2. 小胞体ストレス応答系の役割 鉛は細胞内のカルシウムの恒常性を攪乱することから、小胞体ストレスを惹起することが示唆されているが、それに対する防御応答機構は未解明であった。BAECに鉛を曝露すると、Unfolded protein response (UPR) 経路の1つであるIRE1およびJNKのリン酸化レベルが上昇した。同条件下において、小胞体シャペロンであるGlucose-regulated protein 78（GRP78）およびGRP94の顕著な発現誘導が観察された。そこでJNKの阻害剤SP600125またはAP-1の阻害剤クルクミンを前処理したところ、鉛によるGRP78およびGRP94の発現誘導はいずれも抑制された。更に、GRP78をRNA干渉にてノックダウンしたところ、鉛による細胞毒性が増強された。これらの結果より、鉛は血管内皮細胞において小胞体ストレスを惹起する一方で、細胞は鉛に対してIRE1/JNK/AP-1経路を活性化させてGRP78およびGRP94などの小胞体シャペロンを誘導し、当該金属の毒性に対して防御・応答することを明らかにした。
The future of toxicology will depend on how well cutting-edge technology is transferred and integrated to solve problems in toxicology. Toxicity testing is poised to take advantage of the revolutions in biology and biotechnology. In 2007, the National Research Council of the US National Academies published a report, entitled Toxicity Testing in the 21st Century: A Vision and a Strategy, which advocates the use of these new technologies to transform toxicity testing from a system based on whole-animal testing to one founded mainly on in vitro methods that evaluate changes in biologic processes using cells, cell lines, or cellular components, preferably of human origin. This report concluded that a transformative paradigm shift was needed to confront the many issues faced in the toxicity testing of environmental chemicals, drugs, and cosmetics to which humans are exposed. Toxicity testing, as envisioned by this NAS report, involved the interplay of toxicity pathways, targeted testing, chemical characterization, dose-response and extrapolation modeling, population and exposure data, and risk assessment. This lecture will present key elements of this report, along with selected examples of studies and discussions in the scientific literature, which evaluate new approaches in toxicity testing.
Living organisms are constantly subjected to diverse types of stress both external and internal sources. While excessive stress leads to necrotic or apoptotic death, moderate amounts of noxious stimuli may render the cells adaptive or tolerant to ongoing or subsequent insults. Such adaptive survival response normally accompanies de novo synthesis of proteins through activation of distinct stress-responsive signaling. One of the key signaling molecules involved in cellular adaptation or tolerance to a wide array of noxious stmuli is nuclear transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2). Our previous studies have revealed that Nrf2 plays a pivotal role in cellular stress response. Nrf2 is sequestered in the cytoplasm as an inactive complex with the inhibitory protein Keap1. Upon activation, Nrf2 binds to antioxidant responsive element (ARE) or electrophile responsive element (EpRE), leading to the coordinated up-regulation of down-stream target genes that boost cellular antioxidant/cytoprotective potential. Many chemopreventive natural products can induce ARE/EpRE-driven upregulation of antioxidant/phase-2 detoxifying enzymes or other cytoprotective proteins, thereby fortifying cellular defence against oxidative, nitrosative and inflammatory insults. Cysteine thiols present in Keap1 functions as a redox sensor in transcriptional regulation of a distinct set of stress responsive/cytoprotective proteins. Some chemopreventive/chemoprotective natural products can induce ARE/EpRE-driven upregulation of cytoprotective gene expression, thereby fortifying cellular defence against oxidative, nitrosative and inflammatory insults. Supported by the Global Core Research Center (GCRC) grant, National Research Foundation-MEST, Republic of Korea.
