Neurotoxicity is a major adverse effect pre-clinically and clinically. Among types of neurotoxicity, seizure is one of the most serious finding during the drug development process. Seizure evaluation is usually used to in vivo animal models. Recently, multi-electrode array (MEA) systems measuring the electrophysiological neuronal activities have attracted attention as useful for evaluation of seizure risk and alternatives to animal testing. Multiple parameters calculated from the electrophysiological responses are needed to evaluate the seizure of several seizure-induced compounds. In this section, we introduce evaluation methods for seizure risk using multiple parameters.

Alternative approaches to animal testing have been proposed under the movement of 3Rs. Various alternatives to eye-irritation tests have been developed in the field of ophthalmic research. Technologies mimicking in vivo condition, e.g., 3D in vitro culture and organ-on-chip system, have been reported in recent years. We have undertaken to develop in silico prediction or 3D and in vivo ocular genotoxicity evaluation methods. In this presentation, I will introduce recent knowledge and our challenges for in vitro and in silico approaches in ophthalmic drug safety.

In 2035, in vitro culture systems and analytical systems such as AI will be the important items to solve several problems in safety assessment.

This time, I would like to take up MPS, which is attracting global attention both technically and regulatory. Also, I would like to touch on the activity of the AMED-MPS project which is the national project of MPS for drug discovery.

For MPS to be implemented and established as a practical method for safety assessment by 2035, I will introduce each issue in the industry, government, and academia and the efforts for it.

Many pharmaceutical companies are promoting the simultaneous global development of innovative new drugs, and the number of FIH studies has increased in Japan. Therefore, it is crucial for both sponsors and regulators to communicate with each other, and fully discuss the risks and benefits of individual products from a scientific point of view. In this workshop, all participants, including the audience, will be able to exchange opinions through a questionnaire survey, case studies, and panel discussions, and to promote a mutual better understanding of the non-clinical safety assessment.

It is often argued the requirement of the toxicity study or interpretation of the toxicological findings during drug development. Due to short timeline, there are limited opportunities for direct communication between pharmaceutical companies and PMDA. Here, we conducted a questionnaire survey on the differences of opinion in consultations with PMDA, and received responses from 23 companies. The different opinion on guidelines between Japan and other countries were mentioned. We would like to discuss this survey results with participants for mutual understanding between companies and PMDA.

Recently, modality in drug development has become diverse. Since current toxicity test guidelines do not completely cover the new modalities, consultation with PMDA is important. However, there is a “gap” in view or thinking between companies and PMDA due to their different positions, which sometimes leads to reconsider the development. Mutual understanding of the “gap” may be important to activate drug development in Japan.

I would discuss “peptides with non-natural amino acids” as a virtual drug for the theme of this session, and also pick up the questions came up from the questionnaire.

Regulators question the sponsor about any unknown matters in the consultation of the development of new drug and at the submission of new drug approval. However, there are cases in which regulator's thinking is not fully communicated because of differences in position and way of each thinking. In this presentation, we will outline our views on sponsor’s questions through the interpretations of (i) toxicities of virtual drug, (ii) a questionnaire survey and (iii) the accompanying guidelines. Through this presentation, we hope to help bridge the gap between sponsors and regulators.

In this session, we will focus on gastrointestinal (GI) toxicity and present some case studies to demonstrate utility of advanced human in vitro GI models and preclinical and clinical safety biomarkers for addressing drug-related GI toxicity. We will also discuss the current landscape of microbiome research as gut microbiomes have been reported to alter drug safety or efficacy. Overall, the discussion will focus on how novel models and biomarkers are being positioned today and in the future to support drug discovery and development.

Human induced pluripotent stem cells (hiPSCs) are expected to be useful for both toxicity and pharmacokinetics evaluations of drug candidate compounds in drug discovery. For instance, the small intestine plays an important role for oral absorption of drugs, and the use of human primary intestinal epithelial cells (IECs) may be considered in drug discovery. However, there is an issue in securing of stable acquisition of functional human primary IECs. If the functional human IECs is obtained from hiPSCs, the issue will be resolved. Therefore, in this study, I aimed to generate functional IECs from hiPSCs using the three-dimensional differentiation procedure¹⁾ mimicked the in vivo environment.

HiPSCs were induced to differentiate into IECs via definitive endodermal and midgut/hindgut cells by mimicking the intestinal development. The midgut/hindgut cells were cultured by three-dimensional method, and then intestinal organoids including IECs was obtained. Most of intestinal organoids were expressed an IECs marker protein (villin), however, the surrounding cells of intestinal organoids were expressed a mesenchymal cell maker protein (vimentin). So, intestinal organoids were dissociated into single cells and IECs derived from hiPSCs (hiPSC-IECs) were enriched by magnetic-activated cell sorting. After hiPSC-IECs were incubated with a terfenadine (CYP3A/CYP2J2 substrate) or midazolam (CYP3A substrate), the metabolites were generated, respectively. The results suggest that hiPSC-IECs have CYP3A/2J2 enzymatic activity. Monolayered hiPSC-IECs expressed tight-junction marker proteins (Occludin and ZO-1), indicating hiPSC-IECs have intestinal barrier function. Therefore, hiPSC-IECs can be beneficial for the exploration of drug candidate compounds based on oral absorption character.

