seabird species have been reported to ingest marine plastics. Plastic ingestion may cause exposure to additives contained in plastic products. However, sufficient knowledge is still not available on the toxic effects of additives. The aim of this study was to demonstrate the transfer of plastic additives into tissues and explore their toxic effects. In Awashima, Niigata Prefecture in Japan, wild streaked shearwater chicks were exposed to plastics containing a total of five additives: one brominated flame retardant, three benzotriazole UV stabilizers, and one benzophenone UV stabilizer. Dissection and sample collection were performed 16 or 32 days later. Additives were detected at higher levels in most of the exposed group tissues than in the control group tissues, demonstrating the transfer of additives to the tissues. The gene expression analysis was performed using next-generation sequencing and quantitative PCR. Changes in the expression levels were observed for genes related to the thyroid hormone system in the liver and thyroid gland. There was no difference in plasma or liver thyroid hormone levels between the control group and the exposed group. In addition, changes in the expression levels of bile acid synthesis genes in the liver were also observed. Although there were no significant differences in the bile acid concentration in the liver, the variances were significantly different between the control and the exposed groups. There were no significant differences in the expression levels of lipid metabolism system genes, but there were genes with significantly different variances between the control and the exposed groups. In this study, tissue transfer and accumulation of additives contained in plastics were demonstrated, and their effects on the thyroid hormone system and bile acid synthesis system were suggested.

Bangladesh has undergone severe arsenic pollution from groundwater due to the installation of more than 8 million tube wells. We have been examining the relationship between arsenic exposure levels and health effects among the residents exposed to arsenic in western areas of Bangladesh. We found that the prevalence of hypertension and hyperglycemia was elevated by arsenic exposure. Serum analysis showed that the levels of oxidized LDL-C increased while those of HDL-C decreased. Although the BMI values were as low as 21-22, their waist circumference and triceps skinfold increased. Judging from these parameters, the prevalence of metabolic syndromes among arsenic-exposed residents increased. We also found that the muscle mass markers, such as serum creatinine concentrations, were decreased by arsenic exposure, and the muscle mass reduction was associated with elevated insulin resistance. Notably, several epidemiological studies conducted in Asian countries, including Japan, have reported that lower serum creatinine levels resulted in subsequent increases in the risk of diabetes. Our preliminary studies on body composition analysis and muscle power measurements showed that arsenic exposure increased fat percent while decreasing muscle power and mass among the residents in Bangladesh. These results suggest that arsenic exposure might have caused sarcopenic obesity. Thus, our findings in Bangladesh may provide new insight into the roles of muscle and lipid metabolism as novel targets of arsenic toxicity.

Metals and rare earths that support evolving advanced technologies are essential for the science and technology that underpins medical care and industry, and the majority of these underground resources are dependent on the development of resource-rich countries, including African countries. However, environmental pollution is steadily progressing in the region. The WHO and other international organisations have warned that the environmental pollution problem will have a more serious impact on animals and humans. The authors have conducted joint research with government agencies such as the University of Zambia, Zambia Ministry of Health, Ministry of Livestock and Fisheries, Ministry of Mines and Environmental Protection Agency since 2008 and reported high concentrations of lead accumulation in edible parts of livestock and poultry in the Kabwe mining area in Zambia, which threatens food safety. Furthermore, blood lead levels were measured in 300 children, 440 mother-child pairs (880 people) and 500 households (approximately 1250 people) in the area, and revealed a severe exposure situation in the vicinity of the mine, with 100% of the subjects exceeding the values considered to be warning concentrations. In addition to higher blood lead concentrations in children compared to adults, blood lead concentrations were higher in the vicinity of the mine, especially in the western area, which is an area downwind of the wind direction, indicating that not only distance from the mine as a source of contamination, but also direction and age are determining factors in lead concentrations.

Against the backdrop of a rapidly declining working population and global climate change, the era of easy access to edible meat is being overshadowed. Cultured meat is rapidly gaining attention from an SDG perspective as a means of obtaining animal protein. In our previous research, we developed a technology to isolate proliferative somatic stem cells present in mouse and human tissues. In the present study, we investigated an adipogenic induction of bovine skeletal muscle-derived CD29-positive cells with the aim of applying stem cell-based cultured meat technology. Bovine skeletal muscle-derived cells were screened using a flow cytometer and 246 different antibodies available in Japan, and CD29 (anti-rat, clone: Ha2/5), CD44 (anti-mouse, clone: IM7), and CD344/Frizzled-4 (anti When 1x105 CD29-positive cells were cultured on U-bottom plates, they self-aggregated in about 24 h and formed three-dimensional structures (organoids/spheroids) of about 500 µm in diameter. spheroids) were constructed. After a further 5 days of induction of muscle differentiation, meat buds containing both muscle components and mature fat granules were formed after 2 weeks of culture in fat differentiation induction medium and oleic acid. It is estimated that this technique can yield 7.75x1013 cells in 21 days from 100 g of beef. It is expected that the organoids/spheroids can be used not only for nutrient variability but also for various toxicity assessment purposes.

Safety evaluation of environmental chemicals are usually conducted using healthy cell/animal models. Recently, organoid culture methods, which could be enable to maintain non-cancer cells without immortality, have been established, resulting in a significant expansion of capability of development of innovative safety evaluation approaches. To date, healthy organ/tissue-derived organoid culture of epithelial cells using Matrigel, as well as iPSc-derived organoid culture collecting the wisdom of development biology have been conducted. Both types of organoids consist of each organ-specific various differentiated cell. Organoids with re-constructed organ architectures were also introduced, and they are so-called “mini organs”. We have established healthy mouse lung, liver, colon, and mammary tissue-derived organoids, and recently reported an organoid-based chemical carcinogenesis model. In that model, organoids are treated in vitro with test chemicals, and their tumorigenicity are examined after injection to nude mice, with an endpoint of histopathological findings. This system is a potential candidate method for the evaluation of chemicals as it is short-term, and replaces of repeated treatment of chemicals in vivo at maximal tolerated doses to in vitro treatment. In addition, any other toxicological profiles than carcinogenicity could be examined by analysis of cell viability combined with omics-parameters as an endpoint. Human organoids are also expected to be filling the translational gap between animals and humans. Safety evaluation of chemicals using animals are considered to be partly replaced by organoid systems. It is expected to proceed further application research of organoids for chemical safety evaluation, as it turned out, establishment of an internationally standardized test method.

