Determination of cell death induced by lovastatin on human colon cell line HT29 using comet assay

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conditions. Cell viability was evaluated by MTT assay. Moreover an ex vivo study was conducted where Wistar rats received either DOX (3 mg/kg; IP; weekly; for four weeks; n = 3; DOX group) or saline as control (n = 3). At the end, ATMSC were isolated from such animals, cultured up to the fourth passage and, then, it was assessed MTT assay for cell viability, alkaline phosphatase (AP) production, and proliferation kinetics study. Results: The MTT results indicated higher DOX toxicity for ATMSC compared to breast cancer cells (p < 0.05). In the ex vivo study, compared to control, ATMSC from DOX group had similar MTT results, lower PA production and inferior proliferation counts (p < 0.05). The present data showed DOX induced toxicity for ATMSC raising the possibility of autologous stem cell transplantation to treat DOX cardiotoxicity as a non-suitable option for subjects under chemotherapy with such antineoplastic agent. doi:10.1016/j.toxlet.2011.05.416

P1183 Hallucinogenic amphetamines neurotoxicity in cultured hippocampal neurons S. da Costa Araújo 1 , V.M. Costa 1 , K. Ruscher 2 , M.D.L. Bastos 3 , U. Dirnagl 4 , A. Meisel 4 , F. Carvalho 1 , J.P. Capela 1,∗ 1

Department of Biological Sciences/Laboratory of Toxicology, REQUIMTE, Faculty of Pharmacy, University of Porto, Porto, Portugal, 2 Laboratory for Experimental Brain Research, Department of Clinical Sciences, Lund University, BMC A13, S-22184 Lund, Sweden, 3 Department of Biological Sciences, Faculty of Pharmacy, University of Porto, REQUIMTE, Porto, Portugal, 4 Department of Experimental Neurology and Center for Stroke Research, Charité-Universitätsmedizin Berlin, Charitéplatz 1, Berlin, Germany 3,4-Methylenedioxymethamphetamine (MDMA or “Ecstasy”) and 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) are hallucinogenic amphetamines with addictive properties. The hippocampus, which is involved in learning and memory, seems particularly vulnerable to amphetamine neurotoxicity. We evaluated the neurotoxicity of DOI and MDMA in primary neuronal cultures of hippocampal neurons obtained from Wistar rat embryos (E-17 to E-19). Mature cultured neurons with 10 days were exposed for 24 or 48 h either to MDMA (100–800 ␮M) or DOI (10–100 ␮M). Both the lactate dehydrogenase (LDH) release and the tetrazoliumbased (MTT) assays revealed a concentration- and time-dependent neuronal death afer MDMA or DOI treatment. Additionally, both drugs had the ability to increase the activity of caspase-8 and caspase-3 after 48 h exposure. At concentrations that produced similar levels of neuronal death, DOI (50 ␮M) promoted an increase in the activity of both caspases greater than MDMA (400 ␮M). Additionally, Ac-DEVD-CHO, a caspase-3 inhibitor, attenuated neuronal injury promoted by both drugs, as evaluated by LDH assay. In the mitochondrial fraction of neurons exposed 24 h to DOI (50 ␮M) or MDMA (400 ␮M), we found a significant increase in the 67 kDa band of apoptosis inducing factor (AIF) by Western blotting. Our work shows that hallucinogenic amphetamines promote activation of caspase-dependent and independent mechanisms of hippocampal neuronal death, involving both the mitochondria machinery and the activation of cell death receptors. Acknowledgments: This work was supported by “Fundac¸ão para a Ciência e Tecnologia” (FCT) [PTDC/SAU-FCF/102958/2008]-QREN initiative with EU/FEDER financing through COMPETE. VMC and JPC acknowledge FCT for their Post-doc grants (SFRH/BPD/63746/2009 and SFRH/BPD/30776/2006). doi:10.1016/j.toxlet.2011.05.417

P1184 Distribution study of acetylcholinesterase reactivators after application of therapeutic doses in guinea pigs J. Zdarova Karasova 1 , M. Pohanka 2,∗ , M. Pavlík 3 , J. Kassa 4 , K. Musilek 2 , F. Zemek 3 , K. Kuca 2 1