Our bodies must counteract insults originating from the environment. Toxic chemicals (electrophiles) and reactive oxygen species (ROS) activate expression of detoxifying and antioxidant genes through antioxidant responsive element (ARE). Transcription factor Nrf2 is essential for the coordinated induction of cellular defense enzymes through ARE. This notion is best demonstrated in animal models, showing that Nrf2-null mice are sensitive to a wide variety of electrophiles and ROS. Keap1 is identified as a negative regulator of Nrf2. Electrophiles liberate Nrf2 from the repression by Keap1 and provoke nuclear accumulation of Nrf2. Keap1 possesses multiple reactive cysteine residues that covalently bound with electrophiles, indicating that Keap1 acts as a sensor for xenobiotic stresses and we refer this system to as the Cysteine Code. Mouse and zebrafish models demonstrate that multiple sensor functions reside within Keap1. The hinge and latch model proposed for the Keap1-Nrf2 system describes the regulation of nuclear accumulation of Nrf2 by a Cul3- Keap1 E3 ubiquitin ligase-dependent mechanism. We have verified this model through structure biology, mouse/zebrafish genetics and human cancer analyses. In human cancers, missense mutations have been identified in KEAP1 and NRF2 genes. These mutations disrupt the KEAP1-NRF2 complex and result in constitutive activation of NRF2. Elevated expression of NRF2 target genes confers advantages on cancer cells. Transgenic mouse models provide evidence that mutated form of Keap1 analogous to cancer genotypes lose the ability to repress Nrf2 in vivo. Thus, the Keap1-Nrf2 system opens a new avenue to the understanding of the signal transduction and regulatory processes underlying the stress response and cancer progression.
The discovery of cytochrome P450 (P450) was reported in 1962 by R. Sato and T. Omura (J. Biol. Chem. 237, 1375-1376). Since then, this enzyme system has come to be recognized as having a critical role in toxicology. P450s are involved in ～ 3/4 of human enzymatic transformations of drugs and ～ 2/3 of the bioactivation of carcinogens. Bioactivation, induction, and inhibition are important aspects of P450 in toxicology, especially with drugs and drug candidates. Notable examples of P450 involvement in drug toxicity include terfenadine and acetaminophen. The toxicity of the notorious teratogen thalidomide has been revisited in the context of P450 bioactivation. Knowledge of human P450 enzymes has figured prominently in current efforts in molecular epidemiology, pharmacogenomics, chemoprevention, and risk assessment. Current issues related to P450 are predictions of drug toxicity based upon in silico modeling and the role of covalent protein binding. A general need exists to produce more innovative methods of screening for drug toxicity, with the hope of replicating the success seen in predicting metabolism and pharmacokinetics to the areas of pre-clinical toxicity and especially adverse events in humans. In summary, the understanding of P450s has been a remarkable success story in understanding the metabolism and its consequences with drugs, steroids, and carcinogens.
The perfluoroalkyl acids (PFAAs) are a family of organic chemicals consisting of a perfluorinated carbon backbone (4-14 carbons in length) and an anionic head group (sulfonate, carboxylate or phosphonate). These compounds have excellent surface-tension reducing properties and have numerous industrial and consumer applications. However, they are chemically stable, persistent in the environment, ubiquitously distributed, and present in humans and wildlife. Two issues must be considered regarding PFAA toxicology: pharmacokinetics and potency of the chemicals. The rates of PFAA clearance and their body burden accumulation are dependent on carbon-chain length and animal species. In general, the serum half-life of PFAAs increases with chain length in both rodents and humans, but the estimates in humans are markedly higher than those in laboratory animals. Recent studies with laboratory animal models have indicated a number of toxic effects of PFAAs, including tumor induction, hepatotoxicity, developmental toxicity, immunotoxicity, neurotoxicity and endocrine disruption. The modes of PFAA actions are not well understood, but are thought to involve, in part, activation of nuclear receptor signals (such as peroxisome proliferator-activated receptor-α, PPARα). Based on PPARα activation, potency of PFAAs increases with carbon-chain length, carboxylates are stronger than sulfonates, and mouse receptor is more reactive than human receptor. Adverse effects of perfluorophosphonates in mice resemble those described for sulfonates and carboxylates, although potency of this congener appears to be weaker than the other two counterparts. This abstract does not necessarily reflect US EPA policy.