The spread of viral infections has a great impact on society, and many pharmaceutical companies are still actively developing therapeutic drugs. Antiviral therapeutic drugs include anti-inflammatory drugs that mainly treat symptoms, antiviral drugs which inhibit the growth of the virus itself, and neutralizing antibody drugs that directly eliminate the multiplied virus itself. Antiviral drugs that are less affected with viral mutations and act directly on the virus itself are important for preventing the severity of infections as well as preventing outbreaks of infections. There are only five anti-influenza drugs and four anti-coronavirus drugs approved in Japan (as of February 2023). Further increases in therapeutic options are awaited since various safety issues have been reported for these approved drugs. We will summarize and share the characteristics along with the issues of these antiviral drugs.

In the drug development process for Influenza antiviral drugs, chronic toxicity studies and carcinogenicity studies are not required because they are basically intended for short-term use. In addition, there is little need to consider on-target toxicity since the pharmacological target does not exist in mammals. However, it is necessary to ensure that there is a sufficient therapeutic index in vitro, and to consider the effects on human cell polymerases and mitochondrial DNA. In this symposium, we`ll introduce points to consider in the non-clinical safety of drug development of antiviral drugs, taking the anti-influenza drug baloxavir marboxil as an example. In general toxicity studies in rats, baloxavir induced prolongation of prothrombin time and activated partial thromboplastin time, which are thought to be caused by vitamin K deficiency. Since cases of bleeding suspected to be causally related to this drug reported following its launch, bleeding is included in the Japanese drug risk management plan as an important identified risk. Furthermore, in monkey studies, increases in blood chemistry parameters related liver function were observed without histopathological changes; however, there was no significant difference in the incidence of adverse events related to hepatic dysfunction between the drug group and the placebo group, which hepatic dysfunction is listed as an important potential risk. We’ll also discuss the appropriateness of risk assessment about these events in the non-clinical stage in light of the results of clinical trials and post-marketing surveillance.

[Background] Nirmatrelvir is a potent and selective inhibitor of the SARS-CoV-2 Main protease, administered as an oral agent for the treatment of COVID-19 patients in combination with a low dose of ritonavir. Ritonavir inhibits the CYP3A4-mediated metabolism of nirmatrelvir in humans, thereby providing increased and sustained plasma concentrations of nirmatrelvir.

[Pharmacological evaluation] The primary pharmacodynamic properties of nirmatrelvir were evaluated in a series of in vitro enzymatic and viral cell culture assays. In in vivo study using a mouse-adapted virus, SARS-CoV-2-MA10, inoculated intranasally in mice, the anti-viral effect of nirmatrelvir was confirmed. Combination of nirmatrelvir and ritonavir demonstrated improved efficacy and lung tissue protection compared to nirmatrelvir or ritonavir alone.

[Safety evaluation] Nonclinical toxicity studies of nirmatrelvir were conducted in accordance with ICH M3(R2). The NOAELs were the highest dose in general toxicity studies using rat or monkey. The results of genotoxicity studies were negative. Nirmatrelvir had no adverse effects on male or female fertility in rats, fetal morphology or embryo-fetal viability in rats and rabbits, or PPND in rats. No combination toxicity studies of nirmatrelvir with ritonavir were conducted because no overlapping or additive toxicities are expected.

[Conclusion] From these results, nonclinical toxicology package adequately supports the marketing application of nirmatrelvir in combination with ritonavir for the intended clinical usage.

Drug-induced liver injury (DILI) is a major cause of market withdrawal or drug-development discontinuation because of safety concerns. Drug-induced cholestasis (DIC) constitutes a major subgroup of DILI accounting for as much as 50% of all clinical cases of DILI. Bile acid (BA) synthesis occurs in liver cells. As surfactants or detergents, BAs are potentially toxic to cells, and their concentrations are tightly regulated. The disruption of the normal process of bile secretion results in cholestasis. Focusing on cholestasis caused by drug-induced endogenous BAs stasis such as the inhibition of BA transport, we established an in vitro DIC test system using simply two-dimensional cultured HepaRG cells and 12 types of BAs present in the human serum. Based on the investigation using DILI-concern drugs with the bile acid export pump (BSEP) inhibitory potential, the established DIC test system was mostly superior to the Css/BSEP IC₅₀ (＞ 0.1) assessment system, which one of DIC risk criteria. Therefore, it can be widely applicable as a model for the in vitro potential assessment of DIC. In this session, we would like to introduce the established DIC test system and mention to the risk assessment at the drug discovery stage with reference to in-house case studies.

Severe liver injury is a cause of drug development discontinuation or market withdrawal, and there is a need to improve the predictability of adverse hepatic events in the early stages of development. Among these, drug-induced cholestasis has been difficult to reproducibly and sensitively evaluate in vitro because of the difficulty in forming a bile canaliculi network capable of excreting bile using human hepatocyte culture. In addition, since there are species differences in bile excretion, there is a need for an in vitro test system that can evaluate the risk of cholestasis in humans without relying on animal experiments. Based on the above background, we have attempted to establish a culture method to form a bile canaliculi network capable of bile excretion in human cryo-preserved hepatocytes. We first found that long-term culture of iPS cell-derived hepatocytes promotes the formation of bile canaliculi. (Horiuchi et al. Sci Rep. 2022) Using this culture method, we are now analyzing the formation of bile canaliculi in cryo-preserved human hepatocytes from multiple donors and evaluating differences in bile excretion capacity among donors. We have also found that bile ducts are formed in hepatocytes derived from human liver chimeric mice. Based on the above findings, we re-evaluate hepatocytes from the viewpoint of bile excretion, and consider the characteristics of hepatocytes required for the in vitro assasy system as “hepatocyte-ness.”

