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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Background: Acute poisonings remain a major problem in many countries. Timely and effective treatment is important in minimizing the mortalities associated with acute poisonings.
Methods: A Medline search of literature reports relating to insulin euglycemic therapy, intravenous lipid emulsion (ILE), and extracorporeal life support (ECLS) was conducted and summarized.
Results: Insulin euglycemic therapy was mainly used in the management of beta-blocker and calcium channel blocker poisonings and the mechanisms underlying the therapy include increased inotropy, increased intracellular glucose transport, and vascular dilatation. Both animal studies and numerous case reports suggest that high dose insulin is superior to conventional treatments. The usefulness of ILE in human poisonings was first reported in a patient with bupivacaine related cardiac arrest in 2006. In the past 5 years, many severely poisoned cases receiving ILE were reported and the major mechanism underlying ILE is the “lipid sink” theory. Although there are no published controlled trials (RCTs) on ILE, it seems that ILE maybe useful in patients manifesting life-threatening cardiotoxicity from lipophilic toxicants. The application of ECLS in poisoned patients is rarely reported. In a recently reported study, 2/14 (86%) patients receiving ECLS survived, as compared to 23/48 (48%) patients without ECLS (p= 0.02). Given the limited reports, ECLS may benefit patients with hemodynamic instability not responding to conventional measures.
Conclusions: Limited literature reports suggest that several new treatments may be effective in rescuing severely poisoned patients. Well-designed multicenter prospective cohorts studies and/or RCTs are urgently needed to better understand the effectiveness of such treatments.

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The widely used herbicide, paraquat (PQ), is highly toxic and claims thousands of lives from both accidental and voluntary ingestion. The pathological mechanisms of PQpoisoning–induced acute lung injury (ALI) are not well understood, and the role of complement in PQ induced ALI has not been elucidated. We developed and characterized a mouse model of PQ-induced ALI and studied the role of complement in the pathogenesis of PQ poisoning. Intraperitoneal administration of PQ caused dose- and time-dependent lung damage and mortality, with associated inflammatory response. Within 24 hours of PQ-induced ALI, there was significantly increased expression of the complement proteins, C1q and C3 in the lung. Expression of the anaphylatoxin receptors, C3aR and C5aR, was also increased. Compared with wild-type mice, C3-deficient mice survived significantly longer and displayed significantly reduced lung inflammation and pathology after PQ treatment. Similar reductions in PQ-induced inflammation, pathology, and mortality were recorded in mice treated with the C3 inhibitors, CR2-Crry, and alternative pathway specific CR2-fH. A similar therapeutic effect was also observed by treatment with either C3a receptor antagonist or a blocking C5a receptor monoclonal antibody. Together, these studies indicate that PQ induced ALI is mediated through receptor signaling by the C3a and C5a complement activation products that are generated via the alternative complement pathway, and that complement inhibition may be an effective clinical intervention for post exposure treatment of PQ-induced ALI.

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Organophosphate poisoning is the most frequent among acute poisoning with insecticides and can be managed by anti-muscarinic agents and acetylcholinesterase reactivators. A number of researches on organophosphate poisoning have suggested some treatment guidelines through animal studies and acute human poisoning cases. These guidelines generally have rational to treat the acute organophosphate poisoning properly, but we often encounter perplexing and difficult cases to treat in clinical situation, especially when patients ingested very large amount of organophosphate insecticide in suicide attempts. Many study recommendations show considerable variations in atropine doses and are still confounding how much and how long the pralidoxime dose should be given in severe cases. There have been some randomized controlled studies suggesting proper use of pralidoxime, but these results are often inappropriate to decide the dose and duration of treatment. Lipid solubility of organophosphate is one of the important factors influencing rapid distribution and redistribution in the body after ingestion. Furthermore, clinical evaluation of the severe organophosphate poisoning with large amount is not easy and often misleading in the course of treatment. Studies in clinical toxicology have limitation and are different from the clinical trials for new drug development. Even though randomized controlled trials may be better than observational studies, those are not well-controlled studies. Therefore, the treatment course should be guided by frequent acetylcholinesterase monitoring especially in severe acute organophosphate poisoning with large amount.

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Information on the safety profiles of medications that is critical for making decisions in clinical toxicology initially relies on the profile of adverse reactions determined during clinical development. Adverse reactions can be heuristically classified into types A (Augmented pharmacologic effect), B (bizarre or idiosyncratic effects), C (Chronic effects), D (Delayed effects), E (End-of-treatment effects), F (Failure of therapy) or G (Genetic reactions) and of these categories type A reactions can be expected to be of greatest interest during acute overdose. Causality assessments of individual adverse events during clinical trials are often unreliable, as evidenced by cases where an adverse reaction is assessed as being “related” to study drug but on unblinding the subject is found to have been on placebo. The determination of what adverse reactions can be expected depends on careful review of aggregate clinical trial data comparing the adverse events and clinical testing abnormalities for the investigational compound to placebo and active comparators. The Bradford-Hill criteria, the recommendations of CIOMS VI on evaluating clinical safety data and the FDA’s internal guidance for its reviewers on performing Clinical Safety Reviews are useful guides when evaluating clinical trial safety data. The interpretation of clinical trial safety data, selection of safety issues for future monitoring in Risk Management Plans and considerations for selecting information for the Warnings & Precautions and other Adverse Reactions sections of Package Inserts will be presented and discussed.

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Rationale: Antidote therapy is a major element for treating poisoned patients. However, many of them are orphan drugs. Shortage of antidotes compromises the effectiveness to treat the patients though diagnosis is made and line of management is known. The shortage is worldwide, but getting more serious in developing countries. A new system is designed and operates recently in Thailand.
Method: This is a collaborative work among various agencies. A system includes selection and procurement of essential antidotes; distribution and stock specific antidotes according to their urgency and availability; logistics and communication; web-based administration including searching by geographic information system and real-time online stock; education and training of health personnel; and monitoring and evaluation of the system.
Result: In the first year, 176,600 USD were allocated for this program. Six antidotes (sodium nitrite, sodium thiosulfate, methylene blue, dimercaprol, succimer and glucagon) were selected and procured. Cyanide antidotes and methylene blue were distributed and stocked in all general hospitals. Dimercaprol was in tertiary care centers and glucagon in only a poison center. Succimer was in government pharmaceutical organization. More than 800 health personnel participated in the training workshops held nation-wide. During the first year, 57 patients had derived benefit from the program including poisoning from cyanide, methemoglobinemia, hydrogen sulfide, copper and lead.
Summary: A system is able to safe hospitals’ expense for stocking the drugs and secures the country form poisoning problems. It is flexible for rectifying other orphan drugs and potentially sufficient to share with neighboring countries.

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Toxicological data is an important reference to estimate the safety in the early phase of clinical trials. Especially, in the First in Human trials, the role of toxicological data is more crucial and principal investigator has to presume which point should be paid more attention according to the data. On the other hand, in the later phase of trials, it is not so frequent to be paid attentions to the non-clinical data, unless some severe adverse event occurred, which gives us opportunity to look back to it.
In my talk, I would like to examine the possibility to make the most of non-clinical (toxicological) data not only in the early phase of clinical trials, but also later phase or post-marketing stage by comparing safety data between non-clinical and clinical (trial and post-marketing stage) of some drugs. Moreover, I would like to consider the necessity of new biomarker as a more accurate indicator which will link non-clinical data and clinical safety assessment.

