Exposure to arsenic (As) is a major threat to the public health worldwide. Association of As exposure with neoplastic diseases has been well established. Mounting evidence suggest the involvement of As in non-neoplastic diseases. In our study, we explored the association of As exposure with the risk of diabetes and asthma recruiting human subjects from low and high As exposed areas in Bangladesh. We found that As exposure was dose-dependently associated with risk of hyperglycemia including IGT and DM. Females showed greater susceptibility to As exposure-related hyperglycemia than males. In a follow-up study, we found that chronic As exposure was inversely associated with serum creatinine levels. Serum creatinine levels directly reflect the skeletal muscle mass. Arsenic exposure-related increased blood glucose levels were inversely associated with serum creatinine levels suggesting that decreasing skeletal muscle mass may be a potential mechanism of As-related diabetes. In another study, we found that As exposure associated with the risk of the reversibility in airway obstruction and asthma-like symptoms, the two important characteristic features of asthma. Subjects’ As exposure levels were also associated with the elevated levels of serum immunoglobulin E and Th2 cytokins. Arsenic exposure-related reversibility in airway obstruction, asthma-like symptoms and elevated levels of serum IgE and Th2 cytokines suggest that As-exposed people are susceptible to the allergic asthma. Taken together, our results show the risk and underlying mechanism of diabetes and asthma associated with chronic As exposure.
Metabolic disorders are frequently caused by nutritional, behavioral and metabolic factors as well as by diverse chemical exposure. Together with inflammation and endothelial dysfunction, fatty liver is one of the related disorders in metabolic syndrome. Chemicals induce fatty liver by disturbing normal lipid metabolism including enhanced lipogenesis and free fatty acid uptake as well as decreased fatty acid oxidation and VLDL secretion. As a novel mechanism of ethanol- and diet-induced fatty liver, we report that downregulation of 3-phosphoglycerate dehydrogenase (PHGDH), a critical enzyme involved in the serine synthesis, and low hepatic serine level contribute to the development of fatty liver disease through suppressing SIRT1. Autophagy is one of the essential catabolic pathways activated in response to nutrient deprivation. Recently, lipid droplets have been identified as a substrate for autophagy. Some NSAIDs induces hepatic lipid accumulation and fatty liver by disturbing lysosomal degradation of PLIN2, a substrate for chaperone-mediated autophagy. Identification of novel mediators of fatty liver will contribute to the development of strategies for the treatment and prevention of the disease.
Drug-induced liver injury (DILI) remains a major safety concern; it occurs frequently; is idiosyncratic; cannot be adequately predicted; and a multitude of underlying mechanisms have been postulated. Many experimental approaches to predict human DILI have been proposed utilizing in vitro screening such as inhibition of mitochondrial function, hepatobiliary transporter inhibition, reactive metabolite formation, and cellular health, but achieving only minimal success. We have shown that none of the multitude of hypothesized and investigated predictive markers does any better than just avoiding extensively metabolized, poorly soluble compounds, so-called Biopharmaceutics Drug Disposition Classification (BDDCS) Class 2 drugs. Yet, this is not a useful restriction as more than 1/3 of approved drugs are BDDCS Class 2 compounds. Once a clinically relevant dose is known, studies have shown total administered dose (> 50 mg) to be correlated with higher predictability of DILI. It would be best to have a predictive DILI methodology early in drug development, long before the clinical dose is known. We hypothesize that many of the deficiencies of the high-throughput screens employed based on in vitro measures of hepatocyte toxicity can be overcome with long-enduring metabolically competent hepatocyte co-cultures. The long- enduring status has the potential to allow formation of active metabolites and/or accumulation of intracellular toxic substances in hepatocytes due to inhibition of relevant efflux transporters, as well as giving sufficient time for the hepatotoxicity to manifest.
