xii international congress of toxicology. 19-23 july 2010 ... · xii international congress of...

XII International Congress of Toxicology. 19-23 July 2010 Barcelona, Spain. (ABSTRACT ADENDA)

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KL04 WHAT MAKES A DRUG TO BE HEPATOTOXIC?: HOW NEW TECHNOLOGIES CAN HELP TO BETTER UNDERSTAND THE PHENOMENA

J. V. Castell. Unit for Experimental Hepatology. Research Institute of the University Hospital La Fe, Valencia, Spain

Drug-induced liver injury (DILI) represents a major clinical issue that accounts for the majority of acute liver failures, and is also the major cause of withdrawal during drug development. Therefore, in continuing efforts to improve the identification of potential hepatotoxicity in the process of drug discovery, better understanding of the mechanisms of toxicity is necessary to develop new and predictive assays. In vitro systems are an important component of these new assay system applications. Use of in vitro systems is not new to investigative toxicology or lead optimization processes. Drugs can cause liver injuries by many different mechanisms, which involve direct effects on hepatic metabolic routes, generation of reactive drug-derived intermediates, ROS and adduct formation. Chronic as well idiosyncratic toxicity remains largely undisclosed by most screening methods. Unpredictable, low-frequency, idiosyncratic reactions often occur on a background of a higher rate of mild asymptomatic liver injury. Recent and future advances in toxicogenomics and proteomics should improve the identification of risk factors and the understanding of idiosyncratic hepatotoxicity. A major challenge for mechanistic classifications is that DILI is not sufficiently characterized by the initial injury, frequently involves several mechanisms with complex interactions among them, and the quantitative contribution of the different processes leading to DILI in humans remains largely unknown. This explains why drug hepatotoxic potential is often not properly recognized before marketing. A possible approach to this dilemma is a multiparametric strategy integrating information from functional (cytomics) and metabolic parameters (toxico-metabonomics) of the different specific mechanisms of liver cell injury. Deichmann Award EPIGENETICS: THE LAST FRONTIER BEYOND GENETICS

M. Esteller. Bellvitge Institute for Biomedical Research (IDIBELL), Barcelona, x, Spain We are not our genes. Genes are just part of the story. We cannot fully blame our genome for our behavior and susceptibility to disease. We are our proteins (and our carbohydrates, fat, etc…). This more precise definition relates to the central dogma of molecular biology, in that our proteins are generated from our DNA via an RNA intermediate state. These RNA molecules are transcribed into proteins, however some RNA molecules do not encode proteins (the non-coding RNAs) and have developed very specific and important cellular functions. The base pair nucleotide sequence of DNA, the typical subject of study of the classical genetics, cannot completely explain the functionality of our cells, their disruption in complex diseases or the definition of our species. We need something else. Part of the explanation is provided by the field of Epigenetics. Waddington defined Epigenetics in 1939 as "the causal interactions between genes and their products, which bring the phenotype into being". From our current knowledge we can define Epigenetics as "the inheritance of DNA activity that does not depend on the naked DNA sequence". This "inheritance" is most simply understood during mitosis, the process of transmission when a cell divides to produce daughter cells during the cell cycle; or even, an in a more provocative manner, during meiosis, in our germ cells and thus the epigenetic information is transmitted to our progeny. Epigenetics is not a "unmaterial" area as it refers to the dynamic chemical modifications that occur to our DNA, and its subsequent association with regulatory proteins. The most recognized epigenetic modifications or "marks" are the addition of a methyl-group to our DNA, and the post-translational modifications to histones. The recognition of epigenetics to human disease initiation and progression started in oncology, but now includes other disciplines such as neurodevelopment and neurodegenerative disorders, psychiatric entities, cardiovascular diseases and autoimmune pathologies. AWL VENOM INDUCED CONSUMPTION COAGULOPATHY - UNDERSTANDING AN IMPORTANT MANIFESTATION OF SNAKE ENVENOMING

G. K. Isbister. University of Newcastle and Department of Clinical Toxicology and Pharmacology, New South Wales, Australia Discipline of Clinical Pharmacology, University of Newcastle and Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle, New South Wales, Australia, x, Australia Venom induced consumption coagulopathy (VICC) is the most common coagulopathy associated with snake bite and results from the activation of the coagulation pathway by procoagulant snake toxins, including thrombin-like enzymes, prothrombin activators and factor X activators. VICC is often referred to as disseminated intravascular coagulation because of the elevated D-dimer, prolonged prothrombin time and low fibrinogen. However, VICC differs and is not associated with other important features of disseminated intravascular coagulation, such as evidence of systemic microthrombi and end organ failure. Limited information exists on the dynamics of haemostasis in patients with VICC. Recent work has provided insight into the factor deficiencies, time course and role of treatment in VICC resulting from prothrombin activators in Australian elapid venoms. VICC was characterised by depletion of fibrinogen, factor V and VIII, as well as international normalised ratio and activated partial thromboplastin times exceeding the upper limits of detection within 2 hours of snakebite. Curiously, prothrombin levels never fell below 60% of normal, indicating that the toxins were quickly eliminated or inactivated and re-synthesis of clotting factors occurred. The rapid onset of VICC suggests that antivenom cannot be practically administered in time to prevent VICC which is supported by a model of the coagulation pathway. Analysis of the time to recovery of VICC has shown that time to the administration of


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antivenom does not appear to affect the time to recovery of VICC while the administration of factor replacement, mainly fresh frozen plasma, does speed the recovery of VICC. In vitro studies demonstrate that the commercial antivenoms are highly efficacious in neutralising the procoagulant effects of these venoms. At least for Australasian snakebite the role and effectiveness of antivenom and factor replacement in VICC may require reassessment, despite the efficacy of antivenom in neutralising the procoagulant effects of the venoms. W5 NEW LABEL-FREE ASSAYS USING THE XCELLIGENCE SYSTEM IMPROVE EMBRYOTOXICITY AND CYTOTOXICITY TESTING OF HEPATIC CELLS AND CARDIOMYOCYTES. THREE STUDIES WILL BE PRESENTED SHOWING INNOVATIVE POWERFUL WAYS TO INVESTIGATE CYTOTOXICITY Talk 1: Embryotoxic effects of the marine bivalve toxin, okadaic acid, were identified using different murine embryonic stem cells

Alfonso Lampen, Sandra Stempin, Regina Al-Hamwi, and Anke Ehlers. Department Food Safety. Federal Institute for Risk Assessment. Berlin

Okadaic acid (OA), a marine toxin produced by dinoflagellates, can accumulate in various bivalve molluscs. In humans, consumption of OA induces acute toxic effects like diarrhoea, nausea, vomiting and abdominal pain. OA is a potent inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A), enzymes that are known to be critical regulators of embryonic development. To determine the embryotoxic potential of OA, we performed two independent cellular in-vitro assays, both of which are applicable for the detection of teratogenic compounds: i) the validated embryonic stem

cell test (EST) based on the morphological analysis of beating cardiomyocytes in embryoid bodies and ii) the F9 cell assay quantifying the induction of cell differentiation by measuring the emitted luminescence of a reporter gene. In the presence of OA, beating cardiomyocytes in the EST were inhibited and the reporter gene in transiently-transfected F9 cells was activated. Furthermore, OA treatment led to rapid morphological changes including cell rounding, the loss of cell-cell contacts and changed electrical impedance as monitored in real-time by the xCELLigence system. The two independent bioassays (EST and F9 cell test) detected OA as a potential embryotoxic compound, since OA influences the differentiation process of cultured murine embryonic cells. Talk 2: Screening for compound-induced hepatotoxicity by impedance-based real-time cell monitoring

Stefan Kustermann. F.Hoffmann-La Roche Ltd., Pharmaceuticals Division, Basel One of the major reasons for withdrawal of pharmacological compounds from drug development are adverse effects such as organ toxicities. Cell-based assays are a key tool for the assessment of compound-induced toxicities at an early stage of drug development, during compound selction and well before animal testing. Prediction of hepatic toxicity – the most frequent organ toxicity - by using in vitro assays has been a major challenge so far. To date, the cellular assays used are endpoint assays where the drug-effect is measured at a single defined time point. Thus, choosing the right time point for the in vitro assay is critical and some effects may remain undetected because they develop either before or after the endpoint measurement is performed. A tool which enables measuring cellular behavior over the entire length of the experiment therefore bears the potential of generating in vitro data of higher quality. Such a tool is provided by the xCELLigence Real-Time Cell Analyzer (RTCA) System. This microelectronic device enables label- and hands-free dynamic monitoring of cellular events in a 96-well format over an extended period of time based on an impedance readout indicative of cellular alterations such as proliferation, cell death, adhesion and spreading. By using the xCELLigence technology we monitored compound-induced effects on hepatic cells. The information obtained from impedance readouts over the entire length of the experiment allowed timed decisions for the administration of compounds as well as down-stream analysis, such as biochemical assays or gene expression analysis. In many cases, CI proved to be a highly sensitive indicator for drug-induced toxicity at low concentrations and at earlier time points as compared to established standard measurements such as release of lactate dehydrogenase, reduced glutathione or ATP content. Taken together, the xCELLigence technology may significantly improve the quality of toxicological in vitro tests due to extended time resolution and obviously increased sensitivity compared to standard endpoint measurements. Because the impedance measurement is non-invasive, the xCELLigence system does not prevent the use of any additional readout. Impedance measurements therefore could improve the quality of in vitro assays widely used in preclinical development. Talk 3: Cardiotoxic and proarrythmic compounds were tested using stem cell-derived cardiomyocytes to measure rapid kinetic responses and long-term compound effects Yama A. Abassi. ACEA, Cell Biology and Assay Development Cardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse cardiac effects earlier in the drug discovery process. The major bottleneck for in vitro predictive cardiac toxicity screening has stemmed from lack of a reliable cellular model system available in large enough quantities for low to medium throughput assays and the unavailability of a reliable detection system. In this presentation we describe the xCELLigence RTCA Cardio System, which is a 96 well microelectronic-based detection system that can be employed for screening of drug-induced arrhythmia and toxicity using stem cell derived cardiomyocytes. This novel monitoring system can detect both fast (millisecond kinetics) and long-term drug effects (days to weeks) on cardiomyocytes from the same well and the same group of cells. Furthermore, this system can also be used to quantitatively assess endogenous GPCR responses expressed within stem cell-derived cardiomyocytes. We have tested the utility of this approach in in vitro pre-clinical safety assessment by dose-


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response screening of more than 10 cardiotoxic drugs withdrawn from the market due to interaction with hERG channels in addition to screening a number of inhibitors of voltage-gated calcium channel, K channel and sodium channels. In addition to stem cell-derived cardiomyocytes, we will also show data using our microelectronic monitoring system with primary cardiomyocytes isolated from rat and murine systems. In summary, the convergence of stem cell technology together with the microelectronic monitoring system we describe here should facilitate early cardiac safety assessments for screening of pro-arrhythmic drugs as well as drugs affecting the viability of cardiomyocytes. W06 THE MARMOSET AS A MODEL FOR THE EVALUATION OF NOVEL PHARMACEUTICALS AND BIOTECHNOLOGY-DERIVED PRODUCTS

I. Andreini (1)

, D. D. Rees (2)

, G. Oberto (1)

, A. Orsi (2)

. (1)

RTC, Italy, x, Italy; (2)

Salupont Consulting, UK

Over recent years Callithrix jacchus (common name marmoset) has been utilised as a non-rodent species for the development of pharmaceutical products in disease areas such as infection, neurology, immunology and also for biotechnology-derived products, gene therapies and vaccines in general. Marmosets have several characteristics that make them a key species for use in the development of these products. In addition, the ability of the marmoset to be utilised in pharmacodynamic, toxicity and pharmacokinetic studies provides a strong interconnected relationship between these disciplines, providing a more cohesive dataset. Marmosets breed well in captivity with good reproductive efficiencies and their sexual maturity is reached within 18 months of age allowing for rapid expansion of existing colonies. Their relatively small size compared to macaque species may represent a further potential advantage and translates into smaller caging and ease of handling. Furthermore, reduced body mass should substantially decrease material costs and development time. From an ethical perspective there are several benefits in using the marmoset, as their temperament facilitates the provision of a more complex and enriching environment, being less destructive than larger non-human primates (NHPs) such as the macaque. Their greater wellbeing from this environment should translate into more consistent and reliable results and over the last few decades an extensive database has also been generated providing essential background data to support this. Looking towards the future, with the growth of biotechnology-derived products and gene therapies, it is anticipated that the use of NHPs will also grow. The marmoset offers potential benefits over other NHPs in specific circumstances in light of their genotype, phenotype and physiology. W07 ROLE OF IN VITRO AND IN SILICO ALTERNATIVE METHODS AND GLOBAL IMPACT FOR REGULATORY PROCESSES (ROUND TABLE WITH REPRESENTATIVE INSTITUTIONS)

A. Castaño. REMA (Spanish Platform for Animal Alternatives), Spain

The aim of this round table discussion is to analyse and discuss how we can speed up the regulatory acceptance for an integrated alternative testing approach, analyse the strengths and weaknesses and propose a way forward for getting approval by the regulatory community. In vitro alternative methods are at a cross road. The old paradigm of one-to-one replacement, through years of development, had demonstrated to not be fully successful except for topic toxicity, and in vitro methods has not reach complete acceptance as replacement of animals as originally expected. In addition, regulatory demands, like REACH in Europe, have dramatically increased the need for testing of chemicals. In the same line, the European 7th Amendment to the Cosmetics Directive is asking for a replacement of animals in testing of cosmetic finished products and its ingredients. The partial failure of the old paradigm and the new demands are pushing non animal alternative testing in a new direction and towards a new paradigm. Today we are looking at a combination of approaches in testing strategies, including in silico methods like quantitative-structure-activity-relationships, read across and pharmaco-kinetic models, alternative methods based on primitive organisms or early life stages, which are ethically acceptable, and refined in vivo animal test. The process for a testing method to get regulatory acceptance is long and cumbersome and it will likely take many years to reach acceptance for any alternative based testing approach. Experts from the cosmetics, chemical, and pharmaceutical sectors, the European Consensus Platform for Alternatives, and ECVAM will participate in the discussion. CEC-2 REGULATORY TOXICOLOGY AND RISK ASSESSMENT PROCESSES FOR PREVENTATIVE AND THERAPEUTIC VACCINE DEVELOPMENT

L. Segal (1)

, S. Gould (2)

, M. Gruber (3)

, Y. Sun (4)

, M. Matsumoto (5)

, R. Forster (6)

, P. Barrow (7)

, M. Pallardy (8)

, A. Penninks

(9).

(1) GSK Biologicals, Wavre, Belgium, x, Belgium;

(2) Sanofi Pasteur, Marcy L’Etoile, France;

(3) US FDA, CBER, USA;

(4) Paul-Ehrlich Institut, Federal Agency for Vaccines and Biomedicines, Germany;

(5) Office of Biologics, PMDA,

Japan; (6)

CIT, Evreux, France; (7)

Ricera Biosciences, Saint Germain sur l'Arbresle, France; (8)

INSERM 996, Faculte de Pharmacie, Universite de Paris-Sud 11, France;

(9) TNO, Netherlands,

Vaccines are considered one of the safest and most effective medicines today, and are a growing area of interest and development activity in the pharmaceutical industry. Vaccines, which initially were crude preparations, such as vaccinia vaccine now comprise a diverse range of products, including recombinant proteins, live attenuated products, toxoids, and vector based DNA vaccines. Indication for these products include prevention of infectious diseases as well as therapeutic benefit against disorders such as cancer and HIV. In addition, modern vaccine formulations may


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be complex and contain novel adjuvants, excipients, multivalent antigens and be delivered by novel routes and/or delivery systems. The safety evaluation of vaccines is vital, particularly for preventative vaccines, which are often given to healthy children and adults, placing special emphasis on their safety. The diversity of modern vaccine products requires that the safety evaluation must be specifically tailored to the investigational vaccine product under study. This course aims to address the key issues regarding the nonclinical safety assessment required for vaccines, and will provide a clear overview of the current global regulatory guidelines, plus present an overview of the toxicological studies required to support both first time in man studies and further clinical development and/or licensing. The course will provide an overview of study design and species selection for local tolerance, single and repeated dose toxicity, safety pharmacology, biodistribution and integration studies, as well as, fertility, reproductive and developmental toxicity bioassays. In addition, the course will also consider other factors specifically related to the hazard assessment of vaccines, including hypersensitivity and autoimmunity and environmental safety assessment. In addition, the requirements for safety assessment regarding the presence of residuals, contaminants, leachables and extractables will also be considered. The course would be of interest to a wide audience including students, clinicians, consultants, regulators as well as anyone currently employed in the pharmaceutical field with limited familiarity of vaccines. It would provide an introduction into the safety consideration of vaccines from a toxicology perspective. S03-4 ASSESSMENT OF EXPOSURE TO AIRBORNE NANOPARTICLES IN AN OCCUPATIONAL SETTING

G. Kasper. Institut für Mechanische Verfahrenstechnik und Mechanik Karlsruhe Institute of Technology, Germany

Airborne NP are highly dynamic systems which undergo rapid changes in size distribution and concentration between source and human receptor. These changes and their consequences for workplace measurements, for human exposure, as well as for the design of protective devices such as face masks will be analyzed on the basis of several likely exposure scenarios in a typical work place environment. The analysis is based on aerosol dynamic concepts of collisional growth kinetics and dilution and supported by models and actual measurements in a test chamber. The cases discussed will include (1) a relatively strong source of "true" NP (10 nm) at concentrations well above the general background aerosol; (2) the fate of NP emitted at concentrations on the order of or even below the concentration of the normal background in a workplace. It will be shown that binary coagulation between NP and background aerosol particles is generally faster than self-coagulation among emitted primary NP, except in the unlikely case of extremely high source strengths, and then only locally. The general background aerosol is thus an effective scavenger for airborne NP on a relatively short time scale of about 10 minutes.Based on experimental evidence of the fragmentation of such agglomerates, one may assume that - from a toxicological or chemical perspective - such attached NP have not "disappeared", even though they will no longer be detectable in the size range of the original source, nor will their original size be relevant for estimates of lung deposition. Given the typical size range of common aerosol backgrounds (a few hundred nm) (the typical "most penetrating particle size" of the human lung, one has to assume that attached NP enter deeply into the human airways. S03-5 CARBON NANOTUBES - CHALLENGES FOR ASSURING SAFETY OF NANOTECHNOLOGIES

K. Donaldson. MRC Centre for Inflammation Research, University of Edinburgh, x, United Kingdom In order to ensure safe use of carbon nanotubes we need to understand how they act in the body so that regulation can be focused on the harmful fraction (the biologically effective dose). We have been addressing especially the pathogenicity of long and short multi-wall carbon nanotubes (MWCNT) in relation to the unique hazard posed to the pleural mesothelium by asbestos e.g. mesothelioma. There is persuasive evidence that a fraction of all deposited particles reach the pleura and that a mechanism of particle clearance from the pleura exits, through stomata in the parietal pleura. We therefore contend that injection into the pleural space is warranted as a model that mimics the true translocation of a fraction of deposited CNT to the pleural space. We injected the same panel of fibres used in the peritoneal cavity in the Poland studies (Poland et al Nature Nanotechnology 2008), i.e. long and short amosite asbestos samples, two long nanotubes samples and two short/tangled nanotubes samples and nanoparticulate carbon black as a graphene control. Following injection, the pleural cavity was lavaged to determine the inflammatory response and the chest wall examined for evidence of inflammation and fibrosis. We found clear evidence of length-related inflammation in the pleural space with both the long amosite and the two long nanotubes samples causing inflammation while all the other short samples failed to elicit significant inflammation. The retention of long fibres occurs at stomata on the parietal pleura that drain to the mediastinal lymph nodes. Particles and short fibres that reach the pleural space exit through the stomata; however, long fibres cannot negotiate the stomata and are retained there leading to inflammation and pleural pathology including mesothelioma. S06-5 THE ENVIRONMENTAL HEALTH AND CONTAMINANTS IN SOUTH AFRICA

H. B. Röllin, J. Ø. Odland, T. M. Sandanger. South African Medical Research Council, South Africa Food security, global disease patterns and health impacts of exposure to persistent toxic substances may be affected by climate change directly impacting on the living environment and on human health. It is expected that climate change will affect the global distribution of contaminants in the near future, thus increasing their detrimental health


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effects in general populations, affecting particularly their reproductive health. Several multidisciplinary international projects are currently evaluating the sources and levels of contaminants in populations residing in different geographical regions and ascertaining the relationship between levels of these chemicals and health. To date, no comprehensive data exist on the levels of these contaminants in ecosystems and humans in the Southern Hemisphere and l very few studies have measured concurrently the wide spectrum of pollutants in the most susceptible population group, namely delivering mothers. South Africa, located in the southernmost part of Africa, is both a developed and a developing country; hence it is of particular importance to global research. Within this context, findings of the South African studies will be presented. Studies measured levels of contaminants (metals and organic pollutants) in blood of delivering mothers as an indication of the potential risk to the developing foetus and child, in various geographical locations of the country thought to be exposed to different environmental contaminations. Research conducted to date has found statistical differences between the maternal levels of some metals and persistent organic pollutants, in the various geographical areas. Of particular interest are the findings reported from the malaria endemic areas where indoor spraying with DDT is common practice to combat malaria. The results from the South African studies indicate that the coastal areas are the most affected by global and local pollution, and hence the regions most susceptible to the effects of climate change in the near future. S07-4 CHANGES OF THE GENOME DNA METHYLATION IN THE PROGRESS OF BENZO[A]PYRENE-INDUCED TRANSFORMATION

L. Yang, G. Tao, C. Gong, L. Zhou, J. Liu, Z. Zhuang. ShenZhen Center for Disease Control and Prevention, China

In recent years, numerous studies have demonstrated that a close correlation exists between DNA methylation and the carcinogenic process. In this research a typical environment carcinogen, benzo[a]pyrene, was apllied to transform the human bronchial epithelial cell, 16HBE. Genome DNA methylation level was detected by both 5mC immunofluorescence and HPCE methods at seven detect points including the normal cells and BaP transformed cells. The total activity of methyltransferase and the expression level of the three kinds of methyltransferase were also observed in the progress of transformation. The results showed that the genome DNA methylation level was gradually deccreased during the progress of transformation, and the total activity of metyltransferase was also decreased along the process of BaP treating. Also both indexes touch the bottom in the transformed cells. However, the change of DNMT1, 3a, 3b expression level were totally different in the process. The expression level of DNMT3b gene dereased while the DNMT1 gene increased along with the transformation process, and no obvious change of the DNMT3a gene was observed in the treatment process. S08-4 EPOXIDE HYDROLASES: STRUCTURE, FUNCTION, MECHANISM AND TOXICOLOGICAL IMPLICATION

M. Arand. Institute of Pharmacology and Toxicology, University of Zurich, Switzerland, x, Switzerland

Mammalian epoxide hydrolases are enzymes that hydrolyse oxirane derivatives (epoxides) to the corresponding diols. Since many epoxides possess electrophilic reactivity these enzymes have long been regarded as safeguards being exclusively important for the detoxification of genotoxic and thus potentially carcinogenic compounds. However, an increasing number of endogenous epoxides has been discovered to play important roles in the regulation of physiological processes, and epoxide hydrolases themselves therefore also seem to be physiological regulators. The first EH reported to be implicated in such regulatory processes was the soluble epoxide hydrolase (sEH), that through the hydrolysis of arachidonic acid-derived epoxides, so called EETs, is involved in the regulation of e.g. blood pressure, pain perception and angiogenesis. Very recently, we could show that the sister enzyme microsomal epoxide hydrolase (mEH), the work horse in the detoxification of xenobiotic-derived harmful epoxides, also contributes significantly to the turnover of signalling epoxides in the central nervous system. Furthermore, we have identified a new subfamily of human epoxide hydrolases, of which the first member, EH3, has a quite narrow substrate specificity and, so far, hydrolyses exclusively fatty acid-derived epoxides. In contrast to the classic EHs, these novel epoxide hydrolases are not expressed in the liver, in line with a lack of significance in xenobiotic metabolism. Thus, a picture of the EH family arises that is a mirror of the CYP family of enzymes, yet at a smaller scale: while EHs are definitely important players in the detoxification of xenobiotics, the larger fraction of the family unexpectedly seems to serve endogenous functions. In the future, it will be interesting to learn to what extend the interference of xenobiotics with these endogenous EH functions contribute to the toxic effects of such compounds. P08-5 URINARY 1-OHP LEVELS IN CHILDREN ARE MODULATED BY CYP1A1*2C, CYP1B1*3, GSTM1*0 AND NQO1*2 POLYMORPHISMS

M. Sánchez-Guerra (1)

, N. Pelallo-Martínez (2)

, F. Díaz-Barriga (2)

, L. Carrizales-Yañez (2)

, L. Oropeza-Hernández (3)

, G. Martínez-Aguilar

(1), M. Guaderrama-Díaz

(1), S. Rothenberg

(1), B. Quintanilla-Vega

(1).

(1) CINVESTAV-IPN, Spain;

(2) UASLP; Spain;

(3) PEMEX General Hospital, Spain

Polycyclic aromatic hydrocarbons (PAHs) are considered ubiquitous pollutants in urban cities and infant populations are most vulnerable to their toxicity. The metabolite 1-hydroxypyrene (1-OHP) is until now the best biomarker of internal dose of PAHs of recent exposure, and some enzymes involved in their metabolism such as CYP1A1 and CYP1B1 (activation) and NQO1 and GSTM (deactivation) are polymorphic, which may modulate 1-OHP levels. We performed a study in children from Coatzacoalcos, Veracruz, Mexico (in the Golf of Mexico), where 80% of country´s


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petrochemical activity is located, to evaluate the role of PAHs metabolizing enzymes polymorphisms in urinary 1-OHP-levels. A cross-sectional study was conducted in children (6 to 10 years old) from primary schools located in the vicinities of the main industrial complex. Urine and blood samples were collected and urinary 1-OHP levels were determined by reverse-phase-HPLC and genetic polymorphisms CYP1A1*2C, CYP1B1*3, and NQO1*2 by real time-PCR while GSTM1*0 by multiplex PCR. Fifty-eight percent of children had 1-OHP levels higher (0.3736 µmol/mol creatinine, C.I. 0.29-0.47; geometric mean and 95% confidence interval) than those reported in non-occupationally exposed subjects (0.24 µmol/mol creatinine). Significant higher 1-OHP levels (p=0.056) were observed in heterozygous CYP1A1*2C and a trend (p=0.068) in CYP1B1*3 individuals. Likewise, GSTM null genotype favored higher 1-OHP levels (p=0.002), and even more in heterozygous children to CYP1A1*2C or homozygous to CYP1B1*3 (p<0.05). On the other hand, NQO1*2 polymorphism was associated with lower 1-OHP levels (p=0.017), an influence that was kept (p<0.05) in combination with CYP1A1*2C and CYP1B1*3 polymorphisms. These results suggest that CYP1A1*2C, CYP1B1*3, GSTM1*0 and NQO1*2 single polymorphisms and their combinations modulate 1-OHP urinary levels in children environmentally exposed to PAHs that may contribute to develop differential toxic effects in this susceptible population (Supported by CONACyT Grant-87234). S16-3 THE USE OF X-OMICS IN REGULATORY STUDIES

F. Goodsaid. US. FDA Silver Spring MD, United States A decade has passed since the publication of the human genome sequence. The decade has also included sequencing for genomes associated with animal models used in drug safety assessment studies submitted to regulatory agencies. At the beginning of this decade, expectations for the application of genomic and other `omic data in regulatory studies focused on the identification of predictive biomarkers for drug safety assessment. Development of these biomarkers throughout the decade ranged from diagnostic applications to applications circumscribed to internal decision-making in different companies. Successes and failures in the development of these biomarkers can be seen in a number of cases, some of which show how exploratory `omic biomarkers for drug safety assessment have become important biomarkers for regulatory review. S16-4 INTEGRATION OF GENOMICS, PROTEOMICS AND METABOLOMICS TECHNOLOGIES IN DRUG SAFETY ASSESSMENT

D.Masset French Health Safety Agency AFSSAPS, France

Constant progress in the miniaturization and nanotechnologies fields contributes to emergence and development of new technologies which making possible a better apprehension of whole mechanisms contributing to the cellular or tissue activity. Omics and in particular transcriptomic and toxicogenomic are essential like powerful tools for the research of new mechanisms of action and the discovery new therapeutic targets. The exponential increasing number of publications using omics approaches confirms this fact. The identification, the priorisation and the selection of new chemical entities were at this time the main motors which led the pharmaceutical companies to invest in these approaches. More recently the toxicogemic try to impose itself like major compromise tool between the preclinical and clinical development of new active substances with the objective to improve safety profile and optimize the development costs of those. The main challenges to which this tool must answer are: a better comprehension of toxic effects, the extrapolation of these effects at the cellular level, a better evaluation of the integral dose-effect response in relation with kinetic parameters, a better biological response understanding, a harmonization of the evaluation safety end points between Man and animal based on an better correlation and interpretation of the data between those. Today competent authorities in charge of the regulation of the drugs marketing authorisation encourage the development and the use of such data in Clinical trials or marketing authorization application for new drug in particular through publication of recommendations on the voluntary data submission of genomic. Nevertheless, ended up is to note that in spite of these encouragements the tendency of such studies in the application files is rare. One of the main causes of this situation is a lack of reference mark and solid methodological and scientific references and experience for the appropriation of these tools by decisions makers. This quasi-absence is at the origin of unesiness decoding and the appreciation of relevant and useful biological information provide by these studies in the public health risk evaluation process. S21-4 TOXICITY OF ACETAMINOPHEN IN THERAPEUTIC DOSES

P. B. Watkins. Hamner-University of North Carolina Institute for Drug Safety Sciences RTP, NC., United States When healthy adult volunteers consume 4 grams/day of acetaminophen (paracetamol), about one third will develop elevations in serum alanine aminotransferase, aspartate aminotransferases and glutathione-S-transferase indicating mild liver toxicity (JAMA 5:296(1):87-93, 2006). We have used this model to study acetaminophen toxicity in man. We have found that changes in the urine metabolome that occur shortly after starting treatment (resolved by NMR spectroscopy) can distinguish who will develop the toxicity and who will not (Clin Pharmacol Ther. Epub, in press). Predicttive metabolites include both mercapturates and cysteine conjugates of acetaminophen, but these alone are not predictive unless changes in the endogenous metabolome are also considered. We have also found that a genetic variation in CD44 is more prevalent among those volunteers developing the liver toxicity. We developed the


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hypothesis that CD44 may be involved in susceptibility by examining genetic variation underlying differences in susceptibility to acetaminophen toxicity across a panel of inbred strains of mice (Genome Research, (9):1507-15. 2009). The data we are obtaining in subjects with acetaminophen toxicity, which includes analysis of whole blood transcriptome (Hepatology. 51(1):227-36, 2010) and liver derived transcriptome contained in cell-free plasma (Hepatology, epub,in press) is being used to inform the DILI-sim Initiative. This initiative is collaboration between the Hamner Institutes, the US Food and Drug Administration and the company Entelos to use computer modeling to simulate normal liver physiology and how it is perturbed during drug-induced liver injury in mouse, rat and man. Modeling of acetaminophen liver injury is the intitial project and this is already well underway, with functioning models for sulfation, glucuronidation, NAPQI formation glutathione depletion and compartmentalized hepatocyte turnover. S21-5 IDENTIFICATION OF PATIENTS AT RISK OF HEPATOTOXICITY FOLLOWING ACETAMINOPHEN OVERDOSE

M. L. Sivilotti. Department of Emergency Medicine, and of Pharmacology & Toxicology, Queen’s University, Kingston, Ontario, Canada Better risk prediction lies at the core of patient-tailored N-acetylcysteine treatment, and is necessary to answer research hypotheses regarding risk factors and optimal dosing of antidote. While the Rumack-Matthew nomogram continues to govern the binary decision of whether to treat acute overdose patients, its use to quantify risk is limited by its dependence on a known time of ingestion, and by the powerful protective effect of early treatment. Risk prediction becomes even more difficult in chronic overdose and late presenting patients, who are actually at greatest risk of hepatic failure and death despite treatment. We have developed two risk prediction instruments for acetaminophen overdose patients treated with N-acetylcysteine. The first, ψ, is based on the area under the serum acetaminophen versus time curve prior to treatment with N-acetylcysteine. It estimates the amount of toxic NAPQI generated after glutathione depletion in acute overdose patients treated at a known time post ingestion with N-acetylcysteine. The second, the serum APAP multiplied by the serum aminotransferase both at the start of N-acetylcysteine and 12 hours later, applies to any acetaminophen overdose, including chronic and time unknown. It captures both the magnitude and rate of change of the serum acetaminophen and aminotransferases, the two earliest serum markers of hepatic injury. We will discuss the derivation and performance of these two risk predictors using a large dataset of patients hospitalized for acetaminophen toxicity. We will illustrate how they can illuminate some of the important debates regarding acetaminophen toxicity and its treatment. S22-4 CRITICAL ROLES OF microRNAs IN THE EARLY STAGES OF COLON CARCINOGENESIS

H. Nakagama, M. Ochiai, Y. Hippo. National Cancer Center Research Institute, Tokyo, Japan Given that most chemical carcinogens efficiently bind to DNA, it was simply assumed that subsequent genetic mutations would mainly contribute to oncogenesis. This notion, however, has been recently challenged because accumulating evidences imply that epigenetic regulations of miRNAs might play pivotal roles as well, at least in early steps of chemical carcinogenesis in the colon. We previously reported that SND1, instrumental in the maturation process of miRNAs, is not only frequently overexpressed in human colon cancer, but also downregulates tumor suppressor APC in a post-transcriptional manner. Elevation of SND1 with concomitant accumulation of β-catenin was reproduced in the rat colon cancer model induced by PhIP, a food-borne heterocyclic amine (HCA). Strikingly, this was also the case with dysplastic aberrant crypt foci, early lesions of the colon, in this model, strongly suggesting that a subset of miRNAs preferentially processed by SND1 might promote the initiation of cancer through inactivation of APC. On the other hand, we see acute induction of growth suppressive miRNAs including p53-target miR-34a in this model. It is tempting to speculate that PhIP evokes DNA damage response, rather protecting epithelia from malignant transformation in acute phase. Together these findings indicate that various miRNAs, induced in each step with distinct roles, cooperate with genetic mutations, eventually contributing to tumor development by some mechanism. Very recently, to address more direct link between HCA´s carcinogenic potential and miRNAs, we investigated miRNA profiles in the colon after in vivo exposure to each compound. Notably, carcinogenic group were successfully separated from uncarcinogenic group by their miRNA profiles, in line with the likely relevance of miRNAs in determining carcinogenicity. Moreover, this analysis appears to correctly diagnose whether or not it is carcinogenic with miRNA profiles, potentially paving the way to accelerate the carcinogenicity test by predicting the outcome at an earlier time point. S22-5 microRNAS FOLLOWING DNA DAMAGE

M. Bushell. MRC Toxicology Unit, University of Leicester, United Kingdom The miR-34 family of microRNAs are tumor suppressors and are essential components of the DNA damage response yet the physiologically relevant targets of these miRNAs are unknown. There are three family members miR-34a, b and c, while it is known that they are induced following DNA damage it has not been shown whether these miRNAs have overlapping functions or are independently regulated. It has also been shown that the promoters of the miR-34a and the miR-34b/c genes are subject to inactivation by CpG methylation in many human tumor types. We show that following DNA damage the induction of miR-34a is dependent on p53 whereas the induction of miR-34b and c occurs via signalling through the p38 MAPK pathway in a p53-independent manner. As a result of miR-34c induction, there


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is a repression of c-Myc translation in the absence of mRNA destabilization. These two events are linked since inhibition of p38 MAPK signalling prevents both the induction of miR-34c and the translational repression of c-Myc. Moreover, specific anti-sense inhibition of miR-34c and de-regulated c-Myc expression following DNA damage prevents cell cycle arrest in S-phase, gives rise to radio-resistant DNA synthesis and increased DNA damage. Importantly all these effects are completely reversed by depletion of c-Myc alone. These data clearly show that c-Myc is the major physiological target of miR-34c and provide direct mechanistic evidence for its tumour suppressor activity. Our data suggest that miR-34c replacement therapy may be an effective means to achieve c-Myc repression since miR-34 levels are relatively high in normal tissue, therefore likely to be well tolerated, but are often lost in cancers. S23-5 PRESENT AND FUTURE CHALLENGES IN SAFETY ASSESSMENT FOR PHARMACEUTICALS IN IBERO-AMERICA

J. E. Manautou. University of Connecticut, Storrs, CT, USA, Connecticut, United States GLP regulations assure the quality and integrity of non-clinical safety studies. These regulations assure that data from these studies are accurate, reliable, and reproducible. Currently, GLP practices are either not available or not rigorously enforced in some Ibero-American countries. The importance of executing pre-clinical studies that follow robust guidelines to support clinical development cannot be over-emphasized. Irrespective of the country, preclinical scientists (toxicologists) should follow ICH (The International Conference on Harmonisation) guidance in order for the regulatory packages to be accepted during global regulatory submissions. Current challenges on safety assessment these countries face include, the lack of qualified personnel to direct toxicology studies, inadequate research and animal facilities that meet the standards of pertinent accreditation bodies, and lack proper government oversight. In order for Ibero-American countries to successfully compete globally, these countries must address these concerns so as to become important contributors in pre-clinical safety assessment strategies. S27-5 BROMINATED FLAME RETARDANTS: PAST, FUTURE, AND REGULATIONS

L. Birnbaum. NIEHS, Netherlands Brominated flame retardants (BFRs) encompass a broad class of structurally distinct chemicals which have bromine atoms in common. The rapid increase in their use in the last decades of the past century was followed by an increase in their levels in wildlife and in people. This dramatic rise led to a burst in studies concerning the potential for adverse effects in a wide variety of animal species, as well as recently, humans. The majority of these studies focused on polybrominated diphenyl ethers (PBDEs) which were produced commercially as three complex mixtures, known as Penta, Octa, and Deca. While Penta and Octa were banned in the European Union and withdrawn from production in the US in 2004, their environmental persistence is resulting in ongoing exposure to wildlife and people. Deca was banned by the European Court of Justice in 2008, and in several states of the US. However, it remains a high production volume chemical and is used extensively world-wide, especially in Asia. The other major BFRs, tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are not subjected to any regulation. There are many other BFRs, e.g., BTBPE, DBDPE, TBBPA-DBPE, which are now being detected and are unregulated. P101-029 RESPONSE OF NEWZEALAND RABBITS TO DRINKING WATER TREATMENT WITH ALUM AND POLYDADMAC IN TERMS OF CHANGE IN ELECTROYTES

A. Medani, S. Elbadwi, A. Amin. khartoum college of medical sciences, khartoum, Sudan Neazealnd rabbits of both sexes were purchased ,clinically adapted,given doses of polydadmac and alum at dose rates similar to those given by Khartoum Water Plant at flood season for human consumption.Sera were analyzed for electrolyte prior to experiment and there after at a week interval.As for Neazealnd rabbits tested for polydadmac,Sodium levels investigations revealed significant (P< 0.05) increase in the group recieving the highest dose,where as the rest of the groups showed no significance (P>0.05) differences.For potassium, significant (P< 0.05 - 0.01) increases in different groups were observed.Calcium levels showed remarkable (P< 0.05) deviations from normal in the serum of most groups.Significant (P< 0.05 - 0.01) increases and decreases were highlighted when testing the undertest rabbits for their phosphorus levels, whereas the control rabbits showed normal values when tested for the above mentioned electrolytes the thing which can be attributed to the hjepatic damage induced by the chemical.Both test groups showed significantly low values (P< 0.05 - 0.01) in magnesium, iron, calcium, and phosphorus when compared to the control group in case of testing them fo alum. The control group animals showed normal electrolyte values.This can also be attributed to the direct effect of alum.Intestinal wall which was spotted with white (probably with alum causing focal enteritis) was greatly affected with the irritant alum and/or its metabolites. When the resin is precipitated by alum in its preparation, it is more apt to gripe; commonly the salt intensifies its action and the cream of tartar increases the hydragogue effect .This action was very clear on the congested mesenteric blood vessels and symptomatically, by diarrhea and salivation due to nausea.Similar picture was seen when using alum as atest material.Future suggestions were suggested and practical implications were forwarded.