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Currently in Taiwan, the illicit substances used include sedatives, hypnotics, solvents, heroin, amphetamines, and hallucinogens (MDMA, ketamine, marijuana, and LSD). The so-called club or pub drugs include sedatives, hypnotics, solvents, cocaine, amphetamines, and hallucinogens. All these drugs are popular in the adolescents, especially hallucinogens, sedatives and their mixtures. Since late 1990s, their epidemics in many countries/regions, Taiwan as well, are related to problems of globalization, and further wide-spread by media, websites, pubs/parties.
The acute effects of hallucinogens or sedatives are less toxic than narcotics or stimulants. Therefore, cases of hallucinogen or sedatives seemed seldom found clinically. However, cases of acute phenyl alkylamines (MDMA, PMMA, mephedrone, etc) and gamma-hydroxybutyric acid poisoning were sometimes encountered. Nevertheless, cases of ketamine induced uropathy and nitrous oxide induced neuropathy among the young users were increasingly found in these couple years. Besides, these drugs provide as “gate drugs” due to their variability and changeable ingredients and forms of pills, and more choice for the youth. All these raised important public health issues.
Primary care clinicians may encounter substance abusers often but may not always recognize the direct drug effects, their compli­cations, withdrawals, and even its social consequences, including acci­dents, suicide, homicide, drug facilitated sexual assaults, etc. Moreover, psychological consequences of drug abuse, such as aggressive behavior, suicidal ideation, or hallucinosis, or psychiatric co-morbidities often found in drug users. Patients with these presentations should be carefully related to drug use. Frequently, it is urgent and difficult to tell what conditions indicate the evidence of drug use, and which complications need to be suspected in known drug users. Moreover, it’s hard to deal with adolescents with substance abuse, due to less frequent use, less toxic substance. However, it’s important to detect, educate and treat adolescents with substance abuse problems as earlier as possible.

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According to the statistics from the office of Chinese National Narcotic Control Commission (NNCC), the cumulative number of registered drug abusers in mainland China increased from 70 000 in 1990 to 1.55 million by the end of 2010. Heroin continues to be the most commonly abused drug. According to the data from NNCC, heroin abusers made up 69.0% of the total drug abusers in 2010. Recent epidemiologic survey shows that the heroin abuse has leveled off and may actually be declining in most provinces, autonomous regions and metropolis. However, the “synthetic drugs” or new emerging drugs which majority of that are the psychotropic substances such as “ice” (methamphetamine, MA), “shake-head pill” (MDMA or ecstasy), “K powder” (Ketamine) and “shake-head water” have penetrated into China through various channels since 2000. The first reported case of MDMA abuse we surveyed appeared in China in 1999. Since then, methamphetamine and MDMA and ketamine abuse have spread to disco clubs and public entertainment within the urban areas of major cities, and are rapidly epidemic in many areas. Our epidemiologic survey shows that ATS or new emerging drugs abuse produced multiple social, public health and individual consequences.

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Drug abuse is the important social problem to be resolved in Japan and also in the world. In Japan, stimulants such as methamphetamine have been illegally sold and used in the public after the World War 2. Irrespective of the nationwide fight against the use of methamphetamine and other illicit drugs, there still be in the uncontrolled social status. Additionally, stimulant-derived compounds, such as methylenedioxy-derivatives MDMA and MDA, phenethylamines- and tryptamine-derivatives have been distributed widely. Under these social status, Japanese scientists have developed various analytical methods and finally established as the methods just like formulary. Specifically, The Pharmaceutical Society of Japan comprises the Committee on Analytical Methods for Drugs and Chemicals and publishes and distributed timely the deliberated and established methods to public.The newly and currently appearing cathinone and its related derivatives, cannabinoid receptor agonists, such as aminoalkylindoles, cyclohexyphenyl compounds, and THC analogs, in the illicit drug market have been evaluated and publishied by the Committee. .In this lecture, various aspects of drug abuse happened in Japan will be discussed.

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The primary objective of this study was to determine and characterize surrogate biomarkers that can predict nephrotoxicity induced by mercuric chloride (HgCl₂) using urinary proton nuclear magnetic resonance (¹H NMR) spectral data. A procedure for ¹H NMR urinalysis using pattern recognition was proposed to evaluate nephrotoxicity induced by HgCl₂ in Sprague-Dawley rats. HgCl₂ at 0.1 or 0.75 mg/kg was administered intraperitoneally (i.p.), and urine was collected every 24 h for 6 days. Animals (n = 6 per group) were sacrificed 3 or 6 days post-dosing in order to perform clinical blood chemistry tests and histopathologic examinations. Urinary ¹H NMR spectroscopy revealed apparent differential clustering between the control and HgCl₂ treatment groups as evidenced by principal component analysis (PCA) and partial least square (PLS)-discriminant analysis (DA). Time- and dose-dependent separation of HgCl₂-treated animals from controls was observed by PCA of ¹H NMR spectral data. In HgCl₂-treated rats, the concentrations of endogenous urinary metabolites of glucose, acetate, alanine, lactate, succinate, and ethanol were significantly increased, whereas the concentrations of 2-oxoglutarate, allantoin, citrate, formate, taurine, and hippurate were significantly decreased. These endogenous metabolites were selected as putative biomarkers for HgCl₂-induced nephrotoxicity. A dose response was observed in concentrations of lactate, acetate, succinate, and ethanol, where severe disruption of the concentrations of 2-oxoglutarate, citrate, formate, glucose, and taurine was observed at the higher dose (0.75 mg/kg) of HgCl₂. Correlation of urinary ¹H NMR PLS-DA data with renal histopathologic changes suggests that ¹H NMR urinalysis can be used to predict or screen for HgCl₂-induced nephrotoxicity.

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Taiwan’s National Science Council (NSC) is in charge of the funding policy of all basic scientific researches in Taiwan. Department of Life Science in NSC has three missions: first, to promote and support the life science research; secondly, to establish the infrastructure of life science area in Taiwan; third, to cultivate the personnel and talent for life science and biotechnology. Parts of my presentation will be introducing the funding policy as well as the trends of pharmacological/toxicological research in Taiwan.
In addition, I will also present my own research on cancer stem cell. Our recent study revealed that the histone methyltransferase G9a played a crucial role in maintaining typical characteristics of cancer stem cells such as drug resistance, self-renewal and tumorigenicity. Interestingly, some reports had been shown that the level of G9a could be up-regulated or altered by some toxicants or heavy metals. Therefore we would like to discuss the possibility of where some toxicants exposure might nurture the emergence of cancer stem cells by changing the epigenetic regulators, i.e., G9a.

