Abstracts / Toxicology Letters 189S (2009) S57–S273 S155

Y15Biomarkers of nephrotoxicity: Advancing from animal studiesto human clinical trials

Dominic Eisinger 1,∗, Sabine Küsters 2

1 Rules-Based Medicine, Inc., R&D, Lake Placid, United States,2 Rules-Based Medicine, Inc., R&D, Austin, Germany

In June 2008, the EMEA and FDA issued a joint framework allow-ing submission of a single application for seven new urinarybiomarkers of drug-induced kidney damage in rats. Based on pre-vious experience in developing assays and analyzing samples forNovartis as part of the FDA/EMEA submission by the C-Path Pre-dictive Safety Testing Consortium (PSTC), we recently developed apanel of human multiplexed assays that include the correspond-ing EMEA/FDA listed rat biomarkers in addition to several otheranalytes with potential utility as biomarkers of human kidney dam-age. The assays have been validated to clinical laboratory standardsand are being used to advance the qualification of biomarkers ofdrug-induced kidney damage. A summary of recent examples ofdrug-induced kidney injury biomarker studies in rat, mouse, andmonkey will be presented in addition to results supporting theutility of the biomarkers in chronic and acute kidney disease.

doi:10.1016/j.toxlet.2009.06.758

Y16Biomonitoring of metal contaminants in coastal zone of OndoState, Nigeria: Fuzzy logic modelling approach

Foluso Agunbiade 1,2,∗, Bamidele Olu-Owolabi 2, KayodeAdebowale 2

1 Bells University of Technology, Department of Chemical Sciences,Ota, Nigeria, 2 University of Ibadan, Department of Chemistry,Ibadan, Nigeria, Nigeria

The accumulation pattern of 10 metals in living tissues of plant,Eichornia crassipes and two fish species, Hydrocynus forskahlii,Oreochromis mossambicus were modeled with simple fuzzy clas-sification (SFC) in order to assess toxic effects of anthropogenicactivities on the coastal biota. The plant sample was separated intoroot, stem, and leaves while the fishes were divided into bones,internal organ, and muscle and were analysed for As, Cd, Cr, Cu,Ni, Pb, V, Fe, Mn, and Zn after wet oxidation of the dried weightsamples. The metal concentrations obtained were converted intomembership function of five accumulation classes and aggregatedwith the fuzzy operator (SFC). The classification results showedthat there is no metal accumulation in the different parts of theplant from the coast while the fishes are classified into low accu-mulation category. The internal organs of the fish species have highmetal accumulation than the other parts. Generally, the concentra-tion of Fe and Mn ranked highest in the coastal biota and are fromnatural, geological structure of the area but may not constitute sig-nificant risk. Cr has the highest transfer and accumulation from thecoastal water into the aquatic lives and may be indicative of riskprone system being a toxic metal. The metal contamination in thecoastal zone have not significantly accumulated nor affected thebiota making the coastal biota more risk-averse.

doi:10.1016/j.toxlet.2009.06.759

Y17Alterations of MMPs and TIMPs expression levels in an experi-mental rat model of resistive breathing

Olga Noussia 1,2,∗, Dimitrios Toumpanakis 1, Ioanna Sigala 1,Charis Roussos 1, Theodoros Vassilakopoulos 1, StamatiosTheocharis 2,∗∗

1 University of Athens Medical School, Evangelismos Hospital,Department of Critical and Pulmonary Services, Athens, Greece,2 University of Athens Medical School, Department of ForensicMedicine and Toxicology, Athens, Greece

Introduction: Matrix metalloproteinases (MMPs) and their tissueinhibitors (TIMPs) are involved in the pathogenesis of chronicobstructive pulmonary disease (COPD), which is mainly caused bycigarette smoke exposure and air pollution. Resistive breathing, ahallmark of COPD, leads to large negative intrathoracic pressures.MMPs expression in the lung is increased by mechanical stress andsuppressed by nitric oxide (NO). The effect of resistive breathing onMMPs and TIMPs lung expression and the involvement of NO wereexamined in this study.

