From 2018, an AMED research group “Research on regulatory Issues for ensuring quality and safety of next-generation medium-size peptide drugs” (lead by Dr. Demizu) was launched. Two subgroups on quality of substance/formulation and non-clinical safety were constructed with industry and regulatory members, and have been discussing about points to consider for evaluation of peptide with non-natural/modified structures. Although the discussion is still on-going and no conclusion has been reached, the general direction has been building by comparison with peptide drugs with only a natural structure.

To mitigate on-target toxicity at exploratory phase is a difficult challenge. Exploratory toxicology study using knock-out animals for the target genes is widely used to determine if the toxic finding due to the candidate is on-target or not. In case, on-target toxicity is caused by the candidate targeted for unknown gene, alternative approaches are necessary to identify whether this on-target toxicity can be mitigated. In this presentation, our approaches to mitigate on-target toxicity using knock out animals and in vitro assay system at exploratory phase will be introduced and discussed.

It is important to clarify whether toxicity findings emerge from either on-target or off-target mechanism because the mechanism of toxicity has a big impact on the decision making for development of the compound and risk-management in clinical studies. Therefore, target profiling of toxicity is necessary in the drug development. In this presentation, we would like to introduce an example of target profiling for fatty liver noted in nonclinical toxicology studies with rodents because the drug-induced fatty liver in rodents sometimes implies the risk of human liver injury. We also discuss future tasks necessary for the profiling of toxicity findings.

In the non-clinical safety evaluation of antibody drugs, the specific binding mode of action is important for assessing target-based toxicity. Although monkeys are generally used for the non-clinical in vivo evaluation of antibody drugs, in vitro cell-based evaluation and understanding of Fab/Fc-mediated binding are also crucial for considering antibody drug response. In this presentation, I would like to share our process for evaluating toxicity in drug discovery and present case studies in which target-mediated toxicities were observed in cynomolgus monkeys.

Research and development of nucleic acid drugs and genome editing drugs using CRISPR-Cas9 system have been progressing. Although these modalities are designed to have high selectivity to target sequence, they can also bind to non-target genes having homologous sequences even if the sequences are not completely homologous. This is commonly referred to as the hybridization-dependent off-target effect.

This presentation will focus on in silico analysis on off target effects of these modalities. I would like to show examples of an initial evaluation of antisense oligonucleotides and guide RNA of CRISPR-Cas9 and discuss methodologies and difficulties from the perspective of nonclinical toxicologist.

Risk is commonly defined as “Exposure×Hazard” in human and environmental risk assessment. However, few researchers investigate across both scientific fields. In this workshop, focusing on human pharmacokinetic analysis and environmental exposure analysis, we reviewed how simulation and artificial intelligence technology revolutionize those scientific fields. As one example, we would like to show a novel environmental exposure analysis model integrating machine-learning technology into a hydrology-based calculation, that we have developed to estimate chemical concentration in rivers. We hope this workshop will lead to a clue to develop novel technologies on exposure prediction in human and environmental risk assessment.

Laboratory-based studies to date have reported that marine organisms ingesting tiny plastic beads with/without POPs show a variety of damages. To expand their research accomplishments to the actual ocean, we have to monitor and predict the abundance of oceanic microplastics, because unreasonably huge amount of microplastics in the laboratory cannot tell a cogent result regarding marine plastic pollution in the environment. However, uncovering the fate of microplastics (i.e., marine plastic circulation) is not an easy task; difficulty and future view will be presented in the presentation.

A US report “Exposure Science in the 21st Century: A Vision and A Strategy” has reportedly summarized successes of exposures science and development of powerful new approaches. In vitro-in vivo extrapolations of hepatic clearance and disposition have been reported for a wide variety of drugs. Simplified physiologically-based pharmacokinetic (PBPK) models consist of a chemical receptor compartment, a metabolizing compartment, and a central compartment. Subsequently, final parameter values (including standard deviation values) for an animal PBPK model can be calculated to give the best fit to measured blood substrate concentration values. Differential equations can be solved to estimate blood concentrations of substrates and/or metabolites after oral administration. These simplified human PBPK models for industrial chemicals with toxicity concerns were recently developed and successfully used to estimate human pharmacokinetic parameters, based on the pharmacokinetics in humanized liver mice. The phthalate ester concentrations in urine can be ascribed to exposure of phthalate esters and by reverse dosimetry with the human PBPK model, assuming that the reported urinary concentrations had reached steady-state values. These estimated phthalate ester exposures are far less than the daily tolerable intake (C8 phthalate ester, 30-50 μg/kg/day), implying little risk of either compound in humans under average conditions. Using PBPK forward dosimetry could provide the plasma/tissue concentrations of drugs and chemicals after oral doses, thereby facilitating estimates of hepatotoxic potential as a part of risk assessment.