In addition, I will introduce the outcome using hiPSC-IECs in research area of drug metabolism and pharmacokinetics in Shionogi & Co., Ltd. and the literatures regarding characteristic of intestinal organoids derived from hiPSCs and resent toxicity study using intestinal organoids. Based on knowledge from these literatures, I would like to discuss application of intestinal organoids for evaluation of toxicity in drug discovery.

Reference：

¹⁾Spence. JR., Wells, JM., et al. Nature, 470, 105-109, 2011

Drug-induced gastrointestinal (GI) toxicity is commonly observed as clinical adverse effects of therapeutic agents targeting intracellular effectors in cancer treatment. The adverse effects include diarrhea, colitis, and other drug-induced GI toxicity influencing safety and tolerability of the agents. GI toxicity prediction of candidate molecules during drug discovery and development has been challenging due to limitation of in vitro models to recapitulate key structures and functions of human GI tract. Over the past decade more physiologically relevant models have been developed using three-dimensional (3D) cultures for better cell polarization and characteristics of tissues and organs. This talk will highlight our recent experiences of trans-well formatted 3D GI microtissues, which consist of primary human small intestinal cells and recapitulate structural features such as villi. GI toxicity is assessed by informative readouts such as molecular signatures by whole-transcriptome profiling for better understanding the mechanisms of GI toxicity, and transepithelial electrical resistance (TEER) for barrier integrity. In particular, TEER may provide better prediction of test articles to induce diarrhea, which is correlated to intestinal barrier dysfunction. The talk will provide examples from our studies with the GI model demonstrating the effect of a known class of drugs that cause transcriptional disruption of the cell cycle and autophagy pathways, and influence barrier integrity in a target-dependent manner. We will also discuss the benefits and challenges of the GI model for drug safety assessment.

Intestinal toxicities/adverse effects have been frequently reported in non-clinical and clinical studies during drug development. Intestinal toxicities themselves are often not life-threatening, but they can significantly reduce the quality of life for patients and may lead them to give up medications. Meanwhile, there are few well-established intestinal injury biomarkers like there are for kidney or liver, making it difficult to monitor intestinal toxicities in non-clinical in vivo research and to translate any findings into clinical contexts.

In this presentation, I will introduce multiple intestinal injury biomarkers, including those at discovery stage. Additionally, I will describe our investigation of these biomarkers in intestinal injury model rats. Finally, the significance of these novel biomarkers to the evaluation of toxicity will also be discussed.

The human microbiota, whose relationship with health and diseases is becoming clearer one after another, is currently receiving a great attention not only from science but also from industry and can be expected as sources of new innovations. On the other hand, considering that it has become clear that the Japanese microbiota has different characteristics from those of Westerners, it is necessary to acquire and analyze Japanese data together with various health and medical information for industrial applications. From the viewpoint of industrialization to realize them, it is a major premise that there is a reliable foundation such as data reproducibility. For that purpose, standardization of protocols with an eye to industrialization is extremely important. And development of standard substances as validation tools and establishment of validation flow are required.

In April 2017, The Japan Microbiome Consortium (JMBC) was established with the aim of standardizing protocols important for industrialization, acquiring Japanese data, and building a database of healthy people that can be expected to be used in many industries as a reference.

This lecture introduces background and purpose of the establishment and goals of the consortium, as well as the activities and R & D plans through collaborative cooperation between companies.

Furthermore, recent trends toward drug discovery are also presented from the perspective of application to pharmaceuticals. In recent years, the therapeutic effects of fecal transplantation have been reported successively and application to drug discovery has become active especially in Europe and the United States. Several venture companies engaged in microbiota have already been established in Europe and the United States, and some venture companies have announced alliances with major pharmaceutical companies. In addition, many clinical trials are conducting one after another and the pipeline is increasing.

After mentioning these trends of pharmaceutical companies, we would like to discuss what kind of innovative products can be expected from human microbiome research.

In 2011, I was diagnosed with acute promyelocytic leukemia (APL). Although I experienced severe side effects from chemotherapy, it was somewhat expected, and rather unexpected events were that I felt unwell after complete remission. Under such circumstances, I was encouraged by the words of a person with experience of APL like "10 years have passed since remission.". I became able to talk about my experience of fighting disease. In this presentation, I will introduce an overview of APL and the side effects I experienced as well as how to live as a cancer survivor.