Methods: Anesthetized female rats (n = 10, per group) breathedthrough a two-way valve and the inspiratory line was connected toa resistance, setting at mean tracheal pressure 50% of maximum(inspiratory resistive breathing, IRB). Quietly breathing animalsserved as controls (n = 20). NO involvement was studied by sup-plementation of the NO-synthase (NOS) inhibitor, L-NAME, 3 and6 h prior to IRB. MMP-3, MMP-9, (TIMP)-1 and TIMP-2 expressionwas assessed by immunohistochemistry in different lung cell pop-ulations 1, 3 or 6 h following IRB.

Results: MMP-9 expression increased in airway smooth muscleand parenchymal lung cells 1 and 3 h after IRB (p < 0.01). MMP-3was up regulated only after 1 h (p < 0.05). TIMP-1 expression wasincreased at 3 and 6 h after IRB in parenchymal cells (p < 0.01),while TIMP-2 expression was unaffected. L-NAME supplementa-tion further increased MMP-9 expression in lung parenchyma at6 h (p < 0.01) and MMP-3 expression in airway smooth muscle cellsat 3 h following IRB (p < 0.05).

Conclusion: IRB increases MMP-3, MMP-9 and TIMP-1 expres-sion in the lung, and NOS inhibition may prolong this effect.

doi:10.1016/j.toxlet.2009.06.760

Y18NMDA Receptor antagonist memantine suppresses DFP-inducedoxidative/nitrosative stress and dendritic damage in rat brain

Ramesh Gupta 1,∗, Snjezana Zaja-Milatovic 2,3, Dejan Milatovic 2,3

1 Murray State University, Toxicology, Hopkinsville, KY, United States,2 Vanderbilt University, Pediatrics, Nashville, TN, United States,3 United States

Organophosphate (OP) compounds, such as diisopropylphospho-rofluoridate (DFP), exert excitotoxicity by excessive cholinergicand glutamatergic systems’ stimulation, leading to excessive gen-eration of reactive oxygen species (ROS) and nitrogen species(RNS), depletion of cellular energy, causing neurodegeneration.The present investigation evaluates the extent of DFP-inducedcerebral oxidative/nitrosative and neuronal damages, and whetherNMDA receptor antagonist memantine provides neuroprotectionby preventing those changes in rat brain. Rats treated with DFP(1.25 mg/kg, s.c.) developed onset of toxicity signs within 10 min,

S156 Abstracts / Toxicology Letters 189S (2009) S57–S273

which progressed to maximal severity of seizures and fascicula-tions within 60 min. At this time interval, biomarkers of cerebralROS generation (F2-isoprostanes, F2-IsoPs; F4-neuroprostanes, F4-NeuroPs) were elevated to 142% and 225% of control, respectively.In addition, elevation of citrulline (a marker of RNS) by a 4- to6-fold resulted in significant (p < 0.001) depletion of high-energyphosphates (ATP and phosphocreatine). Quantitative morphome-tric analysis of pyramidal neurons of the hippocampal CA1 region1 h post DFP exposure revealed significant decreases in dendriticlengths and spine density to 30% and 58% of control, respec-tively. Pretreatment of rats, 30 min prior to DFP exposure, withmemantine (18 mg/kg, s.c.) in combination with atropine sul-fate (16 mg/kg, s.c.), markedly attenuated DFP-induced increasesin F2-IsoPs, F4-NeuroPs, citrulline and declines in high-energyphosphates and their metabolites. Furthermore, memantine pre-treatment also protected neurons in the CA1 hippocampal areafrom DFP-induced dendritic degeneration. These results suggestthat memantine provides neuroprotection by suppressing cerebraloxidative/nitrosative stress involving multiple mechanisms (sup-ported by NINDS NS057223).

doi:10.1016/j.toxlet.2009.06.761

Y19Cotinine biomarker validation of self reported smoking statusamong Greek adolescents: The HELENA study

Constantine Vardavas 1,∗, Manolis Tzatzarakis 1, Maria Plada 1,Aristeidis Tsatsakis 2, Alina Papadaki 2, Wim Saris 3, LuisMoreno 4, Anthony Kafatos 2

1 University of Crete, Department of Social Medicine, Faculty ofMedicine, Heraklion, Crete, Greece, 2 University of Crete, Departmentof Forensic Sciences & Toxicology, Medical School, Heraklion, Greece,3 University of Maastricht, Nutrition and Toxicology Research inMaastricht (NUTRIM), Department of Human Biology, Maastricht,Netherlands, 4 Universidad de Zaragoza, Escuela Universitaria deCiencias de la Salud, Saragoza, Spain

Question: A major problem in evaluating active smoking status,especially among adolescents, is its accurate verification, as adoles-cents usually underreport their current smoking status. We aimedat validating their self reported smoking status among adolescentsof the HELENA, Heraklion cohort using serum cotinine.