To overcome disadvantages in a bottom-up approach of the in vitro to in vivo extrapolation, we are developing a new analysis framework designated as STADAM (STAtic Drug Absorption Models). STADAM analyzes in vitro and in vivo information of numerous drugs completely simultaneously to adjust biases and errors appropriately in a data-driven manner. Thousands of exposure changes due to drug interactions between various substrates and inhibitors were successfully estimated from in vivo observations of exposure changes of some combinations obtained so far, and observations of in vitro experiments for each drug.

As part of an initiative (“Development of a drug discovery informatics system”), funded by the Japan Agency for Medical Research and Development, we have developed DruMAP (Drug Metabolism and pharmacokinetics Analysis Platform). This system consists of a curated database of drug metabolism and pharmacokinetics (DMPK) parameters for about 30,000 chemical compounds and a web interface to a range of predictive models, built based on these data. We are also enhancing the system, in collaboration with a group of pharmaceutical companies, by integrating propriety data from these companies.

Based on the risk-based approach advocated by US-FDA in “c-GMPs for the 21st Century Initiative” in August, 2002, the GMPs have been fundamentally revised by the global Inspectorate. This series of revisions was completed with the revision of the PIC/S (EU) -GMP in December, 2018. Accordingly, the GMP Ordinance in Japan is expected to be consistent with the above-mentioned revisions, and is currently under consideration.

The requirements to prevent cross-contamination, which is the basic concept of GMP, have been greatly changed by the revision based on such a risk-based approach. The requirements of the dedicated facilities according to the traditional classification of drug efficacy have been deleted, and instead, it is required to evaluate the health hazard to Patient via the cross-contaminated product, according to the cross-contamination limits specified based on pharmacological and toxicological data. In response, the guidelines for setting cross-contamination limits, “GUIDELINE ON SETTING HEALTH BASED EXPOSURE LIMITS FOR USE IN RISK IDENTIFICATION IN THE MANUFACTURE OF DIFFERENT MEDICINAL PRODUCTS IN SHARED FACILITIES” has been published from PIC/S in cooperation with FDA and EMA.

It meets the Scientific Risk Based Approach, which is one of the main concepts of the GMP revision to identify the potential (cross-contamination limit) toxicologically in order to evaluate the risk to cause health hazard to patients via product-A which is mixed with another API- B carried over to the product A.

In this presentation, I will report on the background of GMP requirements revised for preventing cross-contamination as well as on the existing situation and issues in setting cross-contamination limits in Japan.

Over the past 10 years, Quality Assurance groups within the pharmaceutical industry have changed their approach to developing levels of cross-contamination in drug substances and drug products that are considered acceptable. Beginning in 2010 with the ISPE's Risk-MaPP Guidance document, and continuing in 2014 with the EMA guidance, organizations have moved to Health-Based Exposure Limits (HBELs) that need to be calculated on a case-by-case basis. This is a departure from the older, arbitrary process of dividing the therapeutic dose by 1000. This lecture will examine how companies have developed programs over the past 10 years and will also discuss issues that have been encountered along the way.

In clinical diagnosis, in vivo imaging technologies like X-rays, Echo, CT, PET, MRI, SPECT and various other technologies are being developed and used.

On the other hand, in non-clinical studies (drug efficacy and safety evaluation), these technologies are attracting attention as translational research tools from animals to humans.

In this workshop, we will pick up the latest research using animals with focusing on some imaging modalities, and discuss the effective use of these technologies for drug safety evaluation in drug development.

Nuclear medicine imaging by PET and SPECT enables highly sensitive and quantitative analysis of morphology and function in biological tissues. These techniques can provide in vivo evidences on pharmacokinetic profiles of drug candidates and pathophysiological changes in various diseases, which could lead to useful insights for understanding of efficacy and safety in drug research and development. In this workshop, our research on PET and SPECT imaging of biological function and pathology in liver and kidney diseases will be presented, and future application of molecular imaging for safety evaluation will be discussed.

In assessing drug-induced disorders in organs located throughout the body (e.g. skeletal muscles, vessels, bones, etc.), blood biomarkers and histopathological examinations are generally used, but detection sensitivity and profiling accuracy for systemic lesions are not always high. Bio-imaging approaches can provide spatial and temporal information such as “which tissue is damaged” and “how it changes” and are expected to enable more integrated evaluation of systemic toxicity. In this presentation, we will introduce a case of applying a magnetic resonance imaging (MRI) to profiling of skeletal muscle injury and discuss the role of imaging in toxicity evaluation.

