- Email: [email protected]
Spontaneous mutation frequency in normal human mammary gland tissue
S150
Abstracts / Toxicology Letters 211S (2012) S43–S216
Hexylcinnamaldehyde in the conventional test protocol. (B) While in the conventional LLNA the incorporation of 3H-methyl thymidine is measured by beta-scintillation counting as disintegrations per minute, in the non-radioactive LLNA the incorporation of 5bromo-2-deoxyuridine is measured by ELISA. We established and validated the non-radioactive LLNA in our laboratory and approved the robustness as well as reproducibility of this test method. Although the OECD TG 429 does not recommend any aqueous vehicle, we could show for the conventional LLNA that the measurement of hydrophilic substances can be performed by choosing an appropriate vehicle. Furthermore, the non-radioactive protocol provided in the OECD TG 442B could be established and validated in our laboratory. doi:10.1016/j.toxlet.2012.03.545
P23-10 The Cell Screening Facility at Science for Life Laboratory – Stockholm Bo Lundgren, Ulf Martens, Maria Häggblad SciLifeLab Stockholms University, Sweden Science for Life Laboratory – Stockholm (SciLifeLab) is a new Swedish national resource centre dedicated to large scale research in molecular biosciences and medicine. The major funding for SciLifeLab comes from strategic government grants. SciLifeLab – Stockholm is a joint collaboration between three universities, The Royal Institute of Technology (KTH), Karolinska Institute (KI) and Stockholm University (SU). The Cell Screening Facility at SciLifeLab use advanced robotics and micro plate technology for high throughput screening with RNAi or small molecules libraries in cell lines. The use of micro plates and robotics technologies have been extensively used for long time in the pharmaceutical industry mainly for finding, identify and characterize new drug targets. The major objectives for the Cell Screen Facility are to provide this technology to the academic community. Main activities • RNAi high throughputscreens and validation to identify new proteins involved in biological processes • Small molecule screens (inhibition toxicity) • Assay development • Map functional genetic networks. • Preclinical and in vitro toxicology support Major equipment • • • • • •
Janus 3 arm robot with 96 and 384 head Envison plate reader Cell washer and Flexdrop Echo 550 liquid dispenser RNAi libraries Chemical libraries
SciLifeLab – Stockholm could be engage in projects at three different levels: service, Collaborative and internal SciLifeLab projects for more information see contact info Contact info: [email protected] or www.scilifelab.se. doi:10.1016/j.toxlet.2012.03.546
P23-11 High dose -cyclodextrin exposure via continuous or intermittent IV infusion in rats Harmke van Vugt, Ankie Schoenmakers, Maud Wasserman, Bianca van Rozendaal, Harry Emmen NOTOX a WIL Research Company, Netherlands During nonclinical drug safety testing, excipients are often needed to reach sufficiently high dose levels to achieve the required safety margin. -Cyclodextrins are widely used to solubilize drugs, including those intended for intravenous (IV) administration. However, the possible effects of high doses of -cyclodextrins in a nonclinical setting have not yet been fully characterized. In addition, higher doses may be tolerated when given via intermittent infusion. The effects of a high dose -cyclodextrin, given via either continuous or intermittent IV infusion for 7 days, were studied in rats. Male Sprague-Dawley rats were catheterized via the femoral vein into the vena cava, the catheter was tunneled subcutaneously to the neck and externally attached to a harness and tether. 50% -cyclodextrin was administered for 24-h (continuous) or 4-h (intermittent) by IV infusion at a dosing speed of 1.25 or 7.5 mL/kg/h, respectively, both resulting in a dose level of 15,000 mg/kg/day. The intermittent group received saline during the remaining 20 h/day at 0.4 mL/h. Control animals received 24-h continuous IV infusion with saline at 0.4 mL/h. No significant differences were observed between the two dosing scenarios. -Cyclodextrin-related findings included reduced food intake, body weight loss and general poor condition. Main target organs were liver and kidneys, as reflected by changes in hematology, biochemistry and urinalysis parameters, macroscopic pathology, organ weights and microscopic pathology. At both continuous and intermittent IV infusion, the high level of 15,000 mg/kg/day exceeded the tolerated dose at which cyclodextrin can be used as excipient in rats. doi:10.1016/j.toxlet.2012.03.547