The incidence of osteosarcoma is ca. 1-1.5/year/million in Japan. Osteosarcoma is more likely to occur in children and teens aged 10-20 years. Chemotherapy before and after tumor resection has become standard of care with an overall treatment duration of 1 year. However, chemotherapy often causes serious side effects including hair loss, vomitus, and leukopenia.

In this presentation, my experience of anticancer drug treatment to my family; especially adverse reactions and also its supportive care during the 8-month neoadjuvant and 5-month postoperative chemotherapies will be introduced.

Chemotherapy plays a important role in cancer treatment same as surgery and radiotherapy.

Drug A is evaluated the toxicity profile and the safety in early phase, and evaluated efficacy in late phase with confirmatory clinical study. Although strict trials mention above are performed, there are several unexpected remaining adverse events in clinical practice.

Post-marketing surveillance using ePRO or tracing device information seems important to take measures in unexpected adverse events.

To achieve good clinical practice, it is necessary to collaborate each medical companies without competitive element.

Fatigue is the most prevalent symptom of individuals with cancer who receive radiation therapy, cytotoxic chemotherapy, or biological response modifiers. However, cancer-related fatigue remains undertreated and poorly understood. Given the unmet need, new effective cancer-related fatigue treatments are needed. In the present study, we used cancer-free mice to examine the specific influence of chemotherapy on fatigue. Mice were administered a single dose of cisplatin (CDDP; 10 mg/kg, i.p.) or saline as a control, and then were treated with carbohydrate-enriched diet, fat-enriched diet or saline daily for 4 days. At 24 h after the final dose of diets or saline, fatigue-like behavior was assessed by running activity on a treadmill. After administration of CDDP, running activity of mice decreased significantly. Under these conditions, we found that daily intake of carbohydrate-enriched diet increased the running activity of CDDP-treated mice compared to daily intake of fat-enriched diet. Furthermore, CDDP-treated mice were given daily intake of sucrose, fructose, glucose, or olive oil for 4 days. In CDDP-treated mice, daily intake of sucrose and glucose, but not fructose and olive oil, caused a significant and dose-dependent increase of both the liver glycogen content and running activity. These results suggest that maintenance of the liver glycogen content prevented fatigue-like behavior in mice after administration of CDDP.

Cytotoxic anticancer drugs frequently causes various adverse effects, while some of them, such as peripheral neuropathy or oral mucositis, are still uncontrolled. We found that taxanes induce dedifferentiation of Schwann cells, resulting chemotherapy-induced peripheral neuropathy. By screening drugs that has the ability to induce Schwann cell differentiation, we found that cilostazol, a PDE inhibitor, inhibit taxane-induced Schwann cell dedifferentiation and reduced painful responses in animals. Furthermore, I will introduce the effect of Hangeshashinto oral ointment, a Chinese herbal medicine, on chemotherapy-induced oral mucositis. We are now preparing to conduct clinical trials.

Hand-foot syndrome (HFS) is one of the common side effect of cytotoxic agents and multi kinase inhibitors (MKIs), characterized by erythema, edema and erythrodysesthesia of palmar and plantar with or without pain. HFS has a major impact on patient’s quality of life, and, in severe case, it could affect normal activities of daily life because of pain. In addition, frequently, HFS is known to be a dose-limiting toxicity requiring discontinuation, dose reductions, or delays of anti-cancer drug therapy. Therefore, providing early diagnosis and treatment of HFS would contribute to continuation and completion of anti-cancer drug therapy. However, the mechanism of toxicity of HFS still remains unknown, and the information about the animal model of HFS has been limited. The development of mechanism-based treatment and prophylaxis of HFS is awaited.

We have tried to establish animal models of HFS using a cytotoxic agent to investigate the mechanism and mechanism-based effective treatment to prevent HFS. In our experiments, we successfully induced changes in limbs in rats using repeated dose of DOXIL (liposomal formulation of doxorubicin) or Tegafur (cytotoxic agent), and the changes were macroscopically and histopathologically similar to HFS observed in human. In this session, we will give an overview of HFS and introduce the findings obtained in animals.

This workshop is an annual program that offers topics on nurturing toxicologists and maximizing their value. Toxicologists are required to have wide range of expertise and skills such as detection of toxicity, clarification of MOA, and estimation and management of risk to humans. Artificial intelligence (AI) is now able to assist toxicologists in their work. However, blind use of AI often fails to reap its full benefits. First, it is necessary to "AI-Readiness", the preparation to utilize AI. This workshop will provide topics on raising literacy level and mutual understanding with AI engineers. We hope this will be a good opportunity for toxicologists to start using AI and transform themselves into "AI-Powered Toxicologists" who can fully utilize AI. The topics of this workshop will be presented in Japanese.

