<Background>

Studies examined clinical characteristics of Jelly fish known as “Habu-Kurage” (Chironex yamaguchii) stings were limited to Australia, United States, Europe, and Southeast Asia, and those in other area remains unknown.

Also, only a few case reports of Stonefish known as “Oni-daruma-okoze” envenomation have been reported in Japan.

The purpose of our study was to examine the clinical characteristics of jellyfish and stonefish stings in Japan, and also provide the clinical characteristics of patients.

<Method>

Retrospective questionnaire surveys from January 2013 to December 2017 to determine patient characteristics, treatment, and clinical outcomes.

<Results>

Over the 5-year study period, 204 jellyfish sting patients were identified . All patients recovered well including five patients of severe jellyfish stings. Antivenom was not administered.

In the same period, 15 stonefish sting patients were identified from four hospitals. Over 70% of stonefish envenomation occurred in the northern part of Okinawa Main Inland. All patients recovered well, including four patients with severe stonefish envenomation. In two severe cases, length of hospital admission was more than 1 week (8days, and 9days). Antivenom was not administered.

<Conclusions>

Approximately 40 cases of jellyfish stings were annually occurred in Japan, and they recovered well without administration of antivenom.

Fifteen cases of Stonefish stings during same period occurred in Japan, and they recovered well without administration of antivenom.

Cyanotoxin microcystin-LR (MC-LR) induces acute injury of hepatocytes through protein phosphatases PP1/PP2A inhibition. In case of its chronic exposure, MC-LR induces hepato-carcinoma caused by acceleration of cell proliferation. Previously, we demonstrated the molecular basis for hepato-selective cytotoxicity of MC-LR which is taken-up into cells mediated by hepatocyte-specifically expressing transporter OATP1B1/1B3. Recently, we demonstrated a novel function of MC-LR which induced bi-potentially changes including anoikis resistance and cytoskeleton reorganization. In this symposium, I will discuss about association with epithelial-mesenchymal transition of these cells.

Hair testing has been performed extensively in the field of forensic science to obtain firm evidence of drug ingestion. Hair is the only specimen that can provide chronological information on individual drug use in addition to its longer detection windows. Recently, we investigated the time-course changes in drug distribution along single-strand hair at high resolution using MALDI-MS imaging and sectional hair analyses of 1-mm segments to investigate the incorporation pathways of drugs into hair.

The current analytical methods will be presented as well as additional new findings on the drug incorporation into hair and practical cases of hair testing.

Non-invasive diagnosis using externally secreted bodily fluids such as tear, saliva, urine, and sweat has little physical burden and has been promoted as an alternative to blood-based diagnosis to achieve future personalized healthcare. Various types of biosensors have been developed in recent times for non-invasive quantification of biomarkers in these bodily fluids. On the day, we will present recent progress in this research field including our research especially focusing on printed electrochemical biosensors for non-invasive sweat analysis.

Plants are constantly exposed to the threat of herbivorous insects, and many of them produce toxic substances in response to insect damage. In particular, insect species that have not evolved their detoxification mechanisms will be killed when they eat up their food plants. However, if insects can detect and avoid toxins quickly, they are more likely to survive. Therefore, like humans, in order to avoid toxins, they developed a taste reception and/or recognition mechanism that perceives toxic substances as bitter taste substances. Until around 2000, little had been known about the bitter taste receptor of insects. We and an Italian research group functionally identify the bitter taste receptor cell in the fly taste organs almost simultaneously. Indeed, our study, which noticed the involvement of gustatory odorant-binding protein (gustatory OBP) in a toxic substance reception by the bitter taste receptor cell, was the first to elicit an urgent vomiting response in the detection of oral toxin. In addition, the olfactory mechanism for recognizing the odor of the same substance also plays a role in the toxin detection. It has also been found that odor memory maintains appetite decrease for a lifetime. This appetite decrease may seem unhealthy for the fly, but it may be a beneficial reaction if the insect takes toxins to death. If so, plants no longer need to kill insects with lethal toxins until they have eaten themselves. Otherwise, plants can have another defense strategy targeting insect sensory mechanism for toxin detection with only a little bite and bitterness. Some plants have shifted to such a mild defense strategy. In the second half, I would like to add the story of such a plant species that has evolved a new defense strategy.