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P101-030 EVALUATION OF P16 EXPRESSION IN ORAL MUCOSA CELLS OF PEOPLE WITH CYTOGENETIC DAMAGE.

A. L. González-Yebra, B. González, G. Barbosa, C. Mendiola, R. Huerta, K. Wrobel, K. Wrobel. Universidad de Guanajuato, León, Mexico In a previous study cytogenetic damage was found in workers exposed to solvents. From these results, it was necessary to seek the possible association of this damage with the development of oral dysplasia. It was evaluated the expression of p16INK4a in exfoliated cells of oral mucosa, because p16INK4a has recently been proposed as a new marker with potential application for clinical diagnosis in oral dysplasia. It was evaluated the p16 expression in 43 subjects. 33 subjects exposed to solvents with cytogenetic damage and 10 healthy subjects without exposure to solvents. The results confirm no evidence of overexpression of p16INK4a in oral mucosal cells of the subjects tested. However, it was confirmed the presence of damage and high frequencies of condensed chromatin. Based on these results, we can suppose that apoptotic processes avoid further damage and the mechanisms to repair the cells are becoming efficient. However it requires delving into the expression of other markers oncogenic and tumor suppressor genes. P101-031 QUALITY ASSURANCE (QA) AND QUALITY CONTROL (QC) PARAMETERS IN DETERMINATION OF BISPHENOL A (BPA) IN HUMAN TISSUES AND FLUIDS

C. Mclellan, G. Ball, C. Willhite. NSF International, Ann Arbor, United States

Bisphenol A is used in synthesis of polycarbonate plastics and epoxy resins and free (parent) BPA has been implicated as an endocrine disruptor. However, several recent assessments concluded that repeated ingestion of environmentally-relevant BPA doses does not present a risk to human reproductive health. Health Canada is currently considering urinary BPA biomonitoring where - depending upon the data selected - human margins of exposure range from 10 to 58,000 compared to an oral reference dose of 16 ug/kg-day. Unfortunately, published BPA biomonitoring studies often failed to explain details of sample collection, holding times, procedures to avoid hydrolysis of BPA metabolites or to specify analytical limits of detection. The lack of a standardized protocol and analytical method results in calculated "background" BPA daily exposures ranging from 38 ng/kg to doses 1000 times that value. Laboratory contamination and/or hydrolysis of pharmacologically inactive metabolites to free BPA may have contributed to erroneous reports of free BPA in human fluids and tissues. Analytical procedures should prohibit sample contact with plastics, include immediate and continuous refrigeration, should include an internal standard (e.g., d16-BPA) and measurement of at least five replicates. Analytical limits of BPA detection in human blood and urine on the order of 20 ppt can be achieved using HPLC/MS/MS instruments. Only with rigorous steps taken to assure QA/QC of the sample collection and handling can accurate margins of BPA exposure be derived. P102-031 PERSONAL CARE PRODUCTS AND URINARY LEVELS OF PHTHALATES IN MEXICAN WOMEN

M. Romero Franco (1)

, R. U. Hernández-Ramírez (1)

, A. Calafat (2)

, M. E. Cebrián-García (3)

, L. Needham (2)

, S. Teitelbaum

(4), M. Wolff

(4), L. López-Carrillo

(1).

(1) Population Health Research Center. National Institute of Public Health, Mexico, Cuernavaca, Mexico;

(2) Division of

Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, USA; (3)

Toxicology Division. Center for Research and Advanced Studies, México; (4)

Center for Children's Environmental Health and Disease Prevention Research. Mount Sinai School of Medicine, USA Sources of phthalates other than PVC related products are scarcely documented in Mexico. The aim of this study was to explore the association between urinary levels of nine phthalate metabolites and use of personal care products as well as other potential environmental sources. Subjects included 108 women who participated as controls in an ongoing population based case-control study of environmental factors and genetic susceptibility to Breast Cancer in northern Mexico. Direct interviews were performed to inquire about reproductive history, use of personal care products and diet. The following phthalate metabolites were measured in urine by HPLC-MS/MS at the CDC: monoethyl phthalate (MEP), monobenzyl phthalate (MBzP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-carboxypentyl phthalate (MECCP) and, mono-3-carboxypropyl phthalate (MCPP). Detectable urinary concentrations of phthalate metabolites varied from 75% (MEHP) to 100% (MEP, MBP, MEOHP, MEHHP, MECPP). Setting a cut off point of p<0.05, linear regression models showed that anti-age creams´ use was significantly associated with concentrations of MEP (B=0.84) and MCPP (B=0.51), perfume use significantly predicted increasing concentrations of MBP (B=0.36) and MiBP (B=0.5). MBzP concentration was related to the use of hair conditioner (B=0.58) while DEHP metabolites concentrations were significantly associated to deodorant (MEHP B= 0.49) and body cream use (MECPP, MEHHP, B= 0.4 and 0.47). Bottled water consumption was associated to concentrations of MCPP, MEOHP, MEHHP and MECPP (B= 0.39, 0.39, 0.39 and 0.33 respectively). The results of this study suggest that using personal care products contributes to phthalates body burden. Further estimation of dermal and inhalation exposure by specific products is warranted. "The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention."


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P102-038 DESCRIPTION OF THE CLINICAL FEATURES SEEN IN PATIENTS WITH MEPHEDRONE TOXICITY

D. M. Wood, S. L. Green, P. I. Dargan. Guy's and St Thomas' NHS Foundation Trust, London, United Kingdom Introduction: Mephedrone is a synthetic cathinone and there is increasing evidence of its use across Europe, particularly in the UK and Scandinavia. There is one published fatality associated with mephedrone but no data published on patterns of acute mephedrone toxicity. We report here a series of acute toxicity from a single centre in patients with self-reported mephedrone use. Case Series: No cases were seen prior to January 2009. 31 cases were seen subsequent to this (mean±SD age 27.5±8.1, range 15-60years; 77.4% male). Baseline physiological parameters on presentation were: heart rate 96.0±21.9, range 50-158 bpm; systolic BP 142.6±23.0, range 100-192mmHg; temperature 35.9±.2, range 33.0-38.1ºC. The most common clinical feature on presentation was agitation (51.6%); other features included palpitations (25.8%), vomiting (19.4%), self-limiting seizures (9.7%) and headache (3.4%). No patients had skin discolouration or cold peripheries. 16.1% had hypertension (greater than 160mmHg), 45.1% had tachycardia (greater than 100bpm) and 12.9% had severe tachycardia (greater than 140bpm). No patients had clinically significant hyperpyrexia. Creatinine kinase was measured in 8 (25.8%) and raised in 5 of these (16.1% of the whole group and 62.5% of those in whom it was measured), ranging from 297-4134 IU/L (upper limit of normal 229 IU/L). Twenty-five (80.6%) patients were discharged directly from the ED or the short-stay observation ward. Benzodiazepines were used for the management of agitation in 4 (12.9%) patients. All patients survived to discharge (median length of stay less than 7 hours) from hospital with no long-term sequelae on discharge. Conclusions: In this series, toxicity associated with self-reported mephedrone use was generally associated with mild sympathomimetic toxicity similar to that seen with other stimulant recreational drugs such as MDMA. However, there was a minority who had clinically significant effects including tachycardia, hypertension and seizures. Further work is required to determine the true toxicological profile of mephedrone. P103-047 IN VIVO TESTING FOR DIFFERENCES FROM NORMAL METABOLITE VALUES IN RESPONSE TO CHEMICAL TREATMENT OF DRINKING WATER

A. Medani, S. Elbedwi, A. El Amin Khartoum College of Medical Sciences, Khartoum, Sudan Differential in vivo testing for deviations from normal metabolic values was done using Nubian goats and Newzeaknd rabbits as biosystems. Graphic indicators showed the values after an experiment of 2 month during which alum and polydadmac were given at different doses representing the same used at the water plants in Khartoum for daily supply of drinking water to the consumer,sera were analyzed prior to start and at fotinight interval for albumin, urea, total porotien, chloestrol ,bilirubin, glucose and creatinine for both tesesd animals using the tow materials us de in treatment of drinking water in Sudan.In this study, the daily oral doses of polyDADMAC to Nubian goats caused increased concentration of serum bilirubin, indicating hepatic dysfunction and damage or altered membrane permeability .On assessment of the renal toxicity, increase in urea, total protein and creatinine were positive indicators suggestive of the renal damage caused by the polymer and/or its metabolites . Daily routine dosing of alum revealed the necrotic intoxicated lung, proved on atomic absorption by the high pulmonary levels of aluminum, marked pulmonary affections were probably contributed to the development of dysnoea which support the hypothesis that alum is harmful specially with regard to lung fibrosis .On atomic absorption of the processed liver - alum values were negligible, The absence of bilirubinaemia suggests that alum is not expected to interfere with the excretory ability of the liver cells . Renal insufficiency was indicated by increase in urea, creatinine, total protein and decrease in albumin concentrations. In alum-dosed goats Hepatic damage was menifested in increased level of bilirubin. The additive effect of the increased creatinine and urea concentrations in serum indicated damage to renal tubules with the exception of the very high mortality rates in alum-dosed rabbits, all features were similar to those observed in goats. P103-051 HEAVY METALS AND ESSENTIAL ELEMENTS IN BONES AND EGGS OF CHINSTRAP PENGUIN (PYGOSCELIS ANTARCTICA) FROM DECEPTION ISLAND, ANTARCTICA

S. Jerez (1)

, M. Motas (1)

, J. Benzal (2)

, A. Martínez (2)

, F. Valera (2)

, A. Barbosa (3)

. (1)

University of Murcia, Murcia, Spain; (2)

Estación Experimental de Zonas Áridas, CSIC, Spain; (3)

Museo Nacional de Ciencias Naturales, CSIC, Spain Antarctica is considered to be one of the few regions of the planet unpolluted. Nevertheless, Antarctica could be affected by pollution as a global phenomenon or by local activities. Antarctic animals are least investigated compared with animals from other continents. Being circumpolar and having permanent ecological niche and domination among the avifauna in Antarctica, penguins have potential to be a standard biological indicator for use in monitoring programs of Antarctica nearshore ecosystems. The different concentrations of heavy metals and essential elements (Cd, Pb, Hg, Cu, Zn, As, Se) has been determined in bones (n=15) and eggs (n=7) of chinstrap penguin (Pygoscelis antarctica) from Deception Island (63º00’S 60º40’W), collected between 2008 and 2009. Solutions of bones and eggs were prepared by acid-assisted microwave digestion by employing HNO3 and H2O2, and analyzed by inductively coupled plasma mass spectrometry (ICP-MS). These materials were obtained by non-destructive methods. Bones and eggs showed the following order of metal concentrations, respectively: Zn>Se>Pb>Cu>Cd>As>Hg and Cu>Pb>Zn>Se>Hg>As>Cd. The highest concentrations of Zn, Cd and As have been detected in bones (average


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levels, respectively: 216.1 µg/g dry weight; 0.08 µg/g dry weight; 0.07 µg/g dry weight). The rest of elements showed higher levels in samples of eggs. A wide range of Pb concentrations in eggs have been observed (BDL-6.5 µg/g dry weight). A continual environmental biomonitoring could establish a possible trend to contamination in some Antarctic marine areas, fundamentally in zones with frequent human presence, as Deception Island, usually considered unpolluted because of their remoteness. P103-052 AN INTEGRATED CELLULAR DOSIMETRY AND STRUCTURE-PROPERTY RELATIONSHIP MODELING APPROACH TO CONDUCT IN VITRO-IN VIVO EXTRAPOLATION OF THE DOSE-RESPONSE RELATIONSHIP: A CASE STUDY WITH TOLUENE

T. Peyret, K. Krishnan. IRSPUM, Université de Montréal, Montréal, Canada Toxicity testing strategies for the twenty-first century focus increasingly on the use of: i) in vitro high-throughput assays, and ii) quantitative models relating molecular structure with toxicity or dosimetry endpoints. In this regard, the development and validation of modeling tools for conducting in vitro – in vitro extrapolation of toxicity and dosimetry aspects is critically important. The present study describes an effort to conduct in vitro – in vivo extrapolation using a cellular-level dosimetry model combined with a quantitative structure-property relationship (QSPR) model, using toluene as the model substance. The tissue compartments of the in vivo physiologically-based toxicokinetic (PBTK) model consisted of four matrices: cell, interstitial fluid, plasma and erythrocyte; whereas the in vitro dosimetry model contained two matrices: cell and culture medium. The predicted partition coefficients (PCs) for the in vitro and in vivo models were based on matrix:water PCs, which in turn were estimated based on QSPR models for vegetable oil:air, protein:air and water:air PCs together with the fractional content of neutral lipids, neutral phospholipids, water, and proteins. The QSPR-based PCs and published metabolic parameters were then incorporated within the in vivo and in vitro cellular-level dosimetry models to simulate the inhaled concentrations of toluene yielding the same brain cell concentrations as the in vitro SH-SY5Y cytotoxicity study, obtained from the published literature. For LDH leakage and intracellular Ca2+ effects, the in vitro concentrations of 5.64 (NOAEL), 13.1 (LOAEL), 22.5, 46.6, and 76.3 µM corresponded to the human environmental exposure concentrations of 156, 257, 375, 671 and 1036 ppm, as estimated with the integrated model. Overall, the QSPR-cellular dosimetry model developed in this study represents a potentially useful tool in data poor situations for conducting in vitro-in vivo extrapolation of the cellular-level effects and perturbations evaluated in high-throughput assays. (Supported by AFSSET). P103-061 STUDIES ON HEALTH EFFECTS CAUSED BY VOC EMISSIONS FROM PINE WOOD (PINUS SPECIES) AND ORIENTED STRAND BOARDS IN HEALTHY VOLUNTEERS

R. Gminski (1)

, R. Marutzky (2)

, S. Kevekordes (1)

, F. Fuhrmann (2)

, W. Bürger (2)

, D. Hauschke (3)

, V. Mersch-Sundermann

(1), T. Tang

(1).

(1) Department of Environmental Health Sciences, Freiburg University Medical Center, Freiburg, Germany, Freiburg,

Germany; (2)

Fraunhofer-Institut für Holzforschung - Wilhelm-Klauditz-Institut (WKI), Braunschweig, Germany; (3)

Institut für Medizinische Biometrie, Universität Freiburg, Freiburg, Germany, Introduction. Identification of health effects caused by exposure to VOC emissions from pine wood and OSB, which are widely used in house construction and for interiors, is particularly important. The aim of this study was to evaluate acute health effects, e.g. sensory irritation in healthy human volunteers after exposure to VOC emissions from pine wood and OSB. Material and Methods. Healthy non-smokers were exposed to clean air and pine wood emissions (15 volunteers) or OSB emissions (24 volunteers) for 2 h under controlled conditions in a 48 m3 test chamber at different loading factors and VOC concentrations. Lung function parameters, exhaled NO, eye blink frequency, subjective irritation of eyes, nose and throat using VAS, and sensory perception (hedonics) were examined before, after and partly during exposure. Results. Maximum VOC exposure concentrations reached just under 13 mg/m3 for pine wood emissions and 9 mg/m³ for OSB emissions. Emissions consisted predominantly of alpha-pinene, 3-carene and other terpenes from pine wood and additionally of aldehydes (e.g. hexanal) from OSB. No irritating or pulmonary effects were observed during exposure to these emissions. Except for smell, almost all VAS ratings were at a very low level and the medians did not exceed the expression hardly at all. Only the VAS ratings for smell increased significantly during exposure to pine wood or OSB emissions in comparison to clean air. Conclusions. On the basis of our results, no evidence was found of irritating effects of pine wood and OSB emissions to eyes, throat or pulmonary system, even at the highest VOC concentrations tested. However, in view of the uncertainties regarding (i) long-term exposure, (ii) individual susceptibility to irritant substances, (iii) chemical reaction of pine-wood specific terpenes with ozone, from a preventive point of view, exposure to VOC in indoor air should be minimized wherever possible. P104-041 ASSESSMENT OF MODULATION OF URINARY POLYCYCLIC AROMATIC HYDROCARBON METABOLITES BY ENZYME POLYMORPHISMS IN WORKERS EXPOSED TO BITUMEN - RESULTS OF THE HUMAN BITUMEN STUDY

H.P. Rihs, A. Spickenheuer, E. Heinze, B. Pesch, M. Raulf-Heimsoth, J. Angerer, T. Brüning Institute for Prevention and Occupational Medicine of the German Social Accident Insurance - Institute of the Ruhr-Universität Bochum (IPA) Data about the modulating influence of sequence variants of metabolizing enzymes on urinary biomarkers after current exposure to fumes of bitumen in humans are limited. The aim of this study was to assess the influence of 18 single


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nucleotide polymorphisms (SNP) in genes coding for enzymes involved in polycyclic aromatic hydrocarbon (PAH) and amine metabolism regarding their impact on urinary markers 1-hydroxypyrene (1-OHP) and the sum of 1-, 2+9-, 3-, 4-hydroxyphenanthrenes (OHPHE) in workers with bitumen exposure. Based on personal ambient monitoring data for bitumen emissions 218 German workers exposed to fumes of bitumen during a shift and 96 German roadside construction workers without exposure to bitumen but with similar working tasks (part of the German Bitumen Study) were studied. SNP determination based on DNA aliquots isolated from blood samples by Real-time PCR or direct sequencing. The impact of sequence variants on the urinary levels of 1-OHP and sum of OHPHE was estimated with mixed linear models, adjusted for age, creatinine, exposure, smoking, SNP, time of measurement and interaction terms. In the mixed linear model increasing metabolite levels of OHPHE was only significantly modulated by the CC variant of the cytochrome P450 SNP CYP1A1 3801T>C (rs4646903, Factor 1.6, 95%CI: 1.0-2.5, p=0.043). In contrast, GSTM1-carriers showed a borderline decreased influence on OHPHE levels (Factor 0.9, 95% CI: 0.8-1.0, p=0.05). None of the SNP studied displayed a significant effect on 1-OHP.The influence of modulating SNP effects on OHPHE did never exceed factor 1.6 in the adjusted model and was less pronounced when compared with the effects (maximum factor 2.1) observed in a recent study with 170 workers occupationally exposed to PAH in German industries (Rihs et al., Toxicol. Lett. 2005).This may be due to the much lower PAH exposure in the Human Bitumen Study. P104-045 POLYCYCLIC AROMATIC HYDROCARBONS IN ATMOSPHERIC PARTICULATE MATTER (PM2.5) IN THE ARARAQUARA CITY (SE, BRAZIL): INFLUENCE OF SUGAR CANE BURNING ON POTENTIAL CANCER RISK

F. S. Silva (1)

, J. Cristale (1)

, P. H. N. Saldiva (2)

, M. R. R. Marchi (1)

. (1)

Institute of Chemistry, Unesp – Univ. Estadual Paulista, Araraquara, Brazil; (2)

Faculty of Medicine, USP - University of Sao Paulo, Brazil In Brazil, the sugarcane crops are burned to facilitate the harvesting, this process causes environmental pollution from the large amounts of soot that are released into the atmosphere., this material contain numerous organic compounds such as PAHs. In this study, PM2.5 and PAHs concentrations in the air of Araraquara (SE Brazil, with around 200,000 inhabitants and surrounded by sugar-cane plantations) were determined during the sugarcane harvesting (HV) and non-harvesting (NHV) seasons in 2009. The sampling strategy included two campaigns in each season, with 20 samples in non-harvesting season and 56 samples in harvesting one. PM2.5 was collected using a dichotomous sampler (10 L min-1, 24 h) with TeflonTM filters. Ten filters sets were extracted (ultrasonic bath with hexane/acetone (1:1 v/v)) and analyzed by HPLC/Fluorescence. The average concentration for total PAHs was 2.16 ng m-3 (NHV) and 4.72 ng m-3 (HV). At HV season, the concentration of carcinogenic PAHs (benz(a)anthracene, benzo(b) fluoranthene, benzo(k)fluoranthene and benzo(a)pyrene) was 5 times higher than NHV season. B(a)P average concentration was 0.020 ng m-3 and 0.13 ng m-3 for the NHV and HV seasons, respectively. The potential cancer risk associated to inhalation exposure was estimated based on the benzo[a]pyrene toxic equivalence (BaPeq), where the overall toxicity of a PAH mixture is defined by the concentration of each compound multiplied by its relative toxic equivalency factor (TEF). BaPeq average values were 1.6 ng m-3 and 0.5 ng m-3 for HV an NHV season, respectively. Considering the maximum permissible BaPeq in ambient air is 1 ng m-3, related to the increasing of carcinogenic risk, our data suggest a worrying PAH human exposure scenario in cities surrounded by sugarcane crops where the burning process is used. Acknowledgments: FAPESP, CAPES, CNPq, FACTE. P105-046 ECG AS A PROGNOSTIC INDEX IN ACUTE SEVERE ORGANOPHOSPHATE POISONING

S. Shadnia (1)

, A. Okazi (1)

, N. Akhlaghi (1)

, G. Sasanian (2)

, M. Abdollahi (2)

. (1)

Loghman Hakim Hospital Poison Center, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran (Islamic Republic of);

(2) Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran

University of Medical Sciences, Objective: Many cases of poisoning with organophosphates have been reported. This has put organophosphate poisoning under spotlight. Studies on the cardiac effects of organophosphate poisoning are inconclusive. There have been, however, reports regarding the relationship between prolonged corrected QT (QTc) interval and the severity of poisoning. Methods: 42 patients with the primary diagnosis of OPP whom were admitted in ICU of Loghman Hakim Hospital were the subjects of this prospective study. The QTc interval was determined for each patient, based on which two groups of prolonged and normal QTc interval were devised. Results: According to serum cholinesterase (CE) activity, all of the patients were categorized in severe toxicity group. Out of the total of 42 patients, 47.6% were male, 37.5% of the patients died. 59.5% of patients had prolonged QTc interval and 40.5% had normal QTc interval. The mortality rate in the patients with prolonged QTc interval was higher than that of the normal group (P=0.044). Moreover, the average period of hospitalization of patients with prolonged QTc interval was higher than that with normal QTc interval (P=0.022). The average of the required atropine to control the muscarinic signs and symptoms in patients with prolonged QTc interval was 38.5 mg and in the patients with normal QTc interval was 20.02 mg. Statistically the patients with prolonged QTc interval needed more atropine (P=0.013) than other group. Conclusion: In addition to the respiratory complications arising at the initial stages of poisoning as main mortality factor in patients poisoned with organophosphates, cardiac complications can be effective in the treatment and prognosis. Therefore, it is important to consider cardiac signs and symptoms in the initial evaluations. The close observation of the QTc interval along with primary diagnostic and therapeutic measures seems to be helpful in determining the intensity, prognosis and the patients´ therapeutic plan.


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P105-055 SUBSTANCE- P LEVEL IN THE SERUM OF CHEMICAL VICTIMS OF MUSTARD GAS

M. Shohrati, M. Chanei, S. Mahyar. Baqiyatallah University of Medical Sciences, Tehran, Iran (Islamic Republic of) Chronic pulmonary complications like bronchiolitis and asthma are one of the most important problems of chemical victims of mustard gas. Diverse studies suggest that substance P, as a member of tachykinin neuropeptides, has an important role in inflammatory processes of airways and lungs. Specially substance P affects on constriction of bronchioles, chemotaxies and adhesion of immune cells to bronchial tissues, permeability of lung vessels and also, has a major role in neurogenic inflammation of COPD patients´ airways. We have attempted to determine serum level of substance P in chemical victims of mustard gas and compare it with normal ones. The chemical victims were divided into 2 groups: first group with mild pulmonary symptoms and second group with moderate to severe symptoms, the latter group were on corticosteroid therapy (we ethically could not prevent of it). After preparing our samples and doing the stages of substance P kit, we read the results with ELISA reader and then analyzed them. The measured substance P mean level in chemical patients (2.86±1.47)ng/ml had not a significant difference with control group (3.15±1.03)ng/ml.But the patients with moderate to severe symptoms had a level of 2.48±0.92 that was significantly lower than mild ones (3.28+1.73) and control group (3.15+1.03).Although further studies are needed to clarify the roles and mechanism of substance P, substance P may have a role in pulmonary complications of mustard gas. P105-059 PRONOSTIC FACTORS FOR PERSISTENT HEPATIC DAMAGE IN CHILDREN LESS THAN 5 YEARS OLD POISONING BY ACETAMINOPHEN

P. Escalante Galindo. Hospital Juárez de México, Secretaria de Salud, México City, Mexico Objetive: To know the prognosis factors: child age, drug administration time and dose, concomitant association of hepatic microsomal system inducers (HMSI) in the development of persistent hepatic damage (PHD) for more than 72 hours in children younger than 5 years with acetaminophen (ACM) poisoning Material Methods: Realized in M¨¦xico City. Prospective cohort study. We studied 108 patients, were made concentrations trials for acetaminophen (ACM), prothrombin time and hepatic function tests, with clinical evaluation, when ACM levels were toxic, treatment with NAC was given, serological tests for hepatitis A,B,C and a questionary for the variables in study. Results: Of the 99 remainder, 20 had PHD caused by acetaminophen. The mean age in months was 20+/-6. In the bivariate analysis thah had a relative risk >2 and the confidence interval 95% were the use with drugs (HMSI), clinical phase (CP) of toxicity II,III. In the multiple logistic regression the significance of age was from 3 to 12 months and the use of IMHS. In the analysis of connected consolidation of age with the use of IMHS: ¦Á 47 children, 8.5% of probability of PHD in children > 12 months of age; ¦Â: 40 children from 3 to 12 months of age without IMHS, 25% of PHD; and ¦Ã: 12 patients with IMHS, probability 50% for PHD. In the CP, stratum A age > 12 months , CP I, PHD 3.3%, B in CP I or II with age < or > 12 months, PHD 20% and C any age with IMHS or in CP III 57% of PHD. Conclusions: The results indicate that the factors prognosis of persistent hepatic damage in children with acetaminophen intoxication: HMSI medications, clinical phases of toxicity II and III and age. An prognostic index was obtained, that you can apply without necessity of ACM levels. P105-064 DETERMINATION OF ORGANOCHLORINE PESTICIDES IN HUMAN BLOOD OF INHABITANTS OF AN ANDEAN POPULATION OF HIGH AGRICULTURAL PRODUCTION IN MÉRIDA, VENEZUELA

J. Uzcátegui, G. Biscardi, M. Valero, M. Torres, S. González. Universidad de Los Andes, Mérida, Venezuela This work determined the presence of sixteen organochlorine pesticides in human blood samples were evaluated. The pesticide residues were extracted using the solid phase extraction (SPE) technique. The detection and quantification was performed by gas chromatography with a Electron Capture Detector. It was used as reference a standard mixture of organochlorine that contains the followings: alpha-HCH, beta-HCH, gamma-HCH, delta-HCH, heptachlor, Aldrin, Heptaclor epoxide, alpha-endosulfan, p,p´-DDE, dieldrin, endrin, beta-endosulfan, DDD, endrin aldehyde, endosulfan sulphate, DDT, methoxychlor. For the extraction procedure, C-18 cartridges were used passing through a mixture of hexane-ethyl acetate (2:1) v/v. The dynamic interval was established from 10 to 100 µgL-1, for all the analytes, with quantification limits between 0.002 and 0.029 µgL-1. The method exactitude´s was considered making a recovery study. For all the cases the values of recovery percentage´s obtained for fortified samples with the analytes at two levels of concentration (10 and 100 µgL-1), were between 69 and 102% respectively. The method precision´s was determined for solutions of the analytes prepared in hexane and in blood samples of enriched healthy people with the analytes at two levels of concentration (10 and 100 µg L-1). A total of 51 individual blood samples were analyzed, 55% male and 45% female, between 13 and 60 years exposed and not exposed, labor resident of a population of high agricultural production. The results found demonstrate the presence of high levels of some banned organochlorine in 39 of the samples (76.5%). Of the organochlorine studied only four (gamma-HCH, delta-HCH, endrín aldehyde and endosulfan sulphate), are not present in some of the samples. Altogether, 36 samples (66.6%) present unidentifiable organochlorine by the developed method. The organochlorine that appears in greater level of concentration is beta-HCH with 0.239 µg/mL. Significance of this study reveals the presence of these banned pesticides in human serum.


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P105-070 ANESTHESIOLOGIST´S APTITUDE FOR PRE-OPERATIVE DETECTION AND TREATMENT OF LATEX ALLERGY

S. A. Franco Chávez (1)

, C. Cabrera Pivaral (1)

, C. M. D. L. Preciado Serrano (1)

, O. Peña Ortiz (2)

. (1)

Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Jalisco México; (2)

Centro Universitario de Ciencias Exactas e Ingenieras; Universidad de Guadalajara, Jalisco México Background: Latex allergy is the second cause of perioperative anaphylaxis. Anesthesiologists play a key role in opportune identification of risk factors, as well as clinical diagnosis and therapeutic management. Objective: To evaluate the anesthesiologists aptitude to identify and treat latex allergy. Participants and methods: Sixty-six anesthesiologists from five general hospitals located at Guadalajara, Jalisco, Mexico, were evaluated. Aptitude was determined by applying a validated structured instrument. Aptitude levels were measured by using an ordinal scale. Comparisons were performed using Mann Whitney U test. Results: Anesthesiologist´s global aptitude ranged from -2 to 27 with a median of 8 (from a maximum value in the scale of 40); frequencies by each category of the scale were: Random 48 (72.7%), Very bad 11 (16.7%), Bad 4 (6.1%) and Medium only 3 (4.5%). Both Good and Very good categories registered no anesthesiologist. The relationship of this indicator with other variables did not reach statistical significance (KW 6.478; p = 0.16617). Conclusions: A suboptimal aptitude was identified among anesthesiologists regarding identification of latex allergy. A need to establish new strategies for educative intervention in order to improve this issue was identified. P105-071 SAFETY PRECLINICAL EVALUATION FOR HEBERPROT P. A NEW THERAPEUTIC ALTERNATIVE FOR THE TREATMENT OF PATIENTS WITH DIABETIC FOOT ULCER

K. Cosme, D. Bacardí, J. Suárez, D. Berlanga, L. Aldana, C. Valenzuela, L. Hernández, J. Berlanga, R. Silva, L. Herrera. Universidad de la Havana, Cuba, x, Cuba

The recombinant epidermal growth factor (r-EGF) is an endogenous molecule that has a wide range of physiological mechanisms, comprising: the regulation of all digestive system processes, the replacement of the epithelial population of different organs and tissues, the regulation of watch-sleep cycle, spermatogenesis, the stimulation of the healing processes, and the regeneration of internal and peripheral epithelial structures. The objective of the present work was to demonstrate the safety of a new parenteral formulation, with r-EGF as active principle of that product for its further clinical application for the treatment of the diabetic foot ulcer and prevention from amputation, which is the most frequent complication of Diabetes mellitus. Acute Toxicity studies, local tolerance, toxicity to repeated doses, and Safety Pharmacological studies were designed using Sprague Dawley rats and Beagles dogs, treated via sub-cutaneous, with doses reaching 180 times the therapeutic dose, which guaranteed a wide safety. A satellite group was included, inoculated with highest dose. body weight, food consumption, haematological and biochemical determinations, organs weight and macroscopic and histological observations were included. The results showed that parenteral EGF r, in a single administration unique and reiterated for 3 months did not cause damage in the cellular morphology of the organs studied. Locally, just a diffuse inflammatory reaction in the sub-cutaneous tissues was evidenced, attributable to its repeated administration. The results allowed us to conclude that parenteral EGF r does not cause toxic effects and is safe for its application in humans affected by diabetic foot ulcers. P105-072 IS FUNDAMENTAL RESEARCH IN TOXICOLOGY TRANSLATED INTO CLINICAL PRACTICE?

P. J. Meier-Abt. University of Basel, Basel, Switzerland "Toxicology has always been translational at least with respect to its primary focus on human health" (Clin. Pharmacol. Ther. 85; 327-30, 2009). However, the increasing incidence of toxicity induced drug failure in humans indicate that the translation of drug toxicity studies from animals to humans is currently insufficient. This is not at all surprising, if one takes into account, that specific genotypes can vary enormously within varies animal species and between animals and men. For example, some newly discovered drug transporters exhibit different genetic polymorphisms in humans as compared to mice, thus making concrete extrapolation of drug excretory pathways from animals to humans highly uncertain (Pharmacol. Reviews 62; 1-96, 2010). Similar considerations also apply for certain drug metabolizing enzymes, tissue/tumor specific biomarkers and peroxisome proliferator-activated receptor α (PPARα). Hence, for more precise prediction of drug safety in humans it is necessary to identify and characterize specific biosafety biomarkers that have scientifically sound predictive qualities in both animal as well as human studies. For this purpose, the Predictive Safety Testing Consortium (PSTC) between pharmaceutical industry, drug regulatory agencies and academia has been created in 2006 (Methods Mol.Biol. 460; 221-238, 2008). Although PSTC exploits routinely all scientifically sound informations regarding the extrapolation of toxicological data from animal to clinical studies in humans, the complex (epi)genetic basis of some rare adverse drug reactions in humans makes the successful development of preclinically and clinically usefull safety biomarkers an extremely difficult tasks. In fact, more effective translational toxicology requires not only new forms of collaborations between industrial, academic and clinical researchers, but also new systems biology based approaches of modelling and experimentation and a more thorough incorporation of toxicogenomic research into clinical studies (Phases I to IV).