In order to increase the number of opportunities for toxicologists working with AI and other digital technologies, it is important to accelerate the promotion of DX (Digital Transformation) in the organizations to which they belong. Rather than throwing the project to consulting firms or AI vendors, it is necessary to foster a culture in which the entire organization can handle digital technology as a matter of course. At first glance, this may seem like a tremendously difficult task. We have been supporting the transformation of many organizations that are tackling these difficult issues from the perspective of changing the "people" that make up the organization, rather than just changing the top-level measures, with the aim of creating a prosperous digital society.

In this session, we will show the approaches and successful cases adopted by some organizations such as pharmaceutical companies, and explore ways to increase the opportunities for toxicologists.

In recent years, things that were only in the science fiction world 30 years ago, such as "artificial intelligence" and "machine learning," are becoming reality. Among them, not only companies but also individuals are seeking to utilize AI to find new value. However, AI is also a tool, and its effect cannot be obtained unless it is used well.

But here we run into one big problem. It is a skill gap between engineers who develop or master AI and engineers who really want to utilize it. Each other does not know anything about the content or skills of the other person's work. Originally, mutual understanding is necessary to make the best use of the tool of technology.

In this lecture, the lecturer's own experience will focus on how AI engineers who do not know toxicology at all and toxicologists who do not know AI at all or may have a misunderstanding will seek mutual understanding and utilization. I will explain based on. Starting from the basic idea of ​​AI, we will explain not only the cases in the industrial field that we are doing, but also the points for promoting the use of AI in the future, including research cases of utilization in the medical field.

Quantitative risk assessment methods for chemicals, particularly environmental pollutants and industrial chemicals, have advanced significantly over the last 20 years. In the past, a hundred of safety factor for the most case of derivation of acceptable daily intake has been simply applied to a NOAEL for chronic animal study. New approaches (ex. use of various toxicological endpoints other than chronic effects or use of interspecies difference factors in pharmacokinetics) have been introduced to dose response assessment for derivation of health-based guidance values. A benchmark dose (BMD) approach as one of the new approaches, has been used to for establishing a POD, which can be replaced replace the NOAEL in case of no NOAEL establishment. The BMD approach is also being used to derive a POD for the risk assessment of genotoxic carcinogens. Although universal softwares (BMDS and PROAST) are available as internationally standardized software for calculating BMDL, there is no harmonized procedures on which mathematical model should be selected for BMDL calculation by using such software. And this non-harmonized BMD calculation method is considered to be a barrier for the active use of BMD in other government agencies. We have established the optimal and conservative application criteria of BMD method for administrative use. Recently, model averaging methods for BMDL calculation are becoming the mainstream instead of single mathematical model selection, although new problems have emerged. As for the risk assessment of a large number of chemical substances for which toxicity test information, the TTC concept is useful for administrative management. Although noncarcinogenic TTC is less widely used than carcinogenic TTC, we were able to propose an application guidance for the evaluation of leachable substances from food equipment and containers by using the TTC concept. In this presentation, in addition to introducing research on the development of methods for applying the BMD approach and the TTC concept to administrative use, I would like to discuss how we can improve derivation of the quantitative assessment values, such as health-based guidance values.

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Liver lobules have oxygen gradient from 13% (the portal vein) to 1~5% (the central vein). HIF-1alpha is a key transcription factor activated during hypoxia, and ubiquitinated through hydroxylation of proline residues by PHD. Nrf2 is a key factor in defense against oxidative stress and degraded through binding to Keap1. In this study, we investigated the regulation of drug-metabolizing enzymes by treatment of 2,2'-bipyridyl, a PHD inhibitor, or tBHQ, a Keap1 inhibitor, with Hep3B cells. The results of this study suggest that CYP3A family P450s are regulated by hypoxia and oxidative stress.

DHA has been shown to have neuroprotective effects in Parkinson’s disease, but the underlying mechanism has not been fully elucidated. DHA is metabolized to DHA epoxides by P450s and further hydroxylated to the corresponding diols (DHDPs) by sEH. In this study DHA intake in rats improved the motor dysfunction induced by rotenone. However, these effects were eliminated by cosupplementation with the sEH inhibitor. DHA intake increased the amount of 19,20-DHDP in the rat brain. These results suggest that DHA metabolites have an important role in improving rotenone-induced Parkinson’s disease.