P23-12 Spontaneous mutation frequency in normal human mammary gland tissue Katja Schmalbach, Leane Lehmann University of Wuerzburg, Germany Annually, over 57,000 women develop breast cancer in Germany. In particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. Up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (SMF) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. The only test with the potential to determine low SMFs was the Random Mutation Capture (RMC) assay, a genotype selective method. Therefore, the suitability of the RMC assay to determine SMF in p53 gene in normal human mammary gland tissue was evaluated. The RMC assay was optimized concerning (i) DNA isolation, (ii) PCR conditions, and (iii) amount of mammary gland tissue. (i) Genomic DNA from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) The target sequence
Abstracts / Toxicology Letters 211S (2012) S43–S216
was captured with a complementary uracil-containing DNA-probe, followed by magnetic separation from the remaining genomic DNA. The copy number of the target sequence was quantified by competitive PCR. (iii) With 2 g of normal mammary gland tissue a SMF of 2.2 ± 1.4 × 10−7 per base pair was determined indicating RMC assay suitable for SMF determination. In conclusion, the SMF in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling future investigation of factors influencing SMF during breast cancer development. doi:10.1016/j.toxlet.2012.03.548
P23-13 A GC/MS method for measuring cocaine metabolism in human renal cells
S151
Activation of Nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. In both systems Nrf2::GFP accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). But while CAPE is a potent activator of Nrf2 in cells, it has no effect on SKN-1 localisation. Further the effect on the Nrf2 protein amount was investigated by western blot analysis. The expression of target genes can be investigated by differing means: While PCR methods and western blotting are standard for in vitro studies, the vast number of available GFP reporter strains offers opportunities for research using C. elegans. We have performed congruent assays in a cell culture system and the model organism C. elegans to compare antioxidative capacity and effects of polyphenols on Nrf2 signalling. Therefore, depending on the substance tested, C. elegans is a suitable model system to investigate effects of natural compounds in an organism. doi:10.1016/j.toxlet.2012.03.550
Paula Guedes de Pinho 1,2 , Maria João Valente 1 , Félix Carvalho 1 , Maria de Lourdes Bastos 1 , Márcia Carvalhoa 1 1
REQUIMTE-Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal, 2 CEBIMED, Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal Purpose: Development and validation of a gas chromatography/ion trap-mass spectrometry (GC/IT-MS) method for the simultaneous measurement of cocaine and its metabolites benzoylecgonine (BE) and norcocaine (NCOC) in a cellular matrix, and its application to study cocaine metabolism in primary cultured human proximal tubular epithelial cells (HPTECs). Methods: HPTECs were used as a cellular matrix, simultaneously exposed to cocaine, BE and NCOC. The samples were purified and concentrated by solid-phase extraction (SPE), followed by silylation with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) for GC/IT-MS analysis. Results and conclusions of the study: The method was found to be sensitive, specific and linear (r2 > 0.99) for a wide range of concentrations (0–100 g/mL) of all three analytes, with intra-day precision varying between 3.6% and 13.5% and accuracy between 92.7% and 111.9%. The limits of detection (LOD) for cocaine, BE and NCOC were 0.97, 0.40 and 20.9 ng/mL, while the limits of quantification (LOQ) were 3.24, 1.34 and 69.6 ng/mL, respectively. The applicability of the developed method was demonstrated in primary cultured HPTECs exposed to 1 mM cocaine for 72 h, in which all analytes were identified and quantified. This study may represent an important tool for in vitro mechanistic studies, enabling the correlation of elicited toxic effects with local drug metabolism. doi:10.1016/j.toxlet.2012.03.549
P23-14 Nrf2 (SKN-1) signalling in cell culture models and C. elegans Susannah Havermann, Wim Wätjen Heinrich-Heine-University Düsseldorf, Germany Certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the Nrf2 signalling pathway. We have employed cell culture based assays (DCF, western blot, GFP reporters) and further analysed the effects of these substances in the nematode C. elegans (SKN-1: Nrf2 homologue) in vivo. Baicalein and caffeic acid phenethylester (CAPE) protected cells and the nematode from ROS accumulation after application of stress (shown by DCF assay).