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P106-040 TUMOUR DATA FROM SPRAGUE-DAWLEY (IGS) RAT DIETARY AND ORAL GAVAGE TUMORIGENICITY STUDIES, COMPLETED OVER THE PERIOD OF 1998 TO 2009

W. Hooks, A. Bottomley, I. Taylor. Huntingdon Life Sciences, Huntingdon, United Kingdom

A previous review of the mortality and in-life data from 33 Sprague-Dawley, Crl:CD® BR (VAF), rat dietary and oral gavage tumorigenicity studies completed between 1998 and 2009 showed that the mortality values at 2 years are still high (mean±SD: 55±8.3%, males and 67±5.7%, females). Additionally, there was an indication that the terminal mortality for males has slightly increased over time, but there was no indication of any major differences over time for females or between the routes of administration for mortality or in-life data for both sexes. Partnering the latter review, the purpose of this study was to establish if the tumour profile had changed over time and also if there were any differences between these routes of administration. The most prevalent tumours are pituitary adenoma (>40%) and adrenal benign phaeochromocytoma (>15%) in males and pituitary adenoma (>65%) and mammary fibroadenoma (>40%) and adenocarcinoma (>20%) in females. These tumours are also the major pathological factors contributory to death. There was a trend over time (1998-2009) towards slightly higher incidences for pituitary adenoma in both sexes and mammary adenocarcinoma in females, and conversely, lower incidences for mammary fibroadenoma in females. Compared with dietary studies, oral gavage studies showed slightly lower incidences for skin/subcutis fibroma (males) and mammary fibroadenoma (females), and higher incidences for uterus/cervix polyps. The results indicate that the Sprague-Dawley rat has a well-defined tumour profile, with only relatively minor differences over time or between the routes of administration. It can, therefore, be concluded that the Sprague-Dawley rat continues to be a suitable strain for use in tumorigenicity studies. However, as the terminal mortality remains high (particularly in females) it is advisable to employ a strategy, such as increasing group size, to satisfy regulatory requirements of reaching 2 years with an adequate number of survivors. P106-041 WWW.FURETOX.FR : A SEARCH ENGINE OF HUMAN HEALTH TOXICITY VALUES

C. Heyman (1)

, C. Legout (1)

, O. Grard (2)

. (1)

INSTITUT DE VEILLE SANITAIRE, LILLE, France; (2)

DDASS, ARRAS, France Introduction: Toxicology provides useful information for quantitative health risk assessment, mainly the health effects of substances and the Human Health Toxicity Values (HHTVs). A large number of scientific organisations produce these values, and the choice of the HHTVs selected is of great concern for the quality of the health risk assessment. Purpose: FURETOX (Facilitating the Use of Resources in Toxicology) was developed by the French National Institute for Public Health Surveillance [Institut de veille sanitaire] and the Ministry of Health to offer a quick, updated, open access to HHTVs, integrating information from databases available on line. Method: FURETOX operates with a computer program that conducts a weekly review of the databases and is designed for transparent display of the intellectual property of the scientific organisations for the HHTVs they produced. Results: For any given chemical substance, FURETOX retrieves, first,. the CAS Registry Number (or CAS number) from four databases, and provides then rapid access to HHTVs for chronic exposure from eleven databases and the carcinogenicity classification from four other databases. The user can get a table of existing HHTVs (by route of exposure, cancer or non-cancer health effects) and access to background documents for each HHTV. 200,000 CAS numbers have been entered. Discussion: A test, performed on sixty chemicals, shows a very valuable gain of time as well as a very good reliability of the tool. Corrective patches have been made to compensate for some errors related to families of compounds. Available in French since it was launched in late 2007, FURETOX is also available in English since 2010. There were about 40,000 connexions in two years, involving about 700 users a month, mainly from France, but with a growing part from abroad (the UK, The Netherlands, etc...). More than 90% of the connexions are bookmarked. P106-042 JUVENILE STUDIES IN THE MINIPIG: FEASIBILITY STUDY

C. Bouchez, G. Chevalier, R. Forster. CIT, Evreux, France The Göttingen minipig is a potential non-rodent model for the assessment of pediatric toxicity. The present study was undertaken to evaluate the zootechnical requirements and specificities of pregnant, lactating and juvenile animals and to validate the sensitivity of this species by treating the piglets with reference compounds during their neonatal period (first month of life). Three sows were received at CIT in their last month of gestation and were allowed to deliver normally. In the first days after parturition, the piglets were randomized by cross-fostering (litters were mixed and reattributed to the sows). The 20 newborn piglets were then allocated to groups treated with different compounds and submitted for tests in order to collect specific data on juvenile animals for blood chemistry, electrocardiography, neurological evaluation and ophthalmology. All the piglets were sacrificed on day 34 and submitted for macroscopic and microscopic examination. One group of piglets was treated with enalapril (Angiotensin II Converting Enzyme Inhibitor) by oral gavage from day 7 to day 33. Another group was treated with purified water under the same conditions and acted as a control group. Increased uremia and creatininemia associated with increased renal weight and macroscopic and microscopic lesions in the kidneys were observed in the piglets given enalapril. A third group of piglets received sotalol (antiarrhythmic agent) on the day of electrocardiography, inducing decreased heart rate, PQ and QT interval prolongation. This study supports the interest of the Göttingen minipig model for the assessment of toxicity in the pediatric population.


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P106-043 TOXICOLOGICAL ASSESSMENT OF IMPURITIES AND THEIR LIMITS IN THE RISK ASSESSMENT FOR PLANT PROTECTION PRODUCTS

J. Parra, D. Court Marques, F. Istace, M. Tiramani. European Food Safety Authority, Parma, Italy

An integral part of the risk assessment of active substances of plant protection products (PPPs) at European Union (EU) level is the consideration of the chemical composition of technical material (TM), consisting of the active substance and its associated impurities. Impurities might be considered relevant. From a toxicological point of view, relevant impurities might alter (increase or add a hazard) the toxicological profile of the TM in the event of slight changes in their contents, and therefore, limits need to be set. This work reviews the current methodologies reported in official technical documents and literature and develops an assessment strategy as a basis to establish health-based limits for toxicologically relevant impurities. A tiered approach is proposed, consisting of three stages. The first stage is the establishment of the potential toxicological relevance of an impurity. It is basically the hazard identification of the impurity and its comparison with the hazard of the active substance. The second stage is the estimation of the upper limit concentration in the TM for the potentially toxicologically relevant impurity. In this stage, four schemes are defined depending on the nature of the impurity: N-nitrosamine compounds, dibenzodioxins and dibenzofuran compounds, genotoxic impurities and other types of impurities. Finally, the third stage is the estimation of the likelihood that a potentially toxicologically relevant impurity is a toxicologically relevant impurity in the TM placed on the market. The proposed approach is regarded as a tool to be used in regulatory toxicology assessments for the decision making on active substances at EU level and for the authorization of PPPs at national level. P106-044 INTEGRATING MULTIMEDIA ENVIRONMENTAL MODELS AND EFFECTS MODELS ON A COMMON PLATFORM FOR RISK ASSESSMENT

C. Brochot (1)

, T. Tanaka (2)

, E. Johansson (3)

, R. Beaudouin (1)

, F. Zeman (1)

, F. Y. Bois (1)

, P. Ciffroy (4)

. (1)

Institut National de l'environnement industriel et des risques (INERIS), Verneuil-en-Halatte, France; (2)

Istituto di Chimica Agraria ed Ambientale /Università Cattolica del Sacro Cuore;

(3) Facilia, France;

(4) Electricité de France (EDF)

Exposure and dose-response assessments are two pillars of health risk assessment. Human exposure through multiple pathways is classically estimated by multimedia models, calculating the distribution of contaminants among the different routes of exposure (e.g., drinking water, inhaled air, food). Combined with data describing human behaviour (diet composition, time spent outside, etc), such models provide an estimate of the daily quantity inhaled or ingested by humans. Dose-response modelling aims at determining the relationship between the dose and the probability of an effect. The use of the external dose to relate to adverse responses may not reflect the current understanding of the mode of action of a chemical and does not facilitate extrapolations to other scenarios (species, exposure routes...). To overcome these limitations, physiologically based pharmacokinetic (PBPK) models are used to predict internal effective concentrations, i.e. in the target tissues where toxic effects arise. A dose-response model is then applied to link the effective concentration to the adverse effects. The European project 2-FUN (Full-chain and UNcertainty Approaches for Assessing Health Risks in FUture ENvironmental Scenarios) aims at improving the methodologies currently used in exposure and dose-response assessments. In that project, we integrated multimedia and PBPK models on a common platform, Ecolego TM. We show here the application of these integrated risk assessment models to benzo(a)pyrene, a polycyclic aromatic hydrocarbon. We study in particular the propagation of uncertainty and inter-individual variability along the modelling chain. Multimedia models are used to calculate the daily exposure of humans to BaP. The resulting exposure scenario is automatically input in a generic lifetime PBPK model for humans to estimate internal dosimetry. Finally a dose-response model is coupled to the PBPK model to predict the risk of cancer. P106-046 CHEMICAL MIXTURES ASSESSMENT AND GENETIC VARIABILITY

H. R. Pohl. CDC/ATSDR, Atlanta, United States The Agency for Toxic Substances and Disease Registry’s (ATSDR) approach to chemical mixtures risk assessment can be briefly summarized as follows: If no toxicity data are available on the mixture to be evaluated as a whole entity, approaches to evaluate the toxicity of components of the mixture such as the hazard index (HI) are commonly used. The HI approach assumes dose additivity. Therefore, a weight-of-evidence (WOE) evaluation of the potential for non-additive interactions (synergism, antagonism) among the components in the mixture is used to augment the HI. Determination of interaction thresholds provides an important insight for risk assessment of mixtures. One of the factors that can influence interaction thresholds is genetic variability. Genetic variability is considered in risk assessment for single chemicals to be part of the traditional generic inter-individual uncertainty factor (UF). Identification of susceptibility genes provides an opportunity to replace the generic UF with a data-derived factor for each hazardous chemical. The same concerns apply to chemical mixtures. For example, VOCs in mixtures are metabolized using P450 enzymes, specifically CYP2E1, in their respective metabolic pathways. From the mechanistic view, additivity is likely at low levels for most of the binary interactions. Competitive inhibition may exist between these chemicals at CYP isozyme catalytic sites under high exposure conditions when the sites are saturated. Inheritable gene alterations may result in changes of function of these enzymes in different subpopulations. Variations in quantity and/or quality of particular isoenzymes may cause differences in metabolism of VOCs and different thresholds for


17

interactions seen among these subpopulations. As a result, some populations may be more vulnerable to effects induced by exposure to these mixtures. Risk assessment of mixtures should take into consideration these variations. P106-051 SOURCES OF VARIATION IN THE DERIVATION OF REGULATORY RISK LEVELS FOR ORAL EXPSOURE TO MELAMINE

L. Bestervelt, V. Bhat, G. Ball, C. Mclellan. NSF International, Ann Arbor, United States The general population may be orally-exposed to low levels of melamine through the use of melamine-formaldehyde dinnerware and through drinking water since it is used in some materials that convey potable water. In Spring 2008, melamine was also associated with acute renal failure in Chinese infants due to its use to adulterate infant milk powder to increase apparent protein content. A Reference Dose (RfD) of 0.1 mg/kg-day was calculated by NSF International for melamine in 2009. The point of departure was 10% benchmark dose level of 38 mg/kg-day for urinary calculus formation in laboratory rats due to inadequate data in humans, particularly with respect to the NOAEL for melamine-induced pediatric urolithiasis. The RfD incorporated a 300-fold uncertainty factor (10-fold for interspecies and intraspecies extrapolation and a 3-fold database uncertainty factor). In 2008, the World Health Organization (WHO) established (and Health Canada adopted) a tolerable daily intake (TDI) for melamine of 0.2 mg/kg-day also based on urinary calculus formation in laboratory rats but with an additional feed intake–re¬duction factor and an uncertainty factor of 200 (based on a default 100-fold factor plus an extra 2-fold factor to account for sensitivity of infants and for the possible underreporting of bladder calculi due to tissue preparation). The European Food Safety Authority adopted a TDI of 0.5 mg/kg-day in 2008 for melamine which was previously derived in 1986 with limited available details. These oral risk values are collectively all within a half order of magnitude. With the exception of the EFSA TDI of 0.5 mg/kg-day for which the basis of the derivation in unclear, the differences in recent oral risk levels for melamine are primarily the result of subtle differences in point of departure and uncertainty factor application, since the key study and critical effect appear to be the same. P106-052 STRATEGIES FOR THE TOXICOLOGICAL RISK ASSESSMENT OF MULTI RESIDUAL IN CHILEAN MINING INDUSTRIES

L. Börgel, F. Bobadilla, M. Schulthess. SERVITOX / Legal Medicine Department, Universidad de Chile, Santiago, Chile One of the principal economical activities of Chile is the copper mining industry, which generates several wastes that must be evaluated for the determination of its final destination, carrying out the national norms and the international agreements. Its mayor problem is to confront a multi residual of solutions from cooling; corresponding to acid waters with metals in solution, with physic-chemical established characteristics and containing several metals (As, Mo, Cu, Re, Fe) in sulphuric acid solution. The mining industries must take measures for the waste transport or layout in situ; therefore, a toxicological risk assessment is required for the decision making. These tests are of high costs, because they involve times up to 2 years, therefore the stabilization of an alternative for the evaluation of this risks is of high business importance. As noted above, the realization of this evaluation through models based in REACH, EPA, OMS, and ATSDR was raised; the models for human, mammal and ecotoxicologic risk considering the most critical compounds of these multi residuals and its acute and chronic effects were done. The models applied to the transport situation considered the risks of human exposition by different ways, spillage situations, and possible contamination of ground or water fonts as for acute or chronic exposition, as for effects of cancer risk. The estimations for the LD 50 were done, according to the percentage of the various contaminants of this multi residual. The models executed allow establishing the transport risk for these multi residuals and we recommended the handling in situ. These methodologies applied didn´t require the experimental studies and the models adjust to the local realities of the country, allowing the company for taking the decisions in an informed way. P106-053 A 13-WEEK REPEATED DOSE TOXICITY OF EPHEDRA HERBA ADMINISTERED BY ORAL GAVAGE IN F344 RATS

J. I. Huh (1)

, H. Y. Han (1)

, S. Rime (1)

, J. Y. Kim (1)

, S. J. Park (2)

, B. S. Min (3)

, S. J. Kwack (4)

. (1)

Korea Institute of Toxicology, Daejeon, Korea, Republic of; (2)

College of Veterinary Medicine, Kyungpook National University;

(3) College of Pharmacy, Catholic University of Daegu;

(4) National Institute of Toxicological Research,

Korea, republic of Ephedra Herba is perennial shrub and is distributed over the east of Asia. Its roots have long been used as a traditional indigenous medicine to cure asthma and hay fever. The main component of this herb is ephedrine which has been reported as a cause of sudden deaths. We conducted toxicological evaluation of Ephedra Herba in this study using F344 rats. The extract of Ephedra Herba was administered orally to F344 rats at dose levels of 0, 125, 250, 500 and 1000 mg/kg/day for 13 weeks. Three males and 2 females at 1000 mg/kg/day were found dead during the treatment period. Salivation, eye discharge, and/or hypersensitivity were found in these animals. The reason for the deaths is now under investigation. Subdued behavior, hypersensitivity, lacrimation, eye discharge, salivation and staining around mouth were observed in the animals treated at 1000 mg/kg/day. Body weights and cumulative weight gains were significantly lower in all animals at 1000 mg/kg/day compared to those of the control animals. There are no test article-related changes in ophthalmology and urinalysis. The levels of RBC, HGB, HCT, MCV, MCV, MCH, NEU, PT and APTT in males at 500 or 1000 mg/kg/day in hematology were significantly changed. TCHO, PL, ALP, IP, and


18

K were increased in males. BUN, A/G, and ALP were increased in females in the treatment groups. TG was decreased in both males and females at 1000 mg/kg/day. Relative weights of brain, liver, salivary glands, and kidney were increased significantly in males and females at 1000 mg/kg/day. Relative weights of testis and lung were increased in males at the the higher group and relative weight of ovary in females were increased at 1000 mg/kg/day. These differences were dose-dependent and are considered as treatment-related. Further pathological evaluation will be followed to assess safety of this drug. P106-054 IDENTIFYING "NOT CLASSIFIED" SUBSTANCES (ACUTE ORAL LD50 > 2000 MG/KG) USING THE 3T3 NEUTRAL RED UPTAKE CYTOTOXICITY ASSAY: PERFORMANCE IN AN ECVAM EVALUATION

H. Raabe (1)

, P. Prieto (2)

, R. Curren (1)

, M. Whelan (2)

, A. Hilberer (1)

, S. Coecke (2)

, R. Gibson (3)

, A. Compagnoni (2)

, T. Hartung

(4), A. Kinsner-Ovaskainen

(2).

(1) Institute for In Vitro Sciences, MD, USA, x, United States;

(2) IHCP/ECVAM, EC-JRC, Ispra, Italy, IHCP, European

Commission Joint Research Centre, Ispra, Italy, (3)

Health and Safety Laboratory, UK, (4)

Johns Hopkins University, Bloomberg School of Public Health, Baltimore, USA, Recent legislation affecting chemical safety testing such as 7th Amendment of the European Council´s Cosmetic Directive and the new Chemicals Regulation REACH, challenge the toxicology community to develop novel means of determining the toxicity of chemicals using non-animal methods. It has recently been reported that ~87% of the substances in the European Union´s (EU) New Chemical Database (NCD) have acute oral LD50s >2000 mg/kg, placing them in a "not classified" category. Therefore the European Centre for the Evaluation of Alternative Methods (ECVAM) organized a trial of the 3T3 Neutral Red Uptake cytotoxicity assay to assess its ability to discriminate between toxic/hazardous (LD50< 2000 mg/kg) and not classified (LD50> 2,000 mg/kg) substances. Fifty-four coded industrial chemicals and two plant alkaloids were tested in three independent laboratories by slightly different methods. The Health and Safety Laboratory (UK) used a manual procedure of a previously validated protocol while an automated, robotic version of the protocol was performed at the Institute for Heath and Consumer Protection (JRC, Italy). The Institute for In Vitro Sciences (USA) assessed a less costly, abbreviated version of the protocol targeted at resolving toxicities around the 2,000 mg/kg cutoff. Results were very similar among the three laboratories and showed that assay sensitivity and predictive values were up to 100% for identifying industrial chemicals with LD50>2,000 mg/kg. Only two laboratories tested the highly toxic alkaloids; one alkaloid was correctly identified in both labs, while the other was correctly identified in only one of the two labs. Although specificities were relatively low (<45%), the high prevalence of non-toxic substances in the NCD (~87%) indicates that if this in vitro approach was used in the real-world situation, a large number of new chemicals could be correctly identified as non-toxic without animal testing. P107-026 CALIBRATION OF PBPK MODELS BASED ON IN VIVO AND IN VITRO DATA TO DESCRIBE COMPLEX BIOTRANSFORMATION PROCESSES

D. Habka (1)

, A. R. Péry (1)

, C. Aninat (2)

, A. Corlu (2)

, C. Guguen-Guillouzo (2)

, E. Leclerc (3)

, C. Legallais (3)

, C. Brochot

(1).

(1) Institut National de l'Environnement Industriel et des Risques - INERIS, Unité METO, Verneuil en Halatte, France;

(2)

Université Rennes 1, INSERM - U991, IFR 140, Rennes, France, (3)

Université de Technologie de Compiègne, CNRS - UMR6600, Compiègne, France

Toxicity is not always related to parent compounds, but could implicate their metabolites. In such cases, a full description of the biotransformation integrated in a pharmacokinetic model is needed to predict reliably the internal dosimetry of the different substances (parent and metabolites) in target organs where the toxic effects arise. The modeling of complex metabolic reactions requires data on a wide range of concentrations. Typically, human exposures at low dose for chemicals or therapeutic dose for drugs may be authorized. Then all the data needed for modeling are not available for humans and animals may not be representative of humans since the metabolic activities can be quite different between the two species. In that case, an alternative is to complement the human data with in vitro experiments based on human cells describing the metabolic reactions at the organ level. In this study, we propose to study ifosfamide metabolism in humans. Ifosfamide metabolism is a quite complex process since it exhibits a time-dependent increase in the metabolic clearance which is explained by a mechanism of auto-induction. The concentrations in plasma of ifosfamide and major metabolites (responsible of neurotoxicity and nephrotoxicity) have been investigated after intravenous administration of a therapeutic dose in adults. In addition, in vitro experiments on the induction of the enzymatic activity by ifosfamide on HepaRG cells were performed. A physiologically based pharmacokinetic model for ifosfamide in humans was then calibrated based on the in vitro and pharmacokinetic data to estimate the metabolic parameters and few other model parameters (e.g., partition coefficient). Our analysis shows that the combination of pharmacokinetic data with the description of a complex process via in vitro experiments enables to increase the validity range of complex PBPK model. P107-027 HEPATIC AND RENAL XANTHINE OXIDOREDUCTASE ACTIVITY IN OBESE ZUCKER RAT MODEL OF METABOLIC SYNDROME

J. Laakso (1)

, R. Lapatto (2)

, T. Vaskonen (3)

. (1)

Finnish Food Safety Authority, Evira, Helsinki, Finland; (2)

Hospital for Children and Adolescents, Helsinki, Finland; (3)

Institute of Biomedicine, Pharmacology, Finland


19

Aims: Serum urate levels are typically increased in metabolic syndrome. Xanthine oxidoreductase (XOR) is the key enzyme in purine catabolism and catalyses the reaction producing urate. We hypothesized that XOR is induced during development of obesity and later metabolic syndrome. Main methods: Obese and non-obese Zucker rats were followed up for 10 weeks. Additionally one group of animals was treated with valsartan and some and second with an atherogenic diet. XOR activity was measured with a specific assay utilizing radio-labelled xanthine. Key findings: Xanthine oxidoreductase activity but not mRNA level was increased in kidneys and liver of obese animals. The obese rats had twice as high renal XDH and XO activities than the lean Zucker rats. Valsartan, an angiotensin receptor antagonist, decreased hepatic XDH and XO activities to approximately one fourth of the initial levels. Neither valsartan nor atherogenic diet affected renal XDH or XO activities significantly. Significance: These findings extend the role of XOR in the pathology of metabolic syndrome. We have earlier shown that its activity is increased in hypertensive rats. This study provides evidence that the enzyme is also increased in normotensive obesity. Changes in hepatic XOR activities in obese subjects treated with angiotensin receptor antagonists may alter hepatic elimination rate of purine antimetabolites, which are used as immunosuppressants and anticancer drugs. P107-029 IN VITRO METABOLISM OF THE M2 VINCLOZOLIN METABOLITE BY RAT LIVER MICROSOMES

A. Sierra-Santoyo, M. J. Loera-Rosales, F. G. Garcia-Montes De Oca, M. D. L. Lopez-Gonzalez. Departamento de Toxicologia, Cinvestav-IPN, Mexico, Mexico Vinclozolin (V) is an agricultural fungicide. V is non-enzimatically hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2). V, M1 and M2 are antiandrogenic by interacting with the androgen receptor being M2 is the most potent metabolite of them. Data on the M2 biotransformation is limited. The objective of this study was to determine M2 metabolism by rat liver microsomes. M2 (50-600 uM) was incubated in 0.1 M phosphate buffer pH 7.4, 5 mM MgCl2, 2 mg of non-treated adult male Wistar rat liver microsomes and 1 mM NADPH for 30 min at 37ºC. The addition of anti-rat cytochrome P450 (CYP) antibodies to the incubation media permited to determine the CYP isoforms involved in M2 biotransformation. M2 metabolites from incubation media were extracted with acetonitrile and analyzed by HPLC/DAD. M2 was biotransformed to two metabolites, 3',5'-dichloro-2,3,4-trihidroxy-2-methylbutyranilide (M5) and 3,5-dichloroaniline (M3). The KM and VMax values for M5 were 192.4 uM and 0.924 nmoles/min/mg protein, respectively. Anti-rat CYP1A2 and 2E1 antibodies inhibited 42 and 36%, respectively, the formation of M5. For M3 the KM and VMax values were 344.6 uM and 0.201 nmoles/min/mg protein, respectively. M3 formation was inhibited by anti-rat CYP2A and 3A antibodies about 20% each one. The intrinsic clearance (CLint) values for M5 and M3 were 4.802 and 0.584 ul/min/mg, respectively. These results indicate that M5 is the main metabolite of M2 and their biotransformation is CYP-dependent for both metabolites, in addition, support the proposal that M5 may be used as a biomarker of exposition to V. The characterization of V biotransformation pathway may clarify the relationship between toxicity and tissue dosimetry of V and its metabolites. (Funded by Conacyt 45688-Q). P107-032 EFFECT OF TREATMENT PERIOD WITH CHROMIUM PICOLINATE ON THE TISSULAR ZN/CU RATIO OF DIABETIC RATS

M. V. Aguilar, J. M. Laborda, M. J. González, A. Bernao, I. Meseguer, M. C. Martínez Para, C. J. Mateos. Alcalá de Henares University, Alcalá de Henares, Spain

Due to the role of chromium in the utilization of glucose in conjunction with insulin, the administrat ion of dietary supplement of this element has been proposed as aid therapy in the treatment of diabetes. This disease is a risk factor of coronary pathology and the Zn/Cu ratio is a parameter associated with potential cardiovascular alterations. The aim of our study was to examine Zn and Cu levels and the Zn/Cu ratio in insulin target tissues in diabetic Wistar rats in order to determine the influence of the Cr treatment period because the chromium could interact with other mineral elements. Male growing Wistar STZ diabetic rats were fed with basal diet supplied with 500 micrograms/day of chromium (as chromium picolinate) for 7, 21 and 60 days (n=30). Paralely, a control group (n=10) was not treated with this compound. They are sacrified and the target tissues (liver, adipose tissue and skeletal muscle) were removed and Zn and Cu levels were measured by AAS after wet mineralization. The results showed a trend to higher Cu levels during 7 and 21 days (p<0.01) and lower Cu levels to 60 days of treatment and lower Zn levels in liver and skeletal muscle. Zn/Cu ratio decreases in liver and the other organ to 7 and 21 days, to increase to 60 days. There was not a dependent relationship between Zn/Cu ratio and treatment period. However, these results suggest that if the diabetic rats are treated with chromium picolinate for 7 and 21 days the risk of developing coronary pathologies decreases. P108-058 PRELIMINARY RESULTS ON HEAVY METALS CONTENT OF SPONTANEOUS HERBS GROWN IN POLLUTED AND UNCONTAMINATED SITES

M. G. Volpe (1)

, M. Nazzaro (1)

, A. De Mattia (2)

, F. Volpe (2)

, M. Di Stasio (1)

, R. Scudiero (3)

, F. La Cara (4)

. (1)

Istituto di Scienze dell'Alimentazione, Avellino, Italy; (2)

Agenzia Regionale Protezione Ambientale Campania, Italy; (3)

Dipartimento di Scienze Biologiche, Università degli Studi di Napoli Federico II,; (4)

Istituto di Biochimica delle Proteine, Italy The bioaccumulation of heavy metals in plant and animal tissues causes toxic effects in the short and long term, depending on the metal and its toxicological characteristics. Presently no study on the ecotoxicological effects of metals in plants was carried out in Irpinia, a territory located in the South of Italy. The aims of this research were: -Set-up of an integrated monitoring of heavy metals, airborne or accumulated in the soil, through the use of herbs typical of


20

Irpinian territory (Cichorium intybus, Borrago officinalis L. and Sonchus asper L.) as bioaccumulator of these metals. -Definition of a toxicological overview of metals referring to the analyzed area. This is an innovative approach in a complex territory as the Irpinia that is characterized by articulated patterns of dispersed pollutants. For our investigation we have identified seven sites located in different areas of the Irpinian territory characterized by different pollution grade: four well-defined areas with a considerable potential pollutant and three sites considered at minor risk. The results of preliminary ICP analysis on herbs samples showed that the concentrations of physiological metals for the sites with polluted and uncontaminated sites, did not much differ. Only Borrago officinalis L. showed a tendency to accumulate metals slightly higher than the others herbs. In the case of toxic heavy metals such as cadmium, lead and mercury, the experimental data showed a higher concentration values in the sites considered at high level of pollution, compared to the environment at low pollution risk. In high and low risk areas, Borrago officinalis L. and Sonchus asper L. showed metals concentration values higher of Cichorium intybus. The heavy metals experimental values were significantly higher into the high polluted sites samples. P108-063 METALLOTHIONIN EXPRESSION AS A BIOMARKER OF HEAVY METAL POLLUTION AT JEDDAH COAST-KSA

M. Montaser, M. Mahfuz, S. El-Shazly, G. Ibraheim, S. Bakry. Biotechnology Department, Faculty of Science, Taif University, 888 Hawia,Taif, KSA, Taif, Saudi Arabia Industrial development in the developing countries has resulted in heavy metal contamination of local waters. Metal pollution may damage marine organisms at the cellular level and possibly affect the ecological balance. Metallothionein (MT) is a low molecular weight protein that binds heavy metals in marine organisms for this it considered as biomarker of heavy metal pollution in aquatic environments. In this study, we examined the expression of MT production in Sleek Unicorn fish (Naso hexacanthus). Fishes were collected from five sampling stations and two potentially non-contaminated (control) stations, over the coast Jeddah on Red Sea. Weight and length of each sample were measured then dissected, gills and liver of each excised, divided and kept in liquid nitrogen and fixed in buffered formalin for histopathological examinations. Expression of metallothionein mRNA was monitored using semi-quantitative RT–PCR. Mann-Whitney U-rank test was adopted for assessment the differences between the tested groups. Morphometric measurements showed significant decrease in both secondary lamella length (SLL), width (SLW) and interlamellar distance (ILD) in fishes from contaminated areas. Histopathological examination of the liver revealed hepatocytes vacuolation, cellular swelling, nuclear degeneration and congestion of blood vessels. Pathological changes of gills exhibit secondary lamellar disorganization rupture of lamellar epithelium and epithelial lifting. Meanwhile, the expression of metallothionein mRNA, resulted in significant increase in expression of Metallothionein in response to metal pollution. Our study concluded using of MT as biomarker revealed clear metal pollution in the studied areas of Red Sea near Jeddah coast. Therefore, the problem of metal pollution considered among the most serious once that face mankind in the twenty-one century. It is supposed to be one of the greatest national health problems with referring to peoples eating sea foods in KSA, it require special and intense effort at all level individual, groups, national, and international. P108-065 DNA DAMAGE ON CROCODYLUS MORELETTI FROM AN URBAN LAKE, EXPOSURE TO METALS AT SOUTHERN GULF OF MEXICO

M. T. G. Rodríguez (1)

, R. G. Aldeco (1)

, L. S. Osorio (2)

. (1)

Universidad Juárez Autónoma de Tabasco, Villahermosa, Mexico; (2)

Universidad Nacional Autónoma de México At the southern Gulf of México, some crocodile populations have been decreasing their number due to anthropogenic pressures to their habitat. A continuous inlet of pollutants to their aquatic ecosystems, like municipal waste and pluvial waters carrying particulate and oil deposited by auto motors, with risk to health and reproduction capabilities of individuals. Genotoxic effect of environmental metals (like lead) on the DNA of a wild Crocodylus moreletii population of an urban lake, in order to establish subpopulations or individuals at risk, was determinated lead concentration in 28 blood samples and 20 dermal plates from crocodiles. Samples, their respective blanks and standards were analyzed by triplicate with an A.A. spectrometer with graphite furnace (Perkin Elmer 700 ). DNA strands brakes were evaluated with the Single cell electrophoresis assay on peripheral blood leucocytes of wild individuals taken from the lake and compared to a control group from a reproduction farm. DNA migration from the nucleus measured in µm was from 19.5 up to 49.88 and from 0 up to 35 in the wild and control groups respectively. DNA migration was reported as the Tail/head ratio. Wild group presented a mean value of 1.6 + 0.10, while control group was 0.4 + 0.16.The difference was statistically significant (p<0.05). Lead in blood samples were from 6.55 up to 15.34 µg/dl with a mean of 12.01±1.6 µg/dl. Mean Blood Lead concentration was slightly higher in wild organisms (12.01 ± 1.6 µg/dl) than organisms from a crocodile farm (8.15 ± 1.7µg/dl), however this was not statistically significative (p<0.05).Lead concentration in dermal plates was from 4.2 up to 55.29 ppm with a mean value of 22.62 ± 3.2 ppm. level of damage evaluated on wild animals were medium to high. Crocodylus population in this natural habitat is impacted in their genetic material by urban activities. P201-088 DETERMINATION OF ABSORPTION AND BIOTRANSFORMATION OF BENZO[A]PYRENE IN A PERFUSION CO-CULTURE SYSTEM FOR PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS

D. A. Ouattara (1)

, S. H. Choi (2)

, Y. Sakai (2)

, C. Brochot (3)

. (1)

UMR-CNRS 6600, Université Technologique de Compiègne, Compiègne, France; (2)

Institute of Industrial Science, University of Tokyo, Tokyo, Japan;

(3) INERIS, Verneuil-en-Halatte, France


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In vitro systems are widely used to characterize the processes involved in the toxicokinetics of chemicals, i.e. the absorption, distribution, metabolism and excretion. These systems have been investigated as an alternative to the use of animal experiments. In order to define an in vitro system mimicking as much as possible the human body, systems combining different cellular types and providing an environment close to in vivo conditions (e.g., organ interactions and a fluid connecting the different cell types) have been developed. This study proposes to model the absorption and biotransformation of an organic contaminant, the benzo(a)pyrene (BaP), in a perfusion co-culture system developed to mimic the absorption across the small intestine and the biotransformation in the small intestine and the liver. This simple double-layered co-culture system consists of Caco-2 cells on a semi-permeable membrane culture insert and monolayer-cultured Hep G2 cells. The kinetics of BaP and three metabolites (BaP-9,10-diol, BaP-3-OH and BaP-7,8-diol which is the immediate precursor to the ultimate toxic chemical B[a]P-7.8-diol,-9,10-epoxide) in the perfusion system have been investigated for several exposure conditions (e.g., mono- or co-culture, with or without an inducer...) to allow a full description of the their behaviours in the exposure system. A 3-compartement model describing the kinetics of BaP and metabolites in the exposure system was developed. This model was calibrated with the concentrations of the four chemicals in the apical and basolateral sides of the culture system and in the Hep-G2 insert to estimate the permeability of BaP in the small intestine and the metabolic conversion rates of BaP to BaP-9,10-diol, BaP to BaP-3-OH and BaP to BaP-7,8-diol. These in vitro estimates were then converted to be integrated into a PBPK model for BaP and metabolites in humans. P201-089 ASSESSMENT OF CHEMICAL SKIN SENSITIZING POTENCY BY AN IN VITRO ASSAY BASED ON HUMAN DENDRITIC CELLS

N. Lambrechts, I. Nelissen, H. Witters, R. Van Den Heuvel, G. Schoeters, J. Hooyberghs. VITO nv, Mol, Belgium The skin sensitizing potential of chemicals is an important concern for public health and thus a significant endpoint in the hazard identification process. To determine skin sensitizing capacity, large research efforts focus on the development of assays which do not require animals. As such, an in vitro test (VITOSENS®) has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34+ progenitor-derived dendritic cells (CD34-DC) which allows to dichotomously classify chemicals as skin (non )sensitizing. However, skin sensitization is not an all or none phenomenon and up to now the assessment of relative potency can only be derived using the in vivo local lymph node assay (LLNA). In our study we analyzed the feasibility to predict the sensitizing potency, i.e. the LLNA EC3 values, of 15 skin sensitizers using in vitro data from the CD34-DC-based assay. Hereto we extended the in vitro generated gene expression dataset by an additional source of information, the concentration of the compound that causes 20% cell damage (IC20) in CD34-DC. We statistically confirmed that this IC20 is linearly independent from the gene expression changes, but that it does correlate with LLNA EC3 values. In a further analysis we applied a robust linear regression with both IC20 and expression changes of CREM and CCR2 as explanatory variables. For 13 out of 15 compounds, a high linear correlation was established between the in vitro model and the LLNA EC3 values over a range of 4 orders of magnitude, i.e. from weak to extreme sensitizers. P201-093 EVALUATION OF ENVIRONMENTAL INSULT-MEDIATED DAMAGE USING THE STRATATEST® HUMAN SKIN MODEL

C. Rasmussen (1)

, F. Liebel (2)

, M. Southall (2)

, K. Gratz (1)

, A. Comer (1)

, L. Allen-Hoffmann (1)

. (1)

Stratatech Corporation, Madison, United States; (2)

Johnson & Johnson Consumer & Personal Products Worldwide, United States

Skin is continuously exposed to a variety of potentially damaging environmental factors, such as atmospheric pollutants and ultraviolet (UV) irradiation. Three-dimensional in vitro skin models allow direct assessment of the damage caused by these stressors and permit detailed examination of agents formulated to prevent or mitigate environmental-mediated damage in a format that mimics the in vivo environment. The StrataTest® model is a fully-stratified skin tissue, containing both epidermal and dermal components, that recapitulates the biological characteristics of human skin. In the present study, we demonstrate the utility of this skin model to evaluate the skin-damaging effects of three known environmental insults: ozone, cigarette smoke, and UV light. Oxidative damage, as measured by an increase in reactive oxygen species (ROS), was detected after exposure of StrataTest® skin tissues to all three factors. Pretreatment of tissues with the antioxidant parthenolide-depleted (PD)-Feverfew extract prior to smoke exposure prevented this response. An increase in ROS formation in UV-irradiated StrataTest® skin tissues was also mitigated by pretreatment with PD-Feverfew extract, although this protection was not as effective as a full spectrum UVA/UVB sunscreen. UV-mediated cytokine release and cellular DNA damage were also investigated using the StrataTest® human skin model. IL-1alpha and IL-1RA secretion was shown to increase in a dose-dependant manner upon UV irradiation, however proinflammatory cytokine release was abrogated by pretreatment with sunscreen. Immunohistochemical detection revealed a substantial increase in the formation of thymidine dimers in UV-irradiated skin tissue, but this effect was reduced in tissues pretreated with sunscreen. These results demonstrate the ability for the StrataTest® human skin model to accurately reproduce the in vivo response of human skin to environmental insults, and to assess agents formulated to prevent or mitigate environmental-mediated damage.