Chlorogenic acid (CGA), the most abundant polyphenol in coffee, has been reported to have an anti-oxidative activity and various health benefits by activating the nuclear factor-erythroid 2 related factor 2 (Nrf2). However, the mechanism underlying Nrf2 activation by CGA has not been fully elucidated. Nrf2 is mainly regulated at protein levels via proteasomal degradation orchestrated by several E3 ubiquitin ligase adaptor proteins, including Keap1 and β-TrCP, and newly identified WDR23. In order to study the mechanism behind the activation of Nrf2 and identification of its role in lifespan elongation, we used both human hepatocellular carcinoma (Hep3B) cells and nematode Caenorhabditis elegans. In the present study, we showed that treatment of Hep3B cells with CGA increased Nrf2 protein levels and transcriptional activity, leading to enhanced expression of its target genes (HO-1 and NQO1). Knockdown of WDR23, but not Keap1 or β-TrCP, diminished these effects; suggesting that CGA requires WDR23. In C. elegans, CGA increased SKN-1 (Nrf2 homolog) protein levels and the expression of its target genes (gcs-1 and gss-1), prolonged the lifespan of wild-type strain, and did not affect the lifespan of skn-1 mutant strain; indicating that lifespan extension effect of CGA requires SKN-1. The effect of CGA on lifespan was dependent on WDR23, as CGA did not affect lifespan of wdr-23 mutant strain. Taken together, these results suggested that Nrf2/SKN-1 is critical for the CGA-mediated beneficial effect. Additional efforts are needed to dissect the mechanism by which WDR23 is involved in the response to CGA treatment.

Multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage cell death and NLRP3 inflammasome activation leading to granuloma and mesothelioma in rodents; however, it remains unknown how macrophages recognize MWCNTs. By a target screening of phagocyte receptors, we here demonstrated that T cell immunoglobulin mucin 4 (Tim4) and Tim1 recognize MWCNTs. Using Tim4^-/- Tim1^-/- mice, we revealed that Tim4 is involved in MWCNT-associated pathogenesis. Taken together, these results indicate that Tim4 and Tim1 are receptors for MWCNTs.

Clioquinol is regarded as the causative agent of subacute myelo-optic neuropathy (SMON). Yet, the pathogenesis of SMON has not been fully elucidated. In human neuroblastoma SH-SY5Y cells, clioquinol induced the oxidation of ATOX1, suggesting its inactivation and inhibition of copper transport. The secretion of dopamine-β-hydroxylase, a copper-dependent enzyme, was inhibited by clioquinol, along with decreased noradrenaline levels. The disturbance of cellular copper transport by the inactivation of ATOX1 may be one of the mechanisms involved in clioquinol-induced neurotoxicity in SMON.

Cadmium (Cd) reduced GLUT4 (glucose transporter 4 coded by SLC2A4) in HK-2 cells. Cd treatment and SLC2A4 knockdown caused a significant decrease in intracellular glucose levels. Cd significantly decreased the uptake of glucose into the cells. The decrease in intracellular ATP level was observed by Cd treatment and SLC2A4 knockdown. Long-term exposure to Cd decreased gene expression of Slc2a4 in the kidney of mice. These results indicated that Cd renal toxicity is engaged by the decrease in glucose and ATP level by the suppression of cellular GLUT4 level.

Salubrinal sustained phosphorylation of eIF2α and regulate cell death, but the mechanism is not clear. In this study, salubrinal significantly prevented the CdCl₂ induced cell death. ER stress/autophagy were responsed by CdCl₂, and further elevated by co-treatment with salubrinal. The mechanism is that salubrinal sustained phosphorylation of eIF2α, resulting in a simultaneous response to ER stress and autophagy, but the CdCl₂ induced CHOP mRNAs were suppressed by salubrinal, which may have prevented cell death. These results recently reported the cooperation of ER stress response and autophagy.

Exposure of pregnant rats to low level MeHg induced cerebellar synaptic and neuritic remodeling during the perinatal period. We identified suppression of the TrkA pathway and reduced Arc expression in MeHg-exposed pregnant rats during the perinatal period. MeHg-exposed pregnant rats also exhibited increased perinatal plasma corticosterone levels and decreased estradiol levels. These results suggest that exposure of pregnant rats to low level MeHg affected perinatal cerebellar synaptic and neuritic remodeling through modulation of the TrkA pathway and Arc expression which may be caused by MeHg-induced hormonal changes.

We previously reported that mouse C8γ binds to tri-substituted organotin compounds such as triphenyltin (TPT) and may play an important role in the toxicity of TPT. However, in such carrier molecules, their binding properties are often different among species because of species differences in amino acid sequences. We here evaluated binding affinity of chemical substances to human C8γ protein. We found that it also has specific binding to TPT and group 14 compounds with tri-phenyl substituent. These results suggest that C8γ may play an important role in the toxicity of these compounds in human, too.

