今回ほどワクチンが世界の人々にとって「自分事」になったことは今までなかったことですし、感染症や免疫だけでなく、毒性学も巻き込む基礎研究、臨床研究分野にも新しい潮流が生まれてきており、異分野融合が進むことが期待されます。一方、世界を見渡すと、ワクチン忌避や、ワクチン接種が進んでいない国も多くある現実があり、日本はもっと安全で良く効くワクチンを世界に提供しGlobal health coverageに貢献することが期待されています。本講義では「100 Days Mission to Respond to Future Pandemic Threats」やポストコロナのワクチン開発研究の新展開を議論できれば幸いです。
We will all use pharmaceuticals at some stage in our life. During manufacturing and following their use, these substances can be released to the natural environment. As pharmaceuticals are biologically active substances, in recent years there has been increasing interest from scientists and the general public over the potential impacts of these molecules on aquatic and terrestrial organisms and on humans that consume contaminated drinking water and food items. To understand the impacts of pharmaceuticals in the environment it is essential to understand the concentrations of these molecules in the environment. Over the past two years, the University of York have been co-ordinating the Global Pharmaceutical Monitoring project which has quantified levels of pharmaceutical pollution at 1000 locations across 105 countries. Highest levels of pollution are seen in rivers in Africa and Asia receiving inputs from manufacturing plants, raw sewage disposal or trash dumping. Comparison of the data with available ecotoxicological effects data suggest that, at the most contaminated sites, the growth and reproduction of fish and invertebrate populations will be impaired and the levels of antimicrobial resistant bacteria will be enhanced. Solutions are therefore urgently needed to this problem in order to protect the health of ecosystems and human populations across the globe.
Successful implementation of microphysiological systems (MPS) into drug discovery requires close communication between model developers and end-users to connect emerging technologies and context of use to build better systems (1). We have evaluated not only development devices but also commercial devices in the AMED project. Each of these devices has its own characteristics, and it was considered desirable to make the best use of those characteristics. For that purpose, the user needs to understand each device and select the device that suits each purpose.
In this study, we collaboratively evaluated a new MPS as a tool for detecting hepatotoxicity after repeated exposure to chemicals. In addition, we explored the advantages of non-invasive monitoring, such as by optical coherence tomography (OCT), in the long-term culture in the MPS.
We studied a new MPS device, consisting of a circulating small intestine-liver two-organ connection device developed at the Matsunaga Laboratory, Graduate School of Pharmaceutical Sciences, Nagoya City University (2). Human primary hepatocytes were seeded onto micro-patterned culture surfaces coated with mouse 3T3 feeder cells to form liver spheroids. The liver spheroids were exposed to acetaminophen (APAP) for 7 days, and the cytotoxicity of APAP was evaluated by ATP assay. Albumin, and APAP and its metabolites in the culture media were measured. Cell morphology was recorded by a phase-contrast microscopy and OCT. OCT data underwent image analysis.
After APAP treatment, the appearance of the spheroids changed to a black-coloration. APAP concentration-dependent ATP reduction was observed, suggesting that this liver MPS is strongly able to detect hepatotoxicity after repeated exposure to potential toxicants. Image analysis of OCT data succeeded in counting spheroid numbers. In addition, APAP concentration-dependent decreases in volume, height, and surface area were observed. Albumin also showed an APAP-dependent reduction in culture media.
This liver MPS is potentially applicable to the assessment of hepatotoxicity of chemicals after repeated exposure. OCT is useful for non-invasive monitoring of cellular morphology by image analysis.
混入リスクが確認されると、ニトロソアミンの量を懸念の無いレベルまで低減する、あるいはゼロにする管理手法の設定が求められている。低減する場合、M7のコンセプトに則って10万分の1の発がんリスク以下に管理することが求められ、発がん性試験データがない化合物についてEMAはデフォルト値として18 ng/day以下を提示している。このデフォルト値はニトロソアミン類の中でも一番発がん性の強いNDEA等の発がん性データを根拠に設定されているが、その一方ですべてのニトロソアミン化合物が強力な発がん性を示すとは限らないことを示唆する報告もある。ICHM7ガイドラインでは不純物の遺伝毒性評価としてQSARの利用が認められているが、ニトロソ化合物についてはCohort of Concernとして「混入してはいけない不純物」と取り扱われてきた背景から、QSARが最適化されておらず発がんリスクを適切に見積もることが出来ていない化合物クラスとも言える。このため、今回の事象を契機としてこの化合物クラスの変異原性及び発がん性リスク評価を妥当に行うにはどのような情報が必要で、どのように評価を進めるのが適切かを当局と産業界で議論中である。
Since the available scientific evidence on the causal relationship between chloromethylisothiazolinone and methylisothiazolinone(CMIT/MIT) exposure and lung injury is limited, the victims exposed to CMIT/MIT exposure remains unresolved.