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Analysis of the changes in different stage of human cell transformation may yield insights into the multiple events involved in acquisition of the tumorigenic phenotype induced by chemical carcinogens. In this study, we treated human cell lines with known carcinogens and obtained malignant phenotype of cell transformation confirmed by injection subcutaneously in immunodeficient mouse. We then screened differentially methylated genes or miRNAs at different stages of cell transformation using CpG island and miRNA microarray. As a result, we identified that 83 hypermethylated promoters were differentially modified in transformed cells. Among which 17 genes mRNA levels were decreased significantly and methylation at CpG islands of promoters. Particularly, we examined the methylation status of p16INK4α promoter in peripheral blood lymphocytes (PBLs) of 69 polycyclic aromatic hydrocarbons (PAHs) exposed workers. Among the 35 CpG sites we analyzed, 22 were highly hypermethylated in PAHs-exposed group. These 22 hypermethylated CpG sites were positively correlated to levels of urinary 1-hydroxypyrene (1-OHP) and the cytokinesis-block micronucleus (CBMN) in PBLs. In addition, miRNA expression profiles of B(a)P-transformed HBE cells revealed that 12 miRNAs differentially expressed at both pre-transformed and transformed stages. Among these miRNAs, down-regulation of miR-638 was found in 68% (34/50) of human NSCLC tissues. The average expression level of miR-638 in PBLs from 86 PAHs exposed workers increased by 72% compared with control group. Moreover, we demonstrate that miR-638 is involved in the BaP-induced carcinogenesis by targeting BRCA1. Taken together, our findings suggest that aberrant epigenetic regulation can be potentially used as biomarkers for risk assessment.

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The Keap1-Nrf2 system plays a central role in cytoprotection against oxidative and electrophilic insults. Under normal conditions Keap1 serves as a substrate adaptor for Cullin3-based ubiquitin E3 ligase and promotes proteasomal degradation of Nrf2. Keap1 is inactivated upon the exposure to environmental stimuli such as electrophiles, and in this situation Nrf2 is stabilized and activates transcription of cytoprotective genes. Nrf2 knockout mouse (Nrf2^–/–) is very sensitive to toxicants like acetaminophen. On the other hand, Keap1 knockdown mouse (Keap1^flox/flox) in which Nrf2 is activated is resistant to toxicants. Whereas the turnover of Nrf2 has been well analyzed, little is known about the Keap1 turnover or recovery of the Keap1 activity after the electrophilic insults. We found that Keap1 is accumulated when autophagy is impaired in the liver of mice. Treatment of cells with an autophagy inhibitor or inducer markedly increased or decreased the Keap1 level, respectively. The Keap1 degradation was accelerated upon the exposure to certain type of electrophiles, implying that inactivated Keap1 after modification with electrophiles becomes a preferred substrate of autophagy. We also found that the electrophilic challenge enhances the transcription of Keap1 gene, suggesting that the Keap1 activity is recovered by de novo synthesis of Keap1. Consequently, Keap1 protein was kept in constant level even in the presence of electrophiles. These results thus demonstrate that Keap1 is degraded through autophagy and that this degradation machinery in concert with the de novo synthesis of Keap1 maintains the active Keap1 level and the redox homeostasis regulated by Nrf2.

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Garlic has been widely used as a therapeutic agent as well as a spice for more than 2000 years. Besides its well-known cardioprotective effects, garlic is also considered to possess potential cancer preventive properties. Chemopreventive effects of garlic have been attributed to its oil-soluble sulfur ingredients, such as diallyl sulfide, diallyl disulfide, and diallyl trisulfide (DATS), but their underlying molecular mechanisms remain largely unresolved. In the present study, we have compared the effects of aforementioned allyl sulfides on the growth of cultured human breast carcinoma (MCF-7) cells. DATS inhibited the growth of MCF-7 cells to a greater extent than did the other allyl sulfides as determined by the MTT assay. DATS also induced apoptosis in MCF-7 cells, which was mediated through accumulation of reactive oxygen species with subsequent activation of JNK that catalyzes phosphorylation of Bcl-2 at Ser70. In another experiment, DATS prevented tumor formation in a mouse xenograft model. Aberrant upregulation of cycloocygenase-2 (COX-2) has been frequently observed in several types of cancer cells and is considered as a molecular target for cancer chemoprevention. Topically applied DATS inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in dorsal skin of female ICR mice. DATS inhibited the DNA binding activity of AP-1 which is one of key transcription factors regulating the inflammation and expression of COX-2. DATS inhibited TPA-induced phosphorylation of Akt and JNK, which are major MAPKs regulating AP-1. A pharmacologic Akt inhibitor LY294002 and a JNK inhibitor SP600125 abrogated the TPA-induced COX-2 expression, suggesting that suppression of COX-2 expression by DATS in TPA stimulated mouse skin is mediated by blocking the PI3K-Akt and JNK signaling. Topical application of DATS protected against mouse skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene plus TPA. In addition, DATS strongly inibited DNA binding activity of NF-κB compared with other allyl sulfides in human mammary epithelial (MCF10A) cells treated with TPA. DATS inhibited the transcriptional activity of NF-κB, phosphorylation of IκBα, and activity of IKKβ. Inhibition of NF-κB DNA binding activity and IKKβ activity by DATS were blunted by addition of the antioxidant N-acetyl-L-cysteine (NAC) and the reducing agent dithiothreitol. Therefore, anti-inflammatory activity of DATS may be associated with oxidation or covalent modification of thiol groups contained in IKKβ.

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Cigarette smoke is a major risk factor for bladder cancer and contributes to chemoresistance in bladder cancer patients who continue to smoke while receiving chemotherapy. Nicotine has been implicated as a co-carcinogen that promotes lung cancer development through pro-survival pathways and is known to induce chemoresistance in some cancer cells through anti-apoptosis mechanisms. The aims of our study were to investigate the role of nicotine in inducing bladder cancer cells proliferation and chemoresistance. We found that transient nicotine stimulation activates Stat3/ERK1/2 leading to induction of Stat3 and NF-κB DNA binding activity, which is associated with cyclin D1 expression and cell proliferation in bladder cancer cell line T24 cells. Chronic nicotine exposure strongly activated Stat3 leading to cyclin D1 overexpression, cell cycle perturbations, and chemoresistance. Nicotine mobilized cell proliferation/chemoresistance is mainly mediated by Stat3 and its downstream signals via nicotinic acetylcholine receptor. We further found that pterostilbene effectively inhibits the growth of sensitive and nicotine-induced chemoresistant human bladder cancer cells by inducing necrosis, cell cycle arrest, autophagy and apoptosis. Pterostilbene-induced autophagy was triggered by the inhibition of AKT/mTOR/p70S6K pathway and activation of ERK1/2 pathway.

Key words: pterostilbene, bladder cancer, nicotine, chemoresistant, autophagy

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Xenobiotic metabolizing enzymes, such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase 1 (NQO1), play an important role in the detoxification of chemical carcinogens and suppression of oxidative stress. The induction of these detoxifying enzymes is regulated by a cis-acting sequence, referred to as the antioxidant response element (ARE). The most effective transcription factor that binds to ARE is the NF-E2 related factor 2 (Nrf2), a member of the NF-E2 family of basic leucine zipper transcription factors. Many lines of evidence indicate that Nrf2 activation enables adaptation to oxidants and electrophiles. In the present study, various herbal medicines were screened for inducers of Nrf2-regulated enzymes. This assay showed that Notopterygium incisum extract potently increased GST and NOQ1 at both mRNA and protein levels. According to bioactivity-guided cell-based assays, falcarindiol was isolated from the extract and identified as a novel Nrf2 activator. Several experimental models were used to understand effects of falcarindiol on normal cells. Pretreatment with falcarindiol activated Nrf2 in vitro and in vivo and conferred protection against menadione-induced cytotoxicity through enhancement of the menadione detoxification process and carbon tetrachloride-induced liver toxicity through suppression of lipid peroxidation. Dextran sulfate sodium (DSS) has been known to induce intestinal inflammation and nitrosative stress. DSS-induced colitis was suppressed in accordance with Nrf2 activation by falcarindiol. These results demonstrated that falcarindiol has a key role for Nrf2 in controlling the ability to withstand oxidative and electrophilic stress.