Rodent in vivo carcinogenicity bioassays are required for human risk assessment and have been utilized in this capacity for decades. Accordingly, there is an abundance of data that could be accessed and analyzed to better understand the translatability of xenobiotic-induced rodent tumors to human risk assessment. In the past decade various groups have studied and published their assessment of the value garnered by these very lengthy and costly studies. The Expert Working Group (ICH-S1 EWG) was initiated in 2012 to evaluate the predictability of the current testing paradigm and to propose a more comprehensive and integrated approach to human carcinogenicity risk assessment. Results and recommendations by this working group are pending. Key to this effort is the translation of rodent carcinogenicity findings to humans. Some tumors were shown to be rat-specific based on mechanism of action, while others with insufficient evidence remain of potential risk to human. CNS tumors in the rat are very rare and translatability to human remains unknown. This presentation focuses on one type of CNS tumor (microglial cell tumor [MCT]) which is observed specifically in male rats that were exposed to a few pharmaceutical drugs and discusses the classification, nomenclature, potential mechanism of action of this type of CNS tumor, and speculates about its relevance to human.
経済協力開発機構（OECD: Organisation for Economic Co-operation and Development）はEAGMST（Extended Advisory Group on Molecular Screening and Toxicogenomics）というプロジェクトにおいて、化学物質のヒトや野生動物への悪影響に関する知識向上のため、毒性発現経路（AOP: Adverse Outcome Pathway）を開発している。AOPとは、化学物質等が個体または集団に有害性をもたらす経路であり、化学物質の曝露から、吸収・分布・代謝の過程、および分子、細胞、組織・臓器・器官、個体に生じる連続的な事象である。AOPは当初、環境毒性の発現経路を明確にするために、米国環境省（US EPA：United States Environmental Protection Agency）で開発され、以後OECDにて採用され、この情報をもとに環境毒性やヒト健康影響に関するin silicoやin vitro試験の開発を促すためにその概念が発展してきた。このプロジェクトは2012年に設置され、US EPAとEU JRC（Joint Research Centre）が中心となって作成ガイダンスや入力フォームAOP wikiやEffectopedia等の開発、AOPの普及・教育が行われてきた。ただし、これまでに成立したAOPはまだ10に満たず、現在、70程度のAOPが開発中であるに過ぎない。OECDのAOPとは、毒性発現までの過程をなるべくシンプルに、枝分かれ少なく記載しようというものであり、MIE（Molecular Initiation Event）から始まり、KE (Key Event)とKER（Key Event relation ship）からなり、AO（Adverse Outcome）に至る。それにより、証拠の重み付け（WoE: Weight of Evidence）が明確になっていくことが期待される。開発者はEAGMSTに申請した後、入力フォームにそれぞれのAOP案を入力し、そのAOPはEAGMSTによる内部評価を受けた後、外部第三者評価を受けて公定化される。
ヒト結腸がん由来細胞株Caco-2 細胞を用い、化合物の in vitro 透過係数を調べたところ、17種HESS化合物の肝無作用量（NOEL）値と対数変換した透過係数実測値の間に有意な逆相関が認められた。推定物性値を用いる重回帰分析で得られた回帰式より算出した透過係数の予測値は、実測値と良好に相関した。構造情報を活用することで透過係数、さらに化学物質の経口吸収性をも予測しうることが示された。血中化学物質濃度推移の文献・実測値を活用し、ラット各種体内動態パラメータ値を算出し、コンパートメントおよび消化管、肝臓および全身循環からなるPBPKモデルを活用して、仮想投与後の血中または肝臓中濃度推移を予測した。両方法で予測した化合物の血中濃度および時間曲線下面積は一致したが、血中と肝中濃度の相関係数は低下した。1日あたりの肝中濃度時間曲線下面積と7種HESS化合物の肝最小作用量（LOEL）値は有意な逆相関を示した。各化合物の薬物動態３種パラメータ値［吸収速度定数、分布容積および肝代謝消失］を化合物の構造特性値から推定する手法の整備を契機に、薬物動態モデルを用いる化学物質の体内濃度推移と安全性評価技術の統合に期待したい。
生涯を通して受ける様々な環境因子の複合的曝露（エクスポソーム）が疾患発症リスクを評価する新たな指標として注目を集めている。多くの環境化学物質は親電子性が高く、これらは共通して生体分子中の求核物質（タンパク質中のシステイン（Cys）パースルフィドをはじめとする活性イオウ種）と化学的に共有結合し、生体分子に機能修飾を与える。こうしたタンパク質機能修飾の蓄積が生体恒常性の変容を誘発し、個体機能低下を招くことが示唆されている。我々は、ミトコンドリアの品質管理を制御するGタンパク質dynamin-related protein 1 (Drp1)が高いレドックス活性を持つことに着目し、Drp1のレドックス活性がC末端に存在するシステイン（Cys624）残基によって制御されることを見出した。Cys624はミトコンドリアtRNA合成酵素（CARS2）活性依存的にポリイオウ化されることで、Drp1活性を負に調節すること、神経毒性を起こさない極低用量のメチル水銀曝露によりDrp1ポリイオウ鎖がイオウ枯渇することでDrp1依存的なミトコンドリア分裂が起こることをマウス心臓レベルで明らかにした。ミトコンドリア過剰分裂したマウス心臓に圧負荷を加えたところ、心臓の圧負荷に対する抵抗性が顕著に減弱し、突然死率と心不全重症度の顕著な増加が観察された。Drp1のCys624のポリイオウ鎖枯渇を模倣した変異体（Drp1(C624S)）を心筋細胞株に発現させたところ、ミトコンドリア分裂の亢進が認められ、逆にCys624のポリイオウ鎖構造を模倣した変異体（Drp1(C624W)）を発現させた細胞ではMeHg曝露によるミトコンドリア分裂が抑制された。以上の結果は、Drp1タンパク質のポリイオウ鎖が環境化学物質センサーとして機能し、ミトコンドリア品質低下を伴う心疾患発症を制御していることを強く示唆している。