Arsenic is an environmental toxic metalloid responsible for many human diseases and affecting many millions of people all over the world. In the present investigation, the efficacy of curcumin, a naturally occurring polyphenol against arsenic-induced cytotoxic manifestations with insights into biomolecular mechanism/s has been studied in PC12 cells. Results revealed that arsenic (10 μM) treatment in PC12 cells for 24 h induced cytotoxicity by decreasing cell viability and intracellular glutathione level and increasing lactate dehydrogenase activity and DNA fragmentation. Moreover, arsenic-induced apoptotic cell death in PC12 cells, which were confirmed from the flow cytometry results. In addition, arsenic treatment significantly down-regulated the survival proteins; mTOR, Akt, Nrf2, ERK1, Bcl-x, Xiap and up-regulated the cell-death related proteins; ULK, LC3, p53, Bax, cytochrome c, caspase 9, cleaved caspase 3 and ultimately caused autophagic and apoptotic cell death. However, pretreatment of curcumin (2.5 μM) with arsenic (10 μM) protects PC12 cells from arsenic-induced cytotoxicity by increasing cell viability, GSH level and boosting the antioxidant defense system, and limiting the LDH activity and DNA damage. Furthermore, pretreatment of curcumin with arsenic significantly inhibited arsenic-induced toxicity and cell death by reversing the expressions of proteins. Our findings suggested that curcumin showed antioxidant properties through the Nrf2 antioxidant signaling pathway and improves arsenic-induced toxicity in PC12 cells via modulating autophagy/apoptosis.

Maternal exposure to TCDD was suggested to attenuate LH production during the perinatal stage, and the supplementation of α-lipoic acid to TCDD-exposed pregnant dams showed a recovery effect on the attenuated LH production during the gestational period. Therefore, it was expected that α-lipoic acid supplementation would have a recovery effect on the retardation of the male offspring’s sexual immaturity induced by maternal exposure to TCDD. This study suggests that maternal supplementation with α-lipoic acid has a restoring effect on the decreased SDN-POA volume, which is an indicator of sexual differentiation in male offspring.

Our previous study showed that 5 μM sodium arsenite exposure for 3 days in human hepatic stellate cell line LX-2 induced cellular senescence and expression of IL-8, one of the senescence-associated secretory phenotype (SASP). In this study, we examined whether the gene expression levels of other SASP factors besides IL-8 were increased. Our current study showed that 5 or 7.5 μM arsenite exposure for 6 days strongly induced gene expression levels of IL-1β, CXCL1, MMP1 and MMP3 in LX-2 cells. Furthermore, the expression levels of those genes were still maintained for 5 days after removing arsenite from medium.

1,2-Dichloropropane (1,2-DCP) was extensively used in the past in offset colour proof-printing. In 2014, the International Agency for Research on Cancer reclassified 1,2-DCP to Group 1. Prior to the reclassification, cholangiocarcinoma (CCA) was diagnosed in a group of workers exposed to 1,2 -DCP in an offset colour proof-printing company in Japan. In comparison with other forms of CCA, 1,2-DCP-induced CCA was of early onset and accompanied by extensive pre-cancerous lesions in large bile ducts. However, the mechanism of 1,2-DCP-induced CCA is poorly understood. Inflammatory cell proliferation was observed in various sites of the bile duct in the noncancerous hepatic tissues of the 1,2-DCP-induced CCA. The aim of this study was to enhance our understanding of the mechanism of 1,2-DCP-related cholangiocarcinogenesis. We applied an in vitro system to investigate the effects of 1,2-DCP, using MMNK-1 cholangiocytes cultured alone or with THP-1 macrophages. The cultured cells were exposed to 1,2-DCP at 0, 0.1, 0.2, 0.4 and 0.8 mM for 24 hours and then assessed for cell proliferation, cell cytotoxicity, DNA damage, and ROS production. Exposure to 1,2-DCP increased proliferation of MMNK-1 cholangiocytes cultured alone but not those cultured with macrophages. 1,2-DCP also increased LDH cytotoxicity, DNA damage and ROS production in MMNK-1 cholangiocytes/ THP-1 macrophages but not those cultured alone. 1,2-DCP increased TNFα and IL-1β protein expression in macrophages. The results highlight the role of macrophages in enhancing the effects of 1,2-DCP on cytotoxicity, ROS production and DNA damage in cholangiocytes.

Acrylamide (ACR) is a recognized neurotoxicant which produces effects of neuropathies in humans and experimental animals. Sulforaphane (SFN), a phytochemical mostly found in cruciferous vegetables has been implicated to be protective against inflammation, oxidative stress and several neurologic disorders. The present study therefore sought to investigate the effects of SFN against ACR-induced neuropathy in mice.

Mice were exposed to ACR through drinking water at 0, 200 or 300 ppm and co-treated with SFN at 0 or 25 mg/kg body weight in physiologic saline by subcutaneous injections daily for 28 days. Immunohistochemistry for noradrenergic axons, landing foot spread test, RNA-expression and oxidative stress analysis were conducted following exposure of mice.