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P201-094 IN VITRO EXPERIMENTAL MODEL OF ETHANOL INDUCED HEPATOTOXICITY USING ANTI-OXIDANT BIOMARKERS IN HEPG2 CELLS

S. Sathaye, Y. Bagul, S. Gupta, R. Kenjale. Institute of Chemical Technology, Matunga, Mumbai., Mumbai, India

Chronic alcoholism diminishes the cellular antioxidant levels through free radical induced injury causing hepatitis and auto-immune cirrhosis with mortality in severe cases. Despite numerous public education initiatives, alcoholism still remains a leading cause of mortality throughout the world. Ethanol induced hepatotoxicity is mainly due to generation of reactive oxygen radicals. Ethanol interferes with transport of glutathione through cellular membranes, leading to its depletion from mitochondria. This glutathione deficiency may permit mitochondrial damage and cell death by means of unimpeded lipid peroxidation (Bird and Williams., 1988). Considering the free radical mechanism of ethanol induced toxicity and the role of antioxidants, an experimental model simulating such conditions was developed using human hepatocyte cell line HepG2. Cells were incubated in the presence of increasing ethanol concentrations (40-140mM). Cytotoxicity was quantitated spectrophotometrically by the metabolism of the tetrazolium dye MTT. Anti-oxidant biomarkers of ethanol damage, viz., glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and anti-lipid peroxidation potential (LPO) were also estimated. No cytotoxicity was observed at 40mM ethanol whereas a dose-dependent increment was found at higher concentrations (60-100 mM ethanol). At 140 mM ethanol, cell viability after 24 hours was reduced to 52% as assessed by MTT. The SOD, CAT and GSH activities were significantly decreased at 100mM ethanol. The above model was used to examine the protective effects of aqueous extract of leaves of Murraya koenigii and its carbazole alkaloids and tannin fractions, compared to that of positive control, L-Ornithine L-Aspartate which is used to treat complications of alcoholic hepatitis. The modulating effect of the aqueous extract and isolates on liver metabolizing enzymes, reduction in lipid peroxidation and decreased cellular damage were found to contribute to their significant hepatoprotective activity. The method is well standardized as an in-vitro model to screen hepatoprotective activity of various drug substances from different origin. P201-095 TOXIC EFFECTS OF CHLORHEXIDINE MOUTHWASH FOR FIBROBLASTS AFTER ORAL SURGERY

S. Vahabi. SBMU, Tehran, Iran (Islamic Republic of)

Introduction: Chlorhexidine (CHL) is one of one of the most useful mouth rinses to establish chemical plaque control after oral surgeries and fibroblasts play a key role in healing process of periodontal tissues. This study was performed to evaluate the toxic effects of Chlorhexidine on fibroblasts of rat. Methods and Materials: L929 rats fibroblasts in culture media supplemented with FBS were treated by 0.2, 0 .12 and 0.009 percents of CHL concentrations for 30 seconds, 1 minute and 5 minutes. Then media was removed and cells were washed with RPMI for 3 times and were incubated in new culture media with MTT for 4 hours. Cytotoxicity of CHL would affect mitochondrial dehydrogenase of viable cells, so that they could not reduced the MTT to formazan crystals. Then Eliza reader detected optical density of color changes. Results: CHL was toxic in all doses and times. Toxicity between different doses and durations was not significant. Conclusion: Regarding to cytotoxicity of CHL in doses and durations much lesser than clinical application, we recommend conservative use of CHL after oral surgery and more similar studies to determine safe and effective dose and duration of it. P201-096 IMPROVED CELLULAR ANALYSIS FOR THE EARLY DETECTION OF COMPOUND-INDUCED HEPATIC CYTOTOXICITY

M. Schmitz1, A. Seiler

1, S. Kustermann

2, F. Boess

2, M. Watzele

1, L. Suter-Dick

2, A. B. Roth

2

1Roche Diagnostics GmbH, Penzberg, Germany;

2F. Hoffmann-La Roche AG, Basel, Switzerland

In toxicology, the early identification of toxic compounds by in vitro assays is expected to reduce the use of laboratory animals by preventing in vivo testing of suboptimal compounds that would fail at a later stage of drug testing. The combination of cellular assay systems with down-stream applications, such as proteomics and toxicogenomics, is a prerequisite for the generation of a predictive toxicological assessment, eventually strengthening the value of in vitro testing. We tested if the xCELLigence System could be implemented in a well-established toxicology workflow, using primary rat hepatocytes as an in vitro model for liver toxicity. The impedance-based xCELLigence real-time Cell Analyzer (RTCA) instrument, co-developed by Roche and ACEA Biosciences, allows label-free, dynamic monitoring of cellular events such as proliferation and cell death. Compound-induced hepatotoxicity could be continuously monitored by the xCELLigence System, yielding compound- and concentration-dependent impedance profiles. This reveals detailed information on the cell culture conditions over the entire length of the experiment, allowing informed decisions for the administration of compounds as well as the implementation of down-stream assays. Here we demonstrate a comprehensive workflow of in vitro techniques, including real-time cell analysis, biochemical assays, and gene expression analysis, for the assessment of liver toxicity. The herein described workflow application, along with the correlation of the collected data from all systems, highlights the added value of the xCELLigence System. As a consequence, the combination of the xCELLigence system with well-established endpoint assays and gene expression profiling may significantly improve the quality of early safety evaluations. For life science research only: XCELLIGENCE is a trademark of Roche; ACEA BIOSCIENCES is a registered trademark of ACEA Biosciences, Inc. in the US. Other brands or product names are trademarks of their respective holders.


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P201-098 CHARACTERISATION OF CADMIUM CHLORIDE INDUCED MOLECULAR AND FUNCTIONAL ALTERATIONS IN AIRWAY EPITHELIAL CELLS

M. D. P. Prieto-Peraita (1)

, E. Forti (2)

, A. Bulgheroni (3)

, Y. Cetin (4)

, P. Jennings (2)

, W. Pfaller (2)

. (1)

European Commission, Joint Research Centre, Ispra, Italy; (2)

Innsbruck Medical University, Austria, (3)

Polichem SA, Lugano-Pazzallo, Switzerland, ;

(4) Ministry of Agriculture and Rural Affairs, Turkey,

Epidemiological studies show that cadmium (Cd) exposure causes pulmonary damage, such as emphysema, pneumonitis, and lung cancer. However, the mechanisms leading to pulmonary toxicity are not yet fully elucidated. The aim of this study was to further investigate cadmium chloride (CdCl2) induced toxicity using Calu-3 cells as an in vitro model of human bronchial epithelial cells. CdCl2 induced effects following either apical or basolateral exposure were evaluated by Neutral Red Uptake (NRU), Trans-Epithelial Electrical Resistance (TEER), and alteration in Metallothionein 1X (MT1X), Heat shock 70 (HSP70), and Heme oxygenase 1 (HMOX-1) genes. CdCl2 exposure resulted in a collapse of barrier function and the induction of MT1X, HMOX-1 and HSP70 genes, prior to alterations in cell viability. These effects were more pronounced when the exposure was from the basolateral side. Co-administration of N-Acetylcysteine (NAC) exerted a strong protective effect against CdCl2 induced barrier damage and stress related genes, while other antioxidants only attenuated CdCl2 induced HSP70 and HMOX-1 and showed no protective effect on the barrier collapse. These findings indicate that CdCl2 exposure is likely to impair Calu-3 barrier function at non cytotoxic concentrations by a direct effect on adherens junction proteins. The protective effect of NAC against CdCl2 induced MT1X, HSP70 and HMOX-1 genes, demonstrates an anti-oxidant effect of NAC in addition to Cd chelation. P201-101 PHYTOCHEMICALS MODULATING CELL PROLIFERATION AND PRO-INFLAMMATORY CYTOKINES IN VITRO

E. Codorean (1)

, L. Albulescu (1)

, E. Raducan (1)

, I. D. Popescu (1)

, C. Nichita (2)

, C. Tanase (1)

, R. Albulescu (1)

. (1)

"Victor Babes" National Institute of Pathology, Bucharest, Romania; (2)

Natl Inst Chem Pharm. R&D, Bucharest, Romania Background - Many herbs and plant extracts have been identified as a source of various phytochemicals, some of them possessing important biological activities. Aim: to compare in vitro immunomodulatory effect of the Rosmarinus officinalis extracts (Rs) with those of some standardized bioflavonoids. Methods - Bioflavonoid: rich extracts from Rosmarinus officinalis (Rs) - RF1, RF2, RZ2, RF3 and some standardized compounds: rosmarinic acid, apigenin, quercetin, vitexin, genistein, silimarin were applied in range of 0.05µg/mL, 0.5µg/mL, 5µg/mL, 50µg/mL in the cultures of human whole peripheral blood and Jurkat cell line (human lymphoblastic T cells). After 24h the proinflammatory cytokines IL-1beta, TNF-alfa, IL-2, IL-6, IL-8, were quantified in supernatants of human peripheric whole blood cultures by xMAP assay (Luminex® 200™) using fluorescent microspheres, validated by correlation with ELISA method. Cell proliferation response to mitogens - ConA, PHA and LPS were evaluated by MTS test (MTS-Non Radioactive Assay kit). Results - Rs extracts induced anti-proliferative effects on Jurkat cell line (human lymphoblastic T cells) in a dose-dependent manner with optimal effects at 50µg/mL and with poor efficiency below 5µg/mL. Comparative assay with standardized compounds indicates R1 as fraction with optimal modulatory activity. The cytokine panel exhibited similar trends for both Rs extracts and rosmarinic acid, with significantly reduced IL-1beta (p<0.01) and non-significantly reduced TNF-alfa, IL-2, IL-6, IL-8. Conclusions - Rs extracts induced differential antiproliferative effects and modulation of antiinflamatory cytokines in vitro. Rs and rosmarinic acid showed nearly identical antiinflammatory and antiproliferative activities indicating the efficacy / pharmacological effects of each Rs fraction. The xMAP cytokine panels in vitro represent a rapid screening tool testing the natural extracts for standardization of new drug formulations. P201-102 ASSESSMENT OF A MODEL PROTOCOL FOR A HUMAN RECOMBINANT FULL LENGTH ESTROGEN RECEPTOR-ÁLPHA BINDING ASSAY - INITIAL STEPS TOWARDS VALIDATION

A. Freyberger (1)

, V. Wilson (2)

, M. Weimer (3)

, S. Tan (4)

, H. S. Tran (1)

, H. J. Ahr (1)

. (1)

Bayer Schering Pharma AG, Wuppertal, Germany; (2)

U.S. EPA, Office of Research and Development, NHEERL, United States;

(3) German Cancer Research Center, Heidelberg, Ghana;

(4) U.S. EPA, Office of Science Coordination

and Policy, OPPTS, Washington, United States

Despite two decades of research in the field of endocrine active compounds, still no validated human recombinant (hr) estrogen receptor-alpha (ERa) binding assay is available. In a joint effort, US EPA and Bayer Schering Pharma with funding from the European Union (LSHB-CT-2004-503257, ReProTect) developed a model protocol for such an assay. Important features of this assay are the use of a full length hr-ERa and performance in a 96-well plate format. A full length hr-ERa was chosen, as it was considered to provide the most accurate and human-relevant results. Besides three reference compounds [17beta-estradiol, norethynodrel, dibutylphthalate] test compounds with different affinities to the ERa [diethylstilbestrol (DES), ethynylestradiol, meso-hexestrol, equol, genistein, o, p´-DDT, nonylphenol, n-butylparaben, corticosterone] were used to explore assay performance. Three independent experiments per compound were performed, and dilutions of test compounds from deep-frozen stocks, solutions of radiolabeled ligand and ERa were freshly prepared for each experiment. The ERa binding properties of reference and test compounds were well detected. As expected dibutylphthalate and corticosterone were non-binders in this assay. In terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using a human recombinant ERa ligand and binding domain and a full length hr-ERa. Irrespective of its chemical nature, individual IC50-values for a given compound varied by not more than a factor of 2.5. Our data demonstrate that the


24

assay reliably ranked compounds of different affinity to the ERa with high accuracy. It avoids the manipulation and use of animals, i.e., the preparation of uterine cytosol as receptor source, as a recombinant protein is used and thus contributes to the 3R concept (reduce, replace, refine). Furthermore, in contrast to other assays, this assay could be adjusted to an intermediate/high throughput format and thus is a promising candidate for further validation. P201-103 IN VITRO VAGINAL SAFETY SCREENING APPROACH FOR BATH AND BODY WASH PRODUCTS UTILIZING SKINETHIC HUMAN VAGINAL EPITHELIUM (HVE) MODEL

T. Re (1)

, A. Alonso (2)

, B. Bertino (2)

, G. E. Costin (3)

, A. De Brugerolle De Fraissinette (2)

, D. Orak (1)

, H. Inglis (3)

, K. Norman

(3), P. Kazmi

(1), H. Raabe

(3), V. Srinivasan

(1).

(1) L'Oreal USA Products, Clark, NJ, United States;

(2) Skinethic Laboratories, Nice, France,

(3) Institute for In Vitro

Sciences, Inc. (IIVS), Gaithersburg, MD, USA, A common goal of many personal care companies is to assure the safety of their products without animal testing, due to concerns about ethical and animal welfare issues as well as the relevancy of the animal model to humans. To address these issues, we have developed an in vitro testing program to support the safety evaluation of potential vaginal irritation in a number of bath and shower cleanser products. A series of surfactant-containing formulations, diluted to 10% in water to mimic the maximum concentration expected in bath water, were tested. The formulations were applied topically onto the surface of commercially-available SkinEthic HVE three-dimensional human vaginal epithelium tissues over various exposure times. The ET50 (i.e. the exposure time expected to reduce relative viability of the tissues to 50% of controls) for each candidate was determined. The test results were compared to reference formulations and available human clinical data. The vaginal irritancy evaluation and ranking of bath and body wash products based on ET50 values showed a good correlation with the expected irritation potential of individual ingredients. Histopathology confirmed the overall MTT viability results and provided additional information regarding the effect of the tested products on the tissue´s integrity. However, IL-1α; release did not appear to be as sensitive a marker as the MTT viability assessment at the short exposure times used (20 minutes, 1, 2, and 4 hours). This in vitro safety screening approach shows promise for predicting the vaginal irritancy of tested products and in meeting the typical needs of product development groups charged with developing increasingly milder products. P202-054 CYTOGENETIC EFFECT OF MEDROXYPROGESTERONE ACETATE ON BONE MARROW CHROMOSOMES OF MICE

N. Aseem, S. Bakry, A. Abdullah. Zoology Department, Women College, for Arts, Science and Education, Ain Shams University, Heleopolis, Egypt

Among the many hormonal contraceptives approved by the Food and Drug Administration (FDA) in 1992 was Depo-Provera®, which has been used worldwide by more than 90 million women. In this study we aimed to investigate the effect of MPA on the bone marrow chromosomes of mice. Sixty healthy female mice were injected and divided into two groups. Each mouse was injected with a single dose of Depo-Provera (MPA) (0.39 mg/ mouse & 0.78 mg/ mouse). Animals were sacrificed after 10 and 15 days following treatment and bone marrow chromosomes were examined for chromosomal aberrations and mitotic index. The results of this study revealed that Depo-Provera (MPA) induced reduction in the mitotic indices at various doses which showed a highly statistically significant decrease. Also, this drug induced chromosomal aberrations in the metaphase of the bone marrow cells. These aberrations were manifested in numerical aberration as polyploidy and structural aberrations represented by centric fusion, centromeric attenuation, deletion, end to end association, endomitosis, fragment, and ring. These aberrations showed highly statistically significant increase. The present study indicated that Depo-Provera (MPA) could induce reduction in the mitotic activity and several types of chromosomal aberrations in the bone marrow cells of female mice. These aberrations, if transferred to the offspring could cause structural and/or behavioral malformations. P202-055 EXPOSURE TO RESIDENTIAL INDOOR AIR INDUCES HERITABLE DNA MUTATIONS IN MICE

J. Zhou, Y. Zhou, Y. Liu, W. Qiu, A. Li. Nanjing Medical University, Nanjing, China Exposure to the indoor air pollutants released from synthetic materials and chemical products poses a serious public health problem, but little evidence has been provided to clarify whether such pollutants, in a real-world context, produce inheritable germline mutations. In the present study, we first exposed mice to the indoor air of a newly decorated room, and detected somatic genome mutations present in their offspring by three probes, Ms6-hm, Hm-2, and MMS10 by Southern Blot analysis. For all the three assays, the exposed mice demonstrated mutations with a significant elevation (1.5~2.6 fold) over the control group, which is due primarily to an increase in mutations inherited through the paternal germline. Since formaldehyde (FA) is a type of major volatile organic chemical (VOC) present in indoor air, we further exposed mice to different concentrations of FA (0, 2, 20, or 200 mg/m3). Somatic genome ESTR mutations were detected in their offspring. Data indicated that with an increase in FA exposure dose, the mutation rate of three ESTR probes in somatic genome DNA of F1 mice rose significantly in a dose-dependent manner. The ESTR mutation in F0 mice sperm genome DNA was also increased in the group exposed to 200 mg/m3 FA. These results suggest exposure to a mixture of pollutants in indoor air induced ESTR mutations. Humans exposed to polluted indoor apartment air in China may be at risk for developing germline mutations. Single FA exposure might be a useful tool to identify indoor air mixture exposure-induced heritable DNA damage. Further studies need to clarify ESTR-linked chronic effects such as the specific genetic deficiencies on successive generation progenies.


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P202-059 EFFECTS OF THE TOXICOKINETICS (TK) BLOOD SAMPLING ON THE MICRONUCLEUS (MN) EXAMINATION AND COMPARISON OF THE METHODS TO DETECT MN

Y. Nagayama (1)

, S. Sawada (1)

, Y. Hori (2)

, K. Uchida (1)

, A. Suganuma (1)

, T. Aoki (1)

. (1)

Eisai Co., Ltd., Tsukuba-shi, Japan; (2)

Sunplanet Co., Ltd,

[Introduction] ICH S2(R1) genotoxicity step 2 draft guideline recommends the integration of the MN examination into repeated-dose toxicity studies, and TK animals may be used for the MN examination. Effects of the TK-blood-sampling on the MN detection, however, have been insufficient yet. Thus, in the present study, the frequencies of micronucleated-polychromatic erythrocytes(MN-PCE) were determined with and without TK-sampling. While flow-cytometric-analysis (FCM) is recently available for MN examination, it is important to compare the sensitivity to detect MN between the FCM and microscopic examination. Therefore the samples were analyzed by FCM and two microscopic analyses. [Methods] Cyclophosphamide (CP) 10 mg/kg or vinblastine (VBL) 0.5 mg/kg was administered to 8-week-old male SD-rats once, and blood (0.25 ml/point) was collected at 0.5, 2, 4, 8, 24 hours. Blood for MN test was collected at 48 hours and subjected to microscopic analyses and FCM. For microscopy, the blood smear was stained by acridine-orange (AO) and new methylene blue/acridine-orange staining (N/AO). Anti-CD71-FITC based method (MicroFlowPLUS kit, Litron Laboratories) was used for FCM. [Results and Discussion] As expected frequent blood collection increased the PCE ratio, but didn´t alter the frequency of MN-PCE, suggesting that TK-blood-sampling has no effects on the MN detection. In the CP administration group, the ratio of MN-PCE for FCM, AO and N/AO method was 0.82, 0.87 and 0.88% respectively. In the VBL group, the ratio of MN-PCE for FCM, AO and N/AO method was 0.41, 0.61 and 0.60% respectively. Thus, there is a good consistency in the sensitivity among these FCM and microscopy-based analyses. These results support the integration of the MN examination into repeated-dose toxicity study using the blood samples obtained from the TK animals. In addition, these results indicate FCM would be a suitable method because of additional merits of saving time and manpower while keeping the comparable sensitivity. P202-061 DIVALENT METAL TRANSPORTER 1 POLYMORPHISM FREQUENCIES IN TURKISH POPULATION

M. Odabaşı, Z. Kayaaltı, D. Tekin, A. Karakuş, T. Söylemezoğlu. Ankara University, Institute of Forensic Medicine, Ankara, Turkey Divalent metal transporter 1 (DMT1 also known as DCT1, NRAMP2 or SCL11A2) is a membrane-bound divalent metal transporter which is conserved from prokaryotes to higher eukaryotes. It has been postulated to play important roles in intestinal iron absorption at the brush border of duodenal enterocytes, erythroid iron utilization, hepatic iron accumulation, placental iron transfer, and other processes. It is widely expressed in most tissues and cell types as an integral membrane protein (90–100 kDa) modified by glycosylation. DMT1 gene which contains at least four isoforms (1A/+IRE, 1A/-IRE, 2/+IRE and 2/-IRE) is located on chromosome 12q13 in human. DMT1 mediates the transport of a wide range of metals, including the essential metals Fe2+, Zn2+, Mn2+, Cu2+, Co2+, Ni2+ and toxic metals such as Cd2+ and Pb2+ and mediates active proton-coupled from an acidic microenvironment (endosome, duodenal lumen) to the cell cytosol. The intention of this study is to determine that IVS4+44C/A single-nucleotide polymorphism (SNP) in DMT1 gene of Turkish population. For this purpose 262 biological blood samples from volunteers were analyzed (103 Female and 159 male). DMT1 gene was amplified with the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique and 352 bp oligonucleotide was produced. The amplified oligonucleotides were cut with MnlI restriction enzyme according to their polymorphic characteristics. Digested and undigested products were separated on a 2% agarose gel electrophoresis, visualized by ethidium bromide staining under an ultraviolet illuminator. The genotype frequencies of the SNP in Turkish population DMT1 IVS4+44C/A region were determined as follows; 46.9% homozygote typical, 39.7% heterozygote and 13.4% homozygote atypical genotype. The frequency of the C allele was found to as 66.8% and of the A allele as 33.2%. The allele and genotype frequencies were consistent with Hardy Weinberg equilibrium (÷2 = 2.857 Exact P=0.095). P202-066 INVESTIGATION ABOUT ESTROGENIC ACTIVITY OF ORGANIC EXTRACT OF ATMOSPHERIC PARTICULATE MATTER (PM2.5) FROM ARARAQUARA CITY, SOUTHEAST BRAZIL

F.S. Silva, V. Uzana, M. Frigieri, M. R. R. Marchi, E. A.Varanda Institute of Chemistry, Analytical Chemistry Department, São Paulo, Brazil

In Brazil, the sugarcane crops are burned to facilitate the harvesting, this process causes environmental pollution from the large amounts of soot that are released into the atmosphere. This material contain numerous organic compounds such as PAHs. In this study, PM2.5 and PAHs concentrations in the air of Araraquara (SE Brazil, with around 200,000 inhabitants and surrounded by sugar-cane plantations) were determined during the sugarcane harvesting (HV) and non-harvesting (NHV) seasons in 2009. The sampling strategy included two campaigns in each season, with 20 samples in non-harvesting season and 56 samples in harvesting one. PM2.5 was collected using a dichotomous sampler (10 L min-1, 24 h) with TeflonTM filters. Ten filters sets were extracted (ultrasonic bath with hexane/acetone (1:1 v/v)) and analyzed by HPLC/Fluorescence. The average concentration for total PAHs was 2.16 ng m-3 (NHV) and 4.72 ng m-3 (HV). The same extracts were submitted to recombinant yeast assay (RYA) that uses genetically engineered yeast cells to reproduce the natural pathway of genetic control by estrogens in vertebrate cells. A synthetic estrogen, 17- -ethinylestradiol (EE2), was used as the reference estrogenic compound. The extracts concentrations tested were 3.12, 1.87, 1.25, 0.62 and 0.31 ng /well (NHV season) and 6.41, 3.84, 2.57, 1.28 and 0.64 ng /well (HV season). All the samples showed no detectable estrogenicity values. We conclude that in spite of the HV season extracts (128.1 ng/mL) have presented larger amount of smoke and soot than non-harvest season (62.3 ng/mL), as


26

observed for HPAs, those amounts were not enough for induction of positive estrogenic activity. Acknowledgments: CNPq, CAPES, FACTE. P202-068 INVESTIGATION OF THE REDUCTION AND OXIDATION PRODUCTS FROM MUTAGENIC AZO DYES DISPERSE RED 1, DISPERSE RED 13 AND DISPERSE ORANGE 1

M. D. Chequer Farah1, T. M. Lizier

2, R. de Felício

1, M. V. B. Zanoni

2, N. P. Lopes

1, H. M. Debonsi

1, D. P. Oliveira

1

1Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil;

2Universidade Paulista

Júlio de Mesquita Filho, Brazil

A wide variety of azo dyes are used in the food, textile, printing industries and in chemical and biological laboratories. However, most of these dyes are toxic, carcinogenic, and mutagenic as well as their metabolites formed by reductive/oxidative processes. Our group showed that the azo dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1 are mutagenic in different cell system. However, little is known about the products formed by biotransformation of these azo dyes. Objectives: this work aimed to investigate the products formed after oxidation and reduction process, simulating in vivo reactions. Methods: The electrochemical measurements were carried out using a Potentiostat/Galvanostat. Controlled potential electrolysis was carried out on 3.18 x 10-4 mol.L-1 of Disperse Red 1, Disperse Red 13 and Disperse Orange 1 dyes solutions. Moreover, the electrochemical oxidation and reduction of the dyes’ solutions were also monitored by measurement of the absorbance changes at specified time intervals using a diode array spectrophotometer. HPLC-DAD and GC-MS were used for measuring the dyes studied and its metabolites. Results: After both reduction and oxidation step, we observed a modification in the azo bond, clearly demonstrated by the alteration of the UV-VIS absorption bands at 510 nm, 530 nm and 490 nm of the azo group in the dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1, respectively. We identified some products: Sulfate de N-ethyl-(2-hidroxiethyl)-p-phenyldiamine monohydrate, Nitrobenzene, 4-nitro-benzamine and 2-(ethylphenilamine)-ethanol resultants from oxidation and reduction of Disperse Red 1. The same products were found from oxidation and reduction of Disperse Red 13, in addition 2-chloro-4-nitroaniline also was found. Aniline, nitrobenzene, N-phenil-benzenamine and N-phenil-1,4-benzediamine from oxidation and reduction of Disperse Orange 1. Conclusions: These products detected could also be formed after biotransformation reactions performed by the organism, and can cause toxic effects considering that most of them are mutagenic. P202-069 CHARACTERISTICS OF DNA METHYLATION PATTERN OF ADIPOKINE GENES IN POLYBROMINATED DIPHENYL ETHERS-EXPOSED CELLS

C. Y. Hsieh, W. C. Chou. National Tsing Hua University, Hsinchu, Taiwan Increasing obesity prevalence becomes an alarming global health problem. Some evidence points that endocrine disrupting chemicals alter adipose tissue metabolism and endocrine hormone balance. PBDEs (polybrominated diphenyl ethers) are identified as estrogen mimics and wide-used in consumer products as flame retardants. Organic pollutant PBDEs have been detected in human adipose tissue, blood and breast milk. Adipokines (e.g., leptin and adiponectin) regulate energy metabolism and endocrine homeostasis. Previous studies showed that diet and chemical exposure cause DNA methylation and inherit to offspring. Thus, this study was to investigate the changes on DNA methylation pattern of adipokine genes in human breast cells exposed to PBDEs. Penta-BDE (BDE-710), octa-BDE (BDE-798) and deca-BDE (BDE-209S) were used in this study for commonly-used in daily life. Quantitative PCR and ELISA were respectively performed to detect mRNA expression and protein level of estrogen receptor (ER), leptin and adiponectin influenced by various dose in accordance with the weak estrogenicity of PBDEs in ER-positive human breast cancer MCF-7 cells. Moreover, the DNA methylation pattern of leptin and adioponectin was determined by lumina coBRA chip assay. The results showed that low dose (1-100 ng/ml) of PBDEs did not induce ER, leptin and adiponectin expression. Otherwise, high dose (250-1000 ng/ml) of BDE-798 increased the expression of ER-beta, leptin and adiponectin. The preliminary finding suggested that high dose of BDE-798 exposure increased adipokine expression, and we will further investigate the gene methylation and protein release of adipokines. The goal is to elucidate the correlation between gene expression and DNA methylation in adipokines after PBDEs exposure. Such information may clarify the potency of epigenetic effects of PBDEs. P202-070 GENOTOXIC EFFECTS OF DRINKING WATER EXPOSURE TO ARSENIC ON MEXICAN PEOPLE AND INFLUENCE OF GSTO1 AND AS3MT POLYMORPHISMS

A. Sampayo-Reyes (1)

, A. Hernández (2)

, N. El-Yamani (2)

, C. López-Campos (3)

, M. A. Bermudez De León (1)

, R. Marcos Dauder

(2), S. González-Hernández

(1).

(1) Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social (IMSS),, Monterrey, Mexico;

(2)

Universitat Autonoma de Barcelona, Departament de Genetica i de Microbiologia, Bellaterra (Cerdanyola del Vallès) (Barcelona), Spain;

(3) Unidad de Investigación Biomédica, Instituto Mexicano del Seguro Social, Torreón, Mexico

Arsenic is an important environmental human carcinogen that contaminates ground water worldwide. Inter-individual variation in arsenic metabolism due to environmental or genetic factors has been widely described among populations cronically exposed to the element, mostly via drinking water. The genetic factors associated with arsenic metabolism has been the topic of latter reviews, and a better clarification of its actual role may help to identify subjects at a higher risk of developing arsenic-induced health hazards and may render a chance of clinical intervention.


27

The objective of the present study is to asses the current arsenic exposure situation affecting the population living in the northern part of central mexico (Torreon, mexico), and characterize the genetic background potentially modulating arsenic-related susceptibility of such chronically arsenic-exposed individuals. We used the Comet assay to evaluate DNA damage in the somatic cells . Arsenic exposure was estimated through water intake over 24 h. Arsenic excretion was assessed in the first morning void urine. Total arsenic concentrations and drinking water arsenic content were determined using by Hydride Generation Atomic Absorption Spectrometry (HGAAS). GSTO1-1 and AS3MT polymorphisms were identified by Real-Time PCR. Arsenic content in the drinking water of the population was found to be at levels above the WHO permissible exposure limit of 10 ug/L. Positive correlation between total arsenic intake by drinking water/day and the total arsenic concentration in urine (r = 0.50, P < 0.001) was found. Arsenic excreted in urine ranged from 0.9 to 147 ug/g creatinine. Alellic frequencies found for the selected polymrphisms were as follows: GSTO1-1 140Asp 0.20, Glu155del 0.02, 208Lys 0.02, 236Val 0.05, 287Thr 0.09. A positive association was found between the level of exposure and the genetic damage measured as percentage of DNA in tail (p< 0.001). Future work in this direction is guaranteed. P202-071 IN VIVO MUTAGENICITY OF OCHARATOXIN A USING GPT DELTA RATS

Y. Ishii (1)

, D. Hibi (1)

, Y. Suzuki (1)

, M. Jin (1)

, N. Masui (2)

, T. Nohmi (3)

, Y. Konishi (4)

, T. Umemura (1)

, A. Nishikawa

(1).

(1) National Institute of Health Sciences, Division of Pathology, Tokyo, Japan;

(2) Japan SLC, Incorporated,

(3) National Institute of Health and Sciences, Division of Genetics and Mutagenesis;

(4) National Institute of Health and

Sciences, Division of Microbiology,

Ochratoxin A (OTA) is a naturally occurring mycotoxin produced by several fungus species belonging to the general Asperfillus and Penicillium, which is carcinogenic in the kidneys of the rodents. However, the results of various mutagenicity tests have been shown equivocal, so that the mechanisms underlying OTA carcinogenesis remain unclear. In the present study, we examined reporter gene mutations in the kidneys of gpt delta rats given OTA in the diet for 13 weeks. Six-week-old male and female F344 gpt delta rats were given basal diet or 5 ppm OTA in the diet. Diethylnitrosamine (DEN) at a dose of 20 mg/kg by i.p. injection once a week was given as a positive control group. Rats were sacrificed at 4 and 13 weeks and the blood samples were collected for the examination of BUN and CRN levels in serum. Kidneys were extirpated for the use of the examination of histopathology, gpt and Spi- assays. There were no remarkable changes in general appearance, body weight and food consumption during the experiment. Increases of CRN levels were observed in the OTA-treated males at 4 weeks and females at 13 weeks. Histopathologically, apoptosis, karyomegaly and cytoplasmic vacuolation in the proximal tubular epithelium in the outer medulla were observed in all OTA-treated males and females at 13 weeks. gpt mutant frequencies in the kidneys of male rats treated with OTA at 13-weeks were not significantly increased as compared to the control despite the increment in DEN-treated group being significant. The presence of organic anion transporter in the renal proximal tubule cells to OTA might be responsible for the site-specific histopathological changes. Although gpt mutant frequencies were not changed in 13-weeks treatment with OTA, in vivo mutagenicity of OTA will be confirmed judging from the results of Spi- assay in males and both mutation assays in females. P202-072 EVALUATION OF GENOTOXICITY OF EPHEDRAE HERBA THROUGH A BATTERY OF THREE GENETIC TOXICOLOGY ASSAY

J. Y. Kim (1)

, H. J. Cho (1)

, S. M. Back (1)

, J. Y. Yang (1)

, E. J. Oh (1)

, S. J. Park (2)

, B. S. Min (3)

, J. I. Huh (1)

. (1)

Korea Institute of Toxicology, Daejeon, Korea, Republic of; (2)

College of Veterinary Medicine, Kyungpook National University,

(3) College of Pharmacy, Catholic University of Daegu,

Ephedrae herba, called Mahuang in Chinese, has been used in inducing perspiration, healing a cold, relieving cough and asthma, and is included in a number of popular Kampo formulations. Ephedrine which is an active component of Mahuang has been used as a dietary supplement however, there are several reports for sudden deaths after taking the ephedrine-included supplements. In our current study, we investigated its genotoxic potentials in vitro and in vivo. On this purpose, dried Ephedrae herba was extracted at 80~100°C for 2~3 hours in distilled water until the volume of distilled water became half of the original volume and aqueous extract was dried by freeze-drying machine. Mutagenicity of the extract was evaluated in a bacterial reverse mutation assay using histidine-requiring Salmonella typhimurium and tryptophan-requiring Escherichia coli strains. In vitro chromosome aberration assay was conducted to identify whether the test substance causes structural chromosome aberrations in Chinese Hamster Lung (CHL) cells. Finally, animal-based in vivo micronucleus assay was performed to reflect all conditions expected in a physiological system. The extract of Ephedrae herba in the range of 156.3 to 5000 ug per plate did not demonstrate mutagenic effects both with and without metabolic activation. There was no significant increase in the number of metaphases with structural or numerical aberrations in the Ephedrae herba-treated groups at any dose level regardless of S-9 activation as compared to the vehicle control. The incidence of MNPCEs in bone marrow was not increased by oral administration of the extracts at doses up to 2000 mg/kg in both male and female ICR mice. No significant mortality, clinical signs, and body weight loss were observed in these animals. The results of this study suggest that Ephedrae herba is non-genotoxic.