The prediction of biliary excretion is one of the important factors for evaluating the pharmacokinetics and toxicities of pharmaceutical and food-derived compounds in humans. As the properties of biliary excretions differ among species, a highly predictive in vitro method for human biliary excretion is required. Although cryopreserved human hepatocytes have been widely used for this purpose, it is challenging to form functional and mesh-patterned bile canaliculi resembling those in the human body. Recently, Horiuchi et al. reported a new culture method that constructs bile canaliculi-like structures using human iPS cell-derived hepatocytes and commercially available long-term culture medium¹⁾. In this presentation, we would like to introduce the applicability of the culture method to cryopreserved human hepatocytes and discuss its potential to predict the biliary excretion by using known biliary excreted compounds and 21 types of food-related compounds.

Reference: 1) Horiuchi et al. (2022) Sci. Rep., 12: 15192.

Biliary excretion via drug-metabolizing enzymes and transporters is often critical as the hepatobiliary disposition is directly linked to drug efficacy and safety. Biliary excretion of drugs is mediated by transporters localized in the bile canalicular membrane of hepatocytes. Inhibition of the transporters may induce drug interactions and drug-induced cholestasis by parental drugs or their metabolites. Since there are species differences in the process, an in vitro evaluation system using human hepatocytes considered to be useful for drug discovery and development. However, conventional human primary cultured hepatocytes formed the closed bile lumen between hepatocytes, and thus it is technically difficult to collect the biliary-secreted drug. To overcome the challenge, the present study aimed to construct a human hepatocyte culture system that can collect bile components. Screening for a factor promoting bile duct formation in hepatocytes, the adhesion protein claudins were extracted. When human hepatocytes were cultured on a permeable support coated with claudin proteins, open hemi-luminal morphology was newly formed on the surface of the permeable support. This hepatocyte culture model, icHep (induced open-form canalicular hepatocyte), maintained the functions of a wide range of liver-specific drug uptake transporters and metabolizing enzymes. Permeation assay in icHep showed unidirectional transport of typical substrate of biliary transporters from blood to bile side. Furthermore, the estimated biliary drug excretion from the permeability obtained in icHep was well-correlated with the reported human in vivo values. This approach is useful to assess pharmacokinetics and drug safety during the preclinical stage of drug discovery and development.

Off-target effects of gapmers and siRNAs are generally evaluated by methods to analyze the change in mRNA levels, such as microarray and quantitative PCR. On the other hand, the mechanism of action of oligonucleotide therapeutics is not limited to RNA cleavage but is becoming more diverse, including splicing control, miRNA control and RNA editing. In addition, the development of next-generation small molecule drugs that could compete or coexist with oligonucleotide therapeutics, such as PROTAC (TPD therapeutics) and RNA-targeting small molecule drugs, is progressing as related modalities. In principle, methods to analyze mRNA levels cannot be applied to the off-target evaluation of such advanced modalities, and it is necessary to consider evaluation methods based on the characteristics of the mechanism of action of each modality. In this talk, we would like to introduce our attempts of off-target evaluation for diversified modalities.

Oligonucleotides (ONs) are preparations consisting of natural or chemically modified nucleotides. ONs can directly target intracellular molecules such as mRNA and non-coding RNA without mediated gene expression. In order to create a highly safe ONs, it is necessary to evaluate appropriately based on the toxicity characteristics of ONs and optimize the compound. Toxicity of ONs can be classified into on-target toxicity due to excessive pharmacological effects caused by hybridization to the target sequence, off-target toxicity in a narrow sense based on effects caused by hybridization to other than the target sequence, and hybridization-independent off-target toxicity based on the chemical property of ONs or effects other than ONs components. Based on these toxicity mechanisms, hepatotoxicity and nephrotoxicity are major issues for ONs administered systemically, e.g. subcutaneously or intravenously, and central nervous system toxicity are major issues for ONs administered centrally, e.g. intrathecally. Although various candidates of ONs can be easily designed based on the target genes, the cost of synthesis is higher than that of small molecules. Therefore, it is desirable to screen the initial toxicity of ONs in a stepwise and effective manner for a large number of candidate sequences. In this topic, based on our findings for Gapmer-type antisense oligonucleotide, we’d like to introduce each strategy for (1) Safety specifications before and at the time of sequence design, (2) Early exploratory toxicity screening, and (3) Late exploratory toxicity screening.

“New modality” is an emerging area in drug discovery to open new therapeutic fields that were challenging for conventional small molecules. As well as other new modalities such as oligonucleotide or AAV-based gene therapy, a increasing number of non-conventional small molecules were reported with novel mechanism of actions such as RNA modulation or protein degradation. Among them, RNA splicing modulator is one of key fields where a frontrunner compound, Risdiplam, was approved by FDA in 2020. Though published data is still limited for RNA splicing modulators, it would not be surprising that chemical space of RNA modulator is different from one of conventional small molecule due to different chemical properties of RNAs from proteins. Therefore, early de-risking strategy should be updated especially for RNA-targeted small molecules. In this talk, I would like to share chemical properties analysis of known RNA-targeted small molecules and potential in vitro safety screening strategy including assessment for chemical space related safety risks such as hERG inhibition and phototoxicity.

As one of the new modalities, target protein degraders (TPDs), degrade intracellular target proteins are being actively researched. There are approximately 10 compounds which have entered clinical trials, but their toxicity profile in humans has not been completely clarified because most of them are in the Ph1. Since TPD shows degradation activity only when the target protein-compound-E3 ligase ternary complex is in a conformation suitable for ubiquitination, “off-target toxicity observed only in animals" and "off-target toxicity in humans which cannot be detected in animals" can occur. For the latter issue, a platform for off-target toxicity assessment using human cells is now being established, but it has not yet been developed with animal cells. Currently no specific regulatory guidance for non-clinical toxicity evaluation of TPDs. TPD has been considered as small molecules so far, therefore its toxicity package is according to ICH M3 or S9. This means that toxicity study must be conducted by using both rodent/non-rodent species. In selecting animal species, confirmation whether there is on-target degradation in the target tissue is important. However, since the detection method is western blotting or LC/MS/MS, there are large species differences in the feasibility of antibody or adequacy of protein database. Additionally, even the compounds with the similar scaffold and on-target degradation activity in human cells, it has been reported that there may be large species difference on their degradation activities of homologue proteins among species. Consequently, it is considered difficult to determine which research stage/compound is appropriate to assess the degradation and toxicity profile. In this presentation, the current status and issues of non-clinical toxicity evaluation in the drug discovery of TPDs will be summarized.