Relative to SFN-untreated mice, co-treatment of mice with SFN greatly and dose-dependently protected against ACR-induced neurotoxic effects such as degeneration of noradrenergic axons in the somatosensory cortex and sensorimotor dysfunction. Moreover, neuroprotective effects of SFN were associated with a marked induction of Nrf2 and its downstream antioxidants, NQO1, SOD-1, GST-M, GST-M5, TXNRD1 and MT-1, as well as suppression of proinflammatory cytokine genes TNF-alpha and iNOS along with reduced oxidative stress.

The results suggest that oxidative stress and inflammation mediates the neurotoxicity of ACR and that SFN is a potent inducer of the Nrf2-ARE signaling pathway. Altogether, the present study demonstrates that co-treatment of SFN offered significant protection against ACR-induced neuropathy in mice, probably through activation of Nrf2 and its downstream factors.

To determine the effects of nylon on freshwater microalgae, we investigated the effects of micrometer-sized nylon 6 (Ny6) on the microalga Raphidocelis subcapitata. Ny6 in the culture media adhered R. subcapitata cells electrostatically, which disrupted growth and photosynthesis. Metabolomic analysis revealed that many metabolites related to the amino acid catabolic pathway and γ-glutamyl cycle were induced in in algae, which might reflect responses to avoid starvation and oxidative stress. Our study provides important information on the effects of Ny6 on algae in freshwater environments.

Nanomaterials have unique physicochemical properties and are used in various fields. However, the adverse effects of nanomaterials on human health have not been fully evaluated. In this study, we applied the human Cell Line Activation Test (h-CLAT), and evaluated the antigen-presenting cell activation ability of silver nanoparticles and silver nitrate. It was suggested that the activation of antigen-presenting cells by silver nanoparticles was due to the silver ions released from the silver nanoparticles, in other words, the released silver ions have antigenicity.

The Joint Danube Survey (JDS) belongs to the world’s largest river monitoring campaigns and is performed every six years in the second longest European river, Danube. The goal of this study was characterization of mixtures effects from long term exposed passive samplers within the JDS4 by battery of in vitro bioassays.

Silicone rubber sheets for hydrophobic compounds and Affinisep HLB disks for hydrophilic compounds were deployed at nine sampling sites in River Danube for 101 – 105 days from end of May to beginning of September in 2019. Mixture effects were analyzed in these samples by battery of in vitro reporter gene bioassays – together seven assays per sampling site.

We detected dioxin-like activity, adaptive stress response to oxidative stress, peroxisome proliferator-activated receptor-mediated activity, estrogenic, androgenic, and anti-androgenic activities. There was higher frequency of positive responses in bioassays in HLB disks (48% of cases) than in silicone rubber sheets (29% of cases).

The results of the present study provide baseline levels for the in vitro effects of mixtures of organic micropollutants in Danube.

Acknowledgement: Development of USB – International mobility II” [No. CZ.02.2.69/0.0/0.0/18_053/0016975]; Czech Science Foundation [project No. 20-04676X]; the platform CITEPro (Chemicals in the Terrestrial Environment Profiler) funded by the Helmholtz Association; the International Commission for the Protection of the Danube river for supporting the sampling logistics and the NORMAN association for financial support of sampling and sample preparation.

Given the possible toxicity in humans, carbon nanotubes (CNTs) have been very recently added to the SIN (Substitute It Now) list of chemicals by the Swedish non-profit organization ChemSec. Although not all CNTs are harmful, a certain type of long needle-like multi-walled CNTs (MWCNTs) has been shown in animal studies to cause asbestos-like pathogenicity such as fibrosis and cancer. This pathogenesis is considered to be initiated by macrophage recognition of CNTs or asbestos, followed by NLRP3 inflammasome activation and macrophage cell death. However, it remains largely unknown why and how macrophages efficiently recognize these fibers. By biological and computational analyses, we here identified T-cell immunoglobulin mucin 4 (Tim4), a phosphatidylserine (PS) receptor, as a CNT receptor, and revealed the binding mode of Tim4 to CNTs. We reconstituted NIH-3T3 cells with various phagocyte receptors and found that Tim4/NIH-3T3 cells efficiently bound CNTs. Further, WF119/120AA, but not ND121/122AA, Tim4 mutants completely failed to bind CNTs. Molecular dynamics simulations revealed spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. This binding mode was different from that to PS. Taken together, these results suggest that Tim4 recognizes CNTs through aromatic-aromatic interactions.

The chronotoxicity of 6-MP was investigated using mice. When mice were received single intraperitoneal administration of 6-MP at different administration timings, we obtained the results showing that the severity of toxicity of 6-MP was strong from evening to night (body weight change and lethality were used as indicators). Since the dosage form of 6-MP is only a powdered form in Japan, the occupational exposure of 6-MP to healthcare workers, including pharmacists, has become a problem. Our present data can be a basic finding to prevent health care workers from 6-MP health problems.