Thrombosis continues to represent a major cause of death in spite of the advanced medicine and pharmacotherapy of the modern era. Excessive thrombosis can cause life-threatening thrombotic events including deep vein thrombosis, stroke, myocardial infarction and pulmonary embolism. What is worse, it can lead to the exacerbation of the existing cardiovascular diseases through the degranulation of secondary vaso-active mediators and the stimulation of vascular remodeling. Recently, we and several research groups have discovered that xenobiotics can manifest prothrombotic effects and efforts are being directed into the elucidation of the underlying mechanisms. Especially, prothrombotic effects of heavy metals and ROS-generating chemicals, nanomaterials and neurotoxicants are being extensively investigated in an effort to clarify the link between pro-thrombosis and their well-established cardiovascular toxicities. Platelets had been the main tissue of interest due to their major roles in thrombosis through forming platelet aggregates. However, the focus is being migrated into the involvement of erythrocytes and coagulation systems and their interaction with platelets and other cardiovascular tissues. Exemplifying this, arsenicals which can induce platelet aggregation and thrombosis, also induces procoagulant activation in platelets, a series of events that culminate in phosphatidylserine exposure on outer membrane, the enhancement of thrombin generation and ultimately increased clot formation. It has been demonstrated that erythrocytes can also participate in the xenobiotic-induced thrombogenic activation through exhibiting phosphatidylserine exposure and resultant procoagulant activity. Interestingly, phosphatidylserine exposure is a key marker of apoptotic cells and it can increase cell-cell interaction and initiate phagocytosis by tissue macrophages, reflecting that the procoagulant activity induced by xenobiotic might be further related into other biological events like apoptosis and anemia. These studies indicate the urgent need to expand our current understanding of prothrombotic risks of xenobiotics as a narrow scoped platelet aggregation into a general alteration of cardiovascular tissues as a system. In this context, a timely and comprehensive review on this subject will be informative and inspiring to the participants of the 6th Congress of Asian Society of Toxicology.
The ability of biomarkers to improve treatment and reduce healthcare costs is potentially greater than in any other area of current medical research. However, understanding the characteristics of novel biomarkers and developing the robust evidentiary packages to support incorporating them into drug development and clinical practice is an enormous undertaking requiring significant resources and commitment from a wide range of stakeholders, including regulatory, industry and academic scientists. The Predictive Safety Testing Consortium is a unique public-private partnership formed by the Critical Path Institute to identify new and improved safety testing methods and submit them for formal regulatory qualification by the Food and Drug Administration (FDA), European Medicines Agency (EMA) and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). Nephrotoxicity is a serious problem for drug development and the sensitivity and specificity of accessible biomarkers of nephrotoxicity in current use (particularly BUN and serum creatinine) does not allow early detection of drug-induced kidney toxicity. This results in significant risk to patients and the termination of drug development for potentially innovative compounds for unmet medical needs because of the inability to monitor for early toxicity. In 2008, the PSTC obtained the first qualification of seven urinary renal preclinical safety biomarkers for use in rodent studies, and on a case-by case basis for the inclusion into clinical development. These included KIM-1, clusterin, TFF-3, albumin,β2-microglobulin, total protein and Cystatin C. The PSTC has continued to expand this qualification by increasing the number of biomarkers, assessing prodromal and reversibility characteristics and regional specificity for these biomarkers. In addition, these biomarkers are being examined in canine and primate models. Furthermore, the PSTC is collaborating with the FNIH Biomarkers Consortium on a large clinical program to define thresholds and characterize the performance of these new biomarkers in humans in order to enhance decision making in drug development, particularly for drug candidates that exhibit nephrotoxicity. This session will focus on the success of the preclinical renal safety biomarker qualification, the impact this qualification is making on drug development and the translational activities for the progressive qualification of novel renal safety biomarkers which are needed today.
‘Percellome’ database is providing a unique “per cell” readout in mRNA copy number on various organs of mice for more than 100 chemicals (NIHS). Such knowledge makes possible to use dataset without data pre-elimination and compare profiles across experiments. This opens a possibility for the mathematical representation of biological information in a manner treating a cell as an open system, with no molecule ignored. In collaboration with Kanno group we constructed A Geometric Clustering Tool (AGCT) and a comparative genomic analysis tool on human-mouse-rat (SHOE) which goal is to divide an expression profile by unsupervised learning mathematical technique in order to elucidate gene clusters and find transcription regulation network controlling genes. Analysis was held on two toxic congeners TCDD and TCDF, with 0.1 toxicity equivalency factor. They are known to cause birth defects, immunotoxicity and cancer through the activation of Ahr receptor pathway in mice, whilst Ahr receptor is a mediator in the course of growth, development and differentiation. Data of 20,000 probes per cell followed: replicates normalization, circadian effect subtraction, representation by PCA and/or spectral manifold, unsupervised clustering and sorting by validity. The analysis resulted in 498 (3,117 probes) and 369 (3,771 probes) sensible clusters for TCDD and TCDF, respectively, meeting the biological expectations. Clustering and transcription regulation results can be simulated on CellDesigner (SBI) and interactively annotated on Payao (Matsuoka, SBI). Since this year Percellome database is a member of The Garuda Alliance Common Platform, providing the research community with the consistent analysis workflow and biological databases.