P23-15 Quantification of morphine and metabolites in antemortem and postmortem human samples Ana Oliveira 1 , Ricardo Jorge Dinis-Oliveira 2 , Paula Guedes Pinho 1 , Fernando Remião 1 , Félix Carvalho 1 , Rui Medeiros 3 1
REQUIMTE, Portugal, 2 CESPU, Portugal, 3 IPO-Porto, Portugal
Purpose: Morphine is one of the most effective agents for the short- and long-term control of significant pain. In humans, morphine is primarily metabolized to morphine-3-glucuronide (M3G) and, to a lesser extent to morphine-6-glucuronide (M6G). While M6G is a potent opioid receptor agonist with a higher analgesic activity, M3G has no opioid action and it seems to have a role in the side-effects usually described. Although several methods have been described for the simultaneous determination of morphine and its major metabolites, there is still need of methodologies with cleaner extraction, increased sensitivity, specificity and applicability to different matrices. Methods: A reversed-phase high-performance liquid chromatographic method with coulometric and diode-array detection was developed for the simultaneous determination of morphine, M3G and M6G in antemortem and postmortem samples, namely in plasma, serum, whole blood, urine, liver, kidney and brain. Morphine, glucuronides and the internal standard were extracted using Bond-Elut® C18 and Oasis® WCX solid-phase extraction cartridges and the separation was carried out by using a Waters Spherisorb® ODS2 reversed-phase column and 0.01 M potassium phosphate buffer:acetonitrile (85:15, v/v) containing sodium dodecyl sulfate as the mobile phase. Results: The method proved to be specific, accurate and precise across the calibration range with good linearity for all analytes. The quantification limit was set to 1 ng/ml for morphine and 10 ng/mL for M6G and M3G. The proposed method can be successfully applied in the quantification of morphine and metabolites, covering the routes of distribution, metabolism and elimination of morphine. Acknowledgments: FCT grant SFRH/BD/62775/2009. doi:10.1016/j.toxlet.2012.03.551
Abstracts / Toxicology Letters 211S (2012) S43–S216
Hexylcinnamaldehyde in the conventional test protocol. (B) While in the conventional LLNA the incorporation of 3H-methyl thymidine is measured by beta-scintillation counting as disintegrations per minute, in the non-radioactive LLNA the incorporation of 5bromo-2-deoxyuridine is measured by ELISA. We established and validated the non-radioactive LLNA in our laboratory and approved the robustness as well as reproducibility of this test method. Although the OECD TG 429 does not recommend any aqueous vehicle, we could show for the conventional LLNA that the measurement of hydrophilic substances can be performed by choosing an appropriate vehicle. Furthermore, the non-radioactive protocol provided in the OECD TG 442B could be established and validated in our laboratory. doi:10.1016/j.toxlet.2012.03.545
P23-10 The Cell Screening Facility at Science for Life Laboratory – Stockholm Bo Lundgren, Ulf Martens, Maria Häggblad SciLifeLab Stockholms University, Sweden Science for Life Laboratory – Stockholm (SciLifeLab) is a new Swedish national resource centre dedicated to large scale research in molecular biosciences and medicine. The major funding for SciLifeLab comes from strategic government grants. SciLifeLab – Stockholm is a joint collaboration between three universities, The Royal Institute of Technology (KTH), Karolinska Institute (KI) and Stockholm University (SU). The Cell Screening Facility at SciLifeLab use advanced robotics and micro plate technology for high throughput screening with RNAi or small molecules libraries in cell lines. The use of micro plates and robotics technologies have been extensively used for long time in the pharmaceutical industry mainly for finding, identify and characterize new drug targets. The major objectives for the Cell Screen Facility are to provide this technology to the academic community. Main activities • RNAi high throughputscreens and validation to identify new proteins involved in biological processes • Small molecule screens (inhibition toxicity) • Assay development • Map functional genetic networks. • Preclinical and in vitro toxicology support Major equipment • • • • • •
Janus 3 arm robot with 96 and 384 head Envison plate reader Cell washer and Flexdrop Echo 550 liquid dispenser RNAi libraries Chemical libraries