Multiple factors have been associated with the honey bee colony losses in the past 25 years. Accumulating evidence indicates that at sublethal doses, neonicotinoids cause honey bee brain dysfunction and reduce immunocompetence, leading to impaired navigation and olfactory learning and memory, and susceptibility to pathogens. Moreover, such pesticides disturb the reproductive system of queen bees, i.e., the number of eggs and viability of sperm stored in the spermatheca of queen bees, thereby reducing the numbers of adult bees and broods. Pesticide exposure during the larval development stages prolongs larval development and shortens adult longevity. In addition, the density of synaptic units in the calyces of mushroom bodies in the heads decreases; this effect has been further associated with the abnormal olfactory learning ability of adult honey bees exposed to sublethal doses of imidacloprid during the larval stage. Therefore, numerous physiological aspects of honey bees might be altered after exposure to pesticides at sublethal doses, regardless of the developmental stage. We evaluated the impact of the most widely used neonicotinoids—imidacloprid—on different developmental stages of honey bees by profiling the transcriptomes of worker bees with imidacloprid during only the larval stage. Our results confirm that even intaking 1 ppb imidacloprid during only the larval stage would be enough to severely impact a bee’s gene expression. The existence of many differentially expressed genes may reflect or result in honey bee disorder.
Today’s food systems are -to large extent- anchored in chemically-intensified farming operations, and the unguided use of synthetic pesticides has become the norm in many agricultural systems. Aside from undermining ecosystem functioning, triggering biocide resistance and lowering on-farm economic profitability, this overreliance on agrochemicals carries tangible risks for human health. For example, in certain European countries, nearly 100% of citrus fruit is tainted with pesticide residues. In this talk, I will illuminate the largely untapped potential of biological control - i.e., the tactical use of beneficial pest-killing organisms for crop protection – to remediate pesticide misuse and thus eliminate its associated food safety hazards. Drawing upon the case of cassava mealybug (Phenacoccus manihoti; Hemiptera) in tropical Asia, I illustrate myriad benefits of biological control. In late 2008, the above mealybug invaded Thailand, where it inflicted a 27% drop in cassava production and triggered extensive use of pesticides. Mealybug outbreaks were permanently resolved through the introduction of a minute parasitic wasp Anagyrus lopezi (Hymenoptera). In my talk, I reveal how A. lopezi effectively suppressed P. manihoti at a continent-wide scale, delivering pest control services worth US$200-700/ha and virtually abolishing farmers’ need for pesticides. Our work underlines how biological control presents a safe, desirable alternative to synthetic pesticides and can help meet food production needs while benefiting the global economy, the environment and farmers’ pockets.