Volatile small molecule metabolites have been used for the diagnosis of diseases since the old days of BC. However, the details began to be explained scientifically only about 40 years ago. In recent years, with the rapid progress of analytical instruments, more detailed chemical information associated with diseases (particularly, cancer) has been elucidated. Recently, our research group has established for the first time in the world a novel technology to diagnose oral cancer from odorous volatiles excreted in saliva through the analytical identification of extracted 12 volatile metabolites, which are divided into three groups, such as "newly produced", "increased or decreased", and "disappeared" groups. The saliva samples from 12 cancer patients and 8 healthy volunteers were precisely analyzed using a GC/MS. According to the analysis, it was possible to determine the presence or absence of cancer with a reliability of 90% or more. The result of this study was that we were able to clarify the odor information of oral cancer, but it is highly likely that such volatile metabolites related to lung cancer, stomach cancer, etc. can be identified by a similar method. In the near future, we believe that the current study can be a new tool to realize a new medical diagnostic based on odors excreted from the human body. In parallel, we have progressed the development of measuring instruments that can diagnose cancer simply via human breath blowing.

Various chemicals present in food such as additives, pesticides, food contact materials, etc.. Toxicological information is very limited for more than thousands of compounds of these other than additives and pesticide. Recently, in silico methods are used for risk assessment of compounds that have limited toxicity information at trace levels in foods. The Threshold of Toxicological Concern (TTC) approach is a method to set a pragmatic threshold below which concerns for adverse effects become negligible, used for risk assessment of food flavors. in silico evaluation for genotoxicity has been adopted for assessment of pesticide metabolites by JMPR and EFSA.

ILSI Japan has launched and is working on an alternative animal testing promotion project in the area of food safety. Which aims to promote the development of Animal testing alternatives that can be used for food safety risk assessment. We are working on the following three major themes in cooperation with the academia. "Collecting information on the latest technologies", "Promoting research for accelerating the substitution of animal testing with alternative methods", and "Disseminating information for consensus- building". This talk introduces the latest efforts in the project.

A number of scientific approaches using dose-response experimental data have been used. As an alternative method to classical observational method including the use of no observable adverse effect level (NOAEL), the formality of benchmark dose (BMD) method has been elaborated. The BMD method determines the threshold dose by fitting various statistical models to the dose-response curve, which addresses the problems surrounding the use of NOAEL because it can account for the response data across different doses and can help in objectively calculating the point of departure. The benchmark dose lower bound (BMDL), which is the lower (one-sided) limit of the 95% confidence interval of BMD, can yield a point of departure that is comparable to that based on NOAEL. To employ the BMD method, it is critical to select the best performing BMDL by following the statistical fitting procedures of multiple mathematical models. Parameterized models only characterize reality, so multiple models (usually nine or more) are commonly fitted to the same experimental dataset. As a result, many BMDL values can act as the candidate of preferred reference dose. However, the reference dose should be the best performing BMDL and it must be selected, for example, as the one that gives the best fitting results. There are two additional issues in selecting or determining the BMDL. First, the BMD method uses a specified percentile point (e.g. 10% of the benchmark response, abbreviated as BMD10) as the threshold for the reference value, but the 10% percentile point is never strictly objective. Second, some fitted models (e.g. the Weibull model) yield different parameter estimates when restrictions to the range of parameters are imposed in advance of the inference procedure. In this symposium, technical issues and corresponding guidelines are comprehensively reviewed and presented.

The Three Rs―replacement, reduction, and refinement―are guiding principles for the ethical use of animals. In recent years, a number of test methods incorporating these principles have been adopted by the OECD, the ISO, and other international organizations. The Three Rs have been an integral part of a number of ICH guidelines published since the 1990’s, including ICH S2 on genotoxicity testing, ICH S5 on reproductive toxicology, and ICH S10 on the photosafety evaluation of chemicals. Moreover, since 2009, these principles have been incorporated into ICH M3 R2 Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals.

Test developers continue to research new in vitro, in chemico, and in silica test methods, which suggests that the development of alternatives to animal testing that incorporate the three principles of replacement, reduction, and refinement will continue indefinitely into the future.

Reduction, which is one of 3R's, often appears as a result of Replacement or Refinement, and it is difficult to discuss the very idea of Reduction.

The technology of compound synthesis progressed significantly, a vast number of compounds came to be synthesized in pharmaceutical industry. However, the number of animals used has not increased significantly in proportion to the increase in synthetic numbers.

In this symposium, this fact will be re-evaluated and considered to build a function of promote 3R’s “Reduction”. We would consider what is necessary to promote the 3R's in the following points.

1) We reconfirm that it is contributing to the 3R's without knowing it, when look back at the process of drug discovery research from the viewpoint of animal welfare.