MRI and magnetic resonance spectroscopy (MRS) are promising tools used for bridging research for clinical practice, as they can monitor individual disease progress over time. MRS can also evaluate a quantitative tissue substance level.

In this presentation, as a non-clinical application of MRI/MRS technique, we will show the results of rat studies for drug-induced fatty liver and vasculitis, both of which are difficult to monitor sequentially and are therefore among the major challenges in drug development. We would like to discuss their usefulness and the issues in toxicological evaluation.

CT and MRI are essential in a clinical setting but are underutilized in preclinical studies due to little insight into their application. Accumulating practical knowledge based on an understanding of the characteristics of each technique is essential. Diagnostic imaging is appropriate for NHPs since they require individual examination. Along with the recent increase in the pace of oligonucleotide and peptide drug development, the use of imaging in large animals in preclinical research will likely expand.

This presentation will include examples of CT, MRI, and other techniques in NHPs and dogs.

In classical toxicology, a toxic substance reaches the target molecule and induces malfunction. The targets are proteins/enzymes, lipid membranes, DNA, and other components in the cell. 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.

Here, the author would like to introduce a concept of “Sensor-Effector Mismatch” as a discussion material for this metal toxicity workshop.

We are exposed to a variety of environmental electrophiles such as aromatic hydrocarbon quinones in the atmosphere, methylmercury accumulated in fish, cadmium contaminated in rice. In the workshop, I will introduce that environmental electrophiles selectively and covalently modify sensor proteins such as Keap1 and HSP90 at lower concentrations, thereby activating effector molecules such as Nrf2 and HSF1, respectively. Also, role of reactive sulfur species in the regulation of redox signal transduction pathways and toxicity during electrophilic stress will be discussed.

Though organotins have various biological effects, the effects of low concentrations of them are not well studied. We previously reported that exposure to tributyltin (TBT) inhibits the expression and transcriptional activity of nuclear respiratory factor-1 (NRF-1) and causes neurotoxicity. Since NRF-1 works as transcriptional factor of a broad range of genes, NRF-1 is expected to play a key role in organotin-induced toxicity. We clarified lysosomal dysfunction and the following lysosomal gene downregulation induced by both NRF-1 knockdown and TBT.

We have analyzed the mechanism of neurotoxicity evoked by methylmercury (MeHg) and succeeded in identifying the protein disulfide isomerase (PDI) as a possible target for MeHg. MeHg covalently bound to the Cys residue in the PDI active site (irreversible oxidative modification), leading to PDI dysfunction. This inhibition by MeHg resulted in the neuronal cell death through endoplasmic reticulum stress. We next examined whether these responses were also observed in vivo. ER stress signals (UPR) were detected in the brain by either single or long-term administration of MeHg. Interestingly, this signal was neuron-specific and detected earlier than neuropathy. These results suggest that MeHg specifically affects neurons in the brain, causing cell damage via ER dysfunction.

Arsenic is a well-known hazardous metalloid that exists widely in the environment. Various genome-wide screenings revealed that the intracellular glucose metabolic pathways are involved in arsenite toxicity and its defense system. Furthermore, we demonstrated that arsenite induces cytotoxicity through repression of genes involved in the synthesis of ribose-5-phosphate in pentose phosphate pathway and activation of AMPK associated with glucose metabolism. These data suggest that arsenite increased the disturbances of glucose metabolic pathways and its regulating signaling, resulting in a promotion of cytotoxicity.

This workshop is an annual program that provides topics on nurturing toxicologists and maximizing their value. The topics related to the education of toxicologists and the recruitment of toxicologists to areas other than toxicology were well received. This time, from different perspectives (pharmacovigilance, investigator of early clinical trials, hospital pharmacists, and intermediate organization to exchange and collaborate with various multi-stakeholders,), we will talk about expectations for toxicologists involved in drug development. Toxicologists have a wide range of specialized knowledge and techniques, including detection of toxicity, elucidation of mechanism and management of chemical substances. We hope this workshop will be a good opportunity to share and think what you need to do about to make the information produced by toxicologists truly useful.

It is needless to say that the findings from nonclinical toxicity studies are important to ensure the safety of subjects in the drug development. To compile new drug application dossier, the safety of developed compounds is comprehensively evaluated from both nonclinical and clinical data, and then the product is marketed after approval by the regulatory authorities. This is a scheme called “translational research,” the purpose of which is to deliver a new drug to the patients. In this process, there are many situations where nonclinical toxicologists work with the common goal of the approval of the drug.