In recent years, veterinary schools have undergone diverse educational reforms. The profession of veterinary medicine is diverse, and its needs have expanded rapidly to include advanced veterinary medicine, human and animal medicine (drug) development, infectious disease research and countermeasures, food safety, conservation medicine and One Health, which is health medicine that integrates the environment, society, humans, and animals, and animal welfare. In 2010, the World Organisation for Animal Health (OIE) published a draft Minimum Competencies for Veterinary Education, which requires a response from each country. In Japan, the "Standard Curriculum for Veterinary Specialty Education Programs" agreed upon by the National Council of University Representatives for Veterinary Medicine in 2004 continued to be debated; in 2008, the "Research Collaborators Conference on the Improvement and Enhancement of Veterinary Education" was established to discuss educational reform, including core curricula. When the Model Core Curriculum for Veterinary Education (Veterinary Core Curriculum) was created in 2011, so-called Core Curriculum textbooks were published accordingly, and CBT (computer-based testing) and OSCE (objective clinical competence examination) were launched along with the introduction of polycriteria. The Core Curriculum continued to be debated and updated, and in 2019 a new integrated Core Curriculum was created. The Model Core Curriculum for Toxicology states, "To clarify the harmful effects of chemical substances on humans, animals, and the environment, and to understand the role of veterinarians in preventing such effects.” Here we would like to present an overview of the reforms in veterinary education to date.

In the Model Core Curriculum for Veterinary Education (CC), the overall goal of toxicology is to “clarify the harmful effects of chemical substances on humans, animals, and the environment, learn about their toxic effects in living organisms, toxicokinetics, and mechanisms of toxicological manifestations, as well as dynamics of chemical substances on a regional and global scale and their effects on the environment, and understand the importance of comprehensiveness in toxicology as well as risk analysis and regulatory methods”. The overall goal of the toxicology practical training in the CC is "to acquire the necessary knowledge and skills in methods for clarifying the adverse effects of chemical substances on humans and animals, learn about their toxic effects in living organisms, toxicokinetics, and mechanisms of toxicological manifestations, and understand the importance of comprehensiveness in toxicology". On the other hand, it is practically difficult to provide the same practical skills in toxicology training at all veterinary colleges. In addition, recent amendments to animal welfare laws have promoted the spread of animal welfare awareness and stricter standards for the care and keeping of laboratory animals and the proper conduct of animal experiments, making it difficult to implement some of the content of toxicology training. This presentation will outline the current status and challenges of toxicology education at veterinary colleges, and describe the efforts being made at our university to provide education in line with current laws and regulations and the CC.

In a basic course on veterinary education, students learn about the animal body, differences between humans and animals (comparative biology), and pathogenesis and clinical treatment of various diseases. These studies are expected to enable veterinarians to play an active role in various aspects of the drug discovery process.

Veterinary toxicology imparts the basic knowledge required for conducting non-clinical toxicity studies and interpreting data. However, in the field, we observe toxicity and biological reactions induced by new compounds, which have not been described in textbooks. Further learning is essential to understand and apply these findings for society’s benefit, and students should be encouraged to study and think critically by accurately comprehending technical terms in specialized books and academic journals.

In drug discovery, collaborations with experts from different backgrounds, such as chemistry and protein science, are routinely carried out. Findings are expressed in veterinary terminology; however, these terms are not well understood by experts in other fields. Therefore, students need to acquire the skills to effectively communicate with researchers outside their field of expertise using plain language in an understandable manner.

In this session, I will discuss the future role of veterinary medicine and toxicology education in the field of drug discovery.

Veterinarians are widely active in contract research organizations (CROs) and play an important role. In the non-clinical studies on drug development, laboratory animals are widely used. The studies sometime include special procedures involving surgical operations, and CT, MRI, echo, etc. have been used for imaging diagnosis. Such areas have veterinarians performing the operating procedures or conducting experiments under supervision and guidance. The most important endpoint of toxicity studies using laboratory animals is histopathological examination, which is mostly performed by veterinarians who specialize in veterinary pathology. A veterinarian, who is expected to have systematic knowledge, can play an active role as study director, who engages in the planning of non-clinical studies, the interpretation and evaluation of study data, and the preparation of final reports. In recent years, due to the growing momentum for animal welfare, clinical veterinarians actively intervene, and make diagnosis and treatment in laboratory animals when they show anormal conditions, even during the course of studies. In order to fulfill these diverse roles in CROs, the necessary veterinary education is tried to be discussed.

The field of veterinary profession is very broad, with clinical and public health veterinarians as well as the life science field playing an increasingly importance in the safety evaluation of pharmaceuticals, chemicals, and foods.

Based on the Model Core Curriculum for Veterinary Education (Veterinary Core Curriculum) created in 2011, toxicology is one of the fields of applied veterinary education, but toxicology is currently not given much importance because only a few questions are included in the Veterinarian National Examination. Alternatively, there are aspects of veterinary education at universities that do not adequately address the specialized knowledge and skills in toxicology required in society, and students do not have a concrete image of the types of jobs and duties in which toxicology can be utilized, resulting in a shortage of veterinarians in life science fields such as pharmaceutical companies and a gap between supply and demand for human resources.

Regulatory science is described as "science to make accurate prediction, evaluation, and judgment" and has the role of bridging Regulatory research and Regulatory affairs and covers a wide range of areas including pharmaceuticals, medical devices, chemical substances, agricultural chemicals, and foods. Certified Toxicologists by JSOT also need to be able to make comprehensive judgments based on regulatory science, in addition to pharmacology, biology, chemistry, and veterinary science, as well as toxicology. However, there are few opportunities to learn about regulatory science in veterinary education, students are not fully informed about how it relates to veterinary medicine. Here we discuss the importance of regulatory science in toxicology and the efforts and future challenges in veterinary education.

