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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 21^st 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.

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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.

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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.

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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.

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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.

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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 6^th Congress of Asian Society of Toxicology.

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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.

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‘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.

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