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Estimation of toxic effect of acetaminophen metabolite in isolated mitochondria
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Abstracts / Toxicology Letters 229S (2014) S40–S252
tumour settings. In addition to regulating tumour cell viability, BET inhibitors are also associated with gastrointestinal toxicity, though the molecular and cellular mechanisms behind this toxicity have yet to be elucidated. We have found that BET inhibitors induce a dose-limiting duodenal villous atrophy in vivo, accompanied by inappetance and body weight loss. Ex-vivo cultures of intestinal organoids confirm that villous atrophy occurs with multiple chemical classes of BET domain inhibitors and that their toxicity is driven by their primary pharmacology. Intriguingly, the intestinal atrophy occurs in the absence of a proliferative block, and in the presence of cMyc, suggesting that the intestinal effects are not mediated by cMyc inhibition as may have been assumed. We find instead that BET domain inhibitors induce a rapid loss of intestinal stem cells, suggesting that it is a loss of stem-cell self renewal leading to crypt loss and dose-limiting duodenal toxicity. http://dx.doi.org/10.1016/j.toxlet.2014.06.293 P-1.114 Estimation of toxic effect of acetaminophen metabolite in isolated mitochondria Erika Nydlova 1,2,∗ , Martina Vrbova 1,2 , Otto Kucera 3 , Petr Cesla 2 , Zuzana Cervinkova 3 , Tomas Rousar 2,3 1 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, Pardubice, Czech Republic, 2 Department of Analytical Chemistry, Faculty of Chemical Technology, Pardubice, Czech Republic, 3 Department of Physiology, Faculty of Medicine in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic
Acetaminophen (APAP) is frequently used analgetic and antipyretic drug. It is a safe drug at therapeutic doses but the overdose causes hepatotoxicity. The exact cause of cell damage remains unknown. The most important detoxification pathway acting in overdose is oxidation of APAP by cytochrome P450 to a substance, which is detoxified by reaction with glutathione. This APAP-SG conjugate has been considered as detoxification product generally. However, based on the literature and our previous results, we postulated that the conjugate could possess a toxic effect. APAP-SG conjugate was prepared using organic synthesis and purified by preparative liquid chromatography (purity was >98%). The possible toxic effect of the conjugate was tested in isolated mitochondria from rat liver and from kidney cells. The effect of APAP-SG was estimated using of ROS production. We used CMH2 DCFDA molecular probe that is nonfluorescent until oxidized by ROS. We assessed ROS production during incubation with or without substrates for complex I and complex II. We found that rat liver mitochondria treated with APAP-SG produced ROS in significantly higher extent in comparison with controls. For example, the increase of fluorescence in presence of glutamate and malate (i.e. complex I-related) was 1125 ± 18% in 5 mM APAP-SG. The increase of fluorescence in mitochondria treated without complex I substrates was 1252 ± 16%, in comparison to control. Similar results were found in mitochondria isolated from kidney cells. The obtained results show that APAPSG conjugate is likely able to induce mitochondrial impairment substrate-independently. The study was supported by grant NT/14320-3/2013. http://dx.doi.org/10.1016/j.toxlet.2014.06.294
P-1.115 Vasopressin regulates uptake of exosomes by kidney collecting duct cells Wilna Oosthuyzen ∗ , Andrea Caporali, John Pound, Christopher Gregory, David Webb, Matthew Bailey, James Dear Edinburgh University, Edinburgh, UK Exosomes are cell-derived vesicles present in urine. They contain protein and RNA and represent a reservoir for renal toxicity biomarkers. Exosomes can ‘shuttle’ microRNA between cells and represent a new signalling mechanism. To understand the mechanisms of exosome mediated signalling and to develop exosomes as biomarkers of organ-specific toxicity we explored exosome uptake by renal cells. Exosomes were fluorescently labelled then co-cultured with murine cortical collecting duct (mCCD) cells. In a subset of studies mCCD cells were grown on transwell membranes to allow analysis of apical or basolateral compartments. The supernatant exosome concentration was measured by