Two studies have performed and compared responses of the mouse lung to inhaled and intratracheally instilled the CMIT/MIT.
Intratracheal instillation of CMIT/MIT induced increase in the number of eosinophils and neutrophils, and concentrations of T helper 2(Th2) cytokine in BALF. Moreover, CMIT/MIT induced increase the epithelial cytokine expression like IL-25, IL-33 and TSLP in lung tissue. Histopathological analysis revealed increased eosinophilic inflammation, mucous cell hyperplasia, and fibrosis following CMIT/MIT instillation.
Inhalation of CMIT/MIT induced an increase in the number of inflammatory cells and the concentrations of Th2 cytokines in BALF. Epithelial cytokine expression were also increased. However, inhalation of CMIT/MIT did not induce significant histopathological injuries. We suggests that the initial stage of Th2-mediated lung injury was induced in current exposure condition.
Although our data are insufficient to reflect the entire responses induced by CMIT/MIT, this is the first study to demonstrate the association of inhalation exposure of CMIT/MIT with Th2-mediated lung inflammation in an animal model.
Further studies on chronic inhalation exposure are warranted to evaluate the CMIT/MIT–altered histological lung injuries.
This work was supported by grant from the National Institute of Environment Research (NIER-2021-04-03-001).
Polyhexamethylene guanidine phosphate (PHMG-p) was used as a disinfectant for the prevention of microorganism growth in humidifiers, without recognizing that a change of exposure route might cause significant health effects. Epidemiological and toxicological studies indicated that PHMG-p is strongly associated with interstitial lung diseases. Herein, we have outlined the cellular and molecular mechanisms underlying pulmonary fibrosis induced by PHMG-p. The leakage of zwitterionic liposomes, induced by PHMG-p, was more extensive than that of negative liposomes, indicating that PHMG-p adsorption onto lipid head groups via electrostatic interaction cannot fully explain the induced lipid membrane damage. Using PHMG-p-FITC conjugate, we identified that PHMG-p is rapidly located in the endoplasmic reticulum (ER) and causes ER-stress-mediated apoptosis. PHMG-p triggered G1/S arrest and apoptosis through p53 pathway in lung epithelial cells. We assumed that G1/S arrest induced by PHMG-p may precondition cells undergoing EMT, contributing to the expansion of fibroblasts. Epithelial cells at other phases may result in apoptosis. PHMG-p activated the Akt/β-catenin and Notch signaling pathways, resulting in increased ZEB2 expression. The interplay between these pathways induced EMT, resulting in PHMG-p-induced lung fibrogenesis.
Hazard assessment for manufactured nanomaterials: Along with the development of nanotechnology, variety of nanomaterials have been designed and manufactured. However, the hazard of nanomaterials has not been studied fully. Inhalation study is considered a golden standard for hazard assessment of nanomaterials, but alternative method of intratracheal instillation is usually used in academia because of the limitation of facilities.
Safe by design: To develop safer nanomaterials, knowledge how the design of nanomaterials can reduce the hazard of nanomaterials is needed. We compared bare silica nanoparticles and amino or carboxyl-functionalized silica nanoparticles for pulmonary toxicity in mice. The study shows amino- or carboxyl-functionalization of silica nanoparticles reduces the inflammation in lung and toxicity to macrophage, thus providing useful information for development of safer nanomaterials.
Role of Nrf2 in pulmonary effect of manufactured nanomaterials: Nrf2 is known to be a master regulator for anti-oxidative-stress genes. However, recent studies showed Nrf2 negatively regulate expression of proinflammatory cytokines. We compared pulmonary effect of exposure to zinc oxide nanoparticles between Nrf2 null mice and wild type mice. The result shows that deletion of Nrf2 enhances inflammatory response for exposure to zinc oxide nanoparticles in the lung of mice, suggesting inhibitory effect of Nrf2 on zinc oxide-induced pulmonary inflammation.