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We have previously reported that some Kampo medicines such as Kakkonto could be decocted by using a microwave oven at the condition of 500W for 30 min instead of the conventional method.
In this study, we investigated toxic components in decoction of Keishikabushito by using a microwave oven to confirm the safety of this method. We decocted a daily dose of Keishikabushito containing 1 g of processed Aconite root (Bushi) by a microwave oven in 600mL water at 500W for 30 min and the conventional decoction device in 500mL water at 600W for 40 min, respectively. The contents of toxic components from Bushi (aconitine, mesaconitine, hypaconitine), and three other effective as well as toxic components (benzoylaconine, benzoylmesaconine, benzoylhypaconine), the hydrolysates of the three toxic components in heating process, were analyzed with HPLC method.
In both decoctions obtained by these methods, toxic components such as aconitine, mesaconitine and hypaconitine couldn’t be observed because of hydrolysis in heating process. Benzoylaconine, also not observed, may be further hydrolyzed. The contents of benzoylmesaconine and benzoylhypaconine were 0.68±0.06 mg, 4.75±0.15 mg in decoction decocted by a microwave oven, and 0.70±0.01 mg, 4.89±0.12 mg in decoction decocted by the conventional method, respectively.
There was no significant difference of the contents of toxic components between two methods. Decocting Keishikabushito by using a microwave oven is as safe as the conventional method. In addition, the decoction time can be saved. This new decoction method may be applied widely.

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Cancer preventive effects of bioactive phytochemicals have been extensively investigated and well-documented. The most well known botanicals with chemopreventive potential include epigallocatechin gallate (green tea), sulforaphane (broccoli), isoflavones (soy), resveratrol (grapes), lycopene (tomato), curcumin (turmeric), allyl sulfides (garlic), etc. As oxidative stress and inflammatory tissue injury are two major culprits implicated in pathophysiology of a wide array of human malignancies, antioxidant and anti-inflammatory phytochemicals have been considered to be promising chemopreventive agents. Nuclear transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) play a crucial role in regulating induction of expression of antioxidant and pro-inflammatory genes, respectively. Various antioxidants and anti-inflammatory substances derived from dietary and medicinal plants have been found to modulate these two ubiquitous redox-sensitive transcription factors, thereby potentiating cellular antioxidant or detoxification capacity and/or protecting against inflammatory injuries.

References:
1. Surh, Y.-J. (2003) Cancer chemoprevention with dietary phytochemicals. Nature Reviews Cancer 3, 768-780.
2. Na, H.-K. and Surh, Y.-J. (2006) Transcriptional regulation via cysteine thiol modification: a novel molecular strategy for chemoprevention and cytoprotection. Mol. Carcinog., 45: 368-380.
3. Surh, Y.-J. and Na, H.-K. (2008) NF-κB and Nrf2 as prime molecular targets for chemoprevention and cytoprotection with anti-inflammatory and antioxidant phytochemicals. Genes and Nutrition, 2: 313-317.
4. Surh, Y.-J., Dong, Z., Cadenas, E., and Packer, L. (2008) Dietary Modulation of Cell Signaling Pathways. CRC-Taylor & Francis.
5. Kundu, J.K. and Surh, Y.-J. (2012) Emerging avenues linking inflammation and cancer. Free Radic Biol. Med., 52: 2013-2037

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Currently, scientific term “Regulatory Science” has been appeared in scientific fields with various definitions. In Japan, the term “Regulatory Science” has been firstly introduced by Dr. M. Uchiyama, the former Director General of NIHS. At the time of 1987, he has defined that regulatory science is the scientific activities to conduct a harmonized application of the multifaceted subjects or products to human society under the proper and accurate assessing and evaluating by using the sophisticated updating sciences and technologies. His most recent definition is that the science is the research activities that support the effort of regulating and optimizing scientific and technological development according to the objectives geared toward human health. Thus, in the toxicological aspects, the targeting objectives and products by regulatory science are chemicals distributed in the human society, such as foods, pharmaceuticals, agrochemicals, industrial chemicals and so on.
Regulatory science on drug regulation provides a scientific basis for fitting a substance into society and to patients as a drug via the regulation process. With respect to food regulatory science, each country has their own standards in order to protect people from contaminated and unknown food ingredients. In other respect, regulatory science is not simply limited to the areas as described above, but also the science could be expanding to various areas even in the individual’s daily work.
In turn, toxicology provides scientific evidence with toxic manifestations for evaluating chemicals and others.
Traditional toxicology defines toxic manifestations and current modernized toxicology will clarify mechanism of the targeted compound, even epigenetic changes. Therefore, toxicology may play important roles in the development of regulatory science.

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As the research and development institute under KFDA, NIFDS has played the key role in comprehensive researches which are necessary for food and drugs regulation in Korea since its establishment in 1995. It has performed development and evaluation on toxicological and pharmacological methods. NIFDS has lead national initiatives on endocrine disruptor researches and published a number of test method and evaluation reports. NIFDS has also run Korea National Toxicology Program (KNTP) and produced toxicology report on 40 natural compounds.
NIFDS has recognized needs for adopting cutting-edge life science technologies to improve conventional toxicology screening methods and to develop better evaluation tools for regulatory science.
For this purpose, NIFDS has actively employed emerging technologies such as toxicogenomics, systems toxicology and in silico modeling. We have performed more than 40 microarray projects for toxicology studies and released data for public since 2008. Along with toxicogenomic effort, we have recently initiated a predictive toxicology research program. This program is mainly focused on developing in silico model for toxicity prediction using systems toxicology. Developing alternative test methods is another field of interest for NIFDS. NIFDS is running a program for developing and validating alternative test methods based on 3R principle. NIFDS currently runs endocrine disrupter screening test, eye irritation test and skin irritation test with collaboration of ICATM and already published 6 guidelines for alternative test methods. As safety of nano-materials in food and drugs has become controversial, NIFDS has launched a toxicological research program to evaluate their safety concern. Currently NIFDS runs safety studies on silver, gold and zinc oxide with collaboration of OECD.
NIFDS exerts its effort for developing new toxicity test method by actively adapting emerging technologies and actively participate in international workgroup for modernizing regulatory science. NIFDS will continue its effort for developing new technologies for regulatory science in future by actively adapting emerging technologies.