In the last few years, Real World Data (RWD) has been treated as a common language in the pharmaceutical industries in Japan. Although RWD per se is very valuable, it is necessary to create Real World Evidence (RWE) derived from analyses with RWD in order to contribute to patients and public health.

The pharmaceutical industries are constructing evidence that contributes to decision-making by utilizing RWD/RWE in many situations from clinical development to post-marketing stage. As a feedback mechanism of these RWE to nonclinical or early clinical stages to lead to the development of new drugs, “reverse translational research” is advocated. Similarly, it is highly expected that the RWE on patient safety obtained in post-marketing will be provided to the nonclinical toxicologists and improve the quality of the safety evaluation from the aspects of science.

In this session, I would like to speak the collaboration between toxicologists and RWE, including future expectations.

Although non-clinical data are extremely important when performing clinical trials in early stage to secure subjects’ safety, the amounts of information are limited especially in a first in human trial. Investigators will try to get meaningful information from Investigator’s Brochures (IB), and it is not an easy task. We must recall a tragedy of sacrifices in an FIH study performed in France. The IB of the case was insufficient and included many mistakes of table and figures and mistranslation. Generally, IBs, we read, are correct in scientific meaning, but hard to read owing to too correct and too complicated explanations by specialist in the field. The tendency is especially remarkable when studies were done by outsourcing. Investigators are making every effort in securing subjects’ safety and preclinical data are the only direct information of candidate compounds. Investigators will face subjects living their daily lives who should not be harmed. I hope toxicologists to understand the importance of data and to transfer useful information to investigators.

In the late years, the development of the digital technology such as ICT and IoT accelerates remarkably. “Artificial intelligence (AI)” is also employed to make our work more productive in various fields. The AI should be introduced to help our work more supportively in medical site in near future. In the hospital pharmacy point of view, several tasks such as drug dispensing and mixing of chemotherapeutic drugs have been already automated, and then those automated devices should be optimized by using AI. The AI can be useful for organizing and characterizing data that use to take enormous time and money.

We, department of pharmacy in Okayama university hospital, developed drug information-organizing-system, called “AI-PHARMA”, that make organizing and sharing of drug information more efficient. This system adopts natural language processing-AI. It realized sharing of drug information and pharmacists’ experiences and knowledge not only in a hospital but also between hospitals. That worked amazingly for taking knowledge of “real pharmacists’ needs” about drug information.

In this session, I would like to share case examples about drug information, especially about safety- and toxicity-related information, that pharmacists in medical site have been really looking for.

The NANBYO patient groups (PGs) in Japan are often composed of patients and families. Compared to US/EU PGs, Japanese PGs focus almost exclusively on the social welfare area and don't have strong connections with outside experts. On the other hand, Rare disease research and orphan drug development (R&D) will be increasingly required input from patients. However, the relationship between researchers and patients is not yet mature in Japan.

ASrid is an intermediate organization targeting the rare/intractable disease area. Therefore, ASrid has regularly exchanged and collaborated with various multi-stakeholders, e.g. patients and families, medical agencies, universities, industries (including clinical / non-clinical researchers) and government. ASrid is actively involved in international cooperation and participates in many global activities, including UN-certified NGOs and others. Based on these collaboration achievements, we have implemented activities with multi-stakeholders in the PPI (Patient Public Involvement) initiative.

Leveraging the “not stakeholder” position in this area, ASrid exists as a professional of PRO (Patient Reported Outcome) utilization w/a all relevant parties. For example, we conducted a survey about the patients’ impressions of a clinical research/trial (FY2018) and the opinion survey on orphan drug R&D from people involved in corporate R&Ds (FY2016). We will introduce some actual cases and introduce expectations for clinical and non-clinical researchers and future issues/challenges including input from rare disease/ NANBYO patients/families.

In the early discovery stage, oftentimes, the biology of novel drug targets is largely unknown, and toxicologists may need to support project teams with limited information about target safety. Historically, there has been a strong concordance between the phenotypes of genetically engineered rodent models (GERMs) and pharmacology of the human target, which has emphasized the applicability of GERMs in the understanding of potential on-target toxicities to humans. However, germline ablation often proves to be embryonic lethal for many targets and, consequently, only limited information from tissue-specific gene modifications is available. Moreover, therapeutic agents may result in systemic effects not well-predicted by these animal models. Herein, we discuss the value of temporally controlled conditional knockout (cKO) or knockdown (cKD) rodents, where the expression of the gene of interest is systemically deleted or reduced, respectively, from a given time by use of an inducer. In our experience, systemic cKO or cKD animals have been useful for unveiling potential safety liabilities to the intended patient population and, hence, customizing safety strategy for specific targets. Additionally, they can be utilized to investigate mechanisms of toxicity of compounds in development. In this presentation, we focus on how we have leveraged GERMs in safety de-risking and decision-making in the discovery stage as well as potential challenges and pitfalls associated with GERMs that toxicologists should be take into consideration.