nanoparticle tracking analysis and the intracellular exosome concentration was measured by flow cytometry. Confocal microscopy was used for intracellular visualisation of labelled exosomes. Labelled exosomes were internalised by mCCD cells and this was significantly increased by the vasopressin analogue desmopressin (dDAVP) (percentage of cells containing labelled exosomes: dDAVP 38% ± 8.4% vs control 16.7% ± 4.8%, P < 0.001). Tolvaptan, a V2 receptor antagonist, reduced exosome uptake following dDAVP (tolvaptan and dDAVP 15.6% ± 3.7% vs dDAVP 38% ± 8.4%, P < 0.001). Endothelin-1, a peptide that inhibits vasopressin, and dynamin inhibition, reduced exosome uptake following dDAVP stimulation. Using mCCD cell monolayers, the site of dDAVP action was confirmed to be basolateral, but exosome uptake was from apical and basolateral compartments. In conclusion, this study is the first to demonstrate that vasopressin regulates exosome uptake in a V2 receptor mediated process occurring through clathrin-dependent endocytosis. The effect of drug toxicity on this process is now being explored. http://dx.doi.org/10.1016/j.toxlet.2014.06.295 P-1.116 Multiple modes of chemokine regulation by nitro-PAHs in bronchial epithelial cells Johan Øvrevik ∗ , Marit Låg, Magne Refsnes, Per Schwarze, Jørn A. Holme Norwegian Institute of Public Health, Oslo, Norway Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are ubiquitous pollutants characteristic of diesel emissions. Here we summarize our work to elucidate the mechanisms of nitro-PAH induced chemokine responses in bronchial epithelial BEAS-2B cells. 1-nitropyrene (1-NP) and 3-nitroflouranthene (3-NF) induced identical cytokine/chemokine expression patterns dominated by maximum effects on CXCL8 (IL-8). By use of siRNA-transfection, blocking antibodies, pharmacological inhibitors, chelators and antioxidants, we found that that 1-NP induced CXCL8 responses required signaling through the TACE/TGF-␣/EGFR-cascade and also involved signaling through 2-adrenergic receptors (2AR), calcium, and reactive oxygen species (ROS) and/or reactive metabolites. The CYP-inhibitor ANF and knockdown of CYPOR which donates electrons to CYP-enzymes reduced 1-NP-induced CXCL8 responses, suggesting that CYP-mediated metabolism is of impor-
Abstracts / Toxicology Letters 229S (2014) S40–S252
tumour settings. In addition to regulating tumour cell viability, BET inhibitors are also associated with gastrointestinal toxicity, though the molecular and cellular mechanisms behind this toxicity have yet to be elucidated. We have found that BET inhibitors induce a dose-limiting duodenal villous atrophy in vivo, accompanied by inappetance and body weight loss. Ex-vivo cultures of intestinal organoids confirm that villous atrophy occurs with multiple chemical classes of BET domain inhibitors and that their toxicity is driven by their primary pharmacology. Intriguingly, the intestinal atrophy occurs in the absence of a proliferative block, and in the presence of cMyc, suggesting that the intestinal effects are not mediated by cMyc inhibition as may have been assumed. We find instead that BET domain inhibitors induce a rapid loss of intestinal stem cells, suggesting that it is a loss of stem-cell self renewal leading to crypt loss and dose-limiting duodenal toxicity. http://dx.doi.org/10.1016/j.toxlet.2014.06.293 P-1.114 Estimation of toxic effect of acetaminophen metabolite in isolated mitochondria Erika Nydlova 1,2,∗ , Martina Vrbova 1,2 , Otto Kucera 3 , Petr Cesla 2 , Zuzana Cervinkova 3 , Tomas Rousar 2,3 1 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, Pardubice, Czech Republic, 2 Department of Analytical Chemistry, Faculty of Chemical Technology, Pardubice, Czech Republic, 3 Department of Physiology, Faculty of Medicine in Hradec Králové, Charles University in Prague, Hradec Králové, Czech Republic