2) We understand the 3R's award and 3R's day initiatives in Europe and consider what drives 3R's research.

3) We find research in the development of in vitro technology that could contribute to 3R's in the future, and boost it from the viewpoint of animal welfare.

What do you think of when you hear the word "Refinement"?

Is it the improvement of technical skills and knowledge? Or upgrading of instruments used in experiments? Of course, these are also a kind of "Refinement". However, they are "Refinement" from the viewpoint of researchers and technicians.

So, what is “Refinement” from an animal’s perspective?

“Refinement” should be the minimization of the stress of the animals in areas other than procedures absolutely necessary for an experiment. This is the "purpose of Refinement" Russell and Burch wrote about in "The Principles of Humane Experimental Technique". No matter how much experimental techniques are improved, normal biological reactions are unlikely to occur when animals are stressed, making it difficult to obtain reliable experimental results.

However, "Refinement" requires implementing a variety of changes depending on the purpose of the experiment. As Russell and Burch point out, “Refinement presents more formidable difficulties,” and researchers and technicians may often feel that this goal is more than they can attain.

This is where laboratory animal veterinarians and IACUC members come into play.

They have the role of proposing changes for “Refinement” according to the purpose of each experiment. They are constantly studying in order to make proposals that will obtain the desired experimental results while balancing experimental endpoints with animal welfare. If you have concerns about "Refinement" practices, please ask a laboratory animal veterinarian or IACUC member. Together, we can aim for better animal experiments!

We have enjoyed sound lives thanks to life sciences, which were fundamentally supported by animal experiments. In spite of progress in life sciences, many problems remained to be solved to keep us in good health. It is inevitable to continue scientific researches including animal experiments. To proceed scientific activities with wide social collaboration, it is necessary for life scientists not only to have serious consideration on humane care for experimental animals, but also to communicate with public. Because such efforts have been tried by European countries, they are introduced along with the communication handbook published by the European Animal Research Association.

The ICP-mass spectrometry (ICP-MS) is a sensitive and rapid analytical tool for trace-elements in various materials. The ICP-MS technique is now applied to measure both the sizes and number concentrations of nanoparticles (NPs), mainly dispersed in the aqueous solutions. Another important feature of the ICP-MS is the analytical capability for the imaging analysis using a laser ablation sampling technique. Imaging analysis was conducted based on the repeated-line profiling analyses, and the resulting time-dependent signal intensity profiles were converted to the position-dependent signal intensity data using the in house “iQuantNP” software. With the technique developed here, signal intensities for small size fraction (<10nm) were also visualized. More importantly, chemical status of the analytes, whether present as particulates or as dissolved (ionic) forms, can also be derived by the present technique (Triad Imaging : size, position, and chemical status of analytes). The Triad Imaging technique can provide key information to understand the transport, interaction, and decomposition/dissolution features of the metal NPs.

The major drawback of the current ICP-MS system for the detection of NPs is that only single element (isotope) can be monitored, and thus, no elemental and isotopic ratio data can be made from individual NPs. Faced with this, we are currently developing new ion detection technique for the ICP-MS designed for elemental and isotopic analyses on multiple component NPs. The sensitive and rapid imaging technique for the NPs has immediate potential as a reconnaissance method and given increasing improvement in instrumentation will in the future produce benchmark data for the NPs toxicology.

Selenium (Se) and tellurium (Te) belong to Group 16 (chalcogen) of the periodic table, and their compounds exhibit high toxicity. Although a variety of bacteria have been reported to reduce soluble chalcogen oxyanions to form insoluble nanoparticles of elemental Se and Te inside or outside of cells, little is known about the mechanism of formation of biogenic chalcogen nanoparticles. Here, our recent findings on mechanism of reduction of chalcogen oxyanions to form chalcogen nanoparticles in bacteria will be presented.

The studies for collecting precious metals and toxicant elements by using hyper-accumulator algae are being actively conducted due to its low costs and eco-friendliness. However the mechanism of the accumulation has not been cleared so far. We confirmed the accumulation of gold by unicellular algae, Pseudococcomyxa simplex, and elucidated the accumulation mechanism clarifying the chemical forms at their accumulation, and succeeded in visualizing the distributions of gold nanoparticles produced in a cell of algae. When selenite was added to the algae, selenium was much accumulated than those for selenate. The biogenic nanoparticles of Se were visualized by SEM and the chemical form was confirmed as elemental selenium by XAFS analysis.