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China has recognized the importance of establishment of a legal framework for chemical safety management and made remarkable progress in recent years. SFDA, the state drug authority, has took necessary steps to meet its goal of globalization and promulgated regulations, standards procedures and guidance in safety evaluation and administration of pharmaceutical and medical devices. The Ministry of Environmental Protection has promulgated the amended Measures on Environmental Management of New Chemical Substances (MEP Order No 7) which came into force from October 15^th, 2010. Other guidance has been issued since in supporting of the execution of this regulation. Pesticide safety evaluation and registration system has also been further standardized including certify toxicology/environmental testing laboratories under new rules. The Decree 591, another regulation on safety management of dangerous chemicals had been promulgated by the state council and took effect on the 1 December 2011. This keystone regulation opens a new era for chemicals control in China to convergence with REACH, GHS on dangerous chemical control. In general, these are China’s efforts to the format of US, OECD and other international standards. However, there are indeed some philosophical, technical and practical differences between China and rest of the world, giving the considerations of current situation in China.
This presentation will give an introductory overview of the Chinese regulatory framework of safety evaluation, risk assessment and management in comparison with US and other international standards in regulations, procedures and guidelines. In addition, the presentation will also discuss the new changes and trends in regulatory field in China. Finally, the talk will discuss the challenges and opportunities with personal experience both in the US and China.

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Taiwan Food and Drug Administration (TFDA) was inaugurated on January 1^st, 2010 and superseded four bureaus which are Bureau of Food Safety and Sanitation, Bureau of Pharmaceutical Affairs, Bureau of Food and Drug Analysis, Bureau of Controlled Drug. Since then, TFDA has promoted new strategies to improve the review process for new drug application and a complete life cycle management system was set up to ensure the safety of drug release to market.
In this presentation, we will introduce our new policy and strategies to facilitate the new drug and unmet medical need medication, such as orphan drug, into Taiwan market. Quality enhancement as well as post market risk management system, including a well established ADR reporting system, drug relief foundation, will also be discussed.
New biotechnology development has bring a new era for fighting disease, however, it also bring a great challenge to the regulatory system. An effort to bring the outside experience through international collaboration has also been our top priority goal. Actively involved in APEC activity and establishing the bilateral collaboration agreement, such as Austria and China, has set a good example for future bilateral interaction with other countries.

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Regulatory science has been used to develop tools, standards, and process to assure the efficacy, safety and performance of all products regulated. Thai Food and Drug Administration is the major agency to regulate the health products to protect and promote the safety and quality of all products for the good health of Thai population. The main products are food and drug consumed by Thais and for export to other countries. The regulation of all food products is operated under the Food Act B.E. 2522 (A.C. 1779) and amendment followed. Many types of regulation science are applied, the risk analysis process conformed to international procedure, is the major process used. The risk assessment is the scientific basis for creating management options. The risk management is the policy to evaluate and the decision is made to select an appropriate option for the regulation, such as standard setting, registration process and code or guideline of practice, which is (are) suitable for country. For drug regulation, the process is quite complicated under the Drug Act B.E. 2510 (A.C. 1967) and amendment followed. The data and information on the efficacy test is needed to prove the therapeutic benefit of the product. The safety assessment is also based on the best possible science to assure the safety of the drug which needs comprehensive toxicity testing. Before the drug is marketed, the prove of efficacy and safety in human through the clinical trials according to the best guideline must be performed. Based on this process, the regulation on registration and authorization are established.
Both food and drug regulations, the pre-marketing and post-marketing processes are applied. The pre-marketing process is to assure that the product is good quality and safe for the consumer. While the post-marketing process is the system of monitoring and surveillance on unexpected effect and expected efficacy and quality of the product are followed. The adverse drug reaction (ADR) monitoring program and the food safety monitoring program are established. The data from the monitoring program is then used to improve the regulation for the optimum management. Various guidelines to ensure the efficacy, quality and safety of the products are implemented mandatorily, such as good hygienic practice (GHP), good manufacturing practice (GMP), PICS (in the process of drug regulation) and hazard analysis of critical control point (HACCP).
The application of new available regulatory science is used to modernize, improve and ensure the readiness of the agencies to evaluate the innovative emerging technologies. The strengthening of consumers and professionals is important and help the Thai FDA and related agencies to make proper and appropriate decision on the regulation. The other products under the regulation of Thai FDA and other agencies in Thailand are also based on best possible regulatory science available. The future regulation will be harmonized with the regional as ASEAN and global regulatory process.

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Japanese society of regulatory science, named “Society for Regulatory Science of Medical Products”, has been founded in 2010. Since then discussion has been made on this matter. Regulatory Science is necessary for nonclinical drug development as well as clinical and post marketing phases. We can see drug development as a series of decision making for Go/No go. Nonclinical studies provide data necessary for the decision making throughout all drug development phases. There are regulations like ICH guidelines for these nonclinical studies conducted before New Drug Application. Regulatory Science is necessary for preparation, harmonization and implementation of these guidelines. However, we usually need to conduct additional studies other than required ones in order to solve safety-related issues. Investigative nonclinical studies are essential when unexpected safety findings are found in regulatory required nonclinical studies and/or clinical trials to know mechanism(s) of these toxicities. Data derived from these investigative nonclinical studies are critical for the decision making. Thus Regulatory Science is essential to design and perform these studies. In this presentation, significance of Regulatory Science in nonclinical drug development will be discussed with some examples of our investigative studies.

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Toxicity assessment of nanomaterials (NMs) faces difficulties by its purity/ununiformity, lag in measuring technique, unpredictable/unknown ADME, and hence unpredictable toxicologic endpoints. At least, we know that many of the NMs are biopersistent and chemically stable and therefore chronic toxicity may be more significant than acute toxicity. Although limited in number, there is useful toxicity information of particulate matters (PM) that meets the size definition of nanoparticles. They are the asbestos (mesothelioma/ lung adenocarcinoma) and thorotrast (3-10 nm-sized thorium dioxide emulsion; reticuloendothelial system (RES) deposition with in vivo half life of over 20 years). Some multi-wall carbon nanotubes are 10 to 20 micrometers long and 0.1 micrometer wide and induce mesothelioma when administered intraperitoneally to p53 heterozygous mice. A low dose study suggested non-granulomatous chronic inflammatory microlesions as primary foci for atypical mesothelial proliferation. Fullerene molecules form micrometer-sized aggregates by van der Waals force. Phagocytic cells seem to "digested" the aggregates down to sub-micrometer granules and bring them to RES. Currently these two responses are the initial checkpoints for chronic toxicity of a new nanomaterial. In addition to this, information such as pulmonary fibrosis and/or systemic distribution without overt acute or chronic inflammatory reactions is accumulating. In general, acute responses may not be the direct predictors of chronic toxicity. NMs in an available form should be tested for their chronic pathology, possibly at submicroscopic levels, in a case-by-case approach at least for a while until the elements of the difficulties indicated above resolve. (supported by Grants from MHLW, Japan.)

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Carbon nanotubes (CNTs) are a class of new allotrope of carbon. The cytotoxicity of different diametered multi-walled carbon nanotubes (MWNTs) and different structures of carbon nano material including single-wall nanotubes (SWNTs), MWNTs (with diameters ranging from 10 to 20 nm, MWNT10), and fullerene (C-60) were studied. Different functionalized CNTs may vary from their physical and chemical properties to the biological property; we compared the pulmonary toxicity of different functionalized CNTs in vivo. Meanwhile, the translocation and fate of MWNTs or SWNTs were also studied. Our findings indicate that MWNTs in different diameters exhibited quite different cytotoxicity and carbon nanomaterials with different geometric structures exhibit quite different cytotoxicity and bioactivity in vitro. Water-soluble tau-MWNTs in low and medium doses induce slight and recoverable pulmonary inflammation in mice, and are less toxic than the insoluble raw MWNTs. After mice were exposed by intravenous injection, MWCNTs predominately accumulated in liver and retained for long time. The similar results were found for SWNTs in intravenously exposed mice. These results are useful for optimized application of carbon nanotube and nanotechnology safety.

