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Molecular Mechanism of the Synergistic Cytotoxicity between Pentachlorophenol and Copper Complex in Human Liver Cells
protonation to produce a semiquinoneimine-type free radical • (RSFH ) as demonstrated by direct ESR. Spin-trapping studies of incubations containing RSF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase (SOD)• sensitive DMPO/ OOH adduct. Both absorption and fluorescence spectra of RSF in the presence of NADH demonstrated that the • RSFH was further reduced during irradiation with formation of its colorless dihydroquinoneimine form, dihydroresorufin (RSFH2). • Both RSFH and RSFH2, when formed in an aerobic system, are immediately oxidized by oxygen, which regenerates the dye and forms superoxide. Oxygen consumption measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The suppression of oxygen consumption by addition of SOD or catalase further confirms the production of superoxide and hydrogen peroxide. In short, even room light leads to RSF-dependent reactive oxygen species formation in the Amplex Red assay.
studied, their combined cytotoxicity still has been overlooked so far. Here we show that when non- or sub-toxic levels of metam sodium and copper or zinc ions, were combined, a remarkable synergistic toxicity was observed in HepG2 cells, a human liver cell culture model. The synergism was found to be due to the formation of lipophilic metam-copper or metam-zinc complexes which facilitated metal ion transport into the HepG2 cells. Interestingly and unexpectedly, different modes of cell death were observed between metam/copper and metam/zinc: apoptosis for metam/copper as evidenced by decrease of mitochondrial membrane potential and anti-apoptotic proteins expression, cytochrome c release from mitochondria and caspase-3, 9 activation resulting from oxidative stress (GSH/GSSG decrease and ROS accumulation); necrosis for metam/zinc. This is the first report demonstrating different modes of cell death could be induced by a dithiocarbamate compound and copper or zinc ions, which may have interesting biological and environmental implications.
doi: 10.1016/j.freeradbiomed.2010.10.280
doi: 10.1016/j.freeradbiomed.2010.10.282
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Molecular Mechanism of the Synergistic Cytotoxicity between Pentachlorophenol and Copper Complex in Human Liver Cells
Global Profiling of Reactive Oxygen and Nitrogen Species in Cellfree and Model Cellular Systems – Real Time Fluorescence Monitoring and HPLC Measurements
Ben-Zhan Zhu1, and Zhi-Guo Sheng1 1 Chinese Academy of Sciences, PR China Organic and inorganic environmental pollutants often co-exist and these co-occurring toxicants may interact to produce unexpected combination effects. Both pentachlorophenol (PCP) and copper1,10-phenanthroline (Cu(OP)2) have been used as wood preservatives, and found to co-exist near the wood-preserving facilities and body fluids and tissues of people who are not occupationally exposed. We have shown previously that combination of PCP and Cu(OP)2 caused synergistic cytotoxicity in a bacterial model, possibly by forming a lipophilic ternary complex. However, it is not clear whether these synergistic toxic effects could take place in human liver cells, and if so, what is the underlying molecular mechanism. Here we show that PCP and Cu(OP)2 could cause synergistic cytotoxicity in both HepG2 and HL-7702 cells, and HepG2 cells were more susceptible. PCP/Cu(OP)2-induced cytotoxicity was found mainly due to apoptosis as evidenced by decrease of mitochondrial membrane potential (MMP) and anti-apoptotic proteins expression, cytochrome c release from mitochondria and caspase-3, 9 activation, which were found to result from oxidative stress (ROS accumulation vs. GSH/GSSG decrease). Furthermore, X-linked inhibitor of apoptosis (XIAP) mediated by MAPK/AKT played a pivotal role in PCP/Cu(OP)2-induced cytotoxicity difference. These results suggested that combination of non-toxic levels of PCP and Cu(OP)2 results in synergistic cytotoxicity in both HepG2 and normal HL-7702 cells by ROS-mediated apoptosis in a mitochondrial dependent manner.
Jacek Zielonka1, Monika Zielonka1, Adam Sikora2, Jan Adamus2, Joy Joseph1, Micael Hardy3, Olivier Ouari3, and Balaraman Kalyanaraman1 1 2 Medical College of Wisconsin Technical University of Lodz, 3 Poland, Universités d'Aix-Marseille, France • •– Superoxide radical anion (O2 ) reacts with nitric oxide ( NO) with diffusion-controlled rate constant, forming a potent oxidizing and – nitrating species, peroxynitrite (ONOO ). Here we show the application of novel, boronate-based fluorogenic probes for the detection and quantification of ONOO formed in situ in cell-free systems and in RAW 264.7 macrophages stimulated to produce • •– •– O2 and/or NO. The oxidation of the probes under various O2 • and NO fluxes can be monitored simultaneously in real time using 96-well fluorescence plate reader. In a similar manner the – production of ONOO by activated macrophages can be – •– investigated. The formation of ONOO precursors, i.e. O2 and • NO can be monitored using hydroethidine (HE) and diaminofluorescein (DAF-2) probes, respectively. Hydroethidine forms a superoxide-specific product, 2-hydroxyethidium, while • DAF-2 in the presence of NO and oxidant undergoes conversion to highly fluorescent triazole derivative. The kinetic fluorescence data obtained with those probes will be compared with the results of HPLC analyses, to monitor probes’ uptake and the distribution of the oxidation/nitrosation products. We conclude that the global analysis of ROS and RNS using multiple fluorescence probes described here will help establish the role of ROS/RNS in cell signaling. doi: 10.1016/j.freeradbiomed.2010.10.283
doi: 10.1016/j.freeradbiomed.2010.10.281
275 Mechanism of the Synergistic Cytotoxicity between Metam Sodium and Copper (II) or Zinc (II) in Human Liver Cells: Apoptosis vs Necrosis Ben-Zhan Zhu1, and Rui-Mei Fan1 1 Chinese Academy of Sciences, PR China Both metam sodium (sodium methyldithiocarbamate, the third most commonly used agricultural pesticide in the US) and copper or zinc compounds have been widely used as fungicides, and therefore may co-occur in our environment. While the individual toxicity of either metam sodium or metal ion has been extensively
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SFRBM/SFRRI 2010
studied, their combined cytotoxicity still has been overlooked so far. Here we show that when non- or sub-toxic levels of metam sodium and copper or zinc ions, were combined, a remarkable synergistic toxicity was observed in HepG2 cells, a human liver cell culture model. The synergism was found to be due to the formation of lipophilic metam-copper or metam-zinc complexes which facilitated metal ion transport into the HepG2 cells. Interestingly and unexpectedly, different modes of cell death were observed between metam/copper and metam/zinc: apoptosis for metam/copper as evidenced by decrease of mitochondrial membrane potential and anti-apoptotic proteins expression, cytochrome c release from mitochondria and caspase-3, 9 activation resulting from oxidative stress (GSH/GSSG decrease and ROS accumulation); necrosis for metam/zinc. This is the first report demonstrating different modes of cell death could be induced by a dithiocarbamate compound and copper or zinc ions, which may have interesting biological and environmental implications.