It is known that organisms use a variety of mechanisms to avoid toxicity caused by non-essential metals and essential metals beyond their nutritional requirements. As the detoxification of harmful metals, organisms induce and bind the metals to metal-binding proteins and peptides such as metallothioneine and phytochelatin, regulate the expression of transporters for an influx and an efflux the metals to control the intracellular concentrations, or enhance a transformation of metalloids. In addition to such detoxification mechanisms, my group has recently found a formation of biogenic nanoparticles in animal cells and plants, when there is exposure to ionic metals. Further, nanoparticle formation of mercury selenide was experimentally confirmed in mammalian cells when the cells were co-exposed with mercury and selenium, which are classically known to show antagonism. The mechanism of the generation of biogenic nanoparticles and their toxicological significance will be discussed.

In a past decade, advances in genome editing technologies have provided a possibility ofa novel gene therapy not only in vitro but also in vivo. The genome editing is the technology to cause either the knock-out of target gene or modification of abnormal gene by homologous recombination of target gene (HDR) through the double strand break of target DNA sequence. The targeting ability of genome editing is dependent on the recognition by specific protein to be able to bind target DNA sequence (Zinc-Finger nuclease; ZFN or transcription activator-like effector nuclease ; TALEN) or binding of a single-guide RNA (sgRNA)of which form the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated proteins (Cas) complex.

Many clinical study protocols using genome editing tools have been approved and been ongoing.Advantageous characters of genome editing are utilized to exploit either the knockout technology for thetarget gene by HNEJ or modification of hereditary abnormal genewith normal one by HDR. On the other hand, genome editing technologies have become known to cause DSB on un-target gene (off-target effect), or yield insertions and deletions, or template repair from a separate donor DNA molecule (on-target mutation). Therefore, to overcome the undesirable effects of genome editing should be continuing to develop the analytical methods or new technology.

Science Board in PMDA has established the Committee for Genome Editing to discuss about the quality and safety issues of technologies. In this meeting I would like to discuss to refer this paper.

Gene-edited cellular therapy products (ex vivo gene-edited products) are categorized as cellular and tissue-based products in Japan. However, PMDA requests their safety assessment according to the guidelines for not only cellular and tissue-based products, but also gene therapy products.

In this presentation, I will outline the basic concepts related to the preclinical safety assessment of the cellular and tissue-based products and the gene therapy products, and then introduce the general principles for nonclinical safety evaluation of ex vivo gene-edited products, focusing on safety concerns specific to this technology.

The placenta is an important organ for the development of fetuses. The placental dysfunction and injury have adverse effects on the maintenance of pregnancy and fetal development. However, the placental weight change is only one of the indexes of placental toxicity in developmental toxicity studies. Detecting chemical-induced histopathological placental damages in rats provides a valuable clue to the mechanisms of fetal toxicity in safety evaluation. In this presentation, I will introduce the histopathological placental changes in the rats exposed to cisplatin, ketoconazole, tamoxifen, etc.

We aim to identify factors affecting species differences in fetal drug transfer. Species differences between human and rat in fetus-to-mother concentration ratio of digoxin and indomethacin were cancelled by using the unbound concentration ratio in rat. This can be explained from the fact that the lower albumin concentration in fetal plasma compared with maternal plasma is observed in rat but not in human. Organic anion transporter 4 found only in primates and mediates the transport of olmesartan, which may cause adverse pregnancy outcomes especially occurred in humans. These information enhance possibilities of improving the extrapolation of fetal drug transfer data from rodents to human.

Reproduction involves tightly regulated series of events and the immune system is involved in an array of reproductive processes. Disruption of well-controlled immune functions leads to placental inflammation and dysfunction, resulting in various pregnancy complications, including preeclampsia (PE).

Inflammasomes are involved in the process of pathogen clearance and sterile inflammation. They are large multi-protein complexes that are located in the cytosol and play key roles in the production of the pivotal inflammatory cytokines, interleukin (IL)-1β. The NLRP3 inflammasome is a key mediator of sterile inflammation induced by various types of damage-associated molecular patterns (DAMPs).

Recent evidence indicates that the NLRP3 inflammasome is involved in pregnancy dysfunction, including PE. Many DAMPs (uric acid, palmitic acid, high-mobility group box 1, advanced glycation end products, extracellular vesicles, cell-free DNA, and free fatty acids) are increased and associated with pregnancy complications, especially PE. This presentation will focus on the role of the NLRP3 inflammasome in the pathophysiology of PE.