Acetaminophen (APAP) is frequently used analgetic and antipyretic drug. It is a safe drug at therapeutic doses but the overdose causes hepatotoxicity. The exact cause of cell damage remains unknown. The most important detoxification pathway acting in overdose is oxidation of APAP by cytochrome P450 to a substance, which is detoxified by reaction with glutathione. This APAP-SG conjugate has been considered as detoxification product generally. However, based on the literature and our previous results, we postulated that the conjugate could possess a toxic effect. APAP-SG conjugate was prepared using organic synthesis and purified by preparative liquid chromatography (purity was >98%). The possible toxic effect of the conjugate was tested in isolated mitochondria from rat liver and from kidney cells. The effect of APAP-SG was estimated using of ROS production. We used CMH2 DCFDA molecular probe that is nonfluorescent until oxidized by ROS. We assessed ROS production during incubation with or without substrates for complex I and complex II. We found that rat liver mitochondria treated with APAP-SG produced ROS in significantly higher extent in comparison with controls. For example, the increase of fluorescence in presence of glutamate and malate (i.e. complex I-related) was 1125 ± 18% in 5 mM APAP-SG. The increase of fluorescence in mitochondria treated without complex I substrates was 1252 ± 16%, in comparison to control. Similar results were found in mitochondria isolated from kidney cells. The obtained results show that APAPSG conjugate is likely able to induce mitochondrial impairment substrate-independently. The study was supported by grant NT/14320-3/2013. http://dx.doi.org/10.1016/j.toxlet.2014.06.294
P-1.115 Vasopressin regulates uptake of exosomes by kidney collecting duct cells Wilna Oosthuyzen ∗ , Andrea Caporali, John Pound, Christopher Gregory, David Webb, Matthew Bailey, James Dear Edinburgh University, Edinburgh, UK Exosomes are cell-derived vesicles present in urine. They contain protein and RNA and represent a reservoir for renal toxicity biomarkers. Exosomes can ‘shuttle’ microRNA between cells and represent a new signalling mechanism. To understand the mechanisms of exosome mediated signalling and to develop exosomes as biomarkers of organ-specific toxicity we explored exosome uptake by renal cells. Exosomes were fluorescently labelled then co-cultured with murine cortical collecting duct (mCCD) cells. In a subset of studies mCCD cells were grown on transwell membranes to allow analysis of apical or basolateral compartments. The supernatant exosome concentration was measured by nanoparticle tracking analysis and the intracellular exosome concentration was measured by flow cytometry. Confocal microscopy was used for intracellular visualisation of labelled exosomes. Labelled exosomes were internalised by mCCD cells and this was significantly increased by the vasopressin analogue desmopressin (dDAVP) (percentage of cells containing labelled exosomes: dDAVP 38% ± 8.4% vs control 16.7% ± 4.8%, P < 0.001). Tolvaptan, a V2 receptor antagonist, reduced exosome uptake following dDAVP (tolvaptan and dDAVP 15.6% ± 3.7% vs dDAVP 38% ± 8.4%, P < 0.001). Endothelin-1, a peptide that inhibits vasopressin, and dynamin inhibition, reduced exosome uptake following dDAVP stimulation. Using mCCD cell monolayers, the site of dDAVP action was confirmed to be basolateral, but exosome uptake was from apical and basolateral compartments. In conclusion, this study is the first to demonstrate that vasopressin regulates exosome uptake in a V2 receptor mediated process occurring through clathrin-dependent endocytosis. The effect of drug toxicity on this process is now being explored. http://dx.doi.org/10.1016/j.toxlet.2014.06.295 P-1.116 Multiple modes of chemokine regulation by nitro-PAHs in bronchial epithelial cells Johan Øvrevik ∗ , Marit Låg, Magne Refsnes, Per Schwarze, Jørn A. Holme Norwegian Institute of Public Health, Oslo, Norway Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are ubiquitous pollutants characteristic of diesel emissions. Here we summarize our work to elucidate the mechanisms of nitro-PAH induced chemokine responses in bronchial epithelial BEAS-2B cells. 1-nitropyrene (1-NP) and 3-nitroflouranthene (3-NF) induced identical cytokine/chemokine expression patterns dominated by maximum effects on CXCL8 (IL-8). By use of siRNA-transfection, blocking antibodies, pharmacological inhibitors, chelators and antioxidants, we found that that 1-NP induced CXCL8 responses required signaling through the TACE/TGF-␣/EGFR-cascade and also involved signaling through 2-adrenergic receptors (2AR), calcium, and reactive oxygen species (ROS) and/or reactive metabolites. The CYP-inhibitor ANF and knockdown of CYPOR which donates electrons to CYP-enzymes reduced 1-NP-induced CXCL8 responses, suggesting that CYP-mediated metabolism is of impor-