We aimed to improve an automated cognitive behavioral test system for mice, IntelliCage, for the application in assessing developmental neurotoxicity using juvenile mice. Our novel method demonstrated that mice immediately after weaning performed cognitive behavioral tasks as efficiently as adult mice and exhibited significant prenatal methylmercury toxicity. Therefore, we conclude that this advanced method can track mice’s cognitive behaviors through their developmental time frame and to study developmental toxicity. This work was supported in part by Kakenhi JP18H03036.

The present study examined alteration in gene expression of EBT-8 human CD8+ T cell line exposed to chrysotile A (CA), chrysotile JAWE and crocidolite (CR) over a month by transcriptome analysis. The analysis compared 29,638 transcripts, showing that 4 times or more alterations in expression were found in five, including IFN-γ, while 78 transcripts showed double amount. In contrast, the part of transcripts in CR-exposed cell line showed negative correlation in expression with CA-exposed that. Those obtained results suggest the common genes and the difference in those between chrysotile and crocidolite exposure.

It is known that various endocrine disrupting chemicals, including vinclozolin and glyphosate induce epigenetic transgenerational inheritance (ETI) in rats. ETI is the germline-mediated epigenetic transmission of information between generations in the absence of direct environmental influences that lead to phenotypic variations.

ETI has been shown to be induced by various environmental factors, including various nutritional conditions and even the nursing status off their mother. In ETI experiment by chemical substances, chemicals were administered at the stage of PGC sof developmental embryos in mice and rats.The importance of PGCs on ETI will be discussed.

The δ-aminolevulinic acid dehydratase (ALAD) is a polymorphic enzyme that catalyzes the second step of heme synthesis and has a high affinity for the metal lead (Pb). Previous studies link the methylation and polymorphism of the ALAD gene with negative impacts on lead-exposed people. Kabwe town had a long history of uncontrolled Pb-Zn mining that operated for almost a century and has poisoned generations of children. To date, no data exist regarding the influence of lead exposure on gene polymorphism and DNA methylation level in children from Kabwe. In this study, we conducted a case-controlled study to determine the influence of ALAD G177C genotypes and CpG methylation status in 140 children, aged 2 to 10 years old, exposed to lead.

The mean BLL (± SD) was 19.4 ± 10.6 μg/dL. All the children near the mine dumpsite had BLLs that exceeded the CDC guideline value that raises health concerns; 5 μg/dL. All children were homozygous for the ALAD 1 allele, indicating bioavailable lead in the children’s blood. The methylation frequencies of ALAD CpG and their associations with the risk of lead poisoning showed a significant difference between the areas. The methylated ALAD gene was associated with an increased risk of Pb poisoning (OR = 7.84, p < 0.001) compared with the unmethylated status. In conclusion, lead exposure increases the ALAD methylation, which might be involved in the mechanism of lead toxicity. Moreover, the bioavailable lead, due to the ALAD 1 homozygotes, can transit to soft tissues and the brain. Further larger population-based studies are warranted.

In the present study, we developed an in vitro culture device, and investigated the assessment method using it for the purpose of constructing a rapid evaluation system for compound-induced peripheral neuropathy.

It is required for a high throughput and accurate developmental toxicity test to evaluate a large number of chemicals. Developmental processes are regulated by interaction of signal transduction. Thus, we focused on the disruptions of these signalings caused by a chemical exposure in human iPS cells. Here, the disruption was monitored RTK-SRF signal which regulates limb/digit formation. The prediction showed high accuracy in the case of using chemicals which cause in limb/digit malformation. These results indicate signal disruption based test is useful for high throughput teratogenicity test.

Environmental chemical exposure and drug administration can cause central nervous system (CNS) toxicity. Addressing the higher brain functions is of critical importance. Such brain functions depend on neural circuits. However, neural circuit activity is challenging to detect with conventional physiological methods. VSD imaging methods have been developed since the 1970s to grasp neural circuit activity but are not suitable to evaluate the activity quantitatively. We proposed voltage-sensitive dye (VSD) imaging to detect subtle neural activity modification in the neural circuits quantitatively on a large scale. We used the hippocampal slice preparation as a representative neural circuit responsible for the brain's learning and memory functions. We examined the acute effects of the environmental chemical bisphenol-A and its related substances (BBMTBP and MBMTBP) on hippocampal activity. We tested the hippocampal trisynaptic responses by applying electrical stimulation to Schaffer collateral, mossy fiber, and perforant path and examined the response with VSD imaging with two different stimulation strengths. The excitatory and inhibitory components were examined using a GABA-A receptor blocker (SR95331) to the system. The results were accumulated and subjected to the statistical analysis in whole circuit activity for two different age groups. The result indicates higher risks with BBMTBP and MBMTBP than with bisphenol-A.