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P203-035 PREVENTION OF OXIDATIVE STRESS AND ALTERATION IN HEPATIC AND TESTICULAR MRNA EXPRESSION GENES DURING AFLATOXICOSIS

S. H. Abdel-Azim, M. A. Abdel-Wahhab. National Research Center, Cairo, Egypt

Aflatoxins (AFs) are among the most potent naturally occurring carcinogens and AFB1 is classified as a group I carcinogen. Since the ingestion of aflatoxins-contaminated food is associated with several liver diseases, the aim of the present study was to evaluate the whey protein (WP) and Korean ginseng extract (KGE) singly or in combination against micronuclei formation, DNA fragmentation, gene expression and oxidative stress in rats fed AFs-contaminated diet for four weeks. Eighty male Sprague-Dawley rats were divided into eight groups included the control group, the group fed AFs-contaminated diet (2.5 mg/kg diet), the group treated orally with WP (0.5 ml/rat/day), the group treated orally with KGE (20 mg/kg b.w), the group treated orally with WP plus KGE and the groups fed AFs-contaminated diet and treated orally with WP, KGE or WP plus KGE. Bone marrow micronucleus, DNA fragmentation, gene expression and oxidative stress were assayed in liver and testis. The results indicated that treatment with AFs resulted in significant increase in Mn-PCEs in bone marrow, DNA fragmentation, FAS mRNA expression and lipid peroxidation in liver and testes accompanied with a significant decrease in PCEs/NCEs ratio in bone marrow, PHGPx gene expression and GSH in liver and testes. Treatment with WP and/or KGE have no significant effect on Mn-PCEs or PCEs/NCEs ratio in bone marrow however; KGE or KGE plus WP increased PHGPx gene expression and GSH accompanied with a significant decrease in Lipid peroxidation in live and testes and FAS mRNA expression. The combined treatment with AFs plus WP, KGE or WP and KGE succeeded to restore all the tested parameters towards the control values also these treatments did not normalize them. It could be concluded that the genotoxicity of AFs could be prevented by dietary supplementation with WP, KGE or their combination. P203-038 THE COFFEE DITERPENE KAHWEOL INHIBITS INVASION AND ANGIOGENESIS VIA MODULATION OF STAT3 ACTIVATION

H. G. Kim, H. G. Jeong. Chungnam National University, Daejeon, Korea, Republic of Kahweol, the coffee-specific diterpene, has been reported to have anti-carcinogenic properties. Animal data support such a chemopreventive effect of coffee. However, the precise underlying protective mechanisms are poorly understood. In this study, we examined the anti-angiogenic and anti-metastatic activities of kahweol. Kahweol effectively inhibited IL-6-induced production of vascular endothelial growth factor (VEGF), migration and tube formation in human umbilical vein endothelial cells (HUVECs). Kahweol also inhibited cell migration, production of VEGF, matrix metalloproteinase-2 (MMP-2) and MMP-9 in MDA-MB231 breast cancer cells. Moreover, kahweol inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3), suggesting that STAT3–responsive regions in VEGF and MMPs promoter may be involved in the inhibitory effect of kahweol. Furthermore, transient expression of constitutively active STAT3 significantly reduced the inhibitory effect of kahweol on cell migration and VEGF and MMP-9 expression. Taken together, these observations indicate that kahweol inhibits metastasis and angiogenesis partly through the disruption of STAT3-mediated transcription of genes, including VEGF and MMPs. P203-042 DIFFERENTIAL EFFECT OF CASIOPEINAS IN NEUROBLASTOMA CELL LINES

P. M. Monroy-Romo (1)

, T. Garcia-Gasca (2)

, L. Ruiz-Azuara (3)

, M. E. Bravo-Gómez (3)

, C. Mejia (4)

, R. Ferriz Martínez

(1).

(1) Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México y Facultad de Ciencias

Naturales, Universidad Autónoma de Querétaro, México, D.F., Mexico; (2)

Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Querétaro, Mexico;

(3) Facultad de Química, Universidad Nacional Autónoma de México,

Mexico,D.F., Mexico; (4)

Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, D.F., Mexico Cancer represents a public health problem and its treatment demand more complex strategies. This fact has stimulated the design, synthesis and evaluation of new compounds with less toxic effects. Casiopeinas® are coordinated compounds based on cupper that have shown antineoplasic activity. Between nervous system cancers, neuroblastoma represents a clinical challenge since the particular characteristics of tumor. In order to test the cytotoxic effect of different Casiopeinas on neuroblastoma, SK-N-SH and SK-N-AS cells were used. Different concentrations of three Casiopeinas: IIgly, IIIEa and IIIia were tested in both cell lines and were compared with Cisplatinum. Differential cytotoxic effect was observed on both cell lines with the treatments tested. SK-N-SH cells showed sensibility to Cisplatinum>Casiopeina IIIEa>Casiopeina IIgly>Casiopeina IIIia where Cisplatinum LC50 was 2, 7 and 100 times lower than the observed for Casiopeina IIIEa, Casiopeina IIgly and Casiopeina IIIia, respectively. In the case of SK-N-AS cells, LC50 observed with Casiopeina IIIia was practically the same that the observed with Cisplatinum, but 4 and 6 times lower than the obtained for Casiopeina IIIEa and Casiopeina IIgly, respectively. Our results suggest that Casiopeinas can differentially affect neuroblastoma cells survival with similar potency than Cisplatinum, depending on cell type. Ongoing studies are focusing on the mechanism of action in order to evaluate the potential use of Casiopeinas against neuroblastoma tumors. Acknowledgments: This study was supported with CONACYT contract´s 55464 and 60085 and FOMIX QRO-2007-C01-78221.


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P203-044 MECHANISMS OF INHIBITION OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION BY POTENTIAL TUMOR PROMOTERS

M. Odabasi (1)

, A. Wilke (2)

, H. Boke (2)

, L. Keinow (2)

, E. Kumar (2)

, J. E. Trosko (2)

, B. Upham (2)

. (1)

Ankara University, Institute of Forensic Medicine, Ankara, Turkey; (2)

Department of Pediatrics & Human Development, and the National Food Safety & Toxicology Center, Michigan State University, Although closure of gap junction channels by the chronic exposure to tumor promoters, oncogenes or compensatory release of growth factors is a common phenotype in the nongenotoxic steps of cancer, the signal transduction mechanisms that inhibit gap junctional intercellular communication (GJIC) vary. Currently, two pathways controlling GJIC are known, one dependent on the mitogen activated protein kinase regulator, MEK, or phosphatidyl choline specific phospholipase C (PC-PLC). As to which pathway potential tumor promoters and growth factors inhibit GJIC is generally not known. We screened various chemical toxicants as to which pathway was involved by pre-treating the cells with either a specific inhibitor of MEK, U0126, or PC-PLC, D609, followed by the addition of the promoter/growth factor in question, and then assessing GJIC in a rat liver epithelial cell line with oval cell characteristics using the scrape load-dye transfer assay. Epidermal growth factor, 12-`tetradecanoylphorbol-13-acetate and lindane regulated GJIC through a MEK-dependent mechanism, whereas lower molecular weight PAHs, DDT, PCB 153, dicumylperoxide and perfluorodecanoic acid inhibited GJIC through PC-PLC. Both MEK and PC-PLC were involved in the inhibition of GJIC induced by perfluorooctanoic acid and R59022. Inhibition of GJIC induced by perfluorooctane sulfonic acid, pentachlorophenol and alachlor was independent of both, MEK and PC-PLC. Resveratrol, an abundant antioxidant found in red wine and peanut products, has been implicated in the prevention of cardiovascular diseases and cancer. Resveratrol prevented the inhibition of GJIC by compounds that dysregulated gap junctions either through MEK or PC-PLC. Except for alachlor, resveratrol did not prevent dysregulation of GJIC by compounds regulating through PC-PLC-independent and MEK- independent pathways, which also indicated at least two other, yet unidentified, pathways are involved in the regulation of gap junctions. P203-045 ENVIRONMENTAL EXPOSURE TO PESTICIDES MAY ENHANCE THE RISK OF BLADDER CANCER

T. Parron (1)

, R. Alarcon (2)

, M. D. M. Requena (1)

, A. Hernandez (3)

, A. Pla (3)

. (1)

Delegacion Provincial de Salud de Almeria, Spain; (2)

Universidad de Almeria, Spain; (3)

Universidad de Granada, Spain Bladder cancer is the fourth most common malignancy in men from developed countries. This often occupationally-related neoplasm is also related with tobacco consumption and chlorination by-products in treated water. It is hypothesized that bladder cancer mortality risks may increase from drinking water contaminated with low levels of pesticides. In Almeria (southeastern Spain) the use of pesticides has significantly increased over recent decades to improve crop yield, although there is a great variability within this province. This offers the opportunity to study potential relationships between the increased use of pesticides and ill health in the exposed populations. We designed a descriptive epidemiological study to collect data from all cases of bladder cancer for the study period 1998-2009 through the hospital minimum dataset from the central reference Hospital of Almeria province. This province was then classified in two different geographical areas according to the number of hectares devoted to intensive agriculture: high exposure areas (>1,000 hectares of greenhouses) and low exposure areas (less than 1.000 hectares). Of the 2868 bladder cancer cases extracted, 2337 corresponded to subjects living in high exposure areas (rate of 42.98 cases per 100,000 population) and 531 cases occurred in areas of low exposure (rate of 34.83 per 100,000 population). When both areas were compared, a higher risk for bladder cancer was significantly observed in those areas with high pesticides use (OR 1.24, 95%CI: 1,12-1,36). In conclusion, although this study is not designed to establish causal links, it supports that populations with high environmental exposure are at a 20% increased risk for developing bladder cancer with respect to populations living in low exposure areas. P203-046 DIFFERENTIAL ROLES OF CYTOCHROMES P4501A1 AND 1A2 IN LUNG CARCINOGENESIS MEDIATED BY POLYCYCLIC AROMATIC HYDROCARBONS (PAHS) IN MICE: IMPLICATIONS FOR LUNG CANCER IN HUMANS

B. Moorthy, L. Wang, W. Jiang. Baylor College of Medicine, Houston, United States Humans are constantly exposed to polycyclic aromatic hydrocarbons (PAHs). Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to DNA-binding metabolites, which in turn mediate carcinogenesis in target organs such as lung. In this study, we tested the hypothesis that CYP1A1 and 1A2 have reciprocal roles in PAH-mediated tumorigenesis. Eight week-old female wild type (WT) (A/J) mice or mice lacking the gene for CYP1A1 or CYP1A2 on the A/J background were treated with a single dose of MC (40 µmol/kg), benzo[a]pyrene (BP), or vehicle (corn oil), and liver and lung tumors were studied after 28 weeks. While 100% of WT or Cyp1a2-null mice exposed to MC showed lung tumors after 28 weeks, about 80% of the Cyp1a1-null mice showed lung tumors. However, there were striking differences in the Cyp1a1-null and Cyp1a2-null mice in regard to lung tumor multiplicities. The WT mice treated with MC had about 15 lung tumors/animal. On the other hand, the Cyp1a2-null mice displayed about 40 lung tumors/animal, while the Cyp1a1-null mice showed about 2-3 tumors/mouse. Although BP was less potent in inducing lung tumors in the WT mice compared to MC, it was similar to MC In that tumors were suppressed in the Cyp1a1-null mice and augmented in the Cyp1a2-null mice. Immunohistochemical studies showed that CYP1A1 was overexpressed in the tumorous portions


30

of the lung in the Cyp1a2-null or WT mice, compared to non-tumorous regions, These results suggest that CYP1A1 contributes to the formation of tumorigenesis by PAHs, while CYP1A2 protects against carcinogenesis mediated by PAHs, presumably through it role in PAH detoxification. In conclusion, our results strongly suggest that CYP1A1 and 1A2 could be novel candidates for cancer prevention and therapy though either inhibition of CYP1A1 or induction of CYP1A2. (Supported by NIH grant ES009132.) P203-051 PEER REVIEW AND PWG: SCIENTIFIC SOLUTION FOR CONFLICTING PATHOLOGICAL DATASETS

S. Gröters, C. Wiemann, W. Kaufmann. BASF SE, Ludwigshafen, Germany During EU re-registration of a long-time marketed pesticide concerns were raised on potential carcinogenic effects not considered in earlier assessments. Inconclusive results between two datasets from two different manufacturers containing studies that were performed at different times, in different countries and research test facilities using different animal strains were considered the major reason for this upcoming concern. Differences of opinion or inconsistencies in terminology, morphologic diagnoses, and interpretation can significantly impact the toxicologic assessment of compound safety and carcinogenic risk to consumers. Due to the interpretative nature of pathology and the variability of pathology terminology, morphologic diagnoses, and interpretation within and between different laboratories, a peer review was considered necessary. Therefore, the manufacturers decided in a first step: 1. to perform a Peer Review based on re-evaluation of all available organ and tissue slides of the tumor incidences that caused concern and in a second step. 2. to initiate a PWG (pathology working group) to solve discrepancies between the original study pathologist (first assessor) and the peer reviewing pathologist (second assessor) In the following poster the value of the peer review process and the role of the PWG are discussed. In conclusion, the results of the peer review process and the PWG are recognized as an important scientific tool for inconsistent datasets to come to a final, state-of-the-art, conclusive assessment of pathology data. P204-027 INFLUENCE OF AGE ON SUB-CHRONIC TOXICITY OF AQUEOUS LEAVES EXTRACT OF CALOTROPIS PROCERA IN RABBITS

G. B. Pouokam (1)

, D. Christianna Joshua (2)

, A. Hatem Abdel Moniem (3)

, A. Aliyu (4)

, P. Okewole (5)

. (1)

Department of Biochemistry,University of Yaoundé 1, Cameroon; (2)

Toxicology Unit,National Veterinary Research Institute,Vom-Nigeria,

(3) Chemical laboratory,Medicolegal Department,Ministry of Justice-Egypt,

(4) Biochemistry

Division,National Veterinary Research Institute,Vom-Nigeria, (5)

Center Diagnosis,National Veterinary Research Institute,Vom-Nigeria,

Calotropis procera is a major browse toxic plant for grazing animals. Animals undergo different changes throughout their developmental stages and this affects their susceptibility to drugs inducing injury. The aim of this study was to investigate the influence of age on sub-chronic toxicity of aqueous leaves extract of Calotropis procera in rabbits. The twenty five rabbits used during the study were classified in five groups of identical ages; each experimental group had his own controls. Extract was administered every morning by gavage at the dosage of 200mg/kg body weight for 42 days; food and water were given ad libitum. Liver biomarkers (ALT, ALP, AST, Total protein) and kidney parameters (serum creatine and Urea) were assessed. PCV, Hb, WBC, RBC and differential leucocytes count were determined for hematological analysis. Necropsy, gross and histopathology were performed. All rabbits gained weight during the treatment period, with an appreciable gain for smaller animals. There was a significant decrease (p<0.005) of ALT and RBC for the youngest rabbits. A significant increase (p<0.005) of serum creatinine level and lymphocytes were also observed within the group of juvenile rabbits. Necropsy revealed lesions in kidney, liver, lung, small intestine and brain; these lesion were further confirm by histopathology that revealed more pronounced tissue lesion with the youngest animals compare to the control and the adults rabbits. Extract had no significant effect on rabbit´s weight. Major target organs were liver; kidney; brain and haematopoietic system with the youngest animals being more sensitive. *This study was made possible by grants from The Africa Education Initiative (www.nef3.org). P204-030 TOXIC EFFFECTS OF U&DEE SWEET BITTER HERBAL SUPPLEMENT ON THE ACCESSORY SEX ORGANS OF MALE WISTAR RATS.

E. Obi, R. Negedu-Momo, O. Obioma, J. Afonne, O. E. Orisakwe. Nnamdi Azikiwe University, Nnewi, Nigeria Herbal medications are widely believed to be beneficial, however there have been reports of acute and chronic intoxications resulting from their use. Cases of reproductive failure after a prolonged intake of herbal preparations have been anecdotally reported in Nigeria. This study was aimed at investigating the sub-chronic effects of U&DEE Sweet bitter a brown liquid herbal preparation, registered and marketed in Nigeria on accessory sex organs of male Wistar rats. Twenty male Wistar rats allocated to four dose (0.00, 539, 1077, 1616mg/kg, po ) groups of five rats each for 90days. Animals had access to deionized water and were fed ad libitum with rat chow for 90 days. The feed and fluid consumption of the animals were measured on daily basis while the body weight was measured weekly. Animals were anaesthetized after 90 days, bled, sacrificed, epdidymis (E), seminal vesicle (SV) and prostate (P) were excised and weighed, protein, DNA, epididymal sperm number (ESN), epididymal acid and alkaline phosphatase were determined. There was non-significant increase (p>0.05) in feed and fluid intake, and a significant decrease (p< 0.05) in absolute and relative weights of the E, SV and P in all the treated animals when compared with the control. There were significant (p<0.05) decreases in ESN, DNA, protein, acid and alkaline phosphatase levels in all the treated


31

animals when compared with the control. The present work suggests that U&DEE Sweet bitter herbal supplement may have toxic effect on the accessory sex organs of the male Wistar rats. P204-033 RELATIONSHIP BETWEEN URINARY PHYTOESTROGEN LEVELS AND SEMEN QUALITY IN HUMANS

Y. Xia, P. Zhu, M. Chen, C. Lu, S. Wang, X. Wang. Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing, China Phytoestrogens (PEs) are naturally occurring chemical constituents of certain plants that may interact with estrogen receptors to produce estrogenic effects. Limited studies have suggested that male reproductive function might be associated with PEs exposure. To investigate the relationship between urinary PEs levels and idiopathic male infertility and semen quality in Chinese males, 608 eligibility idiopathic infertile men and 469 fertile controls were recruited in our study. By using UPLC-MS/MS, individual PEs exposures were measured as spot urinary concentrations of six PEs or their metabolites (secoisolariciresinol, SEC; daidzein, DAI; equol, EQU; genistein, GEN; naringin, NAR; coumestrol, COU), which were adjusted by urinary creatinine (CR). Subjects with idiopathic infertility were further divided into “normal” and “abnormal” semen quality groups based on their semen volume, sperm concentration, sperm number per ejaculum and motility. Most of the adult males in our study presented high exposure levels of PEs. The CR-adjusted urinary concentrations of SEC, GEN and DAI of case group were significantly higher than those found in control group. With the increased of the SEC, GEN and DAI levels (assessed as quintiles), the ORs of semen parameters show the similar varied trend. From the first to the fourth quintile, there are no notable variations of ORs. However, from the fourth to fifth quintile, the OR values increased remarkably. Subjects with higher concentrations of SEC, GEN and DAI were more likely to have idiopathic male infertility and abnormal semen quality (P-value for trend < 0.01, 0.05 and 0.05, respectively). These results show unique exposure characteristics of PEs in our study population. Increased urinary concentrations of SEC, GEN and DAI were found associated with increased male idiopathic infertility risks, while the idiopathic infertile subjects with abnormal semen quality might be at higher risk. These findings may be of concern due to the ubiquitous exposure of PEs. P204-038 MRI AND PET, PROMISING INNOVATIONS IN DEVELOPMENTAL TOXICITY TESTING. A FEASIBILITY STUDY IN RATS WITH ORGANOTIN COMPOUNDS

D. De Groot1, M. Berk

1, M. Boogaard

1, V. J. De Groot

1, A. Heerschap

2, L. Van der Horst

1, R. Nederlof

1, A. Veltien

2, E.

De Vries3

1TNO Quality of Life, Zeist, Netherlands;

2Radboud Universiteit Medical Center Nijmegen, Netherlands;

3Groningen

University Medical Center/PET Center, Netherlands

The new European guideline for an Extended One-Generation Reproduction Toxicity Study (OECD EOGRTS (draft)) aims to reduce the number of animals for REACH and other testing programs without compromising safety assessment. The protocol includes not only reproduction, but the developing immune and neurological systems as well. Twenty animals/sex/group are needed to test developmental neurotoxicology. In a model study with organotin compounds we tested the hypothesis that this number could be further reduced using combined functional (Positron Emission Tomography (PET)) and structural (Magnetic Resonance (MR)) in vivo brain imaging which, in time, might replace part of behaviour and neuropathology testing. Hereto, rats were exposed daily to di-octyltin-chloride (DOTC; 30mg/kg BW/day) according to the EOGRTS protocol (2 weeks before mating until postnatal day (PND) 70). Two other groups to tri-butyltin-oxide (TBTO; 8mg/kg BW/day): a developmental (TBTOdev) group from gestational day (GD) 6 until PND 10; a juvenile (TBTOjuv) group from PND 21 until PND 70. Auditory startle, functional observational battery, motor activity and neuropathology were performed. Surplus animals were used for MRI (brain volume) and [18F]FDG-PET (brain glucose metabolism). No alterations in behaviour and [18F]FDG uptake were observed in DOTC and TBTO(juv) groups. DOTC animals showed larger brains with MRI and conventional neuropathology. TBTO(dev) group showed reduced auditory startle habituation, impairments in the neuromuscular domain, increased motor activity, smaller brains (MRI and conventional neuropathology) and an altered [18F]FDG uptake pattern. Relative uptake values for different brain regions, however, were the same among control and exposed groups. To conclude, MRI showed good concordance with conventional neuropathology with less than half of the animals. PET, also with fewer animals, showed an altered [18F]FDG uptake pattern in animals with altered behaviour. Our findings demonstrate that in vivo imaging, like MRI and PET, warrant safety testing for developmental neurotoxicology with fewer animals. P204-039 RISK OF CRYPTORCHIDISM IN GEOGRAPHIC AREAS WITH HIGH USE OF PESTICIDES.

T. Parron (1)

, F. Cavieres (2)

, R. Alarcon (3)

, M. D. M. Requena (1)

, A. Hernandez (4)

. (1)

Delegacion Provincial de Salud de Almeria, Spain; (2)

Universidad de Valparaiso, (3)

Universidad de Almeria, Spain; (4)

Universidad de Granada, Spain Cryptorchidism is a typical urogenital malformation in males wherein one or both testicles do not descend into the scrotum but they reside into the normal descending pathway. In addition to its genetic component, this condition might have an exogenous origin related to prenatal exposures to endocrine disrupting compounds, including different pesticides. The province of Almeria in southeast Spain includes a rural area where a large proportion of population is employed in intensive greenhouse cultivation involving 2–4 annual harvests of early fruits and vegetables. This kind of production requires use of pesticides to disinfect the soil and the plants. Cryptorchidism cases included all males who underwent an orchidopexy (or for whom orchidopexy was programmed) and were first identified in computerized


32

hospital records (minimum data set). This source yielded a total of 594 cases among males born between 1998 and 2009 in the the three health districts of the province of Almeria. These districts were classified into high or low "pesticide impact" based on the criteria of the number of hectares devoted to intensive agriculture (plastic greenhouses). Districts with greenhouse surface above a cut-off of 1000 hectares were classified as "high impact" for pesticide exposure (two districts) whereas those below this cut-off were classified as "low impact" (one district). Of the 594 cases, 84.6% corresponded to children residing in areas with a greater number of hectares (rate of 9.25/100,000 population) versus 15.4% corresponding to children residing in areas of low exposure (rate of 5.96/100,000 population). When comparing both areas, an OR of 1.55 (95% CI 1.23-1.95) was observed for the high exposure area, which supports the association between congenital cryptorchidism and fetal exposure to pesticides acting as endocrine disruptors, alone or in association with other chemicals. P205-038 ARECA NUT EXTRACT ENHANCES THE DEVELOPMENT OF MYELOID-DERIVED SUPPRESSOR CELLS IN OVALBUMIN-SENSITIZED MICE

W. Chia-Chi School of Veterinary Medicine National Taiwan University,Taiwan Areca nut chewing is an etiological factor contributing to oral cancer and pre-cancers, whose pathophysiology has been linked to aberrant immunological and inflammatory reactions. It has been recently demonstrated that areca nut extract (ANE) affected the functionality of neutrophils and lymphocytes in vitro; however, the immunomodulatory effect of ANE in vivo remains mostly unclear. The objective of the present studies was to investigate the immunomodulatory effect of ANE in vivo. Ovalbumin-sensitized BALB/c mice were daily administered with ANE (5-50 mg/kg) by intraperitoneal injection for 10 doses. ANE dose-dependently increased the spleen index and the cellularity of CD11b+ cells in the spleen. Morphological examination revealed that ANE increased the granulocytic phenotype in the splenic CD11b+ cells. Furthermore, ANE dramatically increased the population of CD11b/Gr-1 double positive cells in splenocytes, of which greater than 90% of CD11b+ cells were Gr-1+ in the ANE (50 mg/kg)-treated group. ANE significantly enhanced the production of IL-10 by CD11b+ cells and total splenocytes stimulated with lipopolysaccharide (LPS). In addition, LPS-induced iNOS and arginase I mRNA expression by splenocytes was increased by ANE. Taken together, these results suggested that ANE enhanced the development of CD11b+Gr-1+ myeloid-derived suppressor cells in vivo, which may be a critical mechanism contributing to the pathophysiology of areca-related diseases. P205-039 FUNCTIONAL DISORDER OF PRIMARY IMMUNITY RESPONDING TO RESPIRATORY SYNCYTIAL VIRUS INFECTION IN OFFSPRING MICE EXPOSED PERINATALLY TO DECABROMINATED DIPHENYL ETHER (DBDE)

W. Watanabe (1)

, R. Sawamura (2)

, T. Shimizu (2)

, K. Konno (2)

, A. Hirose (3)

, M. Kurokawa (2)

. (1)

School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Japan, Nobeoka, Japan; (2)

School of Pharmaceutical Sciences, Kyushu University of Health and Welfare;

(3) Biological Safety Research Center, National

Institute of Health Sciences Perinatal exposure to decabrominated diphenyl ether (DBDE) was shown previously to increase viral titers in the lungs of respiratory syncytial virus (RSV)-infected offspring on day 5 post-infection, resulting in exacerbation of pneumonia. In this study, the significant increase of pulmonary viral titers was confirmed even on day 1 post-infection and the effect on the primary immune response to RSV infection were examined to assess a mode of DBDE action on developmental immunotoxicity. On day 1 after infection, the secretion of both TNF-alpha and IL-6 decreased significantly in the bronchoalveolar lavage fluid prepared from RSV-infected offspring exposed to DBDE perinatally, but IL-1beta increased. However, in ex vivo lipopolysaccharide stimulation test, the productivity of TNF-alpha in the bronchoalveolar lavage cells, which are mainly primary immune cells responding to RSV infection, prepared from offspring mice exposed to DBDE perinatally was not lower than that in the control. The primary immune cells retained normally the ability of cytokine production after the DBDE exposure. Gene expressions of innate pattern recognition receptors (Toll-like receptor 3 and 4, melanoma differentiation-associated gene-5 and retinoic acid-inducible gene I) in lung tissues were not affected by DBDE exposure. Because the levels of TNF-alpha, IL-6, and IL-1beta are known to be elevated in the lungs of RSV-infected mice, these irregular productions due to perinatal DBDE exposure indicate a disorder of the primary immune response to RSV infection. Thus, perinatal exposure to DBDE was suggested to cause a functional disorder of primary immunity responding to RSV infection. P205-040 ENDOGENOUS GLUCOCORTICOIDS AND PERIPHERAL BENZODIAZEPINE RECEPTOR LIGANDS MODIFY ADHESION MOLECULES EXPRESSION IN RAT LYMPHOCYTES

B. Lima Camila, J. Palermo-Neto, S.H.P. Farsky Faculty of Pharmaceutical Sciences - Laboratory of Experimental Toxicology, São Paulo, Brazil Benzodiazepines (BZD) are one of the most commonly used groups of anxiolytic drugs, whose effects are mediated through binding on neuronal post-synaptic plasma membrane GAGAA receptors. However, a second class of benzodiazepine binding sites, peripheral benzodiazepine receptor (PBR), has been found in many peripheral tissues and cell populations, as immune and endothelial cells. Studies have been shown that BZD modulate immune–inflammatory response, which can be related to PBR present on immune and endothelial cells and to secretion of endogenous glucocorticoids (EG). We evaluated the effects of PBR-binding drugs on expression of adhesion molecules in lymphocytes during in vivo lockade of EG cytosolic receptor. Adult male Wistar rats were divided into 2


33

groups: control (C) and RU 38486 (10mg/kg) - EG cytosolic receptor antagonist - (RU) that were treated i.p. 18h and 1h before blood collection. After, leukocytes suspensions were incubated with PK 11195 or Ro 5-4864 (100nM) for 1h. In addition, these cells were stimulated or not with fMLP (10-9M) in vitro in order to evaluate the expression of L-selectin, β2integrin, ICAM-1, and VLA-4 by flow cytometry. In vivo RU treatment decreased L-selectin expression on lymphocytes treated in vitro with both PK11195 (C=177.2±29.08; RU=126.78±19.94; p<0.05) and Ro 5-4864 (C=131.69±26.65; RU=78.77±26.92; p<0.05). Moreover, in vitro Ro 5-4864 treatment decreased the expressions of L-selectin and VLA-4 compared to cells treated with PK11195 (L-selectin: C+PK=177.2±29.1; C+Ro=131.7±26.7; p<0.05; RU+PK=126.8±19.9; RU+Ro=78.8±26.9; p<0.05) (VLA-4: C+PK=29.2±1.2; C+Ro=24.9±2.9; p<0.05; RU+PK=28.5±0.8; RU+Ro=22.9±3.3; p<0.01). The same pattern of L-selectin expression was detected in cells in vitro stimulated with fMLP. These results showed that PBR ligands modify the expression of L-selectin and VLA-4 that are molecules responsible for leukocyte-endothelial interactions, suggesting an alteration on lymphocyte migration from blood to tissues. Additionally, EG may modulate these effects. The mechanisms involved in these effects will be further investigated. Financial support: Fapesp nº2009/52245-1 P205-041 NONRADIOACTIVE LOCAL LYMPH NODE ASSAY USING FLOW CYTOMETRY FOR ASSESSING THE SENSITIZING POTENTIAL OF CHEMICALS

Y. K. Lee, C. H. Lim, M. Jang, E. J. Kim, Y. N. Yum, S. Sohn, S. Y. Han, S. H. Kim. National Institute of Food and Drug Safety Evaluation, Seoul, Korea, Republic of The local lymph node assay is widely used method to evaluate the skin sensitization potential and is accepted as OECD test guideline 429. The LLNA is alternative method to traditional guinea pig methods offering refinement and reduction benefits. Here, we performed a nonradioactive LLNA in which number of bromodeoxyuridine (BrdU) incorporation cells is used as endpoint instead of radioisotope (RI); this methods termed nonradioactive murine local lymph node assay: flow cytometry test method (LLNA:BrdU-FC). Groups of female Balb/c mice were topically treated on the dorsum of both ears with test chemicals for a vehicle control on days 1, 2 and 3. All groups were injected 100 ul BrdU solution (20 mg/ml in PBS) on day 5. On day 6, the number of BrdU incorporation cells in the draining auricular lymph node was measured as an alternative endpoint by the flow cytometry analysis. A stimulation index (SI) relative to the concurrent vehicle control was determined, and an SI of 3 was set as the cut-off values. We tested skin sensitization potential of 7 chemicals, which are hexylcinnamic aldehyde (HCA), 2,4-dinitrochlrobenzen (DNCB), 4-phenylenediamine (PPD), isoeugenol, eugenol, isopropanol and sodium lauryl sulfate, using LLNA:BrdU-FC. We also compared the results with those of other test methods. The accuracy of LLNA:BrdU-FC versus radioactive LLNA, guinea pig tests and human tests was 100% (7/7), 86% (6/7) and 71% (5/7), respectively. The SI value results from LLNA:BrdU-FC is similar to those of the radioactive LLNA. However, the number of test chemicals we tested insufficient to evaluate efficacy of LLNA:BrdU-FC. Thus, it is necessary to perform LLNA:BrdU-FC to sufficient chemicals (09162KFDA556). P205-042 CELL-BASED ON-LINE MONITORING OF SUSPENSION CELLS IN THE FOCUS OF IN VITRO IMMUNOTOXICITY STUDIES

S. Ortinau, S. Seemann, S. Drechsler, A. Kob, M. Wego, R. Ehret. Bionas GmbH, Rostock, Germany

The Bionas® 2500 analyzing system provides a non-invasive, multi-parametric and label-free monitoring of metabolic parameters like extracellular acidification and oxygen consumption. Until now, the method has been a valuable tool for the investigation of the dynamic cell behaviour of adherent cell lines and primary cells influenced by test compounds, including cytotoxic and dose-dependent as well as regeneration effects after treatment and removal of test compounds. Cells from the immune system, including their progenitors typically grow in suspension cultures. The requirements for the measurement of those cells are very much the same as for adherent cells with only some differences in the preparation. For using cells that do not attach to the chip surface, a biocompatible, low-buffering matrix that permits movement of fluids but prevents immobilized cells from floating, is required. The evaluation of effects of pharmaceuticals and potential immunotoxic compounds are investigated on a cellular level by monitoring the dynamic immune cell behaviour. Changes of acidification activity and respiration rates caused by test substances are hints for effects on cell physiology and presumably the cytotoxic property of the substance used. Usually, changes of the immune cell physiology are indicators of immunotoxicity. Therefore experiments using different concentrations of the anti-inflammatory folate analogue methotrexate (MTX) and the anti-convulsant valproic acid (VPA) on T-lymphocyte Jurkat cells were performed. Substances were added for 24h followed by a 4h regeneration phase without MTX or VPA. Concentrations of 0,5 mM VPA and 0,1 µM MTX and higher resulted in a decrease of the acidification rates and in altered respiration activities of Jurkat cells. No regeneration effects could be observed after the removal of substances from the medium. Taken together, these results show that the usage of agarose-embedded suspension cells to investigate the action of immunotoxic substances is a valuable tool for a multi-parametric data collection. P205-045 INHALED HYDROQUINONE AFFECTS LEUKOCYTE RECRUITMENT INTO LUNG AND ADHESION MOLECULES EXPRESSION

A. L. T. Ribeiro, A. L. B. Shimada, C. B. Hebeda, W. Tavares De Lima, S. H. P. Farsky. University of Sao paulo, Sao Paulo, Brazil


34

Hydroquinone (HQ) is a phenolic compound of cigarette, medicines and foods. Moreover, it is one of the most important toxic metabolite of benzene. Here we investigated the effect of HQ inhalation on recruitment of leukocytes into LPS inflamed lung and on adhesion molecules expression by circulating leukocytes. Male Swiss mice were exposed to 12,5ppm; 25ppm or 50 ppm of aerosolized HQ, 5 days, 1 hour, once a day. Control animals received the same amount of vehicle (saline with 5% ethanol). Lung inflammation was induced by inhalation of LPS (0,1 mg/mL; 10 min) one hour after the last exposures and the bronchoalveolar exudate (BAL) was collected three hours later. Total and differential number of leukocytes was determined in Neubauer chamber and . in staining smears, respectively. Expressions of B3-Integrin, B2-integrin, L-selectin and PECAM-1 were performed on 1x105 circulating leukocytes obtained one hour after the last exposure and were subsequently stimulated or not with fMLP (10-8 M; 10 or 30 min.). Adhesion molecules expression was measured by flow citometry. HQ exposure reduced the number of leukocytes in the BAL (12,5 ppm= 43,6% vs control; 25 ppm= 53,1% vs control; 50 ppm= 61,7% vs control), but the number of circulating leukocytes was not modified in all schedules of HQ exposure. Circulating leukocytes obtained from mice exposed to HQ in absence of fMLP stimulation presented higher expression of B3-Integrin (111,59%) and PECAM-1(252,24%) vs. collected cells from vehicle treated animals. After fMLP stimulation, the expression of B3-Integrin (50.61%) and L-selectin (73.95%) was lower vs expression on respective cell control. These alterations were mainly on polymorphonuclear cells. Results presented show that inhaled HQ impairs neutrophil recruitment into lung induced by LPS and this effect may be due to alterations on adhesion molecules on neutrophils, which have a role on neutrophil migration into inflamed tissue. P205-046 EFFECTS OF HYDROQUINONE INHALATION ON SECRETION OF TRACHEA CELLS