近年、薬物トランスポーターの遺伝子多型または薬物相互作用によって薬物副作用が引き起こされるという臨床知見が蓄積しつつある。その問題を検討・解決すべく2012年3月12日～13日の2日間、米国にてInternational Transporter Consortium Workshop 2（ITCW2）を開催したので、その報告を行う。我々は2010年にMembrane Transporters in Drug Developmentと題してFDA白書をNature Review Drug Discoveryに発表した(1)。しかしながらトランスポーターの薬物相互作用の評価を創薬のどの段階に組み込むべきか？という問題が製薬企業にはある。白書に記述された全てのトランスポーターを全て評価するのは不可能に近い。そこで我々は創薬の初期段階に組み込めるように、化合物の構造に基づいて薬物相互作用するかどうかをin silico判断するQSAR解析方法を開発した。その応用として、胆汁排泄トランスポーターBSEP/ABCB11を阻害して肝内胆汁鬱滞を引き起こす薬・化合物の重要な構造的因子を同定する事に成功した(2)。また、薬物トランスポーターまたは薬物代謝酵素の遺伝子多型によって、薬物体内動態が大きく影響をうけて、一部の患者おいては重篤な副作用が起きる場合がある。その問題を解決すべく、我々は遺伝子多型を迅速に検出する方法を開発した。その基本原理と臨床応用例を紹介する(3)。 (1) Giacomini KM, et al. Nature Review Drug Discovery, 9, 215-236, 2010. (2) Saito H, et al. AAPS J. 11(3), 581-589, 2009. (3) Aw W, et al. Clin. Pharmacol. Ther. 89, 617-620. 2011.
再生医療に用いられる細胞医薬品は、通常は人（自己あるいは同種）由来の細胞を修飾して使用されるため、まずは細胞の採取と感染のコントロールが重要となる。米国FDAは2001年から、細胞組織利用製品の施設登録、細胞組織利用製品をリストアップするための統合システムの作成、危険因子のスクリーニングと感染症検査結果に基づいたドナー組織、細胞などの適格性確認の基準の規定などを進めてきた。2005年5月にcurrent Good Tissue Practice (cGTP)ガイドラインの最終案を施行し、米国内の細胞組織利用製品の製造業者に対し、感染症の感染や感染拡大を予防するための採取、処理、保存、ラベリング、パッケージング、搬送のための規定と、記録管理の手順などを制定した。本講では、細胞医薬品のCMCについて概要を解説する。
急性参照用量は、農薬等の短期間の摂取による健康影響評価の指標として設定される。胎児および出生児の形態あるいは機能の発達分化の多くは、厳密な時間的スケージュールの下で物理的、生化学的あるいは内分泌学的制御を受けて進行していることから、生殖発生毒性試験で認められた影響は、それが反復投与試験であっても急性影響の可能性を考慮する必要がある。実際、FAO/WHO Joint Meetings on Pesticide and Residues (JMPR)でも、比較的多くの剤で生殖発生毒性試験の成績を根拠に急性参照用量が設定されている。 生殖発生毒性試験で認められる毒性影響は、１）親動物に対する影響、２）母動物に対する毒性を介した胚・胎児／出生児への影響、３）胚・胎児／出生児に対する直接影響、に大別できる。本シンポジウムでは、妊娠動物に農薬を単回投与した際の急性影響を例示するとともに、これら３つの観点から急性参照用量の設定についてについて述べたい。 生殖発生毒性試験の成績から急性参照用量の設定を考慮する際には、胚・胎児／出生児の形態あるいは機能の発達分化に対する影響を考察するとともにライフステージによって変化する生理状態と作用機序との関係を考慮して進めることがより適切な設定へのひとつの道筋になるものと考えられる。