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We studied the toxicity of ZnO nanomaterials in terms of physicochemical characteristics and reactive oxygen species (ROS) properties. ZnO nanorods [synthesized at room temperature (ZnO-RT, length; 18.0 ± 4.2 nm) and at 60 °C (ZnO-60, length; 80.5 ± 6.8 nm)] were used to evaluate the potential toxicity upon growth velocity-related particle size and shape. The cytotoxicity of ZnO-60 was higher than that of ZnO-RT. We observed that the toxicity of ZnO-RT and ZnO-60 was related with ROS formation by using electron spin resonance. Also, we found that the source of toxicity was not related to Zn²⁺ ions. Our results indicate that toxicity of ZnO nanorods may be due to the amounts of ROS. Our study strongly suggests that size of nanomaterial is not the sole factor to be considered, thus, the development of appropriate criteria based on morphological/physicochemical characteristics as well as synthesis procedures may be needed to evaluate the precise toxicity.

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Quantum dot 705 (QD705) is a cadmium/selenium/tellurium (Cd/Se/Te)-based nanocrystal. The near-infrared fluorescence emitted by QD705 may render it useful as a probe for detecting vasculature and imaging in vivo. To evaluate the safety of QD705 for clinical application, we conducted a series of toxicology studies in mice. First, we investigated kinetics and tissues distribution of QD705 after intravenous injection. Compared with Cd ion, QD705 had a longer retention time in plasma, spleen, carcass and kidneys, and accumulated in spleen, liver, and kidneys up to 6 months. Tissues distribution of QD705 could be predicted by a physiologically-based pharmacokinetic model. Furthermore, QD705 slowly degraded and released toxic Cd ion in the kidneys. Toxicities were observed in the liver and kidneys on week 12 and 16. Because drug delivery via the lung and lung imaging are potential applications of QDs, we also evaluated the biological effects of QD705 in the lungs of mice. Intratracheal instillation of a single dose of QD705 reduced pulmonary function and persistently induced acute neutrophil infiltration, followed by interstitial lymphocyte infiltration and a granulomatous reaction on day 17 and 90. We further observed that Toll-like receptor pathways were important mechanisms for QD705-induced inflammatory reactions in mouse macrophages. In summary, QD705 tended to accumulate in certain tissues with delayed degradation and toxicities in target organs for weeks later after intravenous injection. The lung was a dangerous route of administration for QD705. While some QD705 remained intact after intratracheal instillation, QD705 induced acute and persistent adverse responses in the lung.

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Accumulating evidence demonstrate that hydrogen peroxide (H₂O₂) plays essential function in physiological signaling pathways including growth, proliferation, differentiation, migration, and apoptosis. The dosage, localization, and/or activities of cellular antioxidants are important in determining biological responses initiated by H₂O₂. In spite of the fact that low dose (from 0.1µM to 100µM) of H₂O₂ is required in maintaining the normal metabolism, more specific dosages of them still induce different effects including: (1) under physiological condition, high dose of H₂O₂ protects against injury and to clean xenobiotics in cells and tissues; (2) moderate dose of H₂O₂ maintains cell cycle progression through regulation of many signaling pathways; (3) in some cases, H₂O₂ is also needed to initiate antioxidative enzymes, such as manganese superoxide dismutase (MnSOD) and catalase (CAT), by activating NF-E2-related factor2 (Nrf2), and to stimulate proliferation and differentiation of cells in early stage of embryonic development, injury repairment and tumour progressing. H₂O₂ is involved in various redox-regulated processes and controlled by “multi-antioxidats chain” or “antioxidative enzymes chain”. In this study, we found the down-regulation of H₂O₂ level in hepatocytes undergoes a transition from proliferation to cell cycle arrest during development. The higher and steady level of H₂O₂ is required for maintaining cell proliferative status. Intracellular H₂O₂ plays an active role in control of liver cell growth and liver development. These results challenge the traditional view which considers liver H₂O₂ as a solely deleterious byproduct and may pave the way for redox control in the therapy of liver diseases.

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Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr-Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMCs proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guide wire injury. Exposure of VSMCS to platelet derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMC, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control-VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. Vice versa, activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblast (MEF), compared to Pin1-wild type MEF cells. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3 weeks (1 mg/kg, two times a week), significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMCs proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.

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A recent study suggested that simultaneous exposure of non-diabetics to high levels of dioxins and mercury increases their risk of insulin resistance. An increased incidence of diabetes existed in patients with documented Minamata disease (methylmercury poisoning) in Japan. Previous in vitro studies have shown that HgCl₂ altered intracellular Ca²⁺ homeostasis in pancreatic β-cells isolated from mouse islets, and decreased insulin secretion from secretion granules isolated from toadfish islets. Mercury is a well-known toxic metal, which induces oxidative stress. The toxicity of mercury in islets is highly related to oxidative stress. It has been shown that 8-hydroxy-2’-deoxyguanosine, a biomarker of oxidative DNA damage, is significantly elevated in urine samples of people from mercury-contaminated areas. Our studies have also shown that submicromolar-concentration HgCl₂ or methylmercury is capable of affecting the islet β-cell function and survival through an oxidative stress pathway in vivo and in vitro. Low-dose mercury induced mouse pancreatic islet β-cell dysfunction through a phosphoinositide 3-kinase (PI3K)-activated or oxidative stress-triggered Akt pathway in cell culture and animal models. Antioxidant N-acetyl-L-cysteine prevented mercury-induced insulin secretion inhibition and Akt phosphorylation. Moreover, methylmercury could induce oxidative stress-triggered β-cell apoptosis and death. HgCl₂ could also be capable of inducing the oxidative stress-related insulin secretion suppression and cell death in pancreatic β-cells. The further evidences indicate that HgCl₂ enters β-cells and triggers oxidative stress to induce cell death through both apoptotic and necrotic pathways. Taken together, these observations provide evidences to confirm the possibility that mercury is an environmental risk factor for diabetes.

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Oxygen serves as an electron acceptor, enabling efficient production of ATP. However, oxygen can also be converted into toxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H₂O₂); ROS can damage a variety of cellular components, including proteins and unsaturated lipids. Detection of ROS (peroxides) is an important step in the oxidative stress response.
Peroxiredoxin (Prx) is a family of ubiquitous peroxidases found in species ranging from Escherichia coli to humans. In many cases, Prx can reduce H₂O₂ and/or alkylhydroperoxides at the expense of electrons from NADPH through the Trx-dependent redox system (Tpx activity). The catalytic cysteine (Cys) residue of Prx is directly oxidized by hydroperoxides. This oxidation event is followed by the formation of a disulfide bond linkage with a resolving Cys of the same molecule of Prx or with a resolving Cys of another Prx molecule (dimer formation). The disulfide bond can be reduced by Trx. We have suggested that, in addition to their function as peroxidases, Prx family proteins may serve as intrinsic receptors of H₂O₂ and possible other hydroperoxide. Additionally, Prx proteins may relay information about the presence of hydroperoxides to the independent target proteins of each Prx.
In this symposium, I will introduce our recent progress in both yeast and mammalian systems that underscores the role of Prx on hydroperoxide and the redox-dependent modulation of the Prx’s target proteins.