Methods: As part of the European HELENA study, 400 adoles-cents from Crete, Greece were randomly selected and contactedout of which 341 agreed to participate, of which 311 were withinthe valid age range (77.8% response rate) and questionnaire datawas collected through personal interviews. Blood samples werecollected from a random sample of 111 adolescents (of the 142randomly selected, 78.2%) of which 106 were analysed for theircotinine concentrations with GC/MS.

Results: In addition to the adolescents who were self reportedcurrent smokers (at least one cigarette during the last month), thecotinine analysis revealed another four participants with cotininelevels above that of the cut-off (cotinine>15 ng/ml), with serumcotinine levels of 28.86, 38.54, 132.61, and 290.97 ng/ml, respec-tively, despite them having reported that they were non-smokers.Moreover, the analysis indicated that 4% of the non-smokingadolescents were actually smokers, despite their self reported non-smoking status.

Conclusions: The applied questionnaire procedure successfullyidentified 96% of the active smokers, with only a small percentageof smokers found to report otherwise. This information provides

support of the procedures validity in assessing the lifestyle habitsof the adolescents participating in the HELENA study.

doi:10.1016/j.toxlet.2009.06.762

Y20Monitoring of the non-specific metabolites of organophosphatepesticide in amniotic fluid of pregnant women in the region ofCrete

Manolis Tzatzarakis 1,∗, Dimitrios Koutroulakis 2, Stavros Sifakis 2,Matthaios Kavalakis 1, Maria Tutudaki 1, Nikitas Mantas 2,Ourania Koukoura 2, Manolis Kokkinakis 1, Ioannis Mataliotakis 2,Aristidis Tsatsakis 2

1 University of Crete, Laboratory of Toxicology, Medical School,Heraklion, Greece, 2 University Hospital, Obstetrics and Gynecology,Fetal Maternal Unit, Heraklion, Greece

Purpose: The levels of the non-specific metabolites of organophos-phate pesticides (DAPs) in amniotic fluid were determined in thepresent study, in order to assess exposure of pregnant womento organophosphate pesticides. The examined metabolites weredimethylphosphate (DMP), diethylphosphate (DEP), diethylthio-phosphate (DETP), and diethyldithiophosphate (DEDTP). Thesemetabolites are polar water soluble substances, can be detected inseveral biological samples like urine, blood, meconium, hair andare considered as biomarkers of exposure to a great number oforganophosphate pesticides.

Method: During 2007–2008, 200 amniotic samples were col-lected from pregnant women, permanent residents of Crete. Thecollection of amniotic samples was performed at 17–24 weekof pregnancy, during amniocentesis for fetal karyotype investi-gation. An amount of 5 ml of amniotic fluid was collected andstored at −20 ◦C until analysis. DAPs were extracted from amni-otic fluid by liquid–liquid extraction, derivitized and analyzed bygas chromatography–mass spectrometry (GC–MS).

Results: The mean values (±SD) and the range of DMP, DEP,DETP and DEDTP in positive amniotic samples were 14.63 ±38.27 ng/ml (0.22–252.01), 8.65 ± 29.54 ng/ml (0.17–356.85),12.60 ± 119.95 ng/ml (0.12–1449.87), and 0.34 ± 0.67 ng/ml(0.05–5.13), respectively. DEP was the metabolite with thehigher frequency of detection (90.5%) followed by DETP (74.0%),DMP (60.0%), and DEDTP (39.5%).

Conclusion: The high frequency of detection of all DAPs is prob-ably an indication of the widespread use of organophosphatepesticides in the region of Crete. The correlation of the detectedlevels of pesticide metabolites in amniotic fluid with a varietyof parameters of pregnancy outcome including congenital abnor-malities, pregnancy-associated complications (intrauterine growthrestriction, preterm labor, and stillbirth), birth weight, and neona-tal morbidity may be valuable in prenatal screening and pregnancysurveillance. Further studies and more data are required before theaforementioned goal is achieved.

doi:10.1016/j.toxlet.2009.06.763