doi: 10.1016/j.freeradbiomed.2010.10.280
doi: 10.1016/j.freeradbiomed.2010.10.282
274
276
Molecular Mechanism of the Synergistic Cytotoxicity between Pentachlorophenol and Copper Complex in Human Liver Cells
Global Profiling of Reactive Oxygen and Nitrogen Species in Cellfree and Model Cellular Systems – Real Time Fluorescence Monitoring and HPLC Measurements
Ben-Zhan Zhu1, and Zhi-Guo Sheng1 1 Chinese Academy of Sciences, PR China Organic and inorganic environmental pollutants often co-exist and these co-occurring toxicants may interact to produce unexpected combination effects. Both pentachlorophenol (PCP) and copper1,10-phenanthroline (Cu(OP)2) have been used as wood preservatives, and found to co-exist near the wood-preserving facilities and body fluids and tissues of people who are not occupationally exposed. We have shown previously that combination of PCP and Cu(OP)2 caused synergistic cytotoxicity in a bacterial model, possibly by forming a lipophilic ternary complex. However, it is not clear whether these synergistic toxic effects could take place in human liver cells, and if so, what is the underlying molecular mechanism. Here we show that PCP and Cu(OP)2 could cause synergistic cytotoxicity in both HepG2 and HL-7702 cells, and HepG2 cells were more susceptible. PCP/Cu(OP)2-induced cytotoxicity was found mainly due to apoptosis as evidenced by decrease of mitochondrial membrane potential (MMP) and anti-apoptotic proteins expression, cytochrome c release from mitochondria and caspase-3, 9 activation, which were found to result from oxidative stress (ROS accumulation vs. GSH/GSSG decrease). Furthermore, X-linked inhibitor of apoptosis (XIAP) mediated by MAPK/AKT played a pivotal role in PCP/Cu(OP)2-induced cytotoxicity difference. These results suggested that combination of non-toxic levels of PCP and Cu(OP)2 results in synergistic cytotoxicity in both HepG2 and normal HL-7702 cells by ROS-mediated apoptosis in a mitochondrial dependent manner.
Jacek Zielonka1, Monika Zielonka1, Adam Sikora2, Jan Adamus2, Joy Joseph1, Micael Hardy3, Olivier Ouari3, and Balaraman Kalyanaraman1 1 2 Medical College of Wisconsin Technical University of Lodz, 3 Poland, Universités d'Aix-Marseille, France • •– Superoxide radical anion (O2 ) reacts with nitric oxide ( NO) with diffusion-controlled rate constant, forming a potent oxidizing and – nitrating species, peroxynitrite (ONOO ). Here we show the application of novel, boronate-based fluorogenic probes for the detection and quantification of ONOO formed in situ in cell-free systems and in RAW 264.7 macrophages stimulated to produce • •– •– O2 and/or NO. The oxidation of the probes under various O2 • and NO fluxes can be monitored simultaneously in real time using 96-well fluorescence plate reader. In a similar manner the – production of ONOO by activated macrophages can be – •– investigated. The formation of ONOO precursors, i.e. O2 and • NO can be monitored using hydroethidine (HE) and diaminofluorescein (DAF-2) probes, respectively. Hydroethidine forms a superoxide-specific product, 2-hydroxyethidium, while • DAF-2 in the presence of NO and oxidant undergoes conversion to highly fluorescent triazole derivative. The kinetic fluorescence data obtained with those probes will be compared with the results of HPLC analyses, to monitor probes’ uptake and the distribution of the oxidation/nitrosation products. We conclude that the global analysis of ROS and RNS using multiple fluorescence probes described here will help establish the role of ROS/RNS in cell signaling. doi: 10.1016/j.freeradbiomed.2010.10.283
doi: 10.1016/j.freeradbiomed.2010.10.281
275 Mechanism of the Synergistic Cytotoxicity between Metam Sodium and Copper (II) or Zinc (II) in Human Liver Cells: Apoptosis vs Necrosis Ben-Zhan Zhu1, and Rui-Mei Fan1 1 Chinese Academy of Sciences, PR China Both metam sodium (sodium methyldithiocarbamate, the third most commonly used agricultural pesticide in the US) and copper or zinc compounds have been widely used as fungicides, and therefore may co-occur in our environment. While the individual toxicity of either metam sodium or metal ion has been extensively
S106
SFRBM/SFRRI 2010