The estrogen level of rodent fetus is much lower than that of human fetus because the rodent placenta doesn’t have aromatase which converts androgen to estrogen. To examine the species differences in estrogen sensitivity between human and rodent fetuses, we have established a humanized-transgenic mouse that expresses aromatase in the placenta using lentiviral gene manipulation. I will talk about the phenotype of this mouse model, and discuss the species differences in the reproductive and developmental toxicity by estrogenic chemicals between human and rodent.

In classical toxicology, a toxic substance reaches the target molecule and induces malfunction. Signal toxicity is caused by a chemical that binds to a receptor. When the signal is abnormal for a cell or an organ in terms of quality, intensity and timing, then the signal will induce adverse effects to an organism. The developing neural systems are volnerable and can result in permanent neurobehavioral anomalies. The testing strategies of the Developmental Neurotoxicity is under debate between in vitro mechanistic frame work and in vivo testing battery plus mechanistic analysis.

We have been developed the screening systems to find the neurobehavioral toxicities induced by early exposure of neuroactive xenobiotics, using behavioral tests and neuro-scientific analyses. Here, we will show the effects on emotional/cognitive behavior and hippocampal gene expression profiles of male adult mice induced by low-dosed exposure of bisphenols during development phase. We also discuss about some issues related to the improvement of global assessment methods for neurodevelopmental toxicology.

The brain is a computational organ that uses membrane potential to compute and exchange information between neurons. Hence, membrane potential measurements are essential to probe brain function. Here, we introduce an optical measurement method using a voltage-sensitive dye (VSD) to assess its vulnerability. We will present a VSD imaging study regarding chronic and acute BPA applications on the hippocampal circuit activities. Also, we will show our single-photon wide-field recording technique can capture rare events, oscillation, and plasticity of a wide area of a neural circuit.

Toxicity of volatile organic compounds (VOCs) in indoor air, such as formaldehyde (FA), xylene (Xy) and p-dichlorobenzene (pDB), at the levels of Sick House/Building Syndrome (SHS) is difficult to assess by the ordinary inhalation animal studies; histopathological endpoints are negative for toxicity at such concentration levels. Here we applied our Percellome Toxicogenomics Project that has been launched to develop a comprehensive gene network for the mechanism-based predictive toxicology using time- and dose-dependent transcriptomic responses induced by a chemical in mice. For this purpose, a normalization method designated as “Percellome” is developed (BMC Genomics 7:64, 2006) to generate mRNA expression values in “copy numbers per one cell” from microarrays and Q-PCR. Here, we report that the Percellome analysis is capable of predicting functional insults by short-term inhalation at SHS-level concentration.

Lung, liver and hippocampus were analyzed. In contrast to the slight transcriptomic changes in lungs and livers, a remarkable finding common to the three chemicals were the strong suppression of gene expression related to neuronal activity in hippocampus, i.e. the immediate early genes (IEGs) including Arc, Dusp1 and Fos. Review of the lung and liver gene profile pointed out a candidate cytokine upstream of IEGs. Our finding may be considered as a first substantial data that would explain the indefinite or unidentified complaint in SHS patients. The analysis of emotional & cognitive behavior induced by these indoor VOCs will be presented.

Neurogenesis occurs throughout adulthood in the mammalian brain by coordinated proliferation and differentiation of adult neural stem cells. Newborn neurons are incorporated into the functional networks of both the olfactory bulb and the dentate gyrus, suggesting significant roles of adult neurogenesis in brain functions. In this review, we discuss potential application of postnatal and adult neurogenesis in the olfactory bulb to test developmental neurotoxicity.

By mass, the cerebellum constitutes about only 10% of the total volume of the central nervous system. However, the number of cerebellar granule cells comprises 60-80% of the total neurons in the central nervous system. During normal development, a massive number of granule cells are generated from granule cell precursors, which is elicited through various intracellular and extracellular signaling cascades. Drug exposure to developing individuals occasionally results in maldevelopment of granule cells. I this talk, I introduce our recent findings on the molecular machinery for granule cell development and discuss the possibility that mouse granule cell development can be used as the in vivo evaluation system for drug exposure.

In vitro screening experiments such as hepatobiliary transporter inhibition, inhibition of mitochondrial function or reactive metabolite formation w/ and w/o covalent binding have been proposed to predict human drug-induced liver injury (DILI). Fasiglifam was an agonist of GPR40 under development for type 2 diabetes. Unfortunately, a development program was terminated late in phase III trials due to liver safety concerns. In this symposium, it would be introduced whether in vitro screening approaches of fasiglifam which were retrospectively conducted could predict DILI risk in a phase III.

