Studies on the Molecular Mechanisms Involved in the Potentiating Effects by Metal Chelators on the Copper Ion-Induced Cytotoxicity

• Main Authors: Sung Ho Chen, 陳松鶴
• Other Authors: Shoei-Yn Lin-Shiau, Ph.D.
• Format: Others
• Language: en_US
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/55327234561641569733

博士 === 國立臺灣大學 === 毒理學研究所 === 89 === Abstract Various metal salts such as copper and pesticides such as dithiocarbamates are increasingly consumed in industrialized countries, contaminant in food and our environment. Dithiocarbamates are not only the widely used pesticides but also the medical fungicides and developing anti-HIV agents. Since dithiocarbamates are metal chelators. Therefore it is important for us to understand the biological properties of dithiocarbamates-metal complex. In this study, we attempted to investigate the cytotoxic effects of dithiocarbamates, metal ions and the complexes. Moreover, other metal chelators, e.g. pyrrolidine dithiocarbamate (PDTC), disulfiram (DSF), neocuproine (NCP), 8-hydroxyquinoline (8-OHQ), ortho-phenanthroline (OP) were also completely studied. In addition to their chelating effects, there is increasing evidence that these chelating agents possess both antioxidant and pro-oxidant effects. The antioxidant effects of chelating agents have been extensively investigated in preventing cell death induced by different insults. However, it still lacks a systemic study on the molecular mechanisms of the cytotoxic effects of the different metal chelators (PDTC, DSF, NCP, 8-OHQ, and OP) on the primary rat cortical cultured astrocytes. Our results indicate that the DSF is the most, and 8-OHQ was the least toxic. The IC50 of PDTC, DSF, NCP, 8-OHQ, OP, CuCl2 were 300, 21, 600, 700, 250 and 180 mM respectively. The mechanism of cytotoxic effects of pyrrolidine dithiocarbamate-Cu2+ complex on various cells The chelating and antioxidant effects of pyrrolidine dithiocarbamate (PDTC) have been extensively investigated for preventing cell death induced by different insults. However, the toxic effects of PDTC have been studied only recently and fewer studies on the toxic effects have been reported. Since the molecular mechanisms of apoptotic process remain to be elucidated, in this study, we attempted to elucidate the death-signaling pathways of PDTC, CuCl2, and PDTC-Cu2+ complex in the cultured rat astrocytes, HL-60 cells and C6 glioma cells. In this study, we demonstrated that both PDTC and Cu2+ alone induced cell death in cortical astrocytes, C6 glioma cells, and HL-60 cells with different sensitivity respectively. Our data showed that both PDTC and Cu2+ alone were rated from only weakly toxic in inducing cell death in cortical astrocytes with IC50 of 300 mM and 180 mM, respectively, in HL-60 with IC50 of 100 mM, 62 mM, but more toxic on C6 cell with IC50 of 7.2 mM, 30 mM. However, PDTC and Cu2+ in the complex form markedly potentiated with each other by about 1000-fold with IC50 of 0.3 mM PDTC plus 10 mM on astrocytes, IC50 of 0.14 mM PDTC plus 1 mM on HL-60 cells, and IC50 of 0.2 mM PDTC plus 2 mM on C6 cells. Primary cultures seemed more resistant to PDTC than cell line cultures. In contrast to Cu2+, other metals at concentrations of 3~10 mM (VO45+, Cr6+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Pb2+, Bi2+, Ba2+, UO2+, Cs+, SeO4-2, La3+ ) had no such obvious potentiating effects on PDTC. PDTC, Cu2+ alone and PDTC-Cu2+ complex could elevate the intracellular copper content in a time- and concentration-dependent manner. The changes of morphology (nuclear condensation), apoptotic body formation and hypodiploidity of DNA suggested that the PDTC-Cu2+ complex induced cell death through an apoptotic process in all three kinds of cells. PDTC-Cu2+ complex could exert DNA ladder more clearly on HL-60 cells than on astrocytes. We also provided evidence that PDTC-Cu2+ complex exerted comet pattern on single cell both in astrocytes and C6 cells. Further studies showed that the PDTC-Cu2+ complex decreased the mitochondrial membrane potential, increased reactive oxygen species production, and depleted GSH and ATP contents. Following the increased oxidative stress, all three kinds of cells showed that PDTC-Cu2+ complex differentially activated the JNKs, ERK and p38 kinase. PDTC-Cu2+complex persistently activated JNKs, but only transiently increased p38, and ERK activity. NF-kB and AP-1 were also activated on HL-60 cells. The caspase 3 activation and PARP cleavage were triggered in a time-dependent manner. PDTC-Cu2+ complex could induce caspase 3 activation about 15 fold on HL-60 cells and 2 fold on astrocytes. All of these effects were consistent with the increased cellular Cu contents and cytotoxicity. The non-permeable copper-specific chelator bathocuproine disulfonate (BCPS) but not the permeable Cu2+ chelator neocuproine abolished all the observed effects. Antioxidants (N-acetyl-cystein, vitamin C), catalase and Cu binding proteins (albumin, hemoglobin and higher serum) reduced the cytotoxic effects of PDTC-Cu2+ complex. Therefore, we concluded that the death signaling pathway of PDTC-Cu2+ complex was mediated by oxidative stress and subsequent JNK activation. These findings imply that PDTC, a widely used pesticide and medicine which is capable of penetrating the blood brain barrier, may directly cause neurotoxicity and immunotoxicity or indirectly through astrocyte dysfunction. Oxidative stress and JNK activation involved in apoptosis of primary astrocytes induced by disulfiram-Cu2+ complex Disulfiram (DSF) is frequently used in treatment of alcoholism. In this study, we found that CuCl2 (1-10 mM) but not other metal ions (Fe2+, Zn2+, Pb2+) markedly potentiated the DSF-induced cytotoxicity by 440-fold in primary astrocytes. Thus, the molecular mechanisms of cytotoxic effects induced by DSF-Cu2+ complex were explored. The changes in morphology (nuclear condensation and apoptotic body formation) and hypodiploid of DNA suggested that DSF-Cu2+ complex induced an apoptotic process. Our studies on the death signaling pathway reveals that decreased mitochondrial membrane potential, increased free radical production, and depleted GSH and ATP contents were involved. Subsequently, DSF-Cu2+ complex activated JNKs and caspase 3 followed by PARP degradation in a time-dependent manner. The copper chelator bathocuproine disulfonate blocked all of these cellular events, suggesting that Cu2+ is essential for the death signaling. The antioxidants, N-acetylcysteine and vitamin C also inhibited the cytotoxic effect. Thus, we conclude for the first report that DSF-Cu2+ complex induces apoptosis perhaps necrosis at late stage mediated by oxidative stress followed by sequential activation of JNK, caspase 3 and PARP degradation. These findings imply that the axonal degeneration and neurotoxicity observed in the chronic administration of DSF are perhaps, at least in part, due to the cytotoxic effect of DSF-Cu2+ complex form endogenously. Molecular mechanisms of cytotoxic effects of by neocuproine-Cu2+ complex in the cultured rat cortical astrocytes Neocuproine (NCP) is a specific Cu2+ chelator frequently used as an inhibitor of oxidative stress. In this study, we provided evidence that CuCl2 at a low concentration of 10 mM dramatically enhanced the cytotoxic effect of NCP by about 10,000-fold in the cultured rat cortical astrocytes. The IC50 is decreased from 600 mM to 0.06 mM. Fe2+, Zn2+, and Pb2+ had no such potentiating effects. NCP-Cu2+ complex induced apoptotic process on primary astrocytes. Studies on the machinery underlying the cell death indicated that the decreased mitochondrial membrane potential, increased free radical generation, and depleted GSH and ATP contents were sequentially induced by NCP-Cu2+ complex. Following this oxidative stress, we have found that JNK is persistently activated, while ERK and p38 are transiently activated to a lessor content. The subsequent activation of caspase 3 and PARP degradation was also evidently. The BCPS reversed all of these events, which was correlated with the marked elevation of Cu content induced by NCP-Cu2+ complex. This finding implies that NCP may induce oxidative neurotoxicity of the brain through damaging the neuroprotective astrocytes. The conventional application of NCP as an inhibitor of oxidative stress should be cautiously notified. Cytotoxic effects of 8-hydroxyquinoline-Cu2+ complex in the cultured rat cortical astrocytes 8-Hydroxyquinoline (8-OHQ) is often used as a preservative in immunobiological preparations of tuberculin (PDD) in medical examination. 8-OHQ-Cu2+ was produced to be pesticides. Therefore people could expose to 8-OHQ in medical use or food or environmental contamination. Recently, 8-OHQ (1 mM) plus Cu2+ (30 mM) has been shown to induced apoptosis in J774.A1 macrophage-cells. We try to explore the cytotoxic effects and mechanisms of 8-OHQ on primary rat cultured astrocytes. Our data showed that only high concentrations of either 8-OHQ or CuCl2 could induce cell death in rat cortical astrocytes; IC50 was 600 ± 46 mM and 180 ± 12.5 mM respectively. Cu2+ alone at low concentration of 1-10 mM was nontoxic, but Cu2+ (10 mM) become highly toxic in the presence of 8-OHQ (1-10 mM). We found that the 8-OHQ-Cu2+ complex induced the cytotoxic effect and hypodiploidic cells in concentration- and time-dependent manner. The IC50 of 8-OHQ was decreased by 10 mM CuCl2 from 600 mM to 2 mM; therefore the potentiation factor is 300-fold. Moreover, 8-OHQ-Cu2+ complex at 10 mM induced cytotoxicity at a much faster rate than that induced by either 1000 mM 8-OHQ alone or 300 mM CuCl2. 8-OHQ-Cu2+ complex, but not BCPS 300 mM, 8-OHQ 3 mM, nor Cu2+ 10 mM alone caused the astrocytes dissociate gradually within 3 h and then progressively detached. 