Public Health, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic, 2 Center of Advanced Studies, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic, 3 Faculty of Military Health Sciences, Hradec Králové, Czech Republic, 4 Toxicology, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic The pharmacokinetic studies of AChE reactivators started by Wilson and Ginsburg investigation. They found that oximes are able to reactivate of alkylphosphate inhibited AChE. This basic information was soon outlined by recognizing of oximes absorption, distribution and elimination. A simple and reliable HPLC method for the determination of plasma levels of acetylcholinesterase reactivators (HI-6 and obidoxime) is widely presented in our study. The pharmacokinetic was evaluated in Guinea pigs. Acetylcholinesterase reactivators’ separation was carried out by high performance liquid chromatography (HPLC) using electrochemical detector. The calibration curves were linear in the range 0.25–50 ␮g/mL. This is well overlaying the detected plasma level ranges of therapeutical doses of acetylcholinesterase reactivators. After administration of therapeutic doses (HI-6 – 45.1 mg/kg; obidoxime – 4.15 mg/kg) of previously mentioned oximes, the same distribution shapes of curves were found. These therapeutic doses corresponded with 5% of LD50 and were previously established in in vivo tests. The maximal plasma concentrations were found in 15 min after application (HI-6 39.05 ± 2.98 ␮g/ml; obidoxime 4.68 ± 0.55 ␮g/ml) and elimination of both oximes were fast. The minimal effective concentration in plasma is 4 ␮g/ml. This concentration limit was broken by oxime HI-6 till 60 min and obidoxime till 30 min after i.m. applications. doi:10.1016/j.toxlet.2011.05.418

P1185 Determination of cell death induced by lovastatin on human colon cell line HT29 using comet assay M. Rezaei 1,∗ , H. Kalantari 2 , M. HashemiTabar 1 , M. Jafari 1 , Z. Bahadori 1 1

Toxicology and Pharmacology, JoundiShapour School of Pharmacy, Ahvaz, Iran, 2 Toxicology and Pharmacology, JoundiShapour Pharmacy School, Ahvaz, Iran Purpose: Apoptosis or programmed cell death is an essential process for elimination of damaged cells. Also, induction of apoptosis is fundamental for treating cancer. The screening for agents that induce apoptosis in tumor cells help in the development of novel agents for cancer treatment. Numerous studies suggest that the exposure of tumor cells to statins can lead to cell death via two separate processes: apoptosis or necrosis. Severe fragmentation of DNA during apoptosis can be readily measured by neutral comet assay. Migration of DNA fragments of apoptotic cells by the electrical field can produce comet-like images. The aim of this study was to determining the type of cell death induced by lovastatin on human colon tumor cells by using neutral comet assay and to evaluate the utility of this method for detection of apoptosis. Methods: HT29 cells were grown in DMEM medium then exposed to different concentration of lovastatin and DNA fragmen-

Abstracts / Toxicology Letters 205S (2011) S60–S179

tation associated with apoptosis was detected by neutral comet assay method. Results: Lovastatin induced an apoptotic response in HT29 cell line and produced comet pattern similar to positive control (Anisomycin, 2 ␮g/ml). In conclusion this study showed that lovastatin can induce apoptosis in HT29 cell line and confirmed the utility of comet assay for detection of apoptosis. doi:10.1016/j.toxlet.2011.05.419

P1186 Capture compound mass spectrometry: Functional proteomics in drug toxicity profiling A.K. Schrey 1,∗ , J.J. Fischer 2 , S. Michaelis 1 , O.Y. Graebner 3 , S. Baumgart 3 , M. Dreger 2 , F. Kroll 1 , H. Koester 4 1

Medicinal Chemistry, Caprotec Bioanalytics GmbH, Berlin, Germany, 2 Biochemistry, Caprotec Bioanalytics GmbH, Berlin, Germany, 3 Analytical Chemistry, Caprotec Bioanalytics GmbH, Berlin, Germany, 4 Caprotec Bioanalytics GmbH, Berlin, Germany