The key tasks in drug discovery safety evaluation at the exploratory stage are to identify toxicity risks, and to plan and implement risk assessment and mitigation. Considering the timeline for drug development, it is desirable to identify toxicity risks as early as possible. Meanwhile, compounds worthy of conducting toxicity evaluation (e.g., having sufficient pharmacological activity to express on-target toxicity) are needed to obtain wet data. However, if we have to wait until the compounds are produced, this might delay the identification of toxicity risks. To avoid this delay, Target Safety Assessment (TSA), by which toxicity risks for pharmacological targets, among others, are predicted from information in the literature and databases, is being conducted in many pharmaceutical companies at the exploratory stage of drug development. However, each company is currently applying it in a trial-and-error approach. In my presentation, the results of a survey on TSA among domestic pharmaceutical companies will be reported, to trigger discussion in this workshop. The results reveal that each company is struggling to decide how much resources they should allocate to TSA and how they should interpret and handle TSA output. After my presentation, speakers will present their attempts to perform TSA in their companies, and the contents of their presentations and issues regarding TSA will be discussed in the panel discussion.

The value of exploratory non-clinical safety evaluation is to decrease safety-related attrition through a focus on candidate quality to provide safe therapeutics for people. As a part of this value, Takeda Discovery Toxicology team is providing a comprehensive assessment of the potential safety liabilities associated by target modulation through the Target Safety Review (TSR) to all projects at Project Start. The TSR includes biological information (e.g., biology, homology, and expression levels in human/animals), on-target safety liabilities (e.g., disease association, transgenic/knockout animal phenotype, and clinical competitors), off-target safety liabilities (e.g., modality-based information), and risk assessment strategy (e.g., potential safety biomarkers and decision flow). The potential adverse outcomes of exaggerated pharmacology associated with a specific target modulation are ranked, and safety risk assessment strategy will be generated to include experimental safety investigations at early project stage. Some examples are described here. Lipase X was a project for NASH indication. A number of potential systemic liabilities had been reported in literatures. On the other hand, since the TSR did not identify any specific risks for liver, PJ team modified their strategy from general small molecule lipase X inhibitor to identifying liver-selective lipase X inhibition by new modality. Kinase Y was also identified for NASH indication. There were strong links to potential hepatotoxicity (including extremely rapid hepatic tumor development in KO animals) identified in TSR. Although TSR itself doesn’t make Go/No-go decision generally, Takeda Discovery Toxicology team recommended not to move forward with this target in this case because there were limited/no good approaches to definitively derisk the liver tumor potential.

Target safety assessment (TSA) is performed at an early stage in drug discovery to estimate what kind of on-target toxicity might occur in normal tissues, and to determine what kind of non-clinical safety assessments would be needed going forward.

The main purpose of a TSA is to identify potential toxicities early so as to avoid the risk of dropout in later stages. Three important points must be considered when performing TSA: 1) information retrieval, 2) species selection, and 3) non-clinical safety assessment strategy suggestion. Information retrieval is the most important step in assessing potential risk. Target information can be obtained from literature, public databases, and in-house databases, and licensed software is useful for quickly sorting through vast amounts of such data when timelines are limited. Since public databases do not have enough information about target expression in various organs/tissues, a TSA relies on the in-house data. In some cases, knockout mice are used to obtain proof of concept. In particular for small molecule, not only metabolite profile/exposure level, but also pharmacological relevancy (expression, potency, pharmacodynamics) is important for non-clinical species selection. When selecting a species for a toxicity study, pharmacological relevancy should be experimentally confirmed by experiments for each project. Moreover, although information retrieval can be outsourced, the non-clinical safety assessment strategy must always be prepared internally by those familiar with the company’s drug discovery strategy. Altogether, TSA is accomplished through a review of public domain information and by leveraging internal company expertise.

In this presentation, we share our TSA strategies and give examples of how we apply TSA to non-clinical safety assessment.

Pfizer has been using Target Knowledge Review (TKR) since the early 2000s to drive a deeper understanding of safety risks for drug R&D programs. TKR is a review that identifies potential safety concerns and proposes mitigation plans based on a systematic evaluation of the drug target based on the information from a number of public and private sources and databases. Specifically, the following information is collected: biological information on the target molecule (genetic information, biological background), species differences between human and nonclinical animals (homology, expression distribution, comparison of target organs, etc.), similar off-target molecules, phenotypes in genetically modified mice or human SNPs, and existing molecules that are expected to interact with the drug target. TKR has been used to improve the success rate of projects both in the early stages of drug discovery and throughout drug development. In addition, it is also utilized to avoid wasting resources by terminating high-risk projects in the early stages of drug discovery. TKR is created when a new drug target is proposed, but are updated periodically thereafter to incorporate the latest information at all stage of drug R&D. The creation of TKR used to require time-consuming manual curation of target data; we have worked recently to automate as much of the process as possible to make it more efficient and standardize the TKR content. In this presentation, I will outline TKR and introduce an example of a decision to not go-forward based on TKR.

Target safety assessment (TSA), which identify toxicity hazards as drug targets and consider countermeasures, in the early stage of drug development is important to efficiently generate development candidates and increase the success rate of drug development. However, in the early stage, there are often no tool compounds, and TSA is limited to in silico TSA using databases and existing search tools. The creation of gene-knockout mice (KO mice) or conditional KO mice (cKO mice) is a possible option, but the former involves the concern of fetal lethality, while the latter issue requires a very long time using the common Cre/loxP system. Therefore, we have developed a technology that can delete target gene easily in adult mice by combining adeno-associated virus (AAV) and genome editing technology. Although there are some limitations on the organs that can be gene-deficient, the technology can generate cKO mice for any gene in a short term. This suggests that it is possible to provide a part of the solution to the issues that have been encountered in the generation of cKO mice. Here, we will introduce the results of gene deletion experiments targeting blood coagulation factor 9, whose function in vivo is known. We would like to discuss the effective use of this technology for in vivo TSA.