A. L. B. Shimada, A. L. T. Ribeiro, C. B. Hebeda, W. Tavares de Lima, S. H. P. Farsky Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, Brazil Hydroquinone (HQ) is obtained from benzene metabolism and it is an important phenolic compound of cigarette, medicines and foods. Respiratory is the main via of HQ absorption and its effects on cells of this system has been evaluated. Trachea is an important secretory tissue, which contains high concentrations of mast cells. Therefore, it has a relevant role on in innate or acquired immunity and contributes to host defense. This work aimed to investigate the effects of HQ inhalation on cytokines secretion of trachea cells. Male Swiss mice were exposed to aerosolized HQ 1.5mg/60mL/1h (25ppm) during 5 days, once a day. Control animals received the same amount of vehicle (saline with 5% ethanol). One hour after the last exposure, the trachea was removed and incubated in culture medium and was stimulated or not with LPS (1µg/2mL) during 24h. Following, the supernatant of tracheal culture was collected for measurement of cytokines (IL-10; TNF-a; IL-6; MCP-1) by ELISA and NO by Griess Reaction. The results of concentration of these mediators were expressed in pg/mg of dry tissue. Supernatant of trachea collected from mice exposed to HQ, in absence of in vitro LPS stimulation, presented higher concentrations of TNF-a; (336,89%) and NO (59,27%) vs. cells collected from vehicle-treated animals. After LPS stimulation, only tissue from control animals secreted high amounts of IL-10 (40,55%), TNF-a; (263,22%), IL-6 (193,08%), MCP-1 (2164,38%) and NO (224,63%). On the other hand, trachea from HQ exposed animals secreted higher amount of IL-10 (57,84%) and lower levels of MCP-1 (78,81%) and NO (60,38%) vs. control tissue stimulated with LPS. Data obtained show that HQ inhalation modifies the abilities of trachea cells to respond to inflammatory stimulus, which may impair the host defense. P205-047 TRANSGENIC OVEREXPRESSION OF HUMAN GATA3 ENHANCES ALLERGIC SKIN INFLAMMATION BY PHTHALIC ANHYDRIDE

H. I. Kang. National Institute of Food and Drug Safety Evaluation, Seoul, Korea, Republic of It has been elucidated that enforced expression of the transcription factor GATA3 induces an imbalance in the ratio of type 1 helper T (Th1) cells to type 2 helper T cells (Th2), which is thought to be indicative of pathogenesis of allergic disease such as asthma and atopic dermatitis. The aim of this study was to investigate the role of human GATA3 on allergic skin inflammation. To achieve this, human GATA3-overexpressing transgenic (Tg) mice were produced by the microinjection of pCMV/hGATA3 constructs into a fertilized mice eggs. To induce allergic skin inflammation, the 1%, 5%, and 10% phthalic anhydride (PA) solution as chemical allergen was repeatedly spread on the ear´s dorsum of Tg mice and their littermate for 3 weeks. Also, to investigate the effect of D-pinitol exerting anti-inflammatory activity, PA-induced mice were injected with D-pinitol each day from day 18 to 20 on consecutive days. Based on an analysis of the allergic skin inflammation, ear thickness, draining auricular lymph node weight, epidermis thickness, inflammatory cell number, and serum IgE concentration were significantly increased in the Tg mice compared with normal mice dependent on concentration of PA. Furthermore, D-pinitol regulates Th1 and Th2 cytokines via directly suppressing human GATA3 expression in the thymus of PA-induced Tg. These findings suggest that the transgenic overexpression of human GATA3 enhances allergic skin inflammation by PA and also can be diminished by the D-pinitol. P205-049 IN VITRO HIDROQUINONE EXPOSURE ALTERS INFLAMMATORY PROPERTIES IN ENDOTHELIAL CELLS

S. Farsky, C. B. Hebeda, F. J. Pinedo, S. A. Teixeira M. N. Muscará Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, Brazil Hydroquinone (HQ) toxicity has been demonstrated in several cell types, nevertheless its effects on endothelial cell has not been described. Therefore, we studied the effects of in vitro HQ treatment on adhesion molecules expression,


35

nitric oxide (NO) production and cytokines secretion by endothelial cells. Primary endothelial cell (EC) cultures were obtained from cremaster muscle from Wistar rats. Cell cultures (DMEM with 20% FSB) were treated with culture medium (control) or HQ at doses of 10 or 100uM in the presence or absence of LPS (2ug/ml). After the treatments, cell viability was measured by propide iodide and annexin V dual staining analyses; ICAM-1, PECAM-1 and VCAM-1 synthesis and expressions were quantified by RT-PCR and flow cytometry, respectively; NO production by Griess reaction; nitric oxide synthase (NOS) activity by enzymatic assay; and cytokines levels by ELISA. No alterations on cell viability were detected; HQ per se up regulated synthesis and expression of VCAM-1, ICAM-1 and PECAM-1 and secretion of TNF-alpha and IL-1beta and reduced the NOS activity and NO production after LPS incubation. Taken together, data obtained show that HQ alters different intracellular pathways in endothelial cells, as shown by increased expression of adhesion molecules of immunoglobulin super family and levels of pro-inflammatory cytokines and impaired NOS activity. Financial Support: FAPESP (07/56299-3); CAPES. P205-050 HIDROQUINONE IMPAIRS FUNCTIONAL ACTIVITIES OF NEUTROPHILS

C. B. Hebeda, F. J. Pinedo, S. A. Teixeira, A. R. Vinolo Marco, R. Curi , M. N. Muscará, S. Farsky Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, Brazil We have previous demonstrated that experimental exposure of male Wistar rats with HQ alters leukocyte recruitment to the lung during innate or acquired immune response. To extend our knowledge, here we investigated the effects of HQ on neutrophil functions. Neutrophils were obtained from naive Wistar rats 4 hours after i.p. injection of oyster glycogen (1%, 10mL). Cells were incubated with medium (RPMI 1640 with 10% FBS; control) or HQ (5 or 10uM) during one hour and latter cultured in absence or presence of LPS (E. coli, serotype 026:B6; 5ug/mL; 18 hours). Supernatant was used to quantify levels of NO by Griess reaction and TNF-alpha, IL-1beta and IL-6 using enzyme linked immunosorbent assay (ELISA). Neutrophils were employed to quantify nitric oxide synthase (NOS) activity by enzymatic assay, gene expression of TNF-alpha, IL-1beta and IL-6 by RT-PCR and nuclear translocation of NF-kappaB by eletromobility shift assay. In addition, neutrophils were used to study phagocytic and killing activities in presence of Candida albicans. Data obtained showed that HQ did not alter neutrophils viability. HQ reduced NO production in absence and presence of LPS. In presence of LPS, HQ inhibited NOS activity; production of TNF-alpha, IL-1beta and IL-6 (mRNA and protein); NF-kappaB nuclear translocation; phagocytic and killing activities of neutrophils. Taken together, data presented show that HQ impairs fundamental pro-inflammatory properties of neutrophils necessary to start host immune defense. The effects were dependent on inhibition of nuclear translocation of NF-kappaB. Financial support: FAPESP (07/56299-3); CAPES. P205-051 GLUTARALDEHYDE BEING A CONTACT SENSITIZER IN THE LOCAL LYMPH NODE ASSAY?

H. O. Ku (1)

, S. H. Jeong (1)

, H. G. Kang (1)

, S. W. Son (1)

, D. Y. Ryu (2)

. (1)

National Veterinary Research and Quarantine Service, Anyang, Korea, Republic of; (2)

College of Veterinary Medicine, Seoul National University, Seoul, Korea, Republic of

Glutaraldehyde(Glu) has been categorized into a moderate contact sensitizer by the local lymph node assay (LLNA). However, recent studies reported that the LLNA is too sensitive. This study was focused on other endpoints that can support its sensitizing potential using gene expression and cytokine profiles in local lymph nodes (LN) using a modified LLNA. CBA/N mice were topically given the dermal application of 1 to 10% Glu for 3 consecutive days. After 24 hrs, ear swelling was measured and local lymph nodes were excised. Glu increased ear swelling in a dose-dependant manner. Also, LN weights and their cell counts showed significant changes but did not show a dose-response between 3% and 10% Glu. Gene transcripts, such as Gzma and Gzmb, biologically up-regulated in 1% Glu but was not in significant changes. Among cytokines, Il1b and Il6 genes were increased in all treated doses of Glu, other genes such as Il2, Il3 and Il4 in 10% Glu, and Ifng gene in 1% Glu. These findings demonstrate that glutaraldehyde was hard to detect as a sensitizers by other endpoints in the modified LLNA. P206-041 ELECTROPHORETIC AND HISTOPATHOLOGICAL STUDIES ON ADULT FEMALE RATS TREATED WITH DEPO-PROVERA (DMPA)

W. Abu Shaeri (1)

, S. Bakry (1)

, E. Khattab (2)

. (1)

Zoology Department, Faculty of Science - Al Azhar University, Nasr City, Egypt; (2)

, Nasr City, Egypt Medroxyprogesterone Acetate (DMPA) is a long-acting, injectable progesterone derivative contraceptive and classified as sex hormone binding globulin (SHBG). In this study the effect of (DMPA) on serum protein patterns, hepatic and renal tissues were investigated on adult female rats. Sixty mature healthy female rats with average body weight ranged (130 – 200 gm), 40 of them were injected IM with DMPA human therapeutic (150 and 300 mg) equivalent to rat doses (2.7 or 5.4 mg/ rat/ day) and sacrificed after four and six weeks of the treatment. Sera total proteins, liver functions and electrophoretic analysis were quantified as well as both liver and kidneys were removed and fixed. The Student's “t”-distribution were adopted for assessment of significant changes occurring between the groups. DMPA was found to reduce the concentrations of both serum total protein and serum protein fractions (Prealbumin, Albumin and globulin ″ά-β & γ″)of the treated rats as well as several band discomfitures. DMPA induced histopathological lesions in liver and kidneys tissues of the treated rats which represented by; Lymphocytic aggregation and infiltration as well as dilated and edematous blood vessels. The majority of liver cells their cytoplasm displayed signs of degeneration represented by cytoplasmic vacuolization and necrosis. Regarding to the effect renal tissues of treated rats DMPA doses induced several lesions which manifested in ; glomerular


36

shrinkage, lymphocytic aggregation, degeneration of the tubules, congestion of renal blood vessel and obliteration of glomeruli. The findings shed more light and traceable to effects of DMPA on the adult female rats and suggests a toxic potential of DMPA which makes this an important issue that is worthy of animal trials studying possible ill-effects. It demands the lowest possible use of effective doses of DMPA to minimize any potential risk. P206-042 APOPTOSIS AND HEAT SHOCK PROTEIN EXPRESSION IN THE LIVER OF RATS WITH A SINGLE ALPHA-NAPHTHYLISOTHIOCYANATE INTOXICATION

Y. Ohta (1)

, T. Matsura (2)

, T. Nishida (2)

, Y. Imai (3)

. (1)

Fujita Health University School of Medicine, Toyoake, Japan; (2)

Tottori University Faculty of Medicine, Yonago, Japan;

(3) Fujita Health University School of Health Sciences, Faculty of Medical Technology,

Purpose: It has been implicated that oxidative stress and neutrophil infiltration are associated with the appearance of apoptotic cell death in the liver of rats intoxicated once with alpha-naphthylisothiocyanate (ANIT). Heat shock proteins (HSPs) are known to modulate apoptotic cell death. Therefore, we examined how oxidative stress and neutrophil infiltration are associated with apoptosis in the liver of ANIT-intoxicated rats and whether this apoptosis is modulated by HSP25, HSP60, and/or HSP70 expressed in the liver of ANIT-intoxicated rats. Methods: Seven-week-old male Wistar rats were intraperitoneally injected with ANIT (75 mg/kg). The animals were killed under ether anesthesia at 12, 24 and 48 h after ANIT intoxication. Isolated livers were used for the assays of caspase-3, a marker of apoptosis, HSP25, HSP60, and HSP70, lipid peroxide (LPO), and myeloperoxidase (MPO). Expressions of HSP25, HSP60, and HSP70 were estimated by Western blot analysis. Results: At 12 h after ANIT intoxication, the liver of the intoxicated rats showed no change in caspase-3 activity but had increased HSP25, HSP60, and HSP70 expressions, MPO activity, and LPO content. At 24 h after ANIT intoxication, the liver of the intoxicated rats had increased caspase-3 activity, further increased MPO activity and LPO content, and the reductions of increased HSP25 and HSP70 expressions but sustained increased HSP60 expression. At 48 h after ANIT intoxication, the liver of the intoxicated rats showed a reduction of increased caspase-3 activity, re-increased HSP70 expression, and further increased MPO activity and LPO content but sustained increased HSP60 expression and decreased HSP25 expression. Conclusions: Apoptosis appears after the occurrence of oxidative stress and neutrophil infiltration in the liver of ANIT-intoxicated rats. Apoptosis is reduced with excessive enhancements of oxidative stress and neutrophil infiltration in the liver of ANIT-intoxicated rats and it is mainly modulated by HSP70 expressed in the tissue. P206-045 HISTOPATHOLOGIC STUDY OF TAMOXIFEN INDUCED LIVER DAMAGE IN RATS AND THE POTENTIAL PROTECTIVE EFFECT OF VITAMINS C AND E

A. Hassanein, F. Adly, N. Shafei, M. Badawy. National Research Centre, Dokki, Cairo, Cairo, Egypt Background: Tamoxifen is widely used to treat oestrogen dependent breast carcinoma . Previous studies have shown tamoxifen induced hepatoproliferative lesions in rats. This study was designed to evaluate the effects of tamoxifen on liver of rats and the possible protective effects of vitamin (C) and/ or (E). Material and methods: A total of 80 adult female albino rats were used . Animals were divided into eight groups; 10 rats each. Drugs (tamoxifen) , vitamins (C) and (E) were daily orally administered ; for two weeks . The first group was kept as control. Second group was orally daily dosed with tamoxifen 20 mg/kg b. w.. Third group was given vitamin C (0.01 g/100 g b w), 15 mins prior to tamoxifen administration. Fourth group was given vitamin E (100 mg/Kg b.w), 15 mins prior to tamoxifen . Fifth group was given combination of the two vitamins (C) &(E) (0.01g/100 g b.w.) and (100 mg/kg b.w.) respectively 15 mins prior to tamoxifen . The remaining three groups were orally daily given vitamin C (0.01 g/100 g b.w.) , vitamin E (100 mg/kg b.w.) and both vitamins C (0.01 g/100 g b.w.) and E (100 mg/kg b.w.) respectively. Histopathological, DNA ploidy, histochemical and electron microscopic studies were performed . Results: Liver of rats treated with tamoxifen alone showed degenerative histopathologic , histochemical changes, DNA hypoploidy and ultrastructural partial nuclear clumping ; all markedly ameliorated in rats treated with vitamin C and/ or vitamin E prior to tamoxifen. Conclusion: Treatment of rats with vitamin (C) and/or vitamin (E) prior to tamoxifen resulted in amelioration of the histopathological , histochemical and ultrastructural liver changes. P206-051 CHRONIC EXPOSURE TO BENZOPHENONE INDUCED PATHOMORPHOLOGICAL AND ULTRASTRUCTURAL CHANGES IN THE LIVER OF ALBINO RAT

U. Dutta. P.G. Department of Zoology Cotton College Guwahati 781001, Guwahati, India Continuous exposure to commonly used xenobiotics constitute one of the greatest hazard to human health. The present study deals with one such xenobiotic component benzophenone (BZ) - a photosensitive UV absorber, benzene derivative diphenyl ketone. It is extensively used in cosmetics preparation, dyes, medicine and food packaging industries. Chronic exposure to sublethal concentration of BZ at a dose of 15mg/ml/kg body weight for 17 weeks was studied to evaluate the effect of the chemical on the liver of albino rat. The assessment was made on the basis of alterations of gross, histopathological, ultramicroscopical and cytochemical levels of liver. Histopathologically, striking changes manifested in the liver were focal areas of necrosis, nuclear-cytoplasmic degenerations, presence of extensive coarse granules in the cytoplasm and loss of intercellular cementing materials of hepatocytes. Disorganization of lobular architecture resulted due to fluid accumulation and ionic shift. Formation of fibrinous thrombus in the blood vessels and arterial hyperplasia were also marked. Presence of fragmented and bizarre nuclear


37

pattern suggested alteration in the nuclear morphology. Bile duct proliferation with the bile canaliculi formation was well marked feature indicating precarcinogenic changes in the hepatocytes. TEM revealed various ultra structural changes in the hepatocytes such as mitochondrial swelling with loss of cristae and their membrane disruption; loss of cytosol as well as dilation of SER. Extensive condensation of nuclei with enlarged nucleopores and disorganization of nuclei by margination of nucleoli to the periphery indicated an active cell proliferation. Cytochemical studies for DNA showed a progressive weak Fulgen reaction in the hepatocytes, whereas proliferated billiary epithelial cells exhibit strongly positive reaction. The results of the present investigation suggest that BZ has a potent hepatotoxic effect, which may eventually bring about irreversible disorder of the liver - the main detoxicating organ of the body. P206-052 PROTECTIVE EFFECT OF RESVERATROL AGAINST OXIDATIVE DNA DAMAGE INDUCED BY 2-NITROPROPANE IN RAT LIVER

E. Bigagli, M. Lodovici, M. Zaid, C. Luceri, P. Dolara. University of Florence, Florence, Italy 2-nitropropane (2-NP) is a constituent of paints, inks and tobacco smoke; it has been widely used as a model compound for studies on oxidation damage in the liver. Resveratrol is a natural phenolic compound with free radical scavenging and antioxidant properties. We aimed to study the effects of resveratrol on oxidative stress and on 2-NP-induced antioxidant defences to determine whether resveratrol administration could reverse oxidation damage induced by chemicals. A group of five rats received resveratrol (100 mg/kg b.w.) in the diet and after 14 days, 2-NP (100 mg/kg) was injected i.p. into two groups of animals while control animals were treated with vehicle alone. Animals were sacrificed 15 h after 2-NP injection. I.p. administration of 2-NP considerably increased the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in DNA liver, while pre-treatment with resveratrol for 14 days before 2-NP administration restored 8-oxodGuo in DNA to levels similar to controls. Although the average protein oxidation was not significantly modified by 2-NP injection, a correlation between carbonyl residues and 8-oxodGuo levels in rats treated with 2-NP was found. 2-NP induced an increase in SOD and XO activities and in the group treated with resveratrol, SOD and XO levels were similar to controls. Lipoperoxidation, was similar in the three groups while 2-NP treatment significantly reduced GSH/GSSG in comparison to controls and this change was partially reversed by resveratrol. No toxic effect on colon mucosa after 2-NP injection, was observed. In conclusion, pre-treatment with resveratrol significantly reduced oxidative DNA damage induced by 2-NP in the liver by modulating SOD and XO activities and partially restoring the GSH/GSSG ratio showing that this compound may protect against liver oxidative DNA damage induced by 2-NP. P206-054 DIMETHYL-DIPHENYL-BICARBOXYLATE (DDB) AS ANTIAPOPTOTIC AGENT TARGETING DEATH LIGAND AND CASPASES SIGNALING PATHWAY IN A MODEL OF HEPATIC ISCHEMIA REPERFUSION INJURY L. G. Mahran, A. A. El-Bahy, L. A. Kassem, O. A. Heikal Faculty of Pharmacy & Biotechnology, The German University in Cairo, Egypt

Background and aim: Dimethyl-Diphenyl-Bicarboxylate (DDB) is a hepatoprotectant used in treatment of chronic viral hepatitis and other liver diseases; it’s a synthesized derivative of Schizandrin C, active component of traditional Chinese herb Fructus Schizandrae. However, the mechanism of DDB against liver injury needs further elucidation. Our objective is to explore whether DDB exerts hepatoprotective effect against hepatic ischemia reperfusion injury (I/R) and to identify its potential effect on apoptotic cell death targeting the extrinsic apoptotic pathway. Methods: Rats were divided into four groups: Group I (sham-operated), Group II (I/R group) upon which I/R was performed, Group III (Pretreated groups): where rats were treated with DDB at 3 dose levels (100mg/kg, 300mg/kg, 500mg/kg) for 10 days, then subjected to I/R and Group IV (treated group): rats were treated with DDB at the same dose levels but without being subjected to I/R. The following parameters were measured: serum ALT & AST, tissue levels of GSH, MDA & TNF-α, activity of signaling Caspases 8 & 9 and effector Caspase 3 as well as expression of nuclear factor-κB. End point of apoptosis was detected by DNA fragmentation. Results: DDB administration before I/R, significantly attenuated the elevated levels of ALT, AST, MDA and TNF-α. It also significantly increased the GSH content. Hepatic apoptosis was inhibited as evidenced by significant decrease in all caspases activities & expression of nuclear factor-κB as well as by DNA fragmentation. DDB administration to normal rats, inhibited lipid peroxidation, enhanced the antioxidant activity, decreased theTNF-α and downregulated the expression of nuclear factor-κB. These effects are dose-dependent. Conclusion: DDB exerts potent antioxidant effect in both normal and injured livers. DDB inhibited hepatic I/R induced apoptosis through inhibiting signaling Caspases 8, 9 and 3, inhibiting TNF-α production which regulates the extrinsic apoptotic pathway and down regulates NF-κB expression in liver tissue. P206-055 POSSIBLE PROTECTIVE EFFECTS OF HONEY AND, GINSENG AGAINST HEPATONEPHROTOXICITYIN RATS

M. Al-Ghazali (1)

, E. S. El-Denshary (2)

, H. Salem (2)

, F. Mannaa (2)

, N. Hassan (2)

, M. Abdel-Wahhab (2)

. (1)

Faculty of Pharmacy, Sanaa University, Sanaa, Yemen; (2)

, Cairo, Egypt Chronic liver diseases represent a major health burden worldwide, with liver cirrhosis being the ninth leading cause of death in Western countries. Chronic viral hepatitis B and C, alcoholic liver disease, non-alcoholic fatty liver disease, and hepatocellular carcinoma are the major entities and many problems remain unresolved. The aim of the current study was to evaluate the protective effects of Sidr honey and/or ginseng against CCl4-induced hepatotoxicity and oxidative stress in rats. Eighty male Sprague-Dawley rats were divided into eight groups and treated for 4 weeks as follow: the control group, the group treated with CCl4 (100 mg/kg b.w), the group treated orally with hony (5 g/kg b.w),


38

the group treated orally with ginseng extract (20 mg/kg b.w), the group treated orally with honey plus ginseng extract and the groups treated with CCl4 plus honey and or ginseng extract. At the end of treatment period, blood and liver samples were collected for biochemical and histological studies. The results indicated that animals treated with CCl4 alone showed severe toxicity typical to those reported in the literature. Both Sidr honey and ginseng extract showed a significant improvement in all biochemical parameters and histological picture of liver and kidney as indicated by the significant improvement in body weight, food intake, serum biochemical parameters, oxidative stress marker, antioxidant status, and the histological and histochemical pictures of liver and kidney. Moreover, the combined treatment of honey with ginseng was more effective than the single treatment in the protective effects. P206-057 HUMAN HEPATOCYTE CULTURES: IMPROVING FUNCTIONALITY USING 3D CULTURE IN STIRRED BIOREACTOR FOR DRUG TESTING APPLICATIONS

S. B. Leite (1)

, R. Tostões (1)

, M. Serra (1)

, C. Brito (1)

, M. F. Sousa (1)

, J. Jensen (2)

, T. Andersson (3)

, P. Björquist (2)

, M. J. Carrondo

(4), P. M. Alves

(1).

(1) IBET/ITQB, Oeiras, Portugal;

(2) Cellartis;

(3) Astrazenneca,

(4) IBET/ITQB-FCT, Portugal

During the last years an increasing number of in vitro models have been developed for drug screening and toxicity testing. Human hepatocyte cultures are a representative and increasingly used approach to perform these studies. However, these cultures have the major drawback of spontaneous cell dedifferentiation in vitro. Trying to overcome this problem novel strategies that allow for extended hepatocyte maintenance in culture are required. In this work, we explored the potential of spheroid cell structures (3D) as they better mimic the in vivo tissue, not only by enabling retention of important hepatic functions but also by allowing for closer contact between cells. On a first approach, different 3D strategies were tested in stirred bioreactors using primary rat hepatocytes, performing mono and co-cultures with fibroblasts. During culture time, cell concentrations and viability were monitored daily and cultures were characterized using IF and RT-PCR tools. In addition specific-liver metabolic activity was evaluated by measuring urea and albumin production and Phase I (ECOD and MS of CYP metabolites) and Phase II (UGT) biotransformation activity. The results obtained showed an increase of hepatocyte functionality and culture time extension when primary rat hepatocytes were cultured as spheroids (3D) in bioreactor stirred systems that allow for a fully controlled environment with further improvements in co-culture. The best culture strategy obtained with rat cells was further selected to develop an efficient 3D system for human hepatocytes culture. Herein, the culture parameters were defined to be closer to physiological conditions: temperature at 37 ºC, pH 7.4 and pO2 at 30.As previously verified with rat cultures, these 3D strategies improved the human hepatocyte function, particularly in bioreactor where an extension of culture time was obtained up to 5 weeks. P206-058 GENES EXPRESSION FROM LIVER TISSUE AFFECTED IN PHTHALATE TREATED MICE, COUPLED WITH STRYCTURAL ANALYSIS OF PHTHALATES JOINTLY ELUCIDATE MECHANISM OF PHTHALATE HEPATOTOXICITY AND FURTEHR POINT TO TOXICITIES IN OTHER ORGANS AND ITS MECHANISMS

G. Apic (1)

, R. Russell (2)

, F. Bonner (1)

, B. Cosovic (1)

. (1)

Cambridge Cell Networks Ltd, Cambridge, United Kingdom; (2)

University of Heidelberg, Heidelberg, Germany The aim of this study was to predict target organ toxicity and elucidate mechanism of action and toxicity of phthalates tested by a novel approach of combining two methods in a systematic way: chemical structure analysis and gene expression analysis from the rat liver sample. Furthermore, based on this study we designed a novel general method for generating hypotheses on mechanism of action by using in silico systems biology approaches which combine gene annotation with read-across the similar chemicals and their target genes and proteins. We analyzed a gene expression profile with 35 significantly disregulated genes from mouse liver tissues treated with diethylhexylphthalate. From the whole genome microarray set, 35 signature genes were selected as significantly dysregulated and predicted which of 1512 pathologies were the strongest indicated. This procedure involves scoring the gene set based on known relationships of genes to the pathologies, in addition to associations with chemicals (or other genes) that, in turn, are related to the pathologies. 26/35could be traced to have a function relevant in liver tissue, and helped shed light about the mechanism. Interestingly, thou only 2/35 had direct association with testis and understandably most had association with liver we managed to predict testicular toxicity and similar mechanism. As a comparison, we analyzed the expression data profile of testis from male rat embryos treated with dibutylphthalate in utero. Despite the fact that only 2/33 dysregulated genes have direct associations with testis atrophy, links of nine genes with 12 chemicals make this among the most strongly indicated pathologies. A similar utility of indirect evidence also increases the signal for testis pathologies when considering the chemical set. This study shows that the incorporation of indirect evidence can suggest a mechanistic picture for many more genes than traditional analyses of gene expression data. P206-060 EVALUATION OF DRUG TOXICITY USING ISOLATED MOUSE LIVER MITOCHONDRIA

M. Porceddu (1)

, N. Buron (1)

, B. Fromenty (2)

, A. Borgne-Sanchez (1)

. (1)

MITOLOGICS S.A.S. Hôpital Robert Debré, Paris, France, (2)

INSERM U991, Faculté de Pharmacie, Rennes, France Mitochondrial dysfunction is a major mechanism of drug-induced liver injury (DILI), which can be severe and even fatal. DILI can thus lead to the interruption of clinical trials, or drug withdrawal after marketing. As the financial losses can be important for pharmaceutical companies, drug-induced mitochondrial dysfunction and DILI should be detected at early development stages. To facilitate this detection, we propose a high-throughput screening platform on isolated


39

mouse liver mitochondria which allows the measurement of several parameters pertaining to mitochondrial function and membrane integrity. Indeed, these multiparametric assays measure: 1) global mitochondrial membrane permeabilization (swelling), 2) transmembrane potential ( m), 3) mitochondrial outer membrane permeabilization (cytochrome c release) and 4) respiration (O2 consumption) in isolated mouse liver mitochondria. The screening of numerous drugs known to be hepatotoxic (Alpidem, Lumiracoxib, Troglitazone…) or not allowed us to established a strong correlation between in vitro mitochondrio-toxicity and DILI in human. A statistical analysis is currently performed to determine the power of predictivity of this screening platform. P207-045 DOMOIC ACID-INDUCED NEUROTOXICITY IS MAINLY MEDIATED BY THE AMPA RECEPTOR: COMPARISON BETWEEN IMMATURE AND MATURE PRIMARY CULTURES OF CEREBELLAR GRANULE CELLS

H. Hogberg (1)

, A. Price (2)

. (1)

The Johns Hopkins University, Baltimore, United States; (2)

European Commission Joint Research Centre, United States Domoic acid (DoA) is a naturally occurring shellfish toxin that can induce brain damage in mammalians. Neonates have been shown to be more sensitivity to DoA-induced toxicity than adults and prenatal exposure has been mainly associated with damage to neurons in different brain regions, decreased brain GABA levels and increased glutamate levels. In this study we evaluated DoA induced toxicity in immature and mature cultures of primary rat cerebellar granule cells (CGCs) by measuring the mRNA levels of selected genes identified as specific glial and neuronal markers. Moreover, we assessed if the induced toxicity was mediated by activation of the AMPA/KA and/or the NMDA receptor. The mRNA levels of all studied neuronal markers (NF-68, NF-200, NMDA receptor and GABAA receptor) were down-regulated after DoA exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment as the effects were observed at lower concentrations and at an earlier time point than for the immature cultures. This could be due to lower expression in immature cultures of the AMPA/KA receptors that are mediating DoA-induced toxicity. Indeed, the DoA effect was prevented by the antagonist of the AMPA/KA receptor (NBQX) indicating that this receptor is involved in DoA-induced toxicity. In contrast the antagonist of the NMDA receptor (APV) only partly blocked the DoA-induced effects. Interestingly, the astrocytic

markers (GFAP and S100�) and the neural precursor marker (nestin) were only affected in the mature cultures.

These effects could partly be prevented by NBQX, APV and the neurotransmitter GABA, indicating that DoA induced toxicity by different mechanisms in mature and immature cultures. P207-048 CHANGES OF MITOCHONDRIAL ULTRASTRUCTURES AND FUNCTION IN NERVE TISSUE OF HENS TREATED WITH TRI-ORTHO-CRESYL PHOSPHATE (TOCP)

X. Xin (1)

, T. Zeng (1)

, D. D. Dou (1)

, S. Zhao (1)

, J. Y. Du (2)

, J. J. Pei (2)

, K. Q. Xie (1)

, X. L. Zhao (1)

. (1)

Institute of Toxicology, Shandong University, Jinan, China; (2)

, Tri-ortho-cresyl phosphate (TOCP), an organophosphorus ester, is capable of producing organophosphorus ester-induced delayed neurotoxicity (OPIDN) in human being and sensitive animals. The mechanism of OPIDN has not been fully understood. The present study has been designed to evaluate the role of mitochondrial dysfunctions in the development of OPIDN. Adult hens were treated with TOCP by gavage at single dosages of 185, 375, 750 mg/kg•bw respectively, and sacrificed on the corresponding time points of 1, 5, 15, 21 day post-dosing. The cerebrum and spinal cord were dissected, homogenized and used for the determination of the mitochondrial permeability transition (MPT), membrane potential (△ψm) and activity of succinate dehydrogenase assayed by MTT reduction. Hens respectively selected from every time point in 750 mg/kg•bw group and control group were perfused, and then the cerebral cortex cinerea and the ventral horn of lumbar spinal cord of were dissected for electron microscopy. Electron micrographs revealed changes of mitochondria structures in hen's nerve tissues, including vacuolation and fission, which increased with time post-dosing. Ca2+-induced MPT was increased both in cerebrum and spinal cord, with the most noticeable increase in spinal cord. Ca2+-induced MPT of the three treated groups was all significantly increased on day 1 post-

dosing compared to control group (P＜0.05) in spinal cord. On day 5 and 15 post-dosing, Ca2+-induced MPT of the

375 and 750 mg/kg•bw groups still kept this trend (P＜0.05). Although the △ψm was decreased both in cerebrum and

spinal cord, there was no significant difference in the three groups as time went on. In addition, MTT reduction reflecting the activity of the mitochondrial respiratory chain also confirmed mitochondrial dysfunctions following development of OPIDN. The results suggested that mitochondria dysfunction might partly account for the development of OPIDN induced by TOCP. P207-049 SSAMYLOID INDUCE APOPTOSIS, MITOCHONDRIAL INJURY, OXIDATIVE STRASS AND P2X7 CELL DEATH RECEPTOR MODULATION IN HUMAN RETINA CELL - ROLE IN AGE-RELATED MACULAR DEGENERATION

P. Rat, B. De Moucheron, F. Massicot, I. Celerier, J. M. Warnet, O. Laprevote. Université Paris Descartes, PARIS, France ßamyloid induce apoptosis, mitochondrial injury, oxidative strass and P2X7 cell death receptor Modulation in human retina cell - role in Age-related Macular Degeneration ß amyloid have been found in the human's eye with AMD (Age-related Macular Degeneration). the degenerative pathway mechanism is not clear.


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To understand the degenerative pathway in retina cell, ß amyloid was incubated with human epithelial retina cells (ARPE 19) and muller cells (MIO-M1 glial cell) during 24, 48, 72 hours. Methods: mitochondrial injury was assessed with trans membrane mitochondrial potential with JC-1 probes. oxidative stress was evaluated with DCFH-DA probes using Cytofluorometry adapted to microplate (Safire/ TECAN). Apoptosis was assessed with caspases et condenstion chromatin evaluation and Yopro-1 tests have permit to evaluate P2X7 cell death receptor activation. IL-6 IL -8 cytokine assessment was performed with immunofluormetric method on cytometry. Results: sensitivity to ßamyloid depend of the cell type. After oxidative stress, a rapid mitochondrial injury is observed. Cell viability decrase was signifcant after 48h incubation. Reactive Oxygen species (ROS) overproduction was observed, associated to superoxide anion production. Mitochondrial injury was observed with transmembrane mitochondrial potential and mitochondrial succinate deshydrogenase decreasing. Cell death is associated to apoptosis with caspases pathway activation and chromatin condensation. Moreover, P2X7 cell death and inflammation receptor are activated with P2X7 pore opening. Different treatment were tested. P2X7 modulator can limit cell death and apoptosis. Conclusion - ß amyloid induces cell death and degenerative pathway on retina cell and glial cell. ß amyloid seems to be associated to Age-related macular degeneration. P2X7 cell death receptor activation seems to be associated to apoptosis and degenerative pathway observed with ß amyloid incubation on human retina cells and glial cell - P2X7 modulation could be an interesting new therapeutic strategy. P207-050 VALPROATE INHIBITS CEREBRAL GLUTAMINE UPTAKE AND OXIDATION: A CARBON 13 CELLULAR METABOLOMIC APPROACH APPLICABLE TO DRUG DEVELOPMENT

M. El Hage, B. Ferrier, G. Baverel, G. Martin. Metabolys Inc., Lyon, France Glutamine is both synthesized in astrocytes and metabolized in neurons. Our objective was to evaluate the brain slices and the cellular metabolomic approach as tools for drug research and development. Slices prepared from rat brain hemispheres were incubated with 5 mM L-[3-13C]glutamine with 0 and 1 mM valproate, an antiepileptic drug. Substrate removal and product formation were measured by enzymatic and carbon 13 NMR methods. Fluxes through the enzymatic steps involved were calculated with an original mathematical model. In the absence of valproate, 3-13C-glutamine was used as substrate by glutaminase but glutamine synthesis also occurred in astrocytes. 3-13C-glutamate accumulation was also observed. GABA was found labeled mainly on its C3 (direct synthesis) and, to a very small extent, on its C2 (indirect synthesis). Accumulation of aspartate, labeled mainly on its C2 and C3, was also observed. Labeling of glutamate and glutamine on carbons other than their C3 reveals that resynthesis of both amino acids occurred. Approximately half of the C3 of the 3-13C-glutamine removed was released as CO2. In the presence of valproate, 3-13C-glutamine removal was slightly inhibited. Aspartate accumulation and labeling on its C2 and C3 were decreased whereas GABA labeling on its C3 was increased by valproate. Valproate also diminished the production of 13CO2 but did not change the cellular ATP level. Flux calculations indicate that valproate slightly inhibited flux through glutaminase and caused an accumulation of GABA by the direct pathway. It also inhibited fluxes through citrate synthase, succinate semialdehyde dehydrogenase plus alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase and aspartate aminotransferase. By contrast, valproate did not alter fluxes through the other enzymes involved. It is concluded that the model used in vitro and the cellular metabolomic approach employed are suitable for studying the beneficial and adverse interactions of drugs with brain energy and intermediary metabolism. P208-025 A STUDY ON HISTOPATHOLOGICAL CHANGES OF GASTRIC PARIETAL CELLS OBSERVED IN BEAGLE DOGS WITH DECREASED FOOD CONSUMPTION

O. Sawamoto, T. Fukuda, Y. Hayami, Y. Nakashima Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc. BACKGROUND: In toxicity studies, vacuolation of gastric parietal cells has been occasionally experienced in the beagle dogs with marked decrease in food consumption. In this poster, we present the histopathological and ultrastructural features of the parietal cells observed in the stomach of beagle dogs with decreased food consumption. MATERIALS and METHODS: Three 8-month-old male beagle dogs were given adequate calories and nutrients by total parenteral nutrition via a venous catheter for 13 days without oral feeding. Two control beagle dogs were intravenously given 0.9% saline under oral feeding conditions. Stomach samples were taken for histopathology and electron-microscopy. RESULTS: In histopathology, the vacuolation of gastric parietal cells (gastric gland) was seen in 2 of the 3 dogs given total parenteral nutrition without oral feeding. Morphological analysis of the parietal cells by TEM showed tightly closed intracellular canaliculus, increase in the tubulovesicle structure, and/or numerous cytoplasmic vacuoles as compared with the control dogs. These vacuoles contained concentric multilayer membrane structure and/or fluffy substance. CONCLUSIONS: It is known that parietal cells of the stomach secretes gastric acid in response to oral feeding, and the cells morphologically change depending the presence or absence of feeding. It is reported that vacuolation of parietal cells is induced when gastric acid secretion is inhibited by surgical treatment. If the vacuolation of gastric parietal cells is observed in toxicity studies, it is necessary to investigate its association with food consumption.