Critical side-effects such as organ toxicity are the foremost reason for drugs failing in late clinical trials or being withdrawn after market release. This is due to the complex toxicity mechanisms and because these side effects often do not show up in animal models. Thus, assays on biological systems from human origin are needed in an early stage of research to prevent such drop-outs. Capture compound mass spectrometry (CCMS) has proven a valuable tool for unbiased assessment of protein-binding profiles of small molecules from cell lysates, subcellular fractions and whole-cell setups. Capture compounds recognize, covalently bind and isolate protein targets via an affinity function derived from the small molecule, a photo-activated crosslink function, and a sorting function. In our research, we have successfully identified new targets for various drug molecules accounting for the mechanisms of toxic side-effects, alternative modes of action for the main indication or unrelated advantageous side-effects. Implications for drug safety assessment are: CCMS as an assay for proteins involved in adverse pathways for a small-molecule drug; a database containing small molecules and their identified targets; meta-analysis of the results to identify targets of so far unknown functions as correlated to toxic side-effects; CCMS protein lists as descriptors in predictive toxicology doi:10.1016/j.toxlet.2011.05.420

P1187 False positive amphetamine findings D. Sutlovic 1,∗ , M. Versic 2 , M. Definis-Gojanovic 1 1

Forensic Medicine, University Hospital and School of Medicine Split, Split, Croatia, 2 Forensic Medicine, Medical School Split, Split, Croatia It has been noticed that the postmortem liver samples gives a positive result for amphetamines, although it is proved that the person has not previously consumed amphetamines. Postmortem liver samples are of great importance since they contain many valuable specimens in higher concentration then in some other biological samples. It was observed that liver tissue from persons found several days after death occur contains amphetamines. Purpose: The purpose of this study is to determine conditions that are responsible for amphetamine false positive finding in postmortem liver samples. Methods: Postmortem liver samples, which should be toxicologically processed, and the previous test gave

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negative amphetamine findings, are divided into several samples with identical weight and were subjected to the influence of different temperature in different time period. At various time intervals samples are being analyzed and determent qualitatively and quantitative by gas chromatography–mass spectrometry technique (GC/MS). Results: Liver samples with amphetamine negative findings, gave positive results after few days at temperature of 32.5 ◦ C. It was noted that the alcohol concentration has an effect on amphetamine occurrence. doi:10.1016/j.toxlet.2011.05.421

P1188 Differential alterations of GABAA receptor (␣1, ␤2, ␥2 subunit) expression and increased seizure susceptibility in rat offspring from morphine-addicted mothers: Beneficial effect of dextromethorphan S. Yang Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Purposes: Although prenatal morphine exposure experimentally induces seizures in rat offspring, underlying mechanisms remain unclear. This study addresses whether prenatal morphine exposure altered subunit compositions of g-aminobutyric acid receptor subtype A (GABAA R) in the hippocampal CA1 area and temporal cortex and increased seizure susceptibility of young rat offspring, at a representative age (postneonatal days 14; P14). Therapeutic efficacy of dextromethorphan (a noncompetitive antagonist of N-methyl-d-aspartate receptors (NMDARs), in such offspring was also evaluated. Methods: From P7 to 14, Sprague–Dawley rat offspring were intra-peritoneally (ip) injected a representative dose of dextromethorphan (3 mg/kg) twice a day. At P14, some offspring were injected (ip) pentylenetetrazol to estimate seizure susceptibility, while the others were studied for GABAA R subunit (␣1, ␤2, ␥2) expression. Results: Prenatal morphine exposure caused the up-regulated ␣1 subunit and down-regulated ␤2/␥2 subunit expression of GABAA R within hippocampus and temporal cortex in rat offspring associated to increase seizure susceptibility. The magnitudes of up-regulated ␣1 subunit and down-regulated ␤2 subunit expression in the hippocampus were greater than which in the temporal cortex. The use of dextromethorphan markedly reversed the prenatal morphine-induced alterations, indicating the possible therapeutic actions of dextromethorphan. These results suggest that the altered subunit compositions (␣1, ␤2, ␥2) of GABAA R in the hippocampal CA1 area and temporal cortex may contribute, at least in part, to the increased seizure susceptibility of rat offspring subjected to prenatal morphine exposure. More importantly, dextromethorphan may be a promising clinical agent acting against these alterations. doi:10.1016/j.toxlet.2011.05.422