Toxicity study with two animal species is mandatory in drug development, with one selected from rodents and the other from non-rodents. Dog, NHPs, pig or rabbit are generally selected as non-rodent. NHPs are selected for about half of the new active ingredient drugs. In particular, the use of NHPs in biopharmaceuticals has reached about 90%, making their use essential as various modalities have been developed. However, since NHP exports from China were cut off in 2020, the global supply of NHP has been in short supply and prices have been soaring, which is starting to hinder the development of pharmaceuticals, especially biopharmaceuticals. In this changing environment, FDA has issued guidelines on the minimize the use of NHPs, and HESI is studying the evaluation of reproductive and developmental toxicity using the WoE approach with the aim of reducing number of NHPs in these toxicity studies. In addition, countries such as the United States and Korea are making national efforts to establish their own monkey colonies to improve their import-dependent monkey supply. In Japan, since 2022, a research team has been set up to strengthen research on alternative methods and promote replacement of NHP with other nonrodent species. We are also considering the need for a research group to investigate the number of NHP needed for overall biomedical research in Japan and the need for domestic breeding colonies for monkeys (as of March 2023). JPMA conducts a questionnaire at the R&D Committee to understand the status of large animal studies, and the Non-clinical Evaluation Expert Committee cooperates with JALAS and other organizations in conducting these group studies. In this session, the current situation is introduced and the survey results to date are introduced as a preliminary report, and the necessity of nonrodent studies and species selection are discussed.

Clinical pathology testing in nonclinical toxicity studies began to use clinical diagnostic drugs and medical devices in the 1960s. In the 1990s, the Japan Pharmaceutical Manufacturers Association, the Japanese Society of Clinical Chemistry, the American Association of Clinical Chemistry, and the American Society of Veterinary Clinical Pathology harmonized the item of clinical pathology in non-clinical toxicity studies internationally as IHCPT¹. And then, the current standard measurement items have been reached. Furthermore, joint research on toxicity biomarkers such as drug-induced nephrotoxicity biomarkers ² has progressed in recent years. Clinical pathology testing has become one of the important tests supporting translational science. This time, I’d like to have the opportunity to think about the selection of animal species by touching upon the characteristics of animal species in clinical pathological testing and issues in sampling, measurement, and evaluation, and introducing the current efforts and future perspective for solving these issues.

1 Weingand K, et al. Harmonization of animal clinical pathology testing in toxicity and safety studies. The Joint Scientific Committee for International Harmonization of Clinical Pathology Testing. Fundam Appl Toxicol. 1996, 29, 198-201.

2 Dieterle F, et al. Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium. Nat Biotechnol. 2010, 28, 455-62.

The ICH guidelines provide a standardized approach to the toxicity studies that should be conducted in the development of pharmaceutical products. Several toxicity studies are generally conducted using two species, non-rodents and rodents. Species should be selected based on the purpose of the evaluation and the characteristics of pharmaceutical products. Especially in biotechnology-derived pharmaceuticals, the species specificity of biological activity may limit the number of pharmacologically appropriate animal species. In such cases, the developer may consider conducting a study using one species that the developer considers appropriate or using other methods for evaluation, in which case the developer should be able to explain the appropriateness of the evaluation policy in terms of feasibility and significance of the study.

The relevancy of test species may be an issue in consultation between the developer and PMDA regarding the toxicity study package for the initiation of clinical trials and the application for marketing authorization, and decisions are made on a case-by-case basis. Deferral of some toxicity studies may reduce developmental cost and accelerate implementation of clinical trials. However, since safety information obtained only through animal studies may exist, the necessity of animal studies should be carefully determined. In this presentation, we would like to introduce the PMDA's approach on the selection of animal species and the necessity of animal studies when relevant animal species are limited, and discuss the toxicity studies TRULY required.

Available safety and efficacy data are limited in clinical trials and even no human data exist in first human studies. The main interest of investigators is securing safety of subjects in early clinical trials and the investigators pay most attention to safety data obtained by studies such as toxicology study and safety pharmacology studies. Such studies are performed mainly in small animals and investigators are used to interpreting data from small animals. Indeed, the safety of subjects has been secured by those data, so many investigators don’t feel urgent necessity of data from big animals except safety pharmacology data and cardiac safety data in dogs. The thought can be extended to pharmacokinetic data since human pharmacokinetics can’t be predicted from animal data even from primates. However, investigators of medical institutes belonging to JACIC (Japan Association of Contract Institutes for Clinical Pharmacology), who perform early clinical trials, reported their interest in nonclinical studies proving efficacy of investigational new drug besides its safety. Pharmacological actions are explored from the level of receptors in cells to biomarkers and biological responses of whole body and investigators may rely on responses in whole body. These responses may be found not only in big animals but in small animals. The point is whether an appropriate model is selected or not. Species of animals may differ according to target diseases. As an investigator in early clinical trials, I’d like to have data from appropriate models which can be extrapolated to human response with explanation of validity of the models. In the workshop, I will show update of the JACIC survey concerning attitudes to nonclinical data in investigators.

Many Molecular Initializing Events of toxicity manifestation are due to binding to biomacromolecules within the body. One evaluation method is an experimental binding assay using an in vitro expression system, but it lacks comprehensiveness. On the other hand, there is molecular docking using protein tertiary structure information as an in silico evaluation method. However, these tertiary structure analyses require large-scale equipment and the number of proteins with analyzed structures is far less than the total number of proteins. In contrast, several homology modeling methods have been proposed to predict protein tertiary structures from amino acid sequences using known structures as templates. However, the challenge has been that a template with high sequence homology is required for high-precision prediction. The situation changed dramatically in 2021 with the development of AlphaFold2 by DeepMind. AlphaFold2 is a deep learning-based high-precision protein tertiary structure prediction algorithm that achieved a high score comparable to experimental observations in the protein tertiary structure prediction contest CASP14. Furthermore, it has explosively advanced its use by comprehensively predicting the tertiary structures of more than 200 million amino acid sequences listed in UniProt and releasing them as AlphaFold Data Base. In this presentation, we would like to discuss the possibility of toxicity evaluation by combining high-precision prediction of protein tertiary structures since 2020, including AlphaFold2, with existing molecular docking and other methods.