Drug-Induced Liver Injury (DILI) is a main leading cause of attrition in drug development or post-marketing withdrawal. Though the importance of risk assessment of DILI is widely recognized, quantitative DILI assessment is difficult because multiple different mechanism and patient variability complicatedly contribute to human DILI. Extrapolating the animal data to human is also difficult. Consequently, our challenges are that the human risk of DILI could be detected based on the DILI mechanisms in the exploratory stage of drug development and predicted quantitatively by integrating various factors.

In the exploratory stages, we conduct in vitro assays (e.g. reactive metabolites, mitochondrial toxicity, and bile salt transporter inhibition) and estimate physicochemical properties to profile DILI risk. Remaining issues of quantitative human DILI risk assessment are integrating multiple in vitro assay results and human variability, although in vitro studies can profile DILI mechanism. To solve these issues, we have investigated the usefulness of DILIsym® developed by DILIsym Services, Inc. in collaboration with FDA, which is cutting-edge modeling and simulation technology.

This session will highlight in vitro/in silico risk profiling of human DILI in exploratory stages of drug development and retrospective evaluation of the drugs with the elevation of blood liver enzymes in the clinical studies.

In this presentation, our approaches to establish in vitro evaluation systems for predicting DILI at early preclinical stage are introduced. To predict intrinsic or idiosyncratic DILI, we show what types of data are collected and referenced. Logically, in the vitro evaluation systems are only "predictive" of in vivo or human DILI, and "validation" step would be essential when "prediction" is made. Our current challenge is to establish not only in vitro evaluation systems but also in silico models for DILI prediction.

Drug-induced liver injury (DILI) is a significant public health problem that has a critical impact on not only patients but also healthcare professionals, pharmaceutical companies and regulatory authorities. It is considered difficult to predict its occurrence due to a complex interaction between chemical properties and patient factors.

Recent technological advances have led to the development of in vitro assays that can improve the predictability of clinical adverse events, and they are now becoming available in the early stages of drug discovery. Based on the concept of "Safety by Design", we have adopted a tiered approach that is stepwise approach from "a simple and high-throughput assay system" to "a complex assay system focused on organ toxicity".

Recently we re-evaluated Takeda legacy compounds including lapaquistat which has been terminated in phase III due to safety concerns in the liver, by the DILI-related assays introduced in the tiered approach, as part of evaluation of the usefulness of these assays for DILI prediction.

In this symposium, we will report the results and would like to discuss the future challenge to improve the predictability of clinical DILI.

Conventional preclinical studies can detect intrinsic hepatotoxicity of candidate compounds but cannot detect idiosyncratic Drug-Induced Liver Injury (iDILI). One of our compounds which has been discontinued by iDILI during clinical trials in the 2000s has forced us to develop a strategy to avoid iDILI. It is mainly based on in vitro covalent binding amounts to classify iDILI compounds, however, includes false positive and negative for the classification. New human in vitro system using T lymphocytes from healthy volunteers to improve the strategy will be discussed in this presentation.

Methylmercury (MeHg) is a widespread environmental pollutant and causes a serious hazard to health worldwide. Macroautophagy (hereafter referred to as autophagy), a major and highly conserved degradation pathway in eukaryotes, prevents the accumulation of misfolded or damaged proteins, protein aggregates, and damaged organelles in the cytoplasm. We previously demonstrated that MeHg exposure promotes autophagy, and Atg5-dependent autophagy serves to protect cells from MeHg cytotoxicity. Moreover, we found that sequestosome1/p62 protects cells against low-dose MeHg cytotoxicity via clearance of MeHg-induced ubiquitinated proteins. Our findings suggest that p62 is crucial for cytoprotection against MeHg-induced toxicity.

Treatment with low-dose MeHg induced the expression of endoplasmic reticulum (ER) stress (CHOP and GADD34). p62KO MEFs exhibited significantly higher CHOP and GADD34 mRNA induction following MeHg treatment compared to their wild-type (WT) counterparts. The endogenous expression of GFP-p62 was fully compensated for the lack of p62 because of the mild induction of ER stress response in p62KO MEFs. These results suggest that p62 plays a critical role not only in the clearance of ubiquitinated proteins but also in attenuation of MeHg-induced ER stress. In this symposium, we will discuss our recent experimental findings of the role of p62 on autophagic response and ER stress following MeHg exposure.