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VOCs are volatile organic compounds ubiquitously present in the environment as well as in various consumer products. Of VOCs, benzene, toluene, ethylbenzene, and xylene (BTEX) are the major chemicals investigated in this study although other critical VOCs are numerous.
In general, humans are exposed to VOCs not by single chemical but by mixture compounds such as BTEX and others. Therefore, risk assessment especially for VOCs should be carried out for mixture exposure scenario in consumer products. However, the real situation to perform the risk assessment for VOCs is entirely based on single chemical exposure due to lack of toxicological data for mixture compounds. Given the limited situation, risk assessment of VOCs showed that correction fluids and adhesives are of concern for benzene exposure exceeding risk limit of 1.0E-06, and ethylbenzene and xylene far exceeded hazard index (HI) of 1, which is a criteria for safe limits of human exposure. DEHP and DBP were also analyzed in cosmetics and human exposure level was assessment. HI values of DEHP and DBP due to exposure to cosmetic products did not exceed tolerable daily intake (TDI) or refenence dose (RfD), showing less than 1 (HI < 1). These data suggest VOCs and EDCs investigated in this study were shown to be safe although continuous efforts need to be made to monitor regularly and development of risk assessment methodology for mixture exposure to VOCs or EDCs.

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Cosmetic products are unique, as diverse as foods and drugs, and have a history covering thousands of years with the use of many ingredients from natural sources but present technology has led these products to be introduced by many synthetic ingredients. Cosmetic products play an essential role in people’s self-esteem by helping them feel confident in appearance from looking good and smelling nice. They have a huge impact on users. Excluding perfumery and decorative cosmetic products, many items of hair care, skin care, oral care and toiletry products are used daily among most population groups. Like foods and drugs, cosmetic products that put on the market must not cause damage to human health when applied under normal or reasonably foreseeable conditions of use. In practice, cosmetic products have rarely been associated with serious hazards but this does not mean these products are safe especially when used on daily basis and extensively over a large part of body. The fact that cosmetic products compose of a number of chemicals and some of them may affect health. Besides, ingredients used in cosmetic products are generally not tested for long-term results. The accumulation from an alarming amount of some chemicals in the products that can be absorbed into skin or breathed into lungs through continuous use may ensue with serious health problems including cancer. With the modern technology dealing with cosmetic products e.g., nanotechnology, it is needed to concern more on the toxicological aspects, both short and long term toxicities, in cosmetic products.

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Background: Nonylphenols (NP) has been proven as an environmental hormone with estrogenic effects. The influence of human NP exposure is still unclear. The aim of this study is to explore the NP exposure effect on pregnancy outcomes. Methods: Urine samples of pregnant women in the first, second, and third trimester of gestation were collected respectively. They were asked to complete a structured questionnaire to collect the demographic and disease history information. Maternal urinary NP concentration was determined by high-performance liquid chromatography coupled with fluorescent detection. Birth outcomes were measured immediately by pediatrician after delivery. Results: The total number of participants was 201. Creatinine-adjusted urinary NP concentration were 7.27 ±10.08 ug/g, 6.43±8.78 ug/g, and 6.10±5.79 ug/g for the first, second, and third trimester, respectively (p >0.05). For 163 out of 201 pregnant women who were completely followed-up until delivery, above- median levels of urinary NP concentration in the second trimester had significantly decreased maternal weight gain (β=-1.39 kg, p=0.04) as well as neonatal birth length (β=-0.55 cm, p=0.02). The odds ratio of small gestational age on pregnant women with below-median levels of urinary NP concentration was 7.28 fold (95% CI=1.03-51.74). Conclusion: Maternal NP exposure levels in the second trimester are associated with a risk of decreased maternal weight gain and neonatal birth outcomes. This study indicates the 2nd trimester of pregnancy may be the most critical stage of protection from endocrine disruptors’ exposure.

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Traditionally in the food safety, risk assessment is based on deterministic endpoints, i.e., use of the no observed adverse effect level (NOAEL) and the mean or high level of exposure. Methods to assess the dose responses of toxicity assays have evolved beyond just determination of a NOAEL. Further, as the available data allow, probabilistic and distributional methods can be used to characterize the hazard(s) as well as the exposure(s). These approaches allow for more description of variability in the population and uncertainty in the risk estimates. Additional risk assessment outcomes are also used and reported, such as the margin of exposure (MOE), which gives a relative indication of the level of health concern without actually quantifying the risk. These expansions of risk assessment tools and the information they provide may require additional consideration on the part of risk managers as they evaluate risk management options. Therefore, the choice was made to include discussion on the factors of a risk assessment outcome which could be taken into account in the choice for a relevant risk management option. The example highlighted the importance of establishing maximum levels for arsenic in rice as new work by the FAO/WHO Food Standard Programm. China as the lead country of the new work had been requested to develop a paper to explain whether the MLs would be for total or inorganic arsenic. The work focused on the following aspects: 1) The analytical methods for total and/or inorganic arsenic currently in use, and collaborating or performance test reports at national or international level. 2) Available raw data for total and/or inorganic arsenic in rice used to produce the distribution curve. 3) It is preferable to set MLs specifically for inorganic As rather than total As.

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Environmental factors are thought to be critical problems for the increase in allergy including bronchial asthma and atopic dermatitis (AD). Especially, environmental chemicals should be an important participant in the environmental factors. For example, phthalates, ubiquitously used as plasticizers in many polyvinylchloride consumer products, have become widespread in the environment. Several epidemiological studies have suggested that exposure to phthalates may be associated with development of asthma, wheezing, and allergic symptoms. Experimental studies have shown that phthalates have an adjuvant effect on Ig production in mice.
In our previous study, we have shown that phthalates aggravate AD-like skin lesions induced by mite antigen in atopic-prone NC/Nga mice and the aggravation is consistent with eosinophilic inflammation, mast cell degranulation, and chemokine expression in inflammatory site. In addition, we have found that in vitro exposure to phthalates enhance the expression of cell surface activation markers on bone marrow-derived dendritic cells and their production of chemokines, as well as their capacity to stimulate mite antigen specific T-cell proliferation and also enhance IL-4 production and cell proliferation of splenocytes. These results demonstrate that phthalates can aggravate AD-like skin lesions and the mechanisms underlying the aggravation might be partly mediated through the activation of dendritic cells and through direct or indirect activation of T-cells. This presentation will focus on the function of immune cells in the aggravation of allergic diseases by environmental chemicals such as phthalates.