Deep-intronic splicing mutations often cause inclusion of a pseudo exon, which in turn produces a premature stop codon leading to genetical inactivation. Number of disease-associated pseudo exonic mutations has dramatically increased because of recent advances in whole genome sequencing and transcriptome analysis. In contrast, mechanisms involved in pseudo exon recognition remain poorly explored. Understanding the mechanism of a disease-related splicing is crucial for finding an effective therapeutic strategy, as characterization of SMN2 exon 7 splicing led to development of Nusinersen for spinal muscular atrophy. In our hands, as previously demonstrated, splice-targeting therapeutics were achieved using small molecule compounds for genetic diseases such as Duchenne muscular dystrophy, NEMO deficiency syndrome, and familial dysautonomia, by targeting splicing regulators. By analyzing genetic information with AI, we established a novel therapeutic strategy for the inherited diseases caused by pseudo exonic mutations with our splicing chemical regulators.

Human body consists of the major elements (ca. 99.3%) such as oxygen (65%), carbon (18%), hydrogen (10%), nitrogen (3.0%), calcium, phosphorus, sulfur, potassium, sodium, chlorine and magnesium, and trace elements which exist in an amount of less than 0.01%. However, the trace elements have crucial and various roles in physiology and toxicology. Since trace elements are, as the name implies, "trace," their presence in living organisms has been studied for the major purpose of quantifing their total contents in the body. In addition, elemental speciation and imaging are also key techniques to understand the physiological and toxicological roles of trace elements. I have been developing four advanced and hyphenated techniques based on inductively coupled plasma mass spectrometry to reveal the roles. The following techniques were utilized for my research in metal/metalloid toxicology, 1) Speciation; ICP-MS was used as a detector of HPLC (LC-ICP-MS), 2) Laser ablation (LA); LA was used for a sample introduction of ICP-MS (LA-ICP-MS), 3) Single particle/single cell ICP-MS; ICP-MS having a high time-resolve mode was applied to an elemental analysis in a single particle/single cell (sp/scICP-MS), 4) Asymmetry flow field flow fractionation (AF4); AF4 is hyphenated with ICP-MS to analyze nanomaterials (AF4-ICP-MS).

Under the supervision of Dr. Yuzo Hayashi at the Pathology Division of National Institute of Health Sciences (NIHS), I participated in the rodent carcinogenesis studies, followed by so-called signal toxicity, Percellome Toxicogenomics Project, and nanomaterials toxicity studies under the support of Dr. Tohru Inoue through Dr. Satoshi Kitajima at the Molecular and Cellular Toxicology Division, NIHS. During that period, I was also involved in the OECD/EDTA, WHO/IPCS, WHO/IARC, EU-Horizon 2020 and other international academic activities such as ASIATOX and IUTOX.

Dr. Yuzo Hayashi’s project was, along with Dr. Takashi Sugimura of National Cancer Center and Dr. Nobuyuki Ito of Nagoya City University, Japanese version of NTP, generating 2-year rodent carcinogenesis study data. Dr. Hayashi used to say that “Even in broken English, when Japan raised hand for comment, all other participant kindly listened carefully expecting some meaningful contents”. I myself experienced when I was assigned to the lead lab for the development of OECD TG440, that having our original data with our interpretation was a strong propelling force for the project.

In terms of IUTOX, initially I was not attracted by its meeting ICT, but had to get involved when Dr. Inoue became the Vice President and passed along to me quite suddenly.

To make a long story short, International Cooperative Relationships results in the following situation. When you need to know something internationally, you know someone in many countries who you can call directly, e-mail or meet remotely. And those people can introduce you someone more appropriate in that country or region. The core is the human relationships!

Covid-19 had forced us to use more Web meetings. Recently Dr. Ghosh of SBI (https://www.sbi.jp/index.htm) taught me an interesting distinction between communication and connection. Web meeting is good for “communication” that is an exchange of data, whereas it is not good for “making connection” among people.

ASIATOX was initiated by 5 societies, Japan, Korea, Thai, Taiwan and China. In the Executive Committee, Taiwan and China sits next to each other. In IUTOX, when two societies from a certain country was not in good mood, I told such story and managed to make them relaxed.

In my award lecture, by showing snapshots taken so far, I would be happy to convey some idea of the current international situation.

In a first-in-human trial in 2006, the CD28 superagonist antibody (Ab) TGN1412 caused severe cytokine release syndrome (CRS) in all 6 human volunteers. They faced life threatening conditions involving multiorgan failure. The clinical signs of CRS correlate with immune cell activation following a cascade of systemic cytokine release, which can be fatal. The first human dose of TGN1412 had a 500-fold safety margin against the no observable adverse effect level (NOAEL) established in cynomolgus monkeys, but the animal study failed to predict the CRS risk. Consequently, in vitro cytokine release assays using human cells are now used to detect the potential risk of CRS. In this work, we investigated the detection and mitigation of the CRS risk caused by Ab pharmaceuticals.

There are two major in vitro assays for detecting CRS risks: one composed of whole blood with aqueous-phase test articles (i.e., the whole blood cytokine assay [WBCA]), and the other composed of peripheral blood mononuclear cells (PBMCs) with solid-phase test articles (i.e., the PBMC assay). First, we determined an appropriate sample size and confirmed the suitability of the WBCA as a hazard identification tool for CRS. Next, we compared the cytokine concentration and cytokine-producing cells after stimulation with the TGN1412 in the WBCA and the PBMC assay. The results showed that the types of cytokines induced by TGN1412 and the cells producing them differed in the two assays. The results emphasize the need for a better understanding of the characteristics of these in vitro assays and for a test method that is optimized according to the mechanisms of therapeutic mAbs.