Methylmercury (MeHg) is known to cause central/peripheral nerve disorders. To elucidate the mechanism of the peripheral neurotoxicity to MeHg, we performed the behavioral analysis, genetic and immunohistochemical analysis of the dorsal root ganglion (DRG). In the MeHg exposed rat, each sensory modality responded in different timings. Microarray data showed that TNF-α and TLR pathways were involved. Moreover, the activation of microglia/macrophage in DRG was observed. These data suggest that MeHg-induced neurotoxicity in peripheral nerve may occur via inflammatory responses.

Methylmercury (MeHg) induces site-specific neurotoxicity in the adult brain. In this study, we investigated the site-specific expression of the signaling cascade related to neural activity in a mouse model of MeHg intoxication showing neurodegeneration only in the deep layer of the cerebral cortex, especially layer IV. We performed time course studies of c-fos and brain-derived neurotrophic factor (BDNF) expression levels which are proper markers of neural activity. We showed that upregulation of both markers preceded the neuronal degeneration in the cerebral cortex. Immunohistochemical analysis revealed the site-specific upregulation of c-fos in the deep layer of the cerebral cortex. Western blot analysis showed that c-fos and BDNF expression was associated with CREB phosphorylation, which was triggered by the activation of the p44/42 MAPK, p38 MAPK and PKA pathways. However, we did not detect any changes in the expression levels of c-fos and BDNF proteins and no signs of neuronal degeneration in the hippocampus and cerebellum, despite the fact that we could detect accumulation of MeHg in these two brain regions. These results suggested an intriguing possibility that MeHg-induced neuronal degeneration was caused by site-specific neural hyperactivity triggered by the activation of MAPK and PKA/CREB pathways followed by c-fos and BDNF upregulation.

MeHg covalently binds to cellular protein thiols, resulting in structure changes in the proteins and occurrence of toxicity. We previously reported that transcription factor Nrf2 plays a role in detoxification and excretion of MeHg through GSH adduct formation, whereas CSE catalyzing formation of reactive sulfur species contributes to capture and inactivation of MeHg through sulfur adduct formation. In addition, with single and double KO mice for Nrf2 and CSE, it is suggested that CSE and Nrf2 are complementary involved in repression of MeHg toxicity.

We recently found that oncostatin M (OSM), a kind of inflammatory cytokine, was induced from microglia in the brain of mouse treated with methylmercury. In experiments using cultured cells, extracellularly released OSM was found to be involved in enhancing methylmercury toxicity by binding to TNF receptor 3 (TNFR3). Moreover, administration of an antibody against TNFR3 directly into the mouse brain suppressed neuronal cell death caused by methylmercury. In this symposium, I will introduce the OSM/TNFR3 signaling pathway, which was suggested to function as a mechanism of methylmercury toxicity.

Recent progress in cancer treatment significantly improved survival of patients with cancer. The novel targeted cancer therapies are associated with a wide spectrum of cardiovascular (CV) complications in patients. To understand CV toxicity screening and mechanisms of cardiotoxicities, it is necessary to establish a cardiotoxicity testing for anti-cancer agents. To date, we have developed a drug-induced proarrhythmia risk evaluation method using human iPS cell-derived cardiomyocytes (iPSC-CMs). We further extended iPSC-CMs to other types of cardiotoxicity evaluation, such as left ventricular dysfunction. Based on high-speed camera and a specific algorithm, we have developed a novel in vitro contractility assay system. Tyrosine kinase inhibitors reduced contractility speed using iPSC-CMs. We are currently planning to perform multi-site study for contractility and biomarkers, in collaboration with HESI cardiac safety committee. In the symposium, I would like to provide new insights on CV safety using iPSC-CMs during anti-cancer drug development.

Human iPS cell-derived cardiomyocyte (hiPSC-CM) is conceptually promising as an unlimited source of human cardiomyocytes for pre-clinical safety testing. However, intra- and interline variations in functional properties remain to be solved completely. In order to quantify the variations, we employed a motion field imaging to assess the contractile functions. The technique revealed cell-to-cell variations of contractile function of hiPSC-CMs. We would like to discuss on the impact of the variation on pharmacological/toxicological assessment.

To improve the cost-efficiency of drug development, both human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and in silico have been recently employed on the evaluations of safety pharmacology and toxicity tests. On the other hand, it has become to know that the electrophysiological properties of hiPSC-CM are different from the original cardiomyocytes. Hereby, we constructed virtual hiPSC-CM (viPS) and found various differences between them in the molecular, cell, and tissue levels. The viPS is very useful for the safety pharmacology and toxicity tests in the future.