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P208-065 TOXICOLOGICAL AND HEALTH HAZAEDS OF ENROFLOXACIN AS AN ANTIMICROBIAL AGENT ON REPRODUCTION OF MALE ADULT ALBINO RATE

K. Abd El – Hady Faculty of Vetrerinary Medicine Suez Canal University, Egypt

This study includes determination of the toxic effect of enrofloxacin as an antimicrobial agent in doses of 250 and 500 mg/kg B. wt. on fertility parameters including some reproductive organs weight, sperm cell count, motility, sperm live and dead percentage, fructose, testosterone level detection of its residues in tissue and histopathological findings on some organs. A number of 90 adult male albino rats were classed into 3 groups each of 30 rats. first group was treated orally with 250mg/kg B.wt. the second group was treated orally with 500 mg/kg B.wt daily for sucessive 8 weeks , while the third group kept as control group. the results revealed sever inhibition on all fertility parameters which confirmed through histopathological findings also the results concluded that enrofloxacin has residues in testis after stoppage of administration by one week P208-066 NEPHROTOXICITY OF MELAMINE, CYANURIC ACID AND THE MIXTURE

L. Choi, E. Y. Han, D. E. Jang, D. H. Kim, J. S. Park, I. Y. Ahn, S. K. Lim, B. M. Lee. Sungkyunkwan University, Suwon, Korea, Republic of Melamine has been widely used for manufacturing melamine resins and cleaning products for a long time. However, melamine was recently reported to produce the renal calculus in the kidneys and renal failure when accompanied with cyanuric acid, the analog of melamine. In the present study, the acute toxicities of melamine, cyanuric acid and the mixture of melamine and cyanuric acid were comparatively investigated in male Sprague Dawley rats and in MDCK and ACHN cell lines. In vivo, the renal calculi were found in rat kidney and the body weight, and absolute and relative kidney weights of rats were significantly decreased. In addition, blood urea nitrogen (BUN), creatinine significantly increased at high dose groups of the mixture (100X, 1000X). It was shown that inflammation was involved in melamine-induced nephrotoxicity as found increasing of the number of total white blood cell (WBC), neutrophil and lymphocyte in vivo and prostaglandin E2 in vitro. Based on these parameters, it could be established that a NOAEL (no observed adverse effect levels) for the nephrotoxicity of the mixture of melamine and cyanuric acid was 3.15 mg/kg b.w./day (melamine) plus 2.5 mg/kg b.w./day (cyanuric acid). The cytotoxicity property of the chemicals was evaluated in vitro, using the MDCK and ACHN cell lines. When IC50 values of melamine, cyanuric acid and the mixture of them were compared in these cell lines, ACHN cell line was shown to be more sensitive than MDCK cell line. The IC50 values of melamine were smaller than in cyanuric acid in both cell lines, suggesting that melamine is more cytotoxic than cyanuric acid. Furthermore, the IC50 value in the mixture group was lowest and this suggests the cytotoxicity of the mixture was higher than the treatment of single compound. P208-069 INHIBITORY EFFECT OF CYP1A1 EXPRESSION BY CAPSAICIN IN HEPA-1C1C7 CELLS

E. H. Han, J. H. Im, H. G. Jeong. Chungnam National University, Daejeon, Korea, Republic of Capsaicin (CPS), a constituent of green and red peppers, has been linked to the suppression of tumorigenesis and carcinogenesis through the transient receptor potential vanilloid type-1 receptor (TRPV1). The influence of CPS on cytochrome P450 1A1 (CYP1A1) expression and the underlying mechanisms remain unclear. In this study, we examined the effect of CPS on 3-methylcholanthrene (3-MC)-induced CYP1A1 in mouse hepatoma Hepa-1clc7 cells. CPS significantly inhibited 3-MC-induced CYP1A1 mRNA, protein, and CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity in a dose-dependent manner. Additionally, CPS enhanced the CCAAT/enhancer-binding protein beta(C/EBP beta) mRNA and protein levels in Hepa-1clc7 cells. Over-expression of a dominant-negative mutant C/EBP abolished the ability of CPS to suppress 3-MC-induced CYP1A1 activity and gene expression. Treatment with capsazepine, a TRPV1 antagonist, did not affect the inductive effects of CPS on C/EBP expression. Finally, CPS reduced the formation of the 7,12 dimethylbenz[a]anthracene (DMBA)-DNA adduct. Our results indicate that CPS suppresses 3-MC-induced CYP1A1 levels, and that the activation of C/EBP beta by CPS contributes to the suppression of CYP1A1 expression. P208-071 TUBULAR ORGANIC ANION TRANSPORTER 1 AND 3 ARE RESPONSIBLE FOR THE RENAL ACCUMULATION AND NEPHROSPECIFIC TOXICITY OF ARISTOLOCHIC ACID

X. Xue, L. K. Gong, Y. Luan, X. M. Qi, Y. Li, J. Ren. Center for Drug Safety Evaluation and Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Aristolochic acids (AAs) are nephrotoxic and carcinogenic nitrophenanthrene carboxylic acids produced by the Aristolochiaceae family of plants. Ingestion of these phytotoxins, especially the major constituent AAI, results in a syndrome known as aristolochic acid nephropathy (AAN), characterized by renal tubulointerstitial fibrosis and upper urothelial cancer. While extensive investigations have been focused on the metabolic pathways of AAs, much less is known about the mechanism of AAs uptake by renal cells and subsequently induction of kidney-specific toxicity. Here, we reported that probenecid (PRB), a well-known organic anion transporter (OAT) inhibitor, effectively protected mice from AAI induced acute tubular necrosis, although with a 1.5-fold or 3.9-fold increase of total body exposure for AAI


42

and its metabolites AAIa, respectively. Accordingly, PRB increased the concentration of AAI and AAIa in liver, but decreased them in kidney by increasing the hepatobiliary excretion and reducing the renal clearance of AAI and AAIa. We further confirmed that AAI induced kidney injury were less severe in both of mOat1 and mOat3 knockout mice than their corresponding wild-type mice. In vitro study with hOAT1- and hOAT3-overexpressing HEK293 cells exhibited a higher uptake of AAI and a greater toxicity than mock. In addition, six kinds of AAs including AAI, AAII, AAC, AA Na, 7-OH AAI and aristolic acid I (AaI), but not aristolactam I (ALI) could inhibit the uptake of a known substrate 6-carboxyl-fluorescence (6-CF) by hOAT1- and hOAT3-HEK293 cells. In summary, AAs are substrates for both nephritic OAT1 and OAT3, which determine the renal accumulation and nephrospecific toxicity of AAs. P208-073 EFFECT OF SUBACUTE CADMIUM INTOXICATION ON OXIDATIVE AND NITROSATIVE STRESS IN MICE

D. Djukic-Cosic (1)

, M. Ninkovic (2)

, Z. Malicevic (2)

, Z. Plamenac Bulat (1)

, V. Matovic (1)

. (1)

University of Belgrade, Faculty of Pharmacy, Belgrade, Serbia; (2)

Military Medical Academy,

Cadmium (Cd) is a known occupational hazard and an environmental pollutant. As a nonessential element, it exerts multiple toxic effects in mammals and has been classified as a human carcinogen by the International Agency for Research on Cancer. It can cause numerous adverse effects in the organisam, oxidative stress being one of them. Although Cd has no redox activity, it can indirectly induce the production of hydroxyl radicals, superoxide anions, hydrogen peroxide and nitric oxide (NO). The aim of this study was to determine the effect of subacute Cd intoxication on lipid peroxidation (LPO), total thiol (SH) content and NO production in mouse kidney and liver, the main targets of Cd toxicity. Experiment was performed on Swiss albino mice divided into two groups: control (not treated animals) and Cd group intoxicated orally, every day for 14 days with 10 mg Cd/kg. Lipid peroxidation as malondialdehyde (MDA) production (assayed in the thiobarbituric acid reaction), the NO production by Griess method and total SH content by Ellman method were measured in the investigated tissue homogenates. The obtained results show that subacute Cd intoxication induces significant increase of MDA and NO content, but decreased concentration of non-protein SH in the kidney, indicating that reactive oxygen and nitrogen species participate in Cd nephrotoxicity. In the liver, two-weeks Cd treatment induced a significant decrease of MDA, NO if compared with control. This decrease might be the result of the defence mechanism activation, predominantly elevated synthesis of metallothionein which reflected in increased total SH content. These results show that subacute Cd intoxication induces organ specific response to oxidative and nitrosative stress in mice. This work was partly supported by the Ministry of Science and Technological Development, Serbia (project - TR 20212A). P208-075 TARGET ORGAN TOXICITY INDUCED BY VEGF INHIBITORS

A. M. Giusti, F. Marchesi, R. Pulci, A. Parazzoli, E. Giannotti, P. Colombo, M. Brughera. Accelera Srl, Nerviano, Italy The discovery of the dependency of neoplasia and metastasis upon angiogenesis, alongside the uncovering of many of the important molecules controlling new vessel formation, prompted many pharmaceutical and biotechnology companies to develop novel treatments aimed at inhibiting these critical processes. Cell surface thyrosine kinase receptors (RTKs) such as vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs) and platelet derived growth factors (PDGFs) proved to be strongly implicated in angiogenesis and the vascularization associated with solid tumors. Although in the healthy adult blood vessels are stable and quiescent in most tissues, disruption of specific VEGF signaling by monoclonal antibodies has resulted in toxicity in specific organs relying on angiogensis for maintenance and development. In particular, physeal dysplasia in long bones, changes in the renal glomeruli and associated proteinuria, hypertension, and a reduction of number of corpora lutea in the ovary have been associated with a specific VEGF-mediated pharmacological target organ effect, having all been observed in nonhuman primates or patients treated with this class of compounds. The development of small molecules Receptor Tyrosine Kinase inhibitors characterized by a lower selectivity (multi-targeted RTK inhibitors) has further widened the spectrum of toxic lesions induced to a number of organ systems. Additional findings reported to occur in different animal species (rats, monkeys and dogs) include periosteal cartilage and bone formation in the femur and sternebra, hemorrhage and necrosis in the adrenals, increase in liver enzymes, dentin degeneration in the teeth, acinar degranulation in exocrine glands and atrophy of the squamous epithelia (tongue, esophagus and vagina). This review describes the effects of some of these new compounds in preclinical studies and their correlation with what has been observed in patients. P208-076 EVALUATION OF NEWLY DEVELOPED ELISAS FOR RAT, MOUSE, DOG AND PIG NGAL

K. Bangert, J. O. Eriksen, L. O. Uttenthal. BioPorto Diagnostics A/S, Gentofte, Denmark

NGAL is a sensitive marker of nephrotoxicity and acute kidney injury (AKI). To facilitate the use of animal models in toxicological studies and AKI research, we have developed specific ELISAs for determining rat, mouse, dog and pig NGAL and here report their initial evaluation. NGAL was captured with a species-specific NGAL monoclonal antibody in precoated microwells and detected with another species-specific biotinylated monoclonal antibody. The assays were calibrated with recombinant full-length NGAL diluted in buffer. The intraassay (n = 6) and interassay (n = 4) variation was determined by repeated measurements of plasma and urine samples. The detection limit was defined as the NGAL concentration corresponding to the absorbance of the blank + 2 standard deviations. Linearity was considered acceptable if the serial dilutions of plasma and urine did not deviate more than 15% from the expected values. Analytical recovery of recombinant NGAL added to plasma, urine or buffer was determined at 4 different levels


43

covering most of the calibration curve. The specificity of the antibodies was ascertained by positive reaction with the peak of recombinant NGAL and the 25-kDa peak of native NGAL from plasma and urine subjected to molecular size exclusion chromatography. The variation ranged from 0.9 - 5.9% (intraassay) and 0.8 - 6.0% (interassay). Detection limits were from 0.23 to 0.81 pg/mL. All assays had acceptable linearity and analytical recovery (90-106%). The newly developed ELISAs have an acceptable performance and can be used to test whether NGAL responses in animals are similar to those in humans. The ELISAs may prove to be valuable tools for investigating AKI in drug discovery, toxicology and disease models. P208-078 LETHAL DILTIAZEM POISONING IN A DONKEY

A. Nowroozi Asl, M. M. Heidari, A. Rowshan Ghasrodashti. Faculty of Veterinary Medicine, Islamic Azad University , Kazerun Branch, Kazerun, Iran (Islamic Republic of) Introduction: Diltiazem is used primarily for control of supraventricular arrhythmias,systemic hypertension,and hypertrophic cardiomyopathy.It also is used for atrial flutter, AV nodal reentry arrhythmias,and other forms of tachycardia.In horses,diltiazem may be effective for atrial fibrillation.However,treated horses had variable results,and some developed hypotension and sinus arrest. Calcium channel blocker (CCB) overdose is associated with significant cardiovascular toxicity, dysrhythmias and atrioventricular (AV) block.,however,experience with CCBs overdose in donkey is limited. Case: Diltiazem hydrochloride,a calcium channel blocker,has been used in a donkey for treatment of sinus arrhythmia by mistake. The field clinician have used 40 mg/kg body weight diltiazem(100 tablets,each tablet 60 mg,Daru Pakhsh,Iran) in a female donkey(aged 4-5 years old and weighting 150 kg ) for treatment of sinus arrhythmia via nasal tube orally. So, the field clinician not only about the usage but also dosage of drug has mistaken. 20 minutes later the sinus arrhythmia was converted to normal sinus rhythm.Thirty minutes after treatment the donkey has recumbent and in ECG she showed second degree AV block.Finally the animal showed third degree AV block and 90 minutes after treatment, she died. Histologically, multifocal myocarditis was seen in slide of heart.The increase in serum glucose concentration was found after treatment. Conclusion: Although CCB overdose is well described in the medical literature,animal data are limited .The results showed that 40 mg/kg body weight, diltiazem hydrochloride is lethal for donkey and cause some arrhythmias. P208-079 COMPARISON OF TWO METHODS OF MERCURY DETERMINATION IN HAIR: HYDRIDE GENERATION ATOMIC ABSORPTION SPECTROMETRY AND ATOMIC ABSORPTION SPECTROMETRY WITH ZEEMAN BACKGROUND CORRECTION AND PYROLYSIS

L. H. Sánchez (1)

, E. Stashenko (2)

, Y. Vargas (3)

, J. Urrego (4)

. (1)

Escuela de Bacteriología y Laboratorio Clínico, Universidad Industrial de Santander, Bucaramanga, Colombia;Departamento de Toxicología, Universidad Nacional de Colombia, Bogotá D.C., Colombia;

(2) Laboratorio de

Cromatografía, Universidad Industrial de Santander, Bucaramanga, Colombia; (3)

Laboratorio Químico de Consultas Industriales, Universidad Industrial de Santander, Bucaramanga, Colombia;

(4) Departamento de Toxicología,

Universidad Nacional de Colombia, Bogotá D.C., Colombia, Hair samples are widely used for biomonitoring of mercury (Hg) exposure. The traditional methods require 100 - 150 hair strands and involve chemical digestion. The main goals of this work were the validation of a methodology for mercury determination in hair samples using Atomic Absorption Spectrometry with Zeeman background correction (ZAAS), and also the comparison between ZAAS and hydride generation technique. ZAAS principle is the mercury atomization by pyrolysis (attachment RP-91C), and then, the determination of the total mercury content by atomic absorption spectrometry. In this work, the precision and the accuracy of both methods were determined using certified material of reference (IAEA-086, 085 and NIES No.13). The results showed a precision (variance coefficient, %) lower than 8%, and an accuracy (percentage recovery) of almost 100%, for both techniques (ZAAS and hydride generation). The analysis of 50 hair samples (0.32-52µg Hg/g) with both techniques showed high agreement, B&A limits of agreement, mean (0,2 μg Hg/g), IC95% (-2.2 , +3.2). Furthermore, there was no significant difference between the results obtained by each analytical technique (t = -1,38, P=0,172; LInf=-0,052, LSup=0,009). In epidemiological studies, it is recommendable to use ZAAS because it is a low cost technique with excellent results, it does not require long time of analysis and besides it is environmentally friendly. P301-004 ANALYSIS OF RETINOIDS AND CORTICOSTEROIDS IN LIGHTENING COSMETICS AND THEIR POSSIBLE INVOLVEMENT IN THE CUTANEOUS LESIONS DEVELOPED BY SAHARAWI WOMEN

M. C. Pellín (1)

, J. Esteban (1)

, C. Pereda (2)

, J. Barril (1)

, C. Gimeno (3)

. (1)

Universidad Miguel Hernandez de Elche, Elche, Spain; (2)

Hospital de los Arcos. Santiago de la Rivera (Murcia), (3)

UCA de La Vila Joiosa Departamento 16 (Alicante), Given the propensity of darker skin to dyschromias, weather conditions in the desert, and saharawi women´s culture, there is a significant consumption of cosmetics by this population. Among the most widely used cosmetics are skin lightening creams, oral steroids, "henna", and "nila". Industrial cosmetics have been considered as the origin of serious health problems in different African countries for more than 30 years, however this has only been recently recognized among saharawi people. Cosmetics with potentially serious side effects are being traded in the Saharawi Arab Democratic Republic (SADR) without a proper labelling. Such cosmetics are also being introduced in Spain by saharawi immigrant population. Due to the irregular origin of these formulations and, in most cases, lack of reliable specifications in the information sheets, there is a need to identify and quantify their active ingredients as they might


44

be responsible for the observed skin lesions. In the present study we analyse the composition of skin lightening creams, henna, nila, and dexamethasone tablets, employed by saharawi women, by means of HPLC-MS. Our results show the presence of many substances, not described in the information sheets, with a great potential for adverse effects if they are misused, such as all-trans-retinoic acid or corticosteroids. Chromatograms also demonstrate different compositions in creams with apparently the same packaging and labelling. P302-071 HYPOGLYCEMIC AND HYPOLIPIDAEMIC ACTION OF BITTER MELON ON NORMOGLYCEMIC AND HYPERGLYCEMIC DIABETIC RATS

A. Abdullah, S. Bakry, A. Abd El-Baky. Biochemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City, Egypt Background: Recent trends in controlling and treating diseases tend to prefer natural rather than synthetic ones. Long history of usage medicinal plants proves success in folk medicine without undesirable side effects of chemicals. Plants are considered to be the nature's which provide drugs to maintain good health of human. Materials and Methods: Thirty two male rats were divided into two groups. Group I (Normal animals) was I.P injection with a vehicle and divided into two subgroups (eight in each subgroup), one of these served as control group and the second subgroup was received extract of Bitter melon (Citrullus colocynthis) seed 50 mg/kg/day and served as normal treated group. Group II was I.P injection with STZ (diabetic rats) and divided into two subgroup (eight in each subgroup), one of these served as diabetic control group while the second subgroup was received extract of Citrullus colocynthis (C-colocynthis) seed (50mg/kg/day ) orally for 50 days and served as diabetic treated group. Results: Collected data showed a significant decrease in blood glucose, lipid peroxidation value (MDA), total cholesterol (TC), triacylglycerol (TAG), total and direct bilirubin, but lacate dehydrogenase (LDH), reduced glutathione (GSH), alanine transaminase (ALT), aspartate transminase (AST), alkaline phosphatase (ALP) and feces total lipid were record a significantly increased in both normoglycemic and hyperglycaemic rats except ALT, AST and ALP in hyperglycaemic rats had no changes. Conclusions: We conclude that the bitter melon (C-colocynthis) exert hypoglycemic, hypolipidaemic and antioxidative influence on normoglycemic and hyperglycemic diabetic rats. P302-072 EFFECT OF DEPOT MEDROXYPROGESTERONE (DMPA) ON SERUM PROTEINS AND LIPID PROFILE IN MICE

A. Hassan, S. Bakry, A. Abdullah, M. Shahat. Center for Genetic Engineering, Al Azhar University., Nasr City, Egypt The objective of this study was to investigate serum proteins and lipid profiles and following treatment of mice with the injectable contraceptive depot medroxyprogesterone acetate, (Depo Provera®; DMPA). Adult female CD1 mice were treated with a single dose of DMPA that is comparable to the weight-corrected dose in humans (0.39 mg/mouse or 0.78 mg/mouse) and sacrificed on day ten or fifteen post treatment. Sera from both treated and control groups studied for serum proteins and lipid profiles using a colorometric method. DMPA was found to reduce the concentrations of serum total protein of the treated mice as well as decrease in the concentrations of the serum protein fractions (Albumin and Globulin). There was increase in total lipids (TL), triglycerides (TG), total cholesterol (TC) as well as low density-lipoprotein cholesterol (LDL-C) and very low density lipoprotein (vLDL-C). On the other hand, a marked decrease was found in high density-lipoprotein cholesterol (HDL-C) in the DMPA-treated groups. Statistical analysis indicated that these changes were significant (p < 0.01) in all treated groups compared to control ones. This study concluded that, although DMPA is the most common injectable contraceptive in the world and many women using this form of contraception makes this an important issue that is worthy of animal trials studying possible ill-effects and this preliminary study indicates a long-term atherogenic role for DMPA. P302-073 HISTOMORPHOMETRICAL STUDY OF SEMINIFEROUS TUBULE IN RATS AFTER USED TRIBULUS TERRESTERIS

A. Esfandiari. Islamic Azad University, Kazerun Branch, kazerun, Iran (Islamic Republic of) We performed histological and morphometrical analysis of seminiferous tubules collected from 21 days old and 60 days old rats orally treated with 75mg/kg of extract from tribulous terresteris (TT) for 14 days. Twenty male wistar rats were selected and randomly divided into four groups: mature and immature treated and untreated groups of 5 each. The number of leydig cells had increment in experimental group when compared with immature control group. Result showed that the thickness of the wall of seminiferous tubule in experimental group significantly increased (P<0.05). Also, TT produced increase in the accumulation of the spermatogenic cells in the seminiferous tubule. In addition, sperm was not observed in immature control group but sperm were also observed to increase in the treatment groups. It is concluded that TT appears to possess aphrodisiac activity due to its spermatogenic and leydig cells increasing property P302-074 DEVELOPMENT OF A PORCINE MODEL FOR EXAMINING THE EFFECT OF DEBRIDING AGENTS

P. Glerup, M. Skydsgaard, J. J. Tovborg, S. Klastrup LAB Research (Scantox), Denmark


45

With the aim of identifying an optimal model for efficacy and safety testing of debriding agents, a study evaluating the wound healing proces of necrotic wounds with necrosis induced by either acid (20% trichloroacetic acid (TCA)) or base (2M sodium hydroxide (NaOH)) in either split- (1.2 mm) of full-thickness wounds was performed. Due to the high similarity between porcine skin and human skin, Göttingen minipigs were used as the test species. After surgery, the wounds were treated with TCA, NaOH or left untreated. All wounds were covered with dressings for two days to allow for full eschar formation. Daily thereafter, the dressings were changed and each wound was observed in detail, including planimetric measurements. After two weeks, the wounds were harvested for 4-hydroxyproline analysis and histopathological examination. NaOH induced marked necrosis, reduced deposition of granulation tissue and delayed re-epithelialisation. In addition, considerable tissue destruction in the surrounding tissues occurred. The effect of the wound healing process was less considerable with TCA, although marked grades of necrosis also developed. As high variation in necrotic areas induced by NaOH occurred, this model was considered less optimal for standardised testing of debriding agents. In comparison, tissue destruction caused by TCA was mainly restricted to the wound tissue. The most accurate planimetric measurements were obtained from split-thickness wounds. The three dimensions of the deeper full-thickness wounds made exact planimetric measuremnts difficult. Therefore, it was concluded that the split-thickness TCA model was the most ideal model for evaluating efficacy and safety of debriding agents. P302-082 A NOVEL SMALL MOLECULE INHIBITOR OF WNT/Â-CATENIN SIGNAL PATHWAY INDUCES CELL CYCLE ARREST IN HUMAN NON-SMALL LUNG CANCER CELL

M.S. Dong, S. B. Lee, Y. D. Gong, Y. I. Park School of Life Sciences and Biotechnology, Korea University, Korea, Republic of

Aberrant activation of the Wnt/â-catenin signal pathway is linked to a high frequency of numerous tumors. In previous study, we screened the chemical library to develop the new drug for lung cancer therapy as a target of Wnt2/â-catenin pathway using a cell-based assay in A549/Wnt2 cells stably expressed wnt2. We chose one compound, KROR-759, which was reduced to below 50% of the luciferase activity at the cell proliferation IC50 concentration was taken. 759 reduced â-catenin-TCF/LEF dependent transcriptional activity in a dose-dependent manner with an IC50 value of about 2uM. To further address the action mechanism of KROR-759, we performed DNA microarray analysis by using Illumina BeadArray Technology, after treatment of A549 cell with 1uM KROR-759 for 24h. The data demonstrated that KROR-759 had a very selective effect on global gene transcription. KROR-759 was down-regulated the expressions of Wnt/â-catenin signaling pathway target genes, such as E2F, cyclin D1 and mini-chromosome maintenance (MCM), which play critical roles in cell growth, proliferation, and differentiation. KROR-759 induced accumulation of sub-G1 phase and arrests in the G1 phase of the cell cycle. Induction of G1 arrest by KROR-759 was correlated with inhibition of E2F, cyclin D1 and MCM expression. Moreover, mini-chromosome maintenance (MCM) proteins were markedly down-regulated in A549 cells treated with KROR-759. Collectively, these results show KROR-759 possesses potential anti-cancer activity against lung cancer cells by inhibiting their proliferation and inducing cell cycle G1 arrest. P302-076 ANTI-INFLAMMATORY EVALUATION AND MECHANISMS OF EUGENOL AND ISOEUGENOL DERIVATIVES IN LIPOPOLYSACCHARIDE-STIMULATED MACROPHAGES

S. F. Liou (1)

, Y. S. Hong (2)

, C. L. Chen (1)

, Y. W. Liu (2)

, J. L. Yeh (2)

. (1)

Chia-Nan University of Pharmacy and Science, Tainan, Taiwan; (2)

College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan During the past decade, various eugenol and isoeugenol derivatives such as eugenolol, isoeugenolol, isoeugenodilol and glyceryl-isoeugenol have been synthesized and investigated in our laboratory. Several studies have demonstrated isoeugenol and eugenol exert anti-inflammatory actions. However, the anti-inflammatory action of these derivatives is still not determined. Therefore we evaluate the anti-inflammatory effects of these derivatives and study the mechanisms. In this study, we demonstrated that eugenol derivative, eugenolol, exhibited stronger inhibition effects on cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), interleukin -1beta (IL-1beta) as well as NO production than those of eugenol in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. We also found that eugenolol reduced LPS-induced nuclear translocation of nuclear factor-kappaB (NF-kappaB) subunit p65 and the DNA binding activity of NF-kappaB more potently than eugenol by blocking phosphorylation of Akt and inhibitor kappaB (IkappaB)alpha as well as the subsequent degradation of Ikappa B alpha. We also revealed that eugenolol repress the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun NH2-terminal kinase (JNK) and p-38. Besides, the anti-inflammatory effects of isoeugenol derivatives are also investigated, and we discovered that glyceryl-isoeugenol is the strongest COX-2, iNOS and TNF-alpha inhibitor of these derivatives by preventing the Akt and Ikappa B alpha phosphorylation and the following nuclear translocation of p65. Moreover, glyceryl-isoeugenol can repress all three MAPKs. Aside from this, we also studied other transcription factor such as activator protein-1(AP-1), cAMP-response element-binding protein (CREB), hypoxia-induced factor (HIF), and found that eugenolol and glyceryl-isoeugenol inhibited the LPS-induced HIF-1alpha expression and AP-1 binding activity. Taken together, eugenolol and glyceryl-isoeugenol can suppress iNOS and COX-2 expression by blocking MAPKs-mediated pathways with the attendant activation of AP-1, HIF-1alpha and CREB, and also by preventing the phosphorylation of Akt and Ikappa B alpha and the subsequent p65 nuclear translocation as well as the NF-kappaB binding activity.


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P302-077 STEREOLOGICAL STUDY OF METHIMAZOLE-INDUCED ADRENAL GLAND ATROPHY

F. Dehghani, N. Ali, M. Panjehshahin, S. Karbalay-Doust. Shiraz University Of Medical Sciences, Shiraz, Iran (Islamic Republic of) Objective: Methimazole is an antithyroid drug which causes adrenal gland atrophy. In this study, the degree of different parts of adrenal gland atrophy was determined by stereological method. Methods: 20 rats were randomly selected into control and experimental groups. Methimazole (30mg/kg/day, for 30dayes, 0.5ml/rat) was orally administered. Normal saline was given to control group with the same manner. Then animals were deeply anesthetized and their adrenal glands removed and fixed. After tissue processing, 0.5 micron slides were prepared and stained with Heidenhain, s Azan staining method. Volumes of different parts of adrenal glands were estimated by Cavalieri principle and Point counting method. Results: Methimazole decrease the adrenal gland volume by 17%, the cortical zone volume was reduced by 18.5% and the volume of fasciculate zone was decrease by 23% which is statically significant (p<0.05). Conclusion: It seems that Methimazole exert its affect mainly on fasciculate zone. P302-087 ANTILEUKEMIC POTENTIAL OF CRUDE EXTRACTS OF ARRABIDAEA CHICA

A. F. C. Ribeiro, M. M. Melo, G. D. Cassali, V. P. Ferraz, G. M. M. Cardoso, T. C. Telles, E. M. Souza-Fagundes. Universidade Federal De Minas Gerais, Belo Horizonte, Brazil Background: Arrabidaea chica (Bignoniacea) is a plant widespread from South Mexico to Guyana and central Brazil. A. chica is widely used in folk medicine for wound healing, treatment of inflammation, blood dysfunction, anemia, hemorrhage and leukemia. Until today despite the large use of A. chica very few is known about its pharmacological properties. The purpose of the present study was to investigate the antileukemic potential of different extracts of A. chica against leukemic cell lines. In addition we investigate the in vivo anti-tumor activity of aqueous and hexane extracts in Ehrlich tumor bearing mice. Methods: Aqueous, hexane and methanol extracts of dried leaves of A. chica were extracted as described previously. Jurkat and HL60 cell lines were incubated for 48 hours with each extract at 50μg/mL. Cell viability was assessed by MTT assay. Mice with solid Ehrlich tumor were treated with aqueous or hexane extracts (1mg/animal daily via gavage) for 10 days. Tumor growth was examined. Results: The insoluble-hexane-fration (IHF) and methanol-citric acid (MCA) extracts of A. chica demonstrated toxicity against HL60 cells, significantly reducing the cell proliferation (82% and 54%, respectively). To Jurkat cells, these extracts inhibited about 50%, comparing with control. The rate of inhibition was very similar to the positive control vincristine used in clinic (80% of cell proliferation inhibition at 1μg/mL) The MTT cytotoxicity test showed that aqueous extracts have minimal cytotoxicity against Jurkat or HL60 cell lines. Data showed that aqueous and IHF extracts reduced the tumor growth only after 10 days of treatment. No adverse side effects due to treatment were observed. Conclusion: Our findings suggest that Arrabidaea chica has antitumor activity thereby exhibiting potential as a new anticancer agent. Nowadays our group has been performing the bioassay-guided fractionation to isolate the active compound(s) responsible for the cytotoxic effect observed. P303-046 FOURTEEN-DAY ORAL TOXICITY STUDY OF SILVER NANOPARTICLES IN BEAGLE DOGS

M. Y. Song. KEMTI, Inchenon, Korea, Democratic People's Republic of The usage of silver nanoparticles in the food industry and biomedical application has been increased in recent years. To investigate the potential toxicity of silver nanoparticles, many studies have been carried out. But information for toxicity is not enough, so silver nanoparticles remain a controversial research area as regards their toxicity to biological systems. In particular, the potential oral toxicity of silver nanoparticles is of particular concern to ensure public and consumer health. Accordingly, this study tested the oral toxicity of silver nanoparticles (60 nm) over a period of 14 days in Beagle dogs with reference to OECD test guideline 409 with GLP application. Six-month-old dogs, weighing about 9.74 kg for the males and 7.92 kg for the females, were divided into four 4 groups (2 dogs in each group of both genders): vehicle control, low-dose group (150 mg/kg), middle-dose group (300 mg/kg), and high-dose group (600 mg/kg). After 14 days of treatment, the blood biochemistry and hematology were investigated, along with a histopathological examination and silver distribution study. All treated groups showed some toxic symptoms such as mucous stool, watery diarrhea, reddish and glucosuria, diarrhea, anorexia. The body weights of male and female dogs were decreased during experimental periods. The decreases in WBC and neutrophil were found in dose of 600 mg/kg male group in hematology. In serum analysis, the level of ALP, AST and ALT was increased and total cholesterol was decreased in all treated groups. In histopathology, focal yellow pigmentation in liver and multifocal/focal inflammation in stomach was found in treated groups. These results indicate that the target organ in the oral toxicity of silver nanoparticle is liver and gastrointestinal system and the no observed adverse effect level (NOAEL) of silver nanoparticle might be below 150 mg/kg in both sex groups.