Systemic effect of particles: Recent epidemiological studies show association between air pollution and cognitive dysfunction. One of hazardous compositions of particulate matter is polycyclyic aromatic hydrocarbons such as benzopyrene. An epidemiological study also suggests neurobehavioral effect of benzopyrene on workers. We are studying neurobehavioral and neurotoxicological effect of benzopyrene in mice by pharyngeal aspiration method, and the result shows harmful effect of benzopyrene on the density of noradrenergic axons and behavior.
Conclusions: Information on effect of particles, not only nanoparticles but also conventional or unintentionally produced particles in humans suggests us the priority of research on manufacture nanomaterials. Not only pulmonary toxicity but also systemic effect of nanomaterials or particles should be studied further.
農薬の環境毒性試験は、ハチに影響を及ぼすと考えられる農薬が対象となる。生物多様性の観点からヨーロッパでは、ミツバチのみならずマルハナバチとソリタリービーも農薬の環境毒性試験が将来的に考えらる。2013年にGuidance Document on the risk assessment of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees) がEuropean Food Safety Authorityから出されている。
The extent of possible effects of chemicals on mollusks came into public regard with the dramatic effects of tributyltin (TBT) compounds, which have broadly been used as antifouling agents for ships. The females of the Dog Whelk (Nucella lapillus) and of at least 160 further species exposed to TBT developed male parts in addition to the female genital organs, a syndrome named ‘imposex’. With a number of approximately.
Therefore, a review paper of the OECD (2010) recommended test procedures for optimization and possible validation for partial life cycle (PLC) test P. antipodarum (freshwater, gastropod), full life cycle (FLC) with L. stagnalis (freshwater, gastropod), and Crassostrea gigas (bivalve, marine). It was also recommended to develop protocols for FLC with P. antipodarum and PLC with L. stagnalis. The OECD (2016 a, b) published a guideline for a reproduction test with L. stagnalis and P. antipodarum. Histopathology was detailed in the guidelines but stated as ‘…other endpoint (e.g., histopathology)…’ (OECD, 2016b) and was mentioned under point 3.3. and in the Annex for possible histopathology evaluation (OECD, 2016a).
The current guidelines target mainly endocrine disruptor effects. However, inflammatory, and degenerative processes or parasitic infestations can mask or mimic endocrine effects. Therefore, a histopathological examination should be performed. The cost and labor are not high.
Recently, the approaches focusing individual susceptibility to drug have been taken toward drug discovery/development. The backgrounds of patients are diverse, and in fact, there are some cases in which idiosyncratic toxicity is suspected as the reason of drug development failure. Given the acceleration of aging in Japan and the diversity of patients' drugs, we could need to update drug safety strategy. Nevertheless, the conventional drug safety assessment is conducted using models such as homogenous cell lines and normal animals, and it is not enough to understand or predict the individual susceptibility to drugs in patients. As an approach for predicting individual susceptibility to drug in patients, (1) deeply understand the background of the patients, (2) create a hypothesis based on that background, and (3) extrapolate the hypothesis experimentally are applied. To promote these stepwise approaches, first, it is necessary to understand the type of disease, medical history or co-/multi-morbidity, and trend of polypharmacy in patients. Then, it is considering susceptibility to drugs in a specific patient, so that preparing an appropriate in vitro model. In particular, regarding in vitro model, recent advanced models such as iPS cell-based platforms have been versatile for some assays and are highly expected to be leveraged for predicting individual susceptibility to drugs in patients. In the presentation, I would like to discuss a concept to assess individual susceptibility to drug, including examples, and expand the future potential.