SciLifeLab – Stockholm could be engage in projects at three different levels: service, Collaborative and internal SciLifeLab projects for more information see contact info Contact info: [email protected] or www.scilifelab.se. doi:10.1016/j.toxlet.2012.03.546
P23-11 High dose -cyclodextrin exposure via continuous or intermittent IV infusion in rats Harmke van Vugt, Ankie Schoenmakers, Maud Wasserman, Bianca van Rozendaal, Harry Emmen NOTOX a WIL Research Company, Netherlands During nonclinical drug safety testing, excipients are often needed to reach sufficiently high dose levels to achieve the required safety margin. -Cyclodextrins are widely used to solubilize drugs, including those intended for intravenous (IV) administration. However, the possible effects of high doses of -cyclodextrins in a nonclinical setting have not yet been fully characterized. In addition, higher doses may be tolerated when given via intermittent infusion. The effects of a high dose -cyclodextrin, given via either continuous or intermittent IV infusion for 7 days, were studied in rats. Male Sprague-Dawley rats were catheterized via the femoral vein into the vena cava, the catheter was tunneled subcutaneously to the neck and externally attached to a harness and tether. 50% -cyclodextrin was administered for 24-h (continuous) or 4-h (intermittent) by IV infusion at a dosing speed of 1.25 or 7.5 mL/kg/h, respectively, both resulting in a dose level of 15,000 mg/kg/day. The intermittent group received saline during the remaining 20 h/day at 0.4 mL/h. Control animals received 24-h continuous IV infusion with saline at 0.4 mL/h. No significant differences were observed between the two dosing scenarios. -Cyclodextrin-related findings included reduced food intake, body weight loss and general poor condition. Main target organs were liver and kidneys, as reflected by changes in hematology, biochemistry and urinalysis parameters, macroscopic pathology, organ weights and microscopic pathology. At both continuous and intermittent IV infusion, the high level of 15,000 mg/kg/day exceeded the tolerated dose at which cyclodextrin can be used as excipient in rats. doi:10.1016/j.toxlet.2012.03.547
P23-12 Spontaneous mutation frequency in normal human mammary gland tissue Katja Schmalbach, Leane Lehmann University of Wuerzburg, Germany Annually, over 57,000 women develop breast cancer in Germany. In particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. Up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (SMF) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. The only test with the potential to determine low SMFs was the Random Mutation Capture (RMC) assay, a genotype selective method. Therefore, the suitability of the RMC assay to determine SMF in p53 gene in normal human mammary gland tissue was evaluated. The RMC assay was optimized concerning (i) DNA isolation, (ii) PCR conditions, and (iii) amount of mammary gland tissue. (i) Genomic DNA from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) The target sequence
Abstracts / Toxicology Letters 211S (2012) S43–S216
was captured with a complementary uracil-containing DNA-probe, followed by magnetic separation from the remaining genomic DNA. The copy number of the target sequence was quantified by competitive PCR. (iii) With 2 g of normal mammary gland tissue a SMF of 2.2 ± 1.4 × 10−7 per base pair was determined indicating RMC assay suitable for SMF determination. In conclusion, the SMF in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling future investigation of factors influencing SMF during breast cancer development. doi:10.1016/j.toxlet.2012.03.548
P23-13 A GC/MS method for measuring cocaine metabolism in human renal cells
S151
Activation of Nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. In both systems Nrf2::GFP accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). But while CAPE is a potent activator of Nrf2 in cells, it has no effect on SKN-1 localisation. Further the effect on the Nrf2 protein amount was investigated by western blot analysis. The expression of target genes can be investigated by differing means: While PCR methods and western blotting are standard for in vitro studies, the vast number of available GFP reporter strains offers opportunities for research using C. elegans. We have performed congruent assays in a cell culture system and the model organism C. elegans to compare antioxidative capacity and effects of polyphenols on Nrf2 signalling. Therefore, depending on the substance tested, C. elegans is a suitable model system to investigate effects of natural compounds in an organism. doi:10.1016/j.toxlet.2012.03.550