During the early stages of the drug discovery process, the primary focus of drug design is the development of target efficacy and optimization of PK/PD properties. Consequently, the integration and application of safety data during these stages may not be prioritized. If we are to reduce the chance of drug attrition due to safety, however, it is important to proactively mitigate safety signals at this stage. In order to achieve this, it is important that any in vitro assay is validated and demonstrated to have some correlation with human safety and ‘added value’ with respect to assays that have already been established. It has been learnt that the traditional validation datasets of new assays, which often include toxicologically potent drugs such as doxorubicin, cisplatin and sunitinb, can be useful in that they provide information on the dynamic range of an assay and translation to human safety. These compounds by themselves, however, may not provide enough evidence to warrant the consideration of the assay in early drug design. In order to influence drug design, in vitro assays should be validated using test sets that are mechanistically relevant to the toxicological endpoint being modeled and are applicable to modern chemical space. Evidence that the assay can differentiate structural analogues with different toxicological profiles, thus reflecting the situation that is often encountered in early drug discovery, would be advantageous. Through the review of in vitro data for the prediction of bone marrow toxicity and hepatotoxicity, it will be shown that structure-based validation studies can provide valuable evidence that can drive the ‘Safety By Design’ concept and the development of cost-effective, risk assessment strategies. Current early safety screening strategies will also be discussed along with their application to small molecule projects.

Systemic changes caused by exposure to test compounds are evaluated using data from in vivo general toxicity studies. But oftentimes, the conventional approach is not sufficient to elucidate the mechanisms of unexpected toxicity findings. Project teams must make agile and well-informed decisions for go or no-go; however, because there is only limited information about target- and structure-related biological responses especially in the discovery phase, it usually takes time to understand the mechanism of toxicity and identify a mitigation plan and screening strategies.

In our early safety strategy, we have proactively incorporated a compound profiling approach using liver tissue/cell samples as representative systems. Our primary objective is not only to understand the perturbed signals and pathways in liver and predict potential liver liabilities but also to gain insights into molecular mechanisms involved in toxicity observed in other tissues and organs. This approach has maximized the value of in vivo general toxicity studies and saved time of project teams to revise the safety strategy.

This presentation will introduce a case example of our approach and discuss how it can be implemented into the safety lead/candidate optimization and support the project decision-making in the discovery phase.

Artificial intelligence (AI) continues to gain prominence in many aspects of our everyday lives. From calculating our car insurance premiums to acting as an opponent in our favourite board game, AI plays an increasingly important role. The field of safety assessment is no different and opportunities to utilise AI are being actively explored. The use of AI in early drug safety assessment is showing promise, but there are several obstacles to overcome before the technology can reach its full potential.

In this talk the current applications of AI in drug safety assessment, along with its limitations and blockers to development will be discussed. The need to expand our use cases and embrace the wider potential of AI are described. Its ability to extract evidence from large and complex data from multiple sources and convert this into useful knowledge promises to improve and accelerate our scientific understanding. Thus, AI can ultimately support better decision making regarding human safety assessment of potential drug candidates.

I have been working on the search for endpoints and markers for highly accurate screening of developmental neurotoxicity (DNT) and carcinogenicity of chemicals for the past 12 years. Regarding DNT, we focused on hippocampal neurogenesis and developed evaluation models by incorporating hypermethylated gene markers using rodents. We also found possibility of evaluating DNT in a scheme of 28-day treatment study. Regarding carcinogenicity, we searched for hypermethylated genes after 28-day hepatocarcinogen-treatment using rats and found multiple signal alterations leading to carcinogenesis.

I am truly honored to have been able to contribute to the JSOT in the following cases:

1)Practice of the systemic basic education and establishment of the examination systems for technicians and/or assistants engaged in toxicity studies.

2)Improvement of the biostatistical methods in toxicology up to the international standard by pointing out incorrect use of the statistical analysis decision tree for detection of the between-group-differences and by proposing a statistical processing method for outliers generated due to arbitrary dose selection.

3)Improvement of toxic-clinical chemistry in Japan up to the international standard by integrating calibration methods for the autoanalyzers to obtain data with small variations.