Since human iPS cells can be differentiated into various cells, they are expected to be applied to toxicity tests, disease mechanism studies, and drug discovery screening. However, pluripotent stem cell-derived cardiomyocytes are closer to fetal myocardium than adult cardiac tissue cardiomyocytes, which hinders their application to toxicity test systems and drug discovery research. We are conducting various approaches to induce cardiomyocyte maturation. Using genome editing technology, we have created iPS cell lines in which we can monitor the expression of troponin I1 and troponin I3, whose expressions change during cardiomyocyte maturation, by observing the expression of a reporter fluorescent dye and have conducted screening using a compound library. Through the screening, compounds that induce cardiomyocyte maturation were identified. Cardiomyocytes treated with these compounds showed gene expression profiles, mitochondria function, and cell morphology closer to mature cardiomyocytes found in adult heart tissues. Mature iPS cell-derived cardiomyocytes are expected to improve disease models and the accuracy of toxicity tests.

In addition, we are conducting pathological model studies of cardiac diseases, such as hypertrophic cardiomyopathy and genetic arrhythmias. Cardiomyocytes derived from hypertrophic cardiomyopathy iPS cells showed cellular hypertrophy and disordered sarcomere structures, and we are looking for compounds that improve these changes.

In this symposium, I would like to report on the development of a method for producing mature cardiomyocytes from iPS cells and the progress of research on the construction of pathological models and toxicity testing systems using iPS cell technology.

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. Therefore, unveiling molecular mechanisms of pseudo exon recognition will provide a novel therapeutic strategy for pseudo exonic diseases.

The rapid advancement in new machine learning architectures in the field of artificial intelligence (AI) coupled with the availability of multi-scale data has opened novel avenues in various domains of biomedicine and healthcare - from drug discovery, clinical applications and toxicology. The talk will explore the various promises and pitfalls of AI, particularly in the context of toxicology, with use cases for success and failures, various areas of applications towards the goal of finding the right balance (optimal dose) for AI applications in biomedicine and healthcare.

Omic analysis is one of the most powerful approaches to understanding the biological responses to various events. In recent years, a wide variety of analytical tools for omics data analysis have become freely available for researchers. On the other hand, each analytical tool has its own unique characteristics, and the interpretation of their outputs is usually not straightforward.

Here, I will present the analytical flow and the results of transcriptome analysis of pentachlorophenol-administered mice [1]; I’ll further discuss the potential issues with the quality control in the interpretation of the outputs obtained by using such analytical tools.

The basis of the data accuracy control for the Percellome Project was established when the Toxicogenomics Project (NIBIOHN) was initiated in 2003 to accumulate rodent gene expression data for over 150 chemicals. We checked the dose-response performance of the microarrays and developed a RNA spiking method and related softwares to generate absolutized copy number of mRNAs per cell. Accuracy control is essential for the newly designed “repeated dosing studies” which monitors a genome-wide alteration in basal gene expression levels. (supported by Health and Labor Sciences Research Grant)

Comprehensive bisulfite DNA methylation analyses and comprehensive histone modification analyses of the liver of male mice repeatedly orally exposed to carbon tetrachloride, valproic acid and clofibrate showed no major changes in DNA methylation status but showed genome-wide histone modification changes. We report the details of these histone modification changes.

Since 2016, NIHS has been working with the Committee for Non-Clinical safety Evaluation of Pluripotent stem cell-derived producT, the Forum for Innovative Regenerative Medicine (FIRM-CoNCEPT) on MEASURE (Multisite Evaluation study on Analytical method for non-clinical Safety assessment of hUman-derived REgenerative medical products). In MEASURE project, we conducted multisite validation of test methods for the assessment of tumorigenicity and biodistribution of CTPs. We have also been discussing the global consensus of tumorigenicity assessment of CTPs with international stakeholders.

Detection of residual tumorigenic cells is important in the development of human cell-based therapeutic products. MEASURE evaluated a method using NOG mice in multiple facilities. iPS cells were transplanted in the abdominal region of mice by single subcutaneous injection and then observed for 20 weeks. As a result of the multisite main studies, calculated 50% Tumor Producing Dose (TPD₅₀) values were between 15 and 100 in both males and females. In conclusion, the results of the multisite studies suggest that the method is useful for detection of undifferentiated iPSCs.