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P303-047 SAFETY ASSESSMENT OF NANOMATERIALS: INFLAMMATORY RESPONSES OF NANOSILICA WITH DIFFERENT SIZES AND SURFACE PROPERTIES

Y. Yoshioka (1)

, T. Morishige (2)

, S. I. Tsunoda (3)

, Y. Mukai (2)

, N. Okada (2)

, Y. Tsutsumi (4)

, S. Nakagawa (2)

. (1)

The Center for Advanced Medical Engineering and Informatics, Osaka University, Osaka, Japan; (2)

Department of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University,

(3) Laboratory of

Pharmaceutical Proteomics, National Institute of Biomedical Innovation, (4)

Department of Toxicology and Safety Science, Graduate school of Pharmaceutical Sciences, Osaka University, Japan Nanotechnology has produced diverse nanomaterials such as a nanosilica (nSP). nSP have been used for many applications, including cosmetics, foods and drug delivery. However, the increasing use of nSP has raised public concern about the potential risks to human health. Therefore it is urgently necessary to investigate the biological responses of nSP for the development of safe forms of nSP. Here, we systematically examined the inflammatory response of nSPs using various sizes and surface properties of nSP. We used nSPs with diameters between 30 and 1000 nm (nSP30, nSP70, nSP300 and mSP1000), and nSP70s with surface functional groups (-COOH and -NH2). At first, we examined the inflammatory responses of nSPs using macrophage cells. nSP30 and nSP70 induced the significant higher level of TNF-alpha production on macrophage cells than non-treated cells, although nSP300 and mSP1000 did not induce the production of TNF-alpha. In addition, we showed that nSP70-induced cytokine production was suppressed by the surface modification. Next, we compared the inflammatory responses of nSPs in vivo. Each nSP was intraperitoneally injected in mice and the number of infiltrating inflammatory cells into the peritoneal cavity was measured. More infiltrating cells were observed in the abdominal lavage fluid of nSP30- and nSP70-treated mice than mSP300- and mSP1000-treated mice, the latter having similar numbers as the control. Furthermore, we showed that nSP70s with surface functional groups did not cause inflammatory responses in vivo. These results indicate that the inflammatory responses of nSPs depended on particle size and surface properties. These findings might provide useful information for constructing novel nSP with safety. P303-049 SAFETY EVALUATION OF AMORPHOUS NANOSILICA WHEN GIVEN AS A SINGLE INTRAVENOUS DOSE TO MICE

T. Yoshikawa (1)

, H. Nabeshi (1)

, K. Nagano (2)

, Y. Abe (2)

, N. Itoh (1)

, S. I. Tsunoda (2)

, Y. Tsutsumi (1)

. (1)

Graduate school of Pharmaceutical Sciences, Osaka University, Suita, Japan; (2)

National Institute of Biomedical Innovation, With recent development of the nanotechnology, nanomaterials have been successfully employed in various industrial applications such as medicine and cosmetics. Nanomaterials show the useful properties such as electronic reactivity and the tissue permeability that were not provided by micromaterials. Thus, nanomaterials are expected as innovative materials for the development of medicine and cosmetics. However, these innovative properties may show unknown biological responses that could not been detected by the conventional toxicity assay. It is urgent to gather information of the biological effects, and to establish the standard safety evaluation method of nanomaterials. In this context, we are analyzing association among property, biodistribution and biological effects of nanomaterials using nanosilicas (nSP) as a standard nanomaterial. The present study was designed to examine the in vivo localization and biological effects of nSP70 after intravenous injection. BALB/c mouse were intravenously injected with amorphous nanosilicas of sizes 70, 300, 1,000 nm (designated nSP70, nSP300 and mSP1000, respectively) and then assessed for survival, blood biochemistry. As a result, injection of only nSP70 caused fatal toxicity, liver damage. Additionally, nSP70 are efficiently taken up by hepatocyts, suggesting that intrahepatic distributions might be involved in nSP70-mediated biological reactions in vivo. Collectively, these results would provide useful information for establishing appropriate regulatory measures and in designing safety guidelines on nanosilicas. P303-050 INTRACELLUAR REACTIVE OXYGEN SPECIES ARE ASSOCIATED WITH SIO2 NANOPARTICLE-INDUCED CYTOTOXICITY AND DNA DAMAGE IN HACAT CEL

Z. X. Zhuang, C. M. Gong, L. Q. Yang, J. J. Liu. Shenzhen Center for Disease Control and Prevention; School of Public Health, Sun Yat-sen University, Shenzhen, China

Nano silicon dioxide (nano-SiO2) is becoming more widely applicated in the fields of industry. The potential toxic effects of nano-SiO2 and its hazard to human health are drawing more attention. Recently, we reported that nano-SiO2 could induce cytotoxicity and protein alterations in HaCaT cells. However, the potential toxic mechanisms of nano-SiO2 remained unknown. Here, we measured the changes of cell viability, cell morphology, reactive oxygen species (ROS), the marker of DNA damage (8-OHdG, ãH2AX, comet assay) as in HaCaT cells exposed to nano-SiO2. The potentional relationship between oxidative stress and cell cytotoxcity and DNA damage was analyzed. Similar to our previous results, exposure to 15-nm, 30-nm or 100-nm SiO2 significantly decreased cell viability in a dose- and size-dependent manner. Morphological examination revealed that the cells exposed to nano-SiO2 suffered from shrinkage and cell wall loss. In addition, exposure to nano-SiO2 significantly increased the levels of intracellular reactive oxygen species (ROS) and induced DNA damage. A positive correlation between intracellular ROS level and cytotoxcity was found (R2=0.85). These data indicated that oxdative stress may be one of the mechanisms that drive nano-SiO2-induced cytotoxicity and DNA damage.


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P303-051 CYTOTOXICITY OF GOLD NANOPARTICLES FUNCIONALIZED WITH CITRATE AND PENTAPEPTIDES GROUPS ON HEP G2 CELLS

H. Faria. Faculty of Pharmacy, University of Porto, Porto, Portugal

During the last years engineered nanoparticles (NPs) have been extensively used in different technologies and consequently many questions have arisen about the risk and the impact on human health following exposure to nanoparticles. Recent advances and progress in nanobiotechnology have demonstrated many nanoparticles as potencial and novel drug delivery vehicles, therapeutic agents, contrast agents and luminescent biological labels for bioimaging. At present, knowledge about the cytotoxicity induced by nanoparticles is still largely incomplete and the emergence of new biomedical applications based on NPs signifies the need to understand, compare and manage their cytotoxicity. Gold nanoparticles are widely used in biomedical imaging and diagnostic tests. In general, gold nanoparticles (AuNPs) are recognized as nontoxic based on their established use in the laboratory and the chemical stability of Au (0). Moreover, there have been some reports on their cytotoxicity which has been shown to depend on nanoparticles physical dimension, surface chemistry and shape. The liver has been viewed as a target site for NPs accumulation after exposure and the human hepatocellular liver carcinoma cell line Hep G2 is thought to be a realistic and relevant model of hepatocyte behaviour. In this context, we have investigated the cytotoxicity induced by 20 nm spheric AuNPs functionalized with different coatings such as citrate and peptides. For this purpose, Hep G2 cells were exposed to AuNPs (0 - 200 µM) for 6 and 24 hours and cell viability evaluated by two different assays, the (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay that measures mitochondrial function, and the LDH leakage assay that assesses cell membrane integrity. The obtained results suggest that the AuNPs do not have significant toxicity for this model, at the evaluated time points. P303-055 OCULAR TOLERANCE OF IBUPROFEN LOADED POLYESTER NANOPARTICLES ASSESSED BY IN VITRO AND IN VIVO METHODS

M. D. Ricart, E. Vega, E. González-Mira, S. Nikolic, M. A. Egea, M. Espina, M. L. García. Faculty of Pharmacy. University of Barcelona, Barcelona, Spain Topical ophthalmic preparations often cause eye irritation mainly due to the drug or excipients such as preservatives or polymers present in the formulations, specially under certain circumstances: prolonged treatment, the state of the cornea or the wearing of contact lenses Topical administration of polymeric nanoparticles provides a long time ocular retention thus improving drug absorption. The evaluation of ocular tolerance and potential eye irritation is essential to ensuring the safety of these delivery system. Ocular irritation is associated with the appearance of conjunctival reddening, corneal opacification and iridal swelling, the severity of which is based upon the reversibility and duration of these effects. The main in vivo test for the appraisal of ocular tolerance is the Draize test, which relies on macroscopic observation and scoring of changes to corneal, iridic and conjunctival tissues consecutive to the instillation of chemicals onto a rabbit eye. In order to refine and improve the sensibility of the Draize test, some technical modifications have been proposed as reduction of animals involved. Alternative tests, based on the different physiological changes associated with ocular irritation have been developed. In vitro tests differ widely in their requirements and ability to predict irritancy potential. This study focuses on the determination of potential ocular irritation induced by Ibuprofen loaded polyesters nanoparticles by in vitro test: the chick chorioallantoic membrane (HET-CAM) assay and the Eytex™, which evaluate the conjunctival inflammatory response and the increase of corneal opacity respectively. The results obtained demonstrated that the tested nanoparticles were well tolerated and the ocular surface tissues remained normal after in vivo exposure. In vitro tests displayed a good correlation with the in vivo data. These results support to the potential use these nanoparticulate systems as drug carriers to treat ocular surface disorders. P305-012 CORRECTION OF COCAINE VALUES BY CREATININE IN ADDICTS FOR ESTABLISHING THE DILUTION FACTOR AND RETAILS

L. Börgel, C. Criollo, A. Carreño. SERVITOX/Legal Medicine Department, Universidad de Chile, Santiago, Chile

Cocaine is an illegal drug of high distribution and consumption in the world, being an actual serious economic and social problem for many countries. In Chile, a recent study by the national council for the control of narcotics, CONACE, revels a considerable increase in the consumption of cocaine during the last 12 years. Due to the problems caused by this drug in the consumers´ health as for the society, different methods for its detection and quantification have been developed during the detoxification phase. The analysis in urine matrix is the most frequently used method for monitoring consume of cocaine, as for the forensic as for the clinical point of view. One of the inconvenient in the quantitative analysis of cocaine is the adulteration by an intentional increase of the liquid drinking, originating a dilution of the urine, and, as a consequence, the fake quantification. Based in above, a correction by creatinine from the cocaine concentrations obtained from consumers was done. The creatinine is a waste molecule originated by the muscular metabolism that is naturally present in urine and its concentration varies according to the amount of ingested liquid during the day. In the present study, positive samples for cocaine from different individuals were analyzed, and the concentration was corrected by creatinine, assessing the real value of the drug present in the body; this allowed establishing the curve of detoxification of each person, abstinence period and retails. This study corresponds to patient


49

in addiction treatment supervised by psychiatrist, and comprehends the period 2008-2009, with a simple size of 56 and 80 for each year, respectively. P307-049 LEVELS OF CHROME, NICKEL, STRONTIUM AND ALUMINUM IN MEAT (COLD MEAT, RABBIT, VISCERA AND RED MEAT) AND FISH TAKEN BY THE CANARIAN POPULATION

C. Rubio Armendáriz (1)

, D. González-Weller (2)

, A. J. Gutiérrez Fernández (1)

, A. Hardisson De La Torre (1)

. (1)

La Laguna University, La Laguna, Spain; (2)

Laboratory and sanitary inspection service. Health department of Tenerife. Canarian health service,

Introduction: Food is the main source of chrome. Nickel is an essential element for human beings, so it probably has no toxic effects in a balanced diet. Water and food are also the main sources of exposure to aluminum and strontium. Toxicity of strontium is related to its capacity to interfere in biologic procedures that depend on calcium. Aluminum is a potentially neurotoxic element. Aims: The aim of this study is to determine levels of Cr, Ni, Sr and Al in meats and fishes consumed by the canarian population. Material and method: Samples were taken between March and July of 2008 in different shopping centers in the Island of Tenerife. A total number of 100 samples were analysed. Levels of Cr and Ni were determined by means of atomic absorption spectroscopy with a graphite furnace (GFAAS). Levels of Sr and Al were determined by means of atomic emission spectroscopy with inductively coupled plasma (ICP-AES). Results: Levels of the analysed metals in mg/Kg found in cold meat, meat, rabbit, viscera, red meat and fish were as follows: 0.50 „b 0.26, 0.22 „b 0.17, 0.20 „b 0.25, 0.15 „b 0.05 y 0.01 „b 0.01 for chrome; 0.06 „b 0.03, 0.03 „b 0.01, 0.09 „b 0.05, 0.05 „b 0.02 y 0.01 „b 0.01 for nickel; 0.79 „b 0.30, 0.13 „b 0.07, 0.20 „b 0.19 y 0.13 „b 0.13 for strontium; and 2.74 „b 1.06, 6.35 „b 2.83, 11.15 „b 6.15, 9.31 „b 4.85 y 3.48 „b 2.34 for aluminum. This last metal is the one with highest content, while nickel is the lowest, except from chrome in fish. Conclusion: Results of this study show us that, it is necessary to asses, know and monitor levels of metals in food in order to determine dietary intake values considered safe for the population P307-052 THE ROLE OF MITOCHONDRIA MEDIATED APOPTOSIS PATHWAY IN SERTOLI-GERM CELL APOPTOSIS INDUCED BY MANGANESE IN COCKS

S. W. Xu, S. Li, M. Y. Bi. College of Veterinary Medicine, Northeast Agricultural University, Haerbin, China The aim of this study was to discuss the role of mitochondria mediated apoptosis pathway in Sertoli-germ cell apoptosis induced by manganese in cocks. Cock Sertoli-germ cell were cultivated in the DMEM for 24 h added with MnCl2, whose final concentration was 0, 2, 3, and 4 mmol/L respectively. The apoptosis was detected by AO/EB double staining. Mitochondrial transmembrane potential was detected by flow cytometer. Expression of Cytochrome c (Cytc) in cytolymph was detected by western blotting. The activities of Caspase-9, 3 were examined by spectrophotography. Compared with control group, the apoptosis index (AI) of cock Sertoli-germ cells was significantly increased (Pƒ0.01), mitochondrial transmembrane potential was significantly decreased (Pƒ0.01), expression of Cytc in cytolymph was increased, and the activity of Caspase-9, 3 were increased (Pƒ0.01) in 2, 3, and 4 mmol/L MnCl2 group. The apoptosis mediated by mitochondria pathway was one of the reproductive toxic mechanisms caused by manganese. P307-054 MICE EXPOSED TO SODIUM ARSENITE SHOW INCREASED EXPRESION OF AQP9 IN LIVER CELLS

C. Valdovinos Flores, M. Torres-Avila, P. Leal-Galicia, L. C. Sáchez Peña, L. M. Del Razo, M. E. Gonsebatt. Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Chronic inorganic arsenic (iAs) exposure via drinking water has been associated with cancer and serious injury in various internal organs. Tissue analysis of human organs has demonstrated a much higher concentration of arsenic in liver than in other organs. Liver is also the primary arsenic metabolizing organ. Similar observations have been obtained in murine models. Furthermore, dose-repeated oral treatment of mice with sodium arsenite have shown a dose-related accumulation of iAs in the liver, while a different pattern was observed in other tissues such as brain or lung. Aquagyceroporins (AQP) are membrane proteins that participate in the transport of solutes such as glycerol, manitol, and metalloids. AQP9 and AQP7 are channels for the uptake of iAs (III) and MMA (III) and are expressed in mammal liver and adipocytes, respectively. Since the up-take of As species by AQP 9 has been clearly demonstrated, we investigated if dose-related accumulation of iAs in murine liver was due to an up-regulation of this transporter. Mice were treated with different concentrations (0, 2.5, 5 or 10mg/kg/day) of sodium arsenite during 1, 3 or 9 days. A significant dose-related, up-regulation of AQP9 mRNA and protein was observed and which was verified by immunohistochemistry in liver sections using specific antibodies. P307-056 ORAL TOXICITY PROFILE OF KAJJALI: A MERCURY BASED AYURVEDIC PREPARATION

R. Kenjale, G. Gurhale, M. F. Shaikh, S. Sathaye. Institute of Chemical Technology, Matunga, Mumbai., Mumbai, India Kajjali, consisting of Mercury and Sulfur, forms an integral part of many Ayurvedic herbo-metallic formulations. It is used as a "Yogavahi" or a bioenhancer that potentiates bioactivity of drugs with which it is combined. The safety of Ayurvedic herbo-metallic preparations has remained a contentious issue over the years. Saper et al, 2004, reported


50

that "users of Ayurvedic medicine may be at risk for heavy metal toxicity". Several published reports have revealed the adverse neurological and other toxic manifestations of Mercury and its compounds (Von Berg and Greenwood, 1991; Clarkson, 1998; Friberg and Nordberg, 1973). According to Ayurvedic literature, metals like mercury are subjected to physico-chemical processing called 'Shodhana', attributed to purification, detoxification and restoration of therapeutic properties (Dash, 1986). Such descriptions in traditional knowledge support the hypotheses that the known toxic forms may not remain in the final formulation that qualifies their use for therapeutic purposes. However, such claims remain to be validated scientifically, as the study employing modern scientific/pharmacological tools for the toxic manifestations has not been conducted. Hence this study was undertaken. The acute oral toxicity of Kajjali in swiss albino mice indicated the LD50 value to be more than 2000mg/kg. The 28-days and 180-days repeated dose studies at the maximum dose of 45mg/kg body weight in wistar rats revealed no significant changes in the hematological, serum biochemistry and histopathological profiles of the experimental animals. Solubility of Kajjali in physiological buffers and solvents like Methanol, DMSO and Acetonitrile, was found to be negligible (in ppb levels). The physicochemical characterization revealed that the particle size is in the sub-micron range, while SEM and XRD analysis confirmed its crystalline nature. Work on the bio-distribution studies of Kajjali is in progress and genotoxicity studies are planned immediately. These results will certainly help make a comprehensive overview of Kajjali toxicity. P307-060 DISTRIBUTIONS OF SOME METALS IN EL EBRAHIMIA CANAL, BENI SUEF - EGYPT

K. Abdou, I. Khadega Department of Toxicology and Forensic Medicine, Faculty of Veterinary Medicine, Beni Suef University, Beni Suef, Egypt

Concentrations of some metals, cadmium (Cd), lead (Pb), iron (Fe), manganese (Mn) and zinc (Zn) in water, Ceratophillyum demersum (C. demersum) aquatic plant and in muscles, gills, liver, kidney and blood of Claries lazera (C. lazera) collected from seven stations, Beni Suef , Elfashn, Beba, Somosta, , Ehnasia, Elwasta and Naser along El Ebrahimia canal in Beni Suef province, Egypt during 2009-2010 were measured using atomic absorption spectrometer. The obtained results revealed that, the examined metals were detected in all examined samples. In water, Pb has the highest concentration among the detected metals in Elfashn, Beba, Ehnasia, Naser and Elwasta; Mn has the highest concentration in Somosta and Fe has the highest concentration in Beni Suef. The concentration of Pb, Fe, and Mn were above the maximum permissible limits in all districts. Cd concentration was above the permissible limits except in Somosta and Naser while Zn concentration was below the permissible limit in the seven districts. Metal levels in water were compared with national and international water quality guidelines as well as literature values were reported for streams and rivers. Comparisons were made of metal concentrations in water and aquatic plant with those in the catfish tissues caught from the water. In C. demersum aquatic plant, distribution of metal concentrations in the seven studied districts were in the order of Mn > Zn > Pb >Fe > Cd and were above its level in water. In fish, metals were accumulated in different examined tissues by various levels but concentrations of metal in muscles (edible part) were below the metal levels in other organs (no-edible) of fish samples. The concentration of Cd, Pb and Fe in the different tissues of fish was above the international standard while the concentration of Mn and Zn was below that level. The high concentrations of these metals in water, aquatic plant and fish in El Ebrahimia canal may are thought to have resulted from anthropogenic activities producing industrial, agricultural, domestic waste discharges as well as accidental pollution incidents. P308-055 IMMUNOTOXIC EFFECTS WITHIN THYMUS INDUCED BY IN UTERO AND LACTATIONAL EXPOSURE TO ENDOSULFAN IN ADULT RATS

A. Caride, T. Cabaleiro, A. Lafuente. University of Vigo, ORENSE, Spain Endosulfan is an insecticide of the cyclodiene group considered a neuroendocrine disrupter, that has been shown to induce alterations at the immune system level. It can be mobilized during pregnancy and lactation, cross the placenta and be secreted through maternal milk, so prenatal and lactational exposure is an important source of endosulfan exposure. The objective of the present work was to evaluate possible immune alterations induced by endosulfan administered throughout pregnancy and lactation. Mothers were exposed orally to two different doses of endosulfan: 0.61 and 6.12 mg/kg/day, from the gestation beginning until the weaning. Male and female offspring rats were sacrificed at 60th day of life, and different lymphocyte populations and subsets in thymus were analyzed by flow immunocytometry. An increase of lymphocyte B+ percentage was observed in both male and female offspring rats exposed to 6.12 mg endosulfan/kg/day. In males, both doses of the insecticide induced an increase of the lymphocyte T+ percentage. In addition, endosulfan decreased CD4+ and CD8+ lymphocyte percentages in both male and female offspring rats with the two studied doses. It was concluded that endosulfan exposure during pregnancy and lactation induced immunotoxicity in male and female offsprings, as different alterations on cellular and humoral immunity were reported. This work was supported by a grant from the Ministry of Education and Science from Spain (AGL2004-04543/ALI). P308-056 GENOTOXIC EFFECTS OF ENDOSULFAN ON GH AND TSH: POSSIBLE IMPLICATIONS OF VARIATIONS ON REDOX BALANCE

A. Lafuente, A. Caride, T. Cabaleiro. University of Vigo, Orense, Spain


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Endosulfan is an insecticide considered such as an endocrine disruptor. Moreover, it induces oxidative stress. Because the developing fetus is especially sensitive to hormonal disruption by exposure to xenobiotics, and health alterations early in life could have important implications in adulthood, the present work was undertaken to investigate possible effects of in utero and lactational exposure to endosulfán on: (a) pituitary gene expression of growth hormone (GH) and thyroid stimulating hormone (TSH); (b) circulating levels of these hormones; and (c) expression of enzymes involved in oxidative stress responses, such as nitric oxide synthase 1 and 2 (NOS1 and NOS2), and heme oxygenase-1 (HO-1) at pituitary level. For this purpose, dams were orally administered with two different doses of endosulfan (0.61 or 6.12 mg endosulfan/kg/day), daily throughout pregnancy and lactation. Offspring male rats were sacrificed at postnatal days (PND) 15, 30 and 60. In these animals, gene expression was evaluated by using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and plasma hormone levels were measured by specific radioimmunoassay methodology. Endosulfan exposure decreased GH and TSH gene expression only at PND 30. GH plasma levels were increased at PND 30 and decreased at PND 60 after this pesticide administration, whereas TSH plasma concentration increased at PND 15 and decreased at PND 30 and 60. Endosulfan increased NOS1, NOS2 and HO-1 mRNA levels in male offspring rats. Concretely, NOS1 mRNA expression was higher at all ages studied, whereas NOS2 expression increased at PND15 and 30, and HO-1 at PND15 and 60. These results suggest that endosulfan alters GH and TSH expression and secretion (mainly at PND 30), and these alterations could be due to changes on expression of genes involved in oxidative stress at pituitary level. This work was supported by a grant from the Ministry of Education and Science from Spain (AGL2004-04543/ALI). P308-059 EFFECT OF DDT ON EXPRESSION OF CYTOCHROME P-450 IN RAT TESTIS

A. Sierra-Santoyo, M. D. L. Lopez-Gonzalez, C. L. Zazueta-Beltran. Departamento de Toxicologia, Cinvestav-IPN, Mexico, Mexico DDT is an endocrine disruptor able to promote hormone dependent pathologies and a liver cytochrome P-450 (CYP) inducer. CYP-depedent metabolism is regulated by age, gender and specific tissue. Information about the effect of DDT on expression CYP isoforms in extrahepatic tissues is limited. The objective of this study was to determine the effect of acute exposure to DDT on CYP isoforms expression in rat testis. Wistar adult male rats (200 ± 20 g) were administered with an oral dose of DDT technical grade (0.1, 1, 5, 10 and 100 mg/kg) dissolved in corn oil. Animals were sacrificed 24 h later and testis removed and processed to obtain microsomes. Protein content of CYP2A2, 2B1/2, 2C11 and 3A2 was assessed by Western blot analysis and testosterone hydroxylase (OHT) activities were determined using [14C]-Testosterone as substrate and the metabolites were separated by TLC and quantified by densitometric analysis. DDT treatment produced a differential on CYP expression in testis. CYP2A2 and 2B1/2 protein content decreased 35 and 15% at the doses of 0.1 and 1 mg/kg, respectively, while at the same dose CYP3A2 increased about 25%. CYP2B1/2 and 2C11 protein content was not affected by DDT exposure at any dose. At the doses from 0.1 to 10 mg/kg DDT significantly decreased 15alpha-, 16alpha- and 7alpha-OHT, at the highest dose these activities were similar to the non-treated group. 6beta-OHT and 17beta-OHEDH activities increased 35 and 71% at the highest dose, respectively. These results indicate that exposure to a single dose of DDT is able to modify CYP isoforms expression in testis affecting the pattern of testosterone metabolism. They also suggest that the selective effect on testis CYP expression may represent an endocrine disruption mechanism. P309-055 FASTING STATUS AND THERMALLY OXIDISED SUNFLOWER OIL INGESTION AFFECT THE INTESTINAL GLUTATHIONE SYSTEM OF MALE WISTAR RATS

M. J. González Muñoz (1)

, R. Olivero David (1)

, F. J. Sánchez Muniz (2)

, S. Bastida (2)

, A. Schultz (2)

, L. González Torres

(2), J. Benedi González

(3).

(1) Dpto. Nutrición, Bromatlogía y Toxicología. Facultad de Farmacia. Universidad de Alcalá, Alcalá de Henares, Spain;

(2) Departamento de Nutrición. Facultad de Farmacia. Universidad Complutense de Madrid., Madrid, Spain;

(3)

Departamento de Farmacología. Facultad de Farmacia. Universidad Complutense de Madrid., Madrid, Spain

Aim: The effect of thermally oxidised sunflower oil ingestion on glutathione system levels, in the small intestine of fed and fasted rats were studied. Methods: For three consecutive days, 12 male Wistar rats received 0.5g unused sunflower oil/100g body weight (controls, C) while another 12 were given 0.5g thermally oxidised sunflower oil/100 g body weight (test group, T). On the night of day 3, 6 rats from each group were fasted (FC and FT, respectively) while the other 6 animals from each group were given free access to food (NFC and NFT, respectively). On day 4, FC and NFC rats received 1g of unused oil/100g body weight, while FT and NFT rats were given 1g of altered oil/100g body weight. Small intestines were extracted after 4-hr exposure to the oils. Results: Reduced glutathione was affected by fasting (P<0.01) and oil alteration (P<0.001). FT rats show lower reduced glutathione values than NFT (P<0.001) and FC rats (P<0.01). The oxidised glutathione levels were affected by oil alteration (P<0.001). The oxidised glutathione levels of NFT and FT animals were greater than those of NFC and FC animals (P<0.001 and P<0.05, respectively. The redox ratio was significantly affected by fasting (P<0.01) and oil alteration (P<0.001). FT rats displayed a higher redox index than NFT (P <0.001) and FC rats (P<0.01). NFT rats presented a higher redox ratio than NFC rats (P <0.01). Conclusion: Consumption of used frying sunflower oil decreased reduced glutathione levels, increased oxidised glutathione concentrations and the redox ratio, suggesting losses in the efficacy of such antioxidant system. This effect was most evident under fasting conditions.Lengthy fasting and consumption of food containing oxidised fat should both be avoided to prevent intestinal oxidative stress.


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P309-057 HISTOMORPHOMETRIC ALTERATION AND ALKALINE PHOSPHATASE SERUM LEVEL OF KNEE ARTICULAR CARTILAGE PRODUCED BY CHRONIC SOY BEAN MEAL SUPPLEMENTATION IN YOUNG FEMALE MICE

S. Fazelipour Department of Anatomy, Islamic Azad University, Tehran Medical Branch, Tehran, Iran.

Administration of soy protein has been reported to produce a beneficial change in the structure of osteoarthritis, demonstrating a chondroprotective efficacy that delays the progression of the disease. The purpose of the present study was to examine the effect of prolonged Soy bean meal supplementation in young female mice on the histomorphometrical alteration of knee joint and serum level of alkaline phosphates. Forty young Balb/C female mice at the age of three weeks, divided into four groups to be fed for six months from four different regimen including: low protein regimen that contained 13% of protein; complete protein regimen without soy bean meal; the third and forth regimen, contained soy bean meal 20% and 40% of total protein, respectively. Then, body weight differences, histologic and histomorphometric analysis, and alkalin phosphatase determination were assessed and compared after six months. The results showed significant increase in serum ALP activity in all soy bean meals groups when compared with others. Significant increase of the cartilage thickness was also observed in soy bean meal regimen when compared to the low protein regimen. Although there were no significant differences regarding the cartilage growth among soy bean meal regimens and no soy bean meal group, the cartilage thickness in soy bean meal regimen had shown superiority when compared to the second (no soy bean meal) regimen. Additionally, number of chondrocyte in 40% soy bean meal regimens increased significantly (P<0.001), when compared to no soy bean meal regimen. There were differences among four regimens regarding the intracellular fluid concentration in tibia which means the most effective regimens after six months supplementation, was those supplemented with soy bean meal but the differences were not significant. Therefore, it is noteworthy to emphasis the effectiveness of soy bean meal supplementation during childhood to protect joints. P309-058 THE EFFECT OF SOY BEEN MEAL ON THE TIBIAL ARTICULAR CARTILAGE GROETH IN MICE AFTER SUCKING PERIOD: RESULT OF HISTOMORPHOMETRIC AND BIOCHEMICAL INVESTIGATION

Z. Tootian Department of Anatomy, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.

Aim: The purpose of this study was to examine the effect of an oral preparation of soy bean meal supplementation on 3 weeks old female mice on the histomorphometrical alteration of tibia cartilage, and serum levels of calcium and alkaline phosphatase, using computer-assisted histomorphometric methods.Materials and Methods: Forty immature female mice Balb/C were selected. The samples were divided to 4 groups. They were fed for 3 months with four different regimens (low protein regimen without soy bean meal, 23% protein without soy bean meal,20% and 40% of total protein were provided by soy bean meals). Then alkaline phosphatase and calcium determination were assessed. after 3 months. Using Computer assisted histomorphometric analysis, Sections of tibial plateau were photographed. In order to measure the thickness and counting the number of chondrocytes of middle part of the cartilage, H&E stain were used. For intensity of the articular cartilage, toluidine blue was used. Results: There were significant increase in the thickness of the cartilage and the number of chondrocytes in serum calcium and alkaline phosphatase activity in all soy bean groups when compared with others. The concentration of extracellular matrix in the groups with soy bean meal regimens was more than the one in the groups without soy bean mea regimens. Conclusions: The present study suggests that soy bean meal supplementation can stimulate alkaline phosphatase production, increase serum calcium, and increase the number of chondrocytes and the thickness of cartilage in the middle part of tibia plateau, specially started during childhood. So it´s important to mention the effectiveness of soy bean meal supplementation in order to protect joints. Key words: Soy bean meal, articular cartilage, histomorphometric, biochemical, mice P309-059 ILSI EUROPE: RESULTS FROM AN EXPERT GROUP ON THE APPLICATION OF THE MARGIN OF EXPOSURE (MOE) APPROACH TO SUBSTANCES IN FOOD THAT ARE GENOTOXIC AND CARCINOGENIC

M. Dinovi. US FDA, College Park, MD, United States Context: Based on the recommendations of the EFSA/WHO international conference with support of ILSI Europe on “Risk Assessment of Compounds that are both Genotoxic and Caecinogenic: New Approaches”, a new ILSI Europe expert group was created. Project on the application of the margin of exposure (MOE) approach to substances in food that are genotoxic and carcinogenic. This group prepared case studies on twelve different chemicals, which were chosen because of different potencies and quality of available data, for which MOEs were calculated. It should be kept in mind that the focus was specifically on oral intake and dietary exposure and therefore, does not provide the total exposure to the chemical under consideration. In addition, a manuscript summarising the results was prepared and published together with the case studies in a supplement of Food and Chemical Toxicology 48 (2010). The purpose of these articles is to provide guidance in discussing the advantages and any limitations of the MOE approach. Project on the data selection for BMD modelling of substances that are genotoxic and carcinogenic. As a follow-up to this project, a new expert group on the "data selection for BMD modelling of substances that are genotoxic and carcinogenic". This project will build on the discussions and results of the previous expert group to investigate further the effect of the quality of carcinogenicity data and way they are analysed on MOE values. The group will discuss: - Biological relevance of an observed tumour for humans/Mode of Action (MoA), - Key steps of the BMD approach: experimental study designs, statistics and BMD modelling, - Approaches for weighing evidence, uncertainty and quality of studies.


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The expected output will be a guidance document on how to select the most appropriate data set to be used in BMD modelling. P309-060 ANTIMICROBIAL ACTIVITY OF ORIGANUM ONITES L. AND ORIGANUM MINUTIFLORUM O. SCHWARZ & P. H. DAVIS ESSENTIAL OILS

M. Odabasi (1)

, Y. Beyatli (2)

. (1)

Institute of Forensic Medicine, Ankara University, Ankara, Turkey; (2)

Department of Biology, Faculty of Science and Art, Gazi University, Turkey

During several decades, there has been a global trend for the revival of interest in the traditional system of medicine. Simultaneously the need for basic scientific investigation of medicinal plants using indigenous medical systems has become ever more interesting and relevant. The antibacterial activity of essential oils has been recognized for a long time. In this study, the chemical composition and the antibacterial properties of the essential oils obtained from the aerial parts of the two Lamiaceae species, wild oregano (Origanum minutiflorum O. Schwarz & P. H. Davis) (endemic in Turkey), oregano (Origanum onites L.) were evaluated. The major constituent of the oils determined by GC and GC/MS (Gass chromatography and Mass spectrometry). O. minutiflorum essential oil has contained the most carvacrol (78,8%). Reported biological effects of carvacrol are as follows: antibacterial, antifungal, antispasmodic, acetylcholine esterase inhibition, lipid peroxidase inhibition, radical scavenging effect, white blood cell macrophage stimulant, and cardiac depressant activity. Due to their antioxidant properties carvacrol-rich oils are recommended for the preservation of food. The aim of this study is to evaluate the antimicrobial activity of two essential oils on 17 contaminant and pathogen bacteria, 11 lactic acid bacteria and 4 fungi by disk diffusion method. Bacillus cereus RSKK 863 strain has been the most susceptible strain (50%), in addition to this, Pseudomonas aeruginosa ATCC 29212 strain has been the most resistant strain (75%). Among lactic acid bacteria, Pediococcus pentosaceous has been the most susceptible strain (50%) and Leuconostoc mesenteroides has been the most resistant strain (62,5%). In this study, Lactic acid bacteria are resistant to O. onites and O. minutiflorum essential oils. These results confirmed the possibility of using two essential oils in food systems to prevent the growth of foodborne bacteria and extend the shelf life of processed foods. P310-019 METHYLGLYOXAL-INDUCED DNA-PROTEIN CROSSLINKS IN ECV304 CELLS VIA A ROS-INDEPENDENT PROTEIN CARBONYLATION PATHWAY

T. S. Wang, Y. F. Chen, C. W. Huang. Chung Shan Medical University, Taichung, Taiwan

Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced from glycolytic intermediates and gluconeogenesis. Elevated MG levels in diabetes patients are believed to contribute to diabetic complications. MG can modify both DNA and proteins and induces a crosslink between DNA and protein; however, its mechanism is still unclear. In this study, we investigated the possible mechanisms of MG-induced protein crosslink (DPC) in ECV304 human endothelial cells. Cells were exposed to various concentrations (0.5-4 mM) of MG for 0.5-3 hours. DPC and protein carbonylation of MG-treated cells were assayed by modified comet assay and immuno-dot blot assay, respectively. The level of intracellular peroxide of MG-treated cells was also detected by DCHF-DA assay using a multiwell fluorescence plate reader. MG treatment caused a time- and dose-dependent increase of DPC in ECV304 cells. MG also caused a dose-dependent increase of protein carbonylation in ECV304 cells. Addition of 2 mM MG resulted in a transient increase in protein carbonylation in ECV304 cells, and this increase reached a peak within 2 hours, and then rapidly decreases. Co-treatment carbonyl scavenging drugs, such as aminoguanidine, N-acetyl-L-cysteine, and glutathione and MG in ECV304 cells significantly inhibited the formation of DPC, whereas co-treatment with antioxidants ascorbic acid did not decrease MG-induced DPC in ECV304 cells. Although MG induced significant increase in DPC and protein carbonylation levels, it did not change the ROS level in ECV304 cells. Taken together, these results suggest that MG induces DPC formation in ECV304 cells may via a ROS-independent protein carbonylation pathway. P310-020 INSECTICIDAL ACTIVITY OF ANT (CREMATOGASTER STRIATULA) VENOM AND ANTIBACTERIAL EFFECT FROM ANT (TETRAMORIUM BICARINATUM) VENOM AGAINST STRAINS WITH SOCIETAL INTEREST.

A. Rifflet, N. Tene, J. Orivel, M. Treilhou, A. Vétillard Centre Universitaire Jean-François Champollion, Albi, France Ants have colonised most ecosystems and with more than 12000 species around the world, they represent the main group in the order hymenoptera. However, compared to bees and wasps, ant’s venoms have been poorly studied. Our team works on venoms of two ants, Crematogaster striatula, an arboreal ant from Cameroon and Tetramorium bicarinatum, terricol ant from Brazil. We investigated the Biological Activities of both; the content extracted from the Dufour gland of C.striatula and content extracted from the venom reservoir of T.bicarinatum. Dufour gland and

reservoir venom contain respectively lipophilic compounds (esters, alcohols, ketons, alkaloids) and protein compounds. Results obtained with the Dufour liquid demonstrated insecticidal activity against termites. Indeed, a paralysis effect and finally death were observed. Dufour gland molecules would be potent neurotoxins. We also showed haemolytic activity on red blood cells with Dufour gland. Tests were run in parallel with venom of Tetramorium bicarinatum on different bacterial strains and showed antibacterial responses. A colorimetric assay, MTT, was performed and IC50 calculated for each strain. T.bicarinatum venom has a broad spectrum of activity


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against gram+ and gram- bacteria. Among strains with societal interest, two staphylococci (Staphylococcus aureus and Staphylococcus xylosus) show different sensibilities to venom. We will investigate further these two staphylococci. The aim is to understand mechanisms of action of the substance(s) at the origin of antibacterial activities. We experiment with crude venom so in parallel, venom fractions are produced using a spin filter followed by LC/MS analysis. Results showed that only three of these isolated fractions possess antibacterial activity. One of these two fractions exhibits two peptidic molecules. The identifications are in process. These venoms could be source of new biotoxins with great interest for humans.

xii international congress of toxicology. 19-23 july 2010 ... · xii international congress of...

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