4)Expansion of the integrated safety evaluation methods for associated risks by suggesting usefulness of multiple analyses applying cluster analysis.

To assess the removal capability of toxic substances from the water environment, Ni-Al or Mg-Fe complex hydroxide at different molar ratios was prepared in this study. The physicochemical characteristics such as XRD patterns, specific surface area, and surface functional groups were investigated and the interaction between amount of toxic substances removed and characteristics of metal complex hydroxide was also evaluated. Ni-Al or Mg-Fe complex hydroxide was useful for removal of toxic substances such as chromium(VI) ion and inorganic nitrogen from the aqueous phase. Our results elucidated the adsorption mechanism by the elemental distribution and binding energy analyses.

In the developmental period, living organisms are more sensitive to exposure to environmental chemicals than in the adulthood. Recent studies on its toxicity have been advanced from an epigenetics viewpoint. Our studies have shown that fetal growth restriction by cadmium exposure is associated with disruption of the ubiquitin-proteasome system, and that methylmercury exposure during development reduces neurite outgrowth due to HDAC-mediated reduction of histone H3 acetylation. We will make further efforts to elucidate the effects of exposure to low amount of chemicals such as biometals in the fetus and moreover those on the neurodegenerative diseases modified by them in the adult.

Bisphenol A (BPA), a monomer used in the formation of polycarbonate plastic, is regarded as an endocrine disruptor that can exert estrogenic effects via estrogen receptors α/β (ERα/β). We investigated the effects of BPA and its related compounds on biological responses of human breast cancer cells. 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an active metabolite of BPA, significantly downregulated ERα expression and stimulated the proliferation of MCF-7 cells through activation of ERβ. BPAF, an analog of BPA, stimulated ERβ without affecting the expression of ERβ in MDA-MB-231 cells.

Cadmium (Cd) reduced mRNA and protein levels of GLUT4 (glucose transporter 4) in HK-2 cells. GLUT4 mRNA level was also significantly reduced by the knockdown of the gene encoding MEF2A（myocyte enhancer factor 2A）transcription factor. Moreover, knockdown of GLUT4 significantly reduced the viability of HK-2 cells. Both of Cd treatment and GLUT4 knockdown caused a significant decrease in intracellular glucose levels. HK-2 cells incubated in glucose free medium showed the decrease in cell viability compared to that incubated in normal medium. In addition, decrease in intracellular ATP level was observed by not only Cd treatment but also GLUT4 knockdown. These results indicated that Cd renal toxicity through the inhibition of MEF2A activity is engaged by the decrease in glucose and ATP level by the suppression of GLUT4 expression.

We recently established the MerB, bacterial organomercurial lyase, expressing HEK293 cells (MerB-cells) and showed that MerB could demethylate MeHg and convert inorganic mercury in mammalian cells and MerB-cells were significantly more sensitive to MeHg exposure compared to the parental cells1). However, the cellular response to inorganic mercury derived from MeHg has yet fully to be elucidated. The objective of this study is to assess the autophagic response in MerB-expressing HEK293 (MerB-cells) exposed to MeHg. The level of membrane-bound LC3-II in MerB-cells exposed with MeHg was increased in a dose-dependent manner and enhanced compared to the parental cells. Next, to investigate whether LC3-II accumulation is ascribable to induction of autophagy or blockade of autophagic degradation steps, we performed an autophagy flux assay using chloroquine (CQ), which prevents fusion between autophagosomes and lysosomes. Compared with CQ treatment alone, cotreatment with MeHg increased the LC3-II levels. Our data suggest that Hg2+ activates autophagy and exhibits a stronger autophagic response than that MeHg.

1) Intracellular demethylation of methylmercury to inorganic mercury by organomercurial lyase (MerB) strengthens cytotoxicity. 2019, Toxicol. Sci.

trans-Fatty acids (TFAs), such as elaidic acid (EA), highly contained in processed foods, have been associated with a variety of age-related diseases including atherosclerosis and NASH. However, the underlying pathological mechanism remains unknown.

Here, we show that EA promoted DNA damage-induced cellular senescence and cytokine production (so called SASP) in a manner dependent on p53. Moreover, dietary intake of TFA induced accumulation of fat and senescent cells in mouse liver. In this meeting, we discuss the novel mechanisms underlying pro-senescence/inflammatory effects of TFAs.