ヒトの多因子疾患の感受性多型はゲノム上に無数に存在する。全ゲノム関連解析（GWAS）から疾患感受性多型を多数同定できるが、治療標的やバイオマーカー探索にはGWAS結果から生物学的知見を導出するpost-GWAS解析が必要である。我々はごく最近、GWAS結果から構築したポリジェニック・リスクスコア（PRS）をヒト細胞で詳細に解析する新規手法「Polygenicity in a dish」を提唱し、薬剤性肝障害（DILI）にて有用性を証明した。様々な薬剤に共通のDILI感受性多型が無数に存在し、その影響の足し合わせが遺伝的DILIリスクであるとの仮説を立て、それを数理モデル化したPRSをDILIの国際コンソーシアム（iDILIC、DILIN）との共同研究から構築した。臨床試験データに加えて、ヒト初代培養肝細胞やiPS 細胞由来肝オルガノイドによる薬剤処理試験においても、遺伝的DILIリスクがDILI発症の一定部分を説明できた。興味深いことに、in vitroでのトランスクリプトーム解析や薬剤処理試験によって遺伝的DILIリスクが酸化ストレス感受性の違いによるものとわかり、GWAS結果からDILIの予防に繋がる知見が導出された。「Polygenicity in a dish」以外にも、疾患感受性多型が非翻訳RNA（特に活性化エンハンサーから転写されるRNA）周辺に集積することに着目し、DNA配列パターンから非翻訳RNAの発現を予測するAIモデルMENTRを開発した。これは、古くより行われてきた配列モチーフ解析から着想を得つつ、AIによるオミクス統合解析から高度化した手法である。これまでに、従来解釈不可能であった極めて稀な多型が非翻訳RNAを介して喘息やアトピー性皮膚炎の発症に影響する機序を解明した。本講演では、このような遺伝統計解析・機械学習を駆使した研究の最新の動向を紹介する。
Inhalation exposure to nanomaterials (NMs) is the most likely route to be unintentionally exposed in all life cycle of products from the manufacturing to being wasted, and greatest concern in harmfulness. With the progress of product development applying NMs, it is desired to develop an evaluation method that can rapidly and easily obtain fundamental and quantitative information necessary for preventing health damage. However, inhalation exposure study of particulate matter including NMs requires specialized equipment such as aerosol generators, particle concentration measuring equipment, and requires a great deal of time and cost. Furthermore, in the chronic inhalation exposure study of particulate matter, there has been controversy regarding, as it is called, overload; the accumulation of particles in the lungs throughout the exposure period. We have been developing an intermittent exposure method as an alternative to a chronic inhalation exposure study that is more efficient than the usual two-year continuous inhalation exposure study. We first initiated a 4-week intermittent exposure inhalation study, mimicking the lung burden of the rat study reported by Kasai et al., 2016. Male C57BL/6 mice were exposed to 53 μm mesh-filtered Mitsui MWNT-7 aerosol by Taquann system (J. Toxicol. Sci. 2013) at the mass concentrations of ca. 2.6 and 5.0 mg/m3, for 6 hours per day every 4 weeks. MMAD was ca. 500 nm. Lung burden at 6 months were ca. 6.4 and 15.2 μg per animal, and at 12 months 22.3 and 45.8 μg per animal, respectively. Histologically, MWNT-7-laden macrophages were found at the terminal bronchioles to alveolar region. Microgranulomas were often observed. MWNT-7 were also found in pleural cavity, local lymphnodes and, distally, in renal glomeruli. Further details will be presented. (Health and Labour Sciences Research Grant, Japan)
Particle inhalation has been causally linked to diseases including cancer, COPD, fibrosis and cardiovascular disease. We have proposed a biological mechanism of action for particle-induced cardiovascular disease causally linking particle exposure to induction of acute phase response, which is a known risk factor for atherosclerosis and cardiovascular disease.
Inhalation and pulmonary deposition of particles induces inflammation, which is proportional to the total surface area of the pulmonary-deposited particles. In mice, inflammation is accompanied by an acute phase response, which is long-lasting for insoluble particles. Acute phase protein Serum Amyloid A (SAA) is among the most differentially expressed genes in lung tissue following particle exposure.
SAA is causally implicated in atherosclerosis and both overexpression and pulmonary dosing of SAA promotes plaque formation in ApoE-/- mice. In humans, controlled exposure to metal oxides and combustion particles induce dose-dependent increases in blood levels of SAA and C-reactive protein. Blood levels of acute phase proteins SAA and C-reactive protein are risk factors for cardiovascular disease in prospective, epidemiological studies.
Nanoparticles have a higher specific surface area than larger particles with similar chemical composition, and therefore, nanoparticles are more hazardous than larger particles of similar chemical composition in relation to cardiovascular disease. This underscores cardiovascular disease as a particle-induced occupational disease.