Although T cell engagers (TE) show promising therapeutic potential as anti-tumor antibodies, it is still difficult to manage CRS in clinical use. We reported that single bolus infusion of anti-GPC3/CD3 TE caused life-threatening CRS, but daily step-up dosing of the TE did not, even at the lethal single dose. Clinical and non-clinical studies suggest that CRS after subsequent doses of CD3 bispecific constructs is less severe than after the priming dose, and that step-up dosing reduces cytokine levels in animals and humans. However, the mechanism underlying the reduced cytokine induction after priming treatment with TE is unclear. We made an in vitro model elucidating how priming treatment decreases cytokine release from human PBMCs after repeated treatment with anti-GPC3/CD3 TE. Priming treatment with TE did not decrease CD3 expression, CD3-mediated signal transduction, T cell activation markers, and cytotoxicity, but did decrease cytokine mRNA expression. Thus, we conducted ATAC-seq to detect accessible (open) chromatin regions. As a result, the priming treatment decreased chromatin accessibility at the transcription regulation region of IL2. In addition, we found a correlation between chromatin accessibility and individual differences in cytokine induction. The measurement of epigenetic states in T cells could be used to monitor CRS tolerance and to estimate the individual difference in CRS. We would like to further advance this research to find strategies for avoiding CRS and biomarkers for measuring individual differences in the risk of CRS.

Arsenite-induced cancer is known to be development after a latent period, even after the exposure is discontinued. Previously, we have shown that arsenite exposure induces premature senescence and senescence-associated secretory phenotype (SASP) factors in hepatocytes. However, it is unclear whether arsenite exposure-induced premature senescence and SASP factors are maintained even after cessation of arsenite exposure. In this study, we examined whether the arsenite exposure-induced premature senescence and SASP factors in hepatocytes persist after cessation of arsenite exposure.

As a result, senescent features such as morphological changes (enlarged and flatten) and changes of mRNA levels of senescence markers (P21 induction and LAMINB1 reduction) were maintained after 100 hours from cessation of 5 μM sodium arsenite exposure for 72 hours in Huh-7 cells. At that time, mRNA levels of SASP factors (MMP1, MMP3, MMP10, GDF15, PAI-1 and IL-6) were also significantly increased. We further confirmed that senescent cells still exist 7 days after cessation of the exposure by performing SA-β-gal staining. Furthermore, almost SASP factors that were upregulated by arsenite exposure in hepatocytes showed a positive correlation between increased expression and poor prognosis in human hepatocellular carcinoma by analyzing TCGA database.

These results showed that arsenite exposure-induced premature senescence followed by SASP induction is maintained in hepatocytes even after cessation of arsenite exposure and the SASP factors can be involved in progression of cancer.

Methylmercury (MeHg) is known to cause serious neurological deficits in humans. In this study, we investigated the occurrence of MeHg-mediated neuropathic pain and identified the underlying mechanism in a rat model of MeHg exposure. Rats were exposed to MeHg (20 ppm in drinking water) for 3 weeks. Neurological damage was observed in the primary afferent neuronal system, including the dorsal column of the spinal cord, and the thalamus, the starting point of the descending pain-inhibitory pathway. The MeHg-exposed rats showed, a significant decrease in the threshold of mechanical pain. Immunohistochemistry revealed the accumulation of activated microglia in the dorsal horn of the spinal cord. Western blot analyses of the spinal cord demonstrated an increase in inflammatory cytokines and a neuronal activation related protein (phospho-CREB). The results suggest that dorsal horn neuronal activation was mediated by inflammatory factors excreted by accumulated microglia. Furthermore, analyses of the cerebral cortex demonstrated increased expression of phospho-CREB and thrombospondin-1, which is known to be an important factor for excitatory synapse formation, specifically in the somatosensory cortical area. In addition, the expression of pre- and post-synaptic markers was increased in this cortex area. These results suggested that the new cortical circuit was wired specifically in the somatosensory cortex. In conclusion, MeHg-mediated dorsal horn neuronal activation with inflammatory microglia might induce somatosensory cortical rewiring, leading to neuropathic pain.

Cadmium (Cd) is one of the toxic heavy metals spread in the environment. The primary effects of Cd poisoning are acute hepatic toxicity and chronic renal and bone toxicity. Cd is daily ingested to humans through diverse foods, such as rice, vegetables, and seafood, and through smoking. Cd accumulates in the kidney and liver due to the dietary exposure over a lifetime, because the biological half-life of Cd is very long (15–30 years). It is concerning that renal dysfunction diagnosed in elderly people may be exacerbated by Cd accumulation. Damage to the proximal tubular cells is the characteristic of Cd-induced renal toxicity. However, the precise molecular mechanism involved in Cd renal toxicity is remaining elucidated. Our previous studies demonstrated that several transcription pathways regulate Cd toxicity in proximal tubular cells. Recently, it is found that retinoic acid (RA) which coordinates the gene expressions as one of the ligands of retinoic acid receptors (RARs), reduced Cd toxicity in human proximal tubular (HK-2) cells. Moreover, the pretreatment with retinol, the precursor of RA, also reduced Cd toxicity in HK-2 cells. However, knockdown of each gene coding for RARs did not affect Cd toxicity. Besides, the treatment with RA did not affect intracellular Cd concentration in the Cd-treated HK-2 cells. Cd renal toxicity is known to involve the apoptosis pathway. Complementarily, Cd induced apoptosis in the HK-2 cells; in addition, RA reduced not only Cd-induced apoptosis but also caspase-3 activation. These results indicate that RA may protect cells from Cd toxicity through the inhibition of apoptosis, without RAR pathway nor Cd accumulation.

On the basis of the evidence that extracellular Zn²⁺ influx induced with AMPA causes Parkinson's syndrome in rats that apomorphine-induced movement disorder emerges, here we used a low dose of AMPA, which does not increase intracellular Zn²⁺ level in the substantia nigra pars compacta (SNpc) of young adult rats, and tested whether intracellular Zn²⁺ dysregulation induced with AMPA is accelerated in the SNpc of aged rats, resulting in age-related vulnerability toParkinson's syndrome. When AMPA (1 mM) was injected at the rate of 0.05 μl/min for 20 min into the SNpc, intracellular Zn²⁺ level was increased in the SNpc of aged rats followed by increase in turning behavior in response to apomorphine and nigral dopaminergic degeneration. In contrast, young adult rats do not show movement disorder and nigral dopaminergic degeneration, in addition to no increase in intracellular Zn²⁺. The present study indicates that intracellular Zn²⁺ dysregulation, which is induced by GluR2-lacking AMPA receptor activation, is accelerated in the SNpc of aged rats after exposure to AMPA, but not after exposure to NMDA, resulting in age-related vulnerability to Parkinson's syndrome.