In toxicity studies, one of the core endpoint is clinical observation including vital signs (VSs). Measurement of VSs over time has already been realized in safety pharmacology in drug development, but not in toxicity studies on general chemical substances. We believe that simple VSs measurement technique can achieve both refinement of toxicity evaluation and reduction of the number of animals. In this study, we developed a wearable pulse oximeter for rats to measure SpO2, heart rate and respiratory rate. We succeeded to measure these VSs from conscious and unrestrained rats.

Juvenile hormones (JHs), which control growth and development of arthropods, induce male offspring production in daphnids by activating JH receptor methoprene-tolerant (Met). However, target genes of Met have been unidentified. In this study, we showed that JH activities were detectable in yeast expressing Daphnia magna Met via upstream E-box/E-box-like elements of Vrille (Vri) gene, which encodes transcriptional activator of sex-determining factor Doublesex1. We also showed that Vri was an early JH-responsive gene in vivo. These observations suggested that Vri is a target of Met in D. magna.

1,2-Dichloropropane (1,2-DCP) was used in the offset colour proof printing. In 2017, IARC reclassified it from Group 3 to Group 1, due to an outbreak of cholangiocarcinoma (CCA) among workers in Japan, who used 1,2 -DCP in their work. 1,2-DCP-induced (occupational) CCA in comparison with other forms of CCA, showed an early onset, accompanied by extensive pre-cancerous lesions in bile ducts. The exact mechanisms leading to occupational CCA remains poorly understood. The study investigated the molecular mechanisms of occupational CCA using human immortalized cholangiocytes MMNK-1 cells and human leukemia-derived THP-1 monocytes.

Following treatment of 100-800μM 1,2-DCP to either monocultured MMNK-1 cells or co-culture of MMNK-1/differentiated THP-1 macrophages for 24 hours, cell viability was assessed using MTS assay. LDH cytotoxicity assay and measurement of ROS production using DCFDA assay were performed.

Results show increased production of ROS in the MMNK-1/THP-1 co-cultured cells compared with monocultured MMNK-1 cells. Cell proliferation was increased in monocultured MMNK-1 cells exposed to 1,2-DCP but not co-cultured MMNK-1/differentiated THP-1 macrophages. There was an increased level of LDH cytotoxicity in co-cultured MMNK-1/differentiated THP-1 macrophages exposed to 1,2-DCP but not monocultured MMNK-1 cells.

The results demonstrated that exposure to 1,2-DCP enhances ROS production in human cholangiocytes co-cultured with macrophages, being accompanied by increase in cytotoxicity. The production of ROS might be a key mechanism involved in the 1,2-DCP-induced CCA.

Thymus is a primary lymphoid organ important for cellular immune response. The size and function of thymus is rapidly shrunken by various biological and environmental stimulation, which is called as thymic atrophy or thymic involution, to increase risks of infectious diseases and tumorigenesis. In the healthy thymus, naïve T cells have some characters like Th1- or Th2-differentiated T cells. This bias of naïve T cell characters, called as polarization, is tentative and could be changed during thymic involution. Indeed, naïve T cells are more polarized to Th2 than Th1 during dietary restriction-induced thymic involution (Razali et al., in press). We here examined how the polarization of naïve T cells is affected during glucocorticoid-induced thymic involution. ICR mice were administered dexamethasone and their thymus was analyzed. The results indicated that naïve T cells were more polarized to Th2 by dexamethasone, like by the dietary restriction. However, the induction of some Th2 markers was not equivalent under these two stresses, indicating different characters of polarized naïve T cells. In contrast to the dietary restriction, the dexamethasone-administration did not up-regulate the expression of synthetic enzymes of prostanoids, suggesting that prostanoids are not critical mediators of glucocorticoid-induced thymic involution.

[Background, Objective] Cyclophosphamide (CPM) is a prodrug of alkylating agent activated by CYP2B6. Clinical trials showed that polymorphism of CYP2B6 affected on both therapeutic effects and adverse effects of CPM. However, drug interaction (DI) between CPM and CYP inhibitor has not been reported. To elucidate potential DIs between representative CYP inhibitors and CPM, we assessed the inhibitory profiles of these inhibitors by in vitro cocktail experiments and analyzed adverse events caused by a combination of these drugs and CPM which is reported in Japanese Adverse Drug Event Report database (JADER) and FDA Adverse Event Reporting System (FAERS).

[Method] Mixed substrates for 8 CYP isoforms (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A) were incubated with representative CYP inhibitors in human liver microsome. Specific metabolites were measured by LC-MS/MS and evaluated inhibitory potentials of the inhibitors. Proportional Reporting Ratio (PRR) was calculated based on JADER (2014 to 2019) and FAERS (2015 to 2019) to estimate DIs between CPM and inhibitor in clinical.