GPR40 is highly and dominantly expressed in pancreatic beta cells and is activated by medium- to long-chain free fatty acids (FFAs) to potentiate glucose stimulated insulin secretion. Fasiglifam was a novel, oral, and selective small molecule agonist of GPR40 under development as a once-daily treatment for type 2 diabetes. Unfortunately, a development program was terminated late in phase III clinical trials due to liver safety concerns. Three important serious liver injury cases were identified among around 9100 patients treated with Fasiglifam; one case was adjudicated to be a clear Hy’s Law case [ALT or AST > 3 x ULN (upper limit of normal) and total bilirubin > 2 x ULN] and the two remaining cases were considered to closely approximate Hy’s Law cases. Fasiglifam-related liver toxicity was also observed in repeat-dose dog toxicity studies. It was characterized by elevation of plasma AST, ALT, ALP, and /or bilirubin and portal/periportal granulomatous inflammation with crystal formation in histopathology. This toxicity was observed in a 4-week study at 1000 mg/kg/day. The toxicity measures were greater than those observed at 150 and 80 mg/kg/day in 13- and 39-week studies, respectively, indicating that the toxicity was both dose- and duration-dependent. We analyzed the composition of foreign body materials observed in dog liver and identified Fasiglifam and Fasiglifam glucuronide. Although elevations of serum bilirubin and ALT were observed in repeat-dose rat studies, no histopathological changes indicative of liver injury were observed in any rat studies even at the lethal dose of 2000 mg/kg/day. In this symposium, the mechanisms for liver toxicity of Fasiglifam in dogs, safety margins in plasma and bile, human relevancy and discussion with FDA and PMDA would be introduced.
Fasiglifam (TAK-875), a GPR40 agonist, which was under development as a type 2 diabetes agent, voluntarily discontinued the development just before NDA in Japan and the US. The cause of discontinuation was the concern of side effects on human liver, which were recognized as Drug Induced Liver Injury (DILI). In fact, various groups have studied the cause of DILI induction by Fasiglifam; however, due to the complexity of its development mechanism, the extrapolation and predictability from animals to humans have not been sufficiently elucidated.
DILI in humans by fasiglifam was not a phenomenon unexpectedly clarified in the clinical trial, but the influence on liver effect of dogs had been confirmed from the early development stage. In fact, FDA asked for a view on the extrapolation of dog hepatotoxicity to humans, and they requested a prospective for human safety after the initiation of Phase 3.
The results of various nonclinical studies eliminated the concern of extrapolation to humans related to the dog hepatotoxicity and the initiation of Phase 3 trial was approved by FDA.
In this symposium, I would like to share the results of nonclinical DMPK studies that wiped out the concerns about human extrapolation on hepatotoxicity in dogs, and consider whether the judgment was appropriate. Also, I would like to consider the relationship between dog hepatotoxicity and adverse effects of human liver in the Phase 3 trial which actually decided to discontinue the development voluntarily.
医薬品の生殖発生毒性試験法ガイドラインの全面改訂作業がICH（International Council for Harmonization of Technical Requirements for Pharmaceutical for Human Use）の専門家作業部会において進んでいる。今回の改定は約20年ぶりとなるが、注目されるのは発生毒性評価に動物実験代替法（以下代替法）を使用するアプローチの容認が予定されていることである。発生毒性試験代替法の開発の歴史は長く，その間に洗練され，いくつかの代表的なin vitro，ex vivo，あるいは非哺乳類を用いる試験系が広く認知され，一般化学物質や医薬品候補化合物のハザード評価あるいはメカニズム解析を主な目的として利用されている。しかしながら，医薬品の非臨床安全性評価にはハザード評価を超えてリスク評価にまで応える必要があるはずである。現状の発生毒性試験代替法がリスク評価を可能とする領域まで到達しているのだろうか，そうでなければ医薬品のリスク評価に対応するために補完する方策は考慮されているのであろうか。本シンポジウムでは，S5(R3)で述べられる規制当局に受け入れられるための考慮点をベースに，発生毒性試験代替法の代表的な試験系に関する最先端の情報を得たうえで，医薬品の発生毒性試験代替法としての各試験系の課題と早急に着手すべきことを考えてみたい。さらに，代替法のデータからヒトリスク評価を考えるチャレンジングなテーマにまで迫りたい。