Paula Guedes de Pinho 1,2 , Maria João Valente 1 , Félix Carvalho 1 , Maria de Lourdes Bastos 1 , Márcia Carvalhoa 1 1
REQUIMTE-Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal, 2 CEBIMED, Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal Purpose: Development and validation of a gas chromatography/ion trap-mass spectrometry (GC/IT-MS) method for the simultaneous measurement of cocaine and its metabolites benzoylecgonine (BE) and norcocaine (NCOC) in a cellular matrix, and its application to study cocaine metabolism in primary cultured human proximal tubular epithelial cells (HPTECs). Methods: HPTECs were used as a cellular matrix, simultaneously exposed to cocaine, BE and NCOC. The samples were purified and concentrated by solid-phase extraction (SPE), followed by silylation with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) for GC/IT-MS analysis. Results and conclusions of the study: The method was found to be sensitive, specific and linear (r2 > 0.99) for a wide range of concentrations (0–100 g/mL) of all three analytes, with intra-day precision varying between 3.6% and 13.5% and accuracy between 92.7% and 111.9%. The limits of detection (LOD) for cocaine, BE and NCOC were 0.97, 0.40 and 20.9 ng/mL, while the limits of quantification (LOQ) were 3.24, 1.34 and 69.6 ng/mL, respectively. The applicability of the developed method was demonstrated in primary cultured HPTECs exposed to 1 mM cocaine for 72 h, in which all analytes were identified and quantified. This study may represent an important tool for in vitro mechanistic studies, enabling the correlation of elicited toxic effects with local drug metabolism. doi:10.1016/j.toxlet.2012.03.549
P23-14 Nrf2 (SKN-1) signalling in cell culture models and C. elegans Susannah Havermann, Wim Wätjen Heinrich-Heine-University Düsseldorf, Germany Certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the Nrf2 signalling pathway. We have employed cell culture based assays (DCF, western blot, GFP reporters) and further analysed the effects of these substances in the nematode C. elegans (SKN-1: Nrf2 homologue) in vivo. Baicalein and caffeic acid phenethylester (CAPE) protected cells and the nematode from ROS accumulation after application of stress (shown by DCF assay).
P23-15 Quantification of morphine and metabolites in antemortem and postmortem human samples Ana Oliveira 1 , Ricardo Jorge Dinis-Oliveira 2 , Paula Guedes Pinho 1 , Fernando Remião 1 , Félix Carvalho 1 , Rui Medeiros 3 1
REQUIMTE, Portugal, 2 CESPU, Portugal, 3 IPO-Porto, Portugal
Purpose: Morphine is one of the most effective agents for the short- and long-term control of significant pain. In humans, morphine is primarily metabolized to morphine-3-glucuronide (M3G) and, to a lesser extent to morphine-6-glucuronide (M6G). While M6G is a potent opioid receptor agonist with a higher analgesic activity, M3G has no opioid action and it seems to have a role in the side-effects usually described. Although several methods have been described for the simultaneous determination of morphine and its major metabolites, there is still need of methodologies with cleaner extraction, increased sensitivity, specificity and applicability to different matrices. Methods: A reversed-phase high-performance liquid chromatographic method with coulometric and diode-array detection was developed for the simultaneous determination of morphine, M3G and M6G in antemortem and postmortem samples, namely in plasma, serum, whole blood, urine, liver, kidney and brain. Morphine, glucuronides and the internal standard were extracted using Bond-Elut® C18 and Oasis® WCX solid-phase extraction cartridges and the separation was carried out by using a Waters Spherisorb® ODS2 reversed-phase column and 0.01 M potassium phosphate buffer:acetonitrile (85:15, v/v) containing sodium dodecyl sulfate as the mobile phase. Results: The method proved to be specific, accurate and precise across the calibration range with good linearity for all analytes. The quantification limit was set to 1 ng/ml for morphine and 10 ng/mL for M6G and M3G. The proposed method can be successfully applied in the quantification of morphine and metabolites, covering the routes of distribution, metabolism and elimination of morphine. Acknowledgments: FCT grant SFRH/BD/62775/2009. doi:10.1016/j.toxlet.2012.03.551