[Results, Discussion] Voriconazole (VCZ) most strongly inhibited CYP2B6 (IC50 : 0.12 μM) among the isoforms. PRR of neutropenia, known as a typical adverse effect of CPM, was calculated as 2.83 (in JADER) and 10.7 (in FAERS). Both PRR values were significantly decreased to 0.71 and 4.58 by treating with VCZ concomitantly. These results suggested that VCZ decreased the adverse effect of CPM by CYP2B6 inhibition. The therapeutic effects of CPM may also be decreased by co-treatment with VCZ.

Rectal prolapse, thickened colon, enlargement of the spleen and mesenteric lymph nodes were observed in all AhrKO mice. Flow cytometry revealed that Th17 cells significantly increased in the spleen and colonic lamina propria, whereas no obvious differences in Treg cells. Importantly, metagenome analysis with feces of colon revealed a facility-specific flora. RRBS using liver DNA showed hypomethylation in the ARNTL/BMAL1 gene. Our finding indicates that Ahr dysfunction is involved in IBD pathogenesis with changes probably in the gut flora producing Ahr-endogenous ligands.

Impairment of the protein nuclear transport system accompanying disruption of the nucleocytoplasmic gradient of Ran GTPase by oxidative stress is one of the causes of cellular senescence. In this study, we found that Thioredoxin-related transmembrane protein 2 (TMX2) regulate the nuclear Ran levels and importin-β-mediated transport of cargo protein. The cysteine 112 residue of Ran was important in its regulation by TMX2 and in the reduced nuclear Ran levels by oxidative stress. These results suggest that nuclear Ran levels are regulated by its redox state via TMX2.

In cancer cells, HIF-1 alpha expression is upregulated by Warburg effect and promotes glycolysis and suppresses TCA cycle and electron transport system. CGA, containing abundantly in coffee, regulates carbohydrate absorption and antiproliferative effect. In this study, we investigated whether CGA regulated dependent on HIF-1 alpha, Nrf2 and the HIF-1 alpha- and Nrf2-dependent enzymes expression in glucose metabolism in Hep3B cells. As a result, we found that all enzyme mRNA levels in glycolysis were dependent on HIF-1 alpha expression level, and Glut1, PGD, TKT and G6PD were dependent on Nrf2.

For the control of emerging infectious diseases, rapid vaccine development is often needed. However, owing to the low immunogenicity of antigens, vaccine adjuvants with high safety and efficacy profiles are needed to induce complete immune responses to vaccines. Although aluminum adjuvants have been widely used in different types of vaccines and they ensure efficacy and safety, the development of a mode of action (MOA)-based adjuvant is desired.

Our group revealed the MOA of influenza vaccine with different types of adjuvants and identified influenza vaccine-specific biomarkers (BMs) that correlate immune response and toxicity, including antibody production, cytokine production, and hematological toxicity. Using these BMs, we established a new method for evaluating the safety and efficacy of influenza vaccine as well as adjuvants. In the present study, we applied a reverse toxicology to identify novel, safe, and effective adjuvants based on the expression of our BMs both in vitro and in vivo. We established an in vitro screening method to determine the BM levels after interaction of different adjuvant candidates with influenza HA antigen and compare the BM levels in response to HA antigen itself. The adjuvanticity of the candidates was determined using these results, and whole inactivated influenza vaccine was used as a toxicity reference for determining the safety of adjuvants. Using our method, we identified a novel adjuvant candidate with a high safety and efficacy profile. In conclusion, our reverse toxicological approach can enable the identification of novel, safe, and effective adjuvants.

Recent advances in analytical techniques using an ICP-MS enable us to detect a trace or ultratrace amount of element in a single cell at a level of femtogram or attogram. In this study, we measured elemental contents in a single cell such as red blood cell, yeast, green algae, and human leukemia cell (K562) by a single cell-ICP-MS. The measured elemental contents of Mg, Zn, P, S, and Fe showed good agreements with the values obtained from conventional solution based ICP-MS analysis after acid digestion of the cells. This analytical technique can be useful strategy for evaluating the metabolism of both endogenous and exogenous elements in a single cell.

The number of cancer patients is increasing due to the global aging society, we focused on cardiotoxicity, which is the most serious side effect of molecular targeted drugs in Onco-Cardiology, and constructed a novel in vivo high-throughput high content imaging screening system. First, a transparent zebrafish (MieKomachi009) that selectively expresses GFP in myocardium was created. About 10 times the throughput was achieved as compared with the manual screening method. This novel high-throughput in vivo cardiotoxicity screen has hit a number of anticancer agents such as sorafenib.