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Cisplatin is one of the most active drugs for the treatment of cancers. However, cisplatin-induced nephrotoxicity is the most common problem. Although the counteracting apoptotic and survival pathways are balanced as the injured cells either undergo apoptosis or proliferate, the switch molecule of this balance is unclear. This study investigated the expression of Phlda3 as a potential biomarker of renal tubular injury induced by cisplatin in vivo and in vitro, and its functional role in cisplatin-induced toxicity. Phlda3 transcript and protein levels were highly up-regulated in the kidney of mice after cisplatin treatment, which preceded increases in BUN or serum creatinine. Particularly, the level of Phlda3 transcript was substantially increased at an early time, and remained elevated. Treatment of NRK52E with cisplatin caused increases in Phlda3 mRNA and protein. Knockdown of Phlda3 reversed the decrease in cell viability by cisplatin. Cisplatin treatment elicited p53 accumulation via Akt/Mdm2 repression. Phlda3 deficiency attenuated a decrease in Akt phosphorylation by cisplatin, and prevented p53 accumulation. Hence, Phlda3, which leads to p53-mediated tubular cell death, may be an early and sensitive biomarker of cisplatin-induced kidney injury. To assess the effect of Phlda3 inhibition on the anti-cancer effect of cisplatin, we determined whether Phlda3 knockdown diminishes its anti-cancer effect. Cisplatin elicited SKOV-3 cell death, which was not reversed by Phlda3 knockdown, implying that the method to inhibit Phlda3 may be of use in decreasing kidney toxicity without losing the anti-cancer effect of cisplatin.

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We have previously demonstrated that long-term low-dose sodium arsenite exposure induced cellular transformation and aberrant gene expression in human urothelial cell lines. Since epigenetic alterations play critical roles in the regulation of gene expression, we performed systematic analysis to compare the DNA methylation profiles between arsenic-exposed human urothelial cells and untreated cells. By aid of the Infinium Assay, we selected 61 genes with promoter methylation differences between parental and arsenic-exposed cells over 75%. Among them, 40 genes showed hypomethylated in arsenic-exposed cells, whereas 21 genes hypermethylated. To identify genes whose expression associated with DNA methylation, we performed the quantitative real-time PCR analysis after treatment of cells with 5-aza-2'-deoxycytidine. The results showed that 76% of hypomethylated gene expression levels in arsenic-exposed cells are higher than parental cells. However, only 42% hypermethylated genes consistently expressed lower levels of transcripts in arsenic-exposed cells than control cells. Bisulfite sequencing analysis was performed to confirm the methylation status in arsenic-exposed cells. In all of the genes analyzed in this study, lipocalin-2 (LCN2), also known as oncogene 24p3 or NGAL, is one of the most highly expressed in long-term arsenic-exposed cells as compared to-untreated cells. By siRNA technique, we found that silencing of LCN2 in arsenic-exposed cells exhibited reduced oncogenic potential and decreased production of pro-inflammatory cytokines. Taken together, our present results showed that long-term low dose arsenic exposure could be through epigenetic mechanism to alter the gene expression profile and hence induce cell transformation.

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MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function primarily as oncogenes and tumor suppressors by mediating translational repression or mRNA degradation via binding target genes. In this study, malignant human bronchial epithelial cells transformed by anti-benzo[a]pyrenetrans-7,8-diol-9,10-epoxide were used to help characterize the possible mechanisms of miRNA function in chemical carcinogenesis. The expression level of miRNAs was measured by microarray analysis and real time PCR. We used the specific miRNA inhibitors and mimics to downregulate or upregulate miRNA activity in malignantly transformed cells to determine the effects of miRNAs on the biological properties of the cell. Silencing miR-106a, miR-494 or miR-22 by transfection with the inhibitor suppressed cell proliferation, induced cell cycle arrest and apoptosis, and inhibited anchorage-independent growth and tumor growth in nude mice. Increasing the expression of miR-106a, miR-494 or miR-22 in malignantly transformed cells by transfection with the mimic gave the opposite results. Bioinformatic analysis showed that tumor suppressor RB1 is one of predictive targets of miR-106a, and PTEN gene is a target of miR-494 and miR-22. We confirmed these targets by western blot and luciferase reporter assays. These findings suggest that miR-106a, miR-494 and miR-22 might function as oncogenes in transformation induced by a chemical carcinogen. Our study on miR-506 revealed that miR-506 acts as an anti-oncogenic miRNA by regulating Ras in malignantly transformed cells. The identification of oncogenic and anti-oncogenic miRNAs could provide new insight into the molecular mechanisms of chemical carcinogenesis.

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Although inorganic arsenic is a well-known carcinogen associated with urinary bladder, liver, skin, and lung cancers in humans, the underlying mechanisms are not well understood, partly due to lack of appropriate animal models. We have demonstrated carcinogenicities of various organic metabolites of arsenic using various rat models. We showed for the first time that dimethylarsenic acid (DMA^V), a major metabolite of arsenic in humans, is a complete urinary bladder carcinogen, and exerts promotion effects on liver carcinogenesis in rats as well. In the comparison study of urinary bladder carcinogenicities of DMA^V with other arsenic compounds, we identified dimethylmonothioarsinic acid (DMMTA^V), a new urinary metabolite. Furthermore, we demonstrated that DMMTA^V is majorly produced from DMA^V by intestinal bacterial in colon and is an ultimate carcinogen in DMA^V-induced rat bladder carcinogenesis. Our findings provide experimental evidence for arsenic carcinogenicity and will facilitate both the understanding of the carcinogenic mechanisms of arsenic and the development of model in arsenic risk assessment.

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A549 and H460 cells are representative cell lines of NSCLC with the same histology and great differences in invasive behavior as well as in response to ionizing radiation exist between two cell lines. Because these cell lines exhibit an intrinsic resistant difference to both radiotherapy and chemotherapy, in this study, we showed that a variety of genes, highly differently expressed by methylation extent of promoter region of this gene in A549 and H460 cells, modulates cell growth, metastasis and sensitization of cells to the detrimental effects of damaging agents such as γ-radiation Therefore, through the genome-wide screening, it is necessary to determine appropriate candidate genes involved in the radiation resistant characteristics to improve therapeutical efficiency and thus result in reduction of malformation risk of normal. EFEMP1, ALDH1, TSPYL5, SM22α and TM4SF4 are representative genes and proteins that determine the characteristics of cancer stem cell like cells. [The Authors wish to acknowledge the financial support of the Ministry of Education, Science and Technology (Nuclear Research & Development program) of the Republic of Korea)

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Environmental radioactivity could arrive from various sources and can incur higher health risks to those receiving the exposure. One of the most atypical and non-conventional exposure from man-made radioactive sources to a general public occurred in Taiwan in early 1980s, which were developed through recycled steels and more than 20,00 tons of radio-contaminated steel parts and rebar, made their ways to civilian constructions like apartments and school classrooms in a few years since their production. More than 10,000 occupants and students were noted to have stayed in these buildings since then until their discoveries by governmental and civilian societies and individuals from 1992. These individuals received untold protracted low dose-rate gamma-irradiation periodically and daily for up to 10 years, and cumulatively with several to more than 1,000 folds of extra ionizing radiation exposure above the background. Researches since then have identified significantly higher risks of specific malignancies, reduced reproductive fecundity, higher frequencies of lens opacities, increased incidences of benigh thyroid enlargement, and much higher frequencies of cytogenetic damages in peripheral the peripheral lymphocytes, shown as chromosomal abnormalities and micronuclei frequencies. Several of these studies evidenced a none- or lower- thresholds of ionizing radiation exposure on human subjects. Discussion will be provided to elaborate their implications for radiation safety and environmental health for the public.

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