8-OHQ-Cu2+ complex triggered apoptotic cell death and necrosis at late stage. 8-OHQ-Cu2+ (10 mM) complex markedly increased the copper content of the subcellular fractions of the plasma membrane, cytosol, and nuclei. The elevation profile of copper contents was closely correlated with the toxic effects detected by MTT reduction test, hypodiploid of DNA, and the morphological changes. 8-OHQ-Cu2+ complex increased free radical production followed by rapidly GSH decreased and ATP content depleted at 3 h. All the morphological and elevation of intracellular copper content and the cellular changes induced by 8-OHQ-Cu2+ complex but not 8-OHQ alone were completely prevented by BCPS. Oxidative stress induced by 1,10-ortho-phenanthroline-Cu2+ complex in the cultured rat cortical astrocytes 1,10-phenanthroline (OP) is a lipophilic metal chelator. OP also possessed antifungal activity and used in determination of nickel, ruthenium, silver and, serum ion and other metals. OP or CuCl2 could leads to cell death in rat cortical astrocytes with IC50 250 ± 31 mM and 180 ± 12.5 mM respectively. The non-toxic concentration of Cu2+ (1~10 mM) could markedly potentiate the cytotoxicity of OP in concentration- and time-dependent manner (0.3~10 mM). Cu2+ (10 mM) potentiated with each other the cytotoxic effect of OP by about 150-fold by decreasing IC50 of OP from 300 mM to 2 mM. OP-Cu2+ complex could dose- and time-dependently induced hypodiploidic cells and could be completely reversed by BCPS, or partially reversed by NAC (3 mM). Application of OP-Cu2+ complex led the astrocyte shrinkage and apoptotic like morphological changes. The Cu2+(300 mM), and OP-Cu2+ (10 mM) complex significantly increase the copper contents of subcellular fractions as well as on DNA component after 3-h incubation. OP-Cu2+ complex increased ROS production in concentration- and time-dependant manner like other chelators. We also found that OP (10 mM)-Cu2+ (10 mM) complex rapidly decreased the GSH and ATP contents at 3h after application of the OP-Cu2+ complex. All the morphology and sequential cellular events induced by OP-Cu2+ complex, but not OP alone could be blocked by BCPS. It reveals that OP-Cu2+ complex, but not OP alone induced the cytotoxic effect by the elevation of intracellular copper contents. Comparism of enhancing cellular Cu2+ uptake in correlation with the cytotoxicity induced by the five different metal chelating agents We try to figure out whether these five chelating trigger the cell death of primary astrocytes through the common signaling pathway even they own the different kind of structures. First of all, we compare the correlation between their cytotoxicity and their ability on enhancing cellular Cu2+ uptake induced by the five different chelating agents-Cu2+ complexes. In contrast to only PDTC, 8-OHQ alone as well as high dose of Cu2+ alone, all of these chelating agents-Cu2+ complexes could concentration- and time dependently elevate the intracellular copper content at membrane, cytosol nuclear and DNA fraction. The elevation of intracellular copper content produced by chelating agents-Cu2+ complexes and toxic dose of copper ion parallel to the cytotoxicity of these chelating agents-Cu2+ complexes and copper ion. Therefore we confirm that all these five chelating agents-Cu2+ complexes induced the cell death of astrocytes through the elevation the uptake and the increase the intracellular copper contents and then triggered the death signaling pathways. Further studies showed that the onset of decreased mitochondrial membrane potential was dependent on the concentration of the chelating agents-Cu2+ complexes. The higher toxic doses triggered the more rapid and large decrease of mitochondrial membrane potential. The decline pattern of mitochondrial membrane potential induced by Cu2+ complexes of PDTC, DSF and NCP is really similar. The chelating agents-Cu2+ complex increased reactive oxygen species (ROS) production in concentration- and time-dependant manner with a rapid onset at 1 h and reached a peak at 3h after application of chelating agents-Cu2+ complex. The most toxic group of DSF-Cu2+ complex induced the most induction of ROS. The less toxic groups of PDTC-Cu2+, 8-OHQ-Cu2+, and OP-Cu2+ complex (PDTC, 8-OHQ, OP, Cu2+ 0.1, 1, 3, 10 mM respectively) caused less and slower increase of ROS. We also found that chelating agents-Cu2+ complexes rapidly decreased GSH contents to about 65 ± 8.1% within 1 h and reached minimum at 6 h. The decline patterns of GSH induced by these chelating agents-Cu2+ complex were almost the same. On the other hand, the chelating agents-Cu2+ complexes also decrease the ATP content in a time-dependant manner with a minor decrease at 1 h and almost decline to about 30 % of control at 3h after application of chelating agents-Cu2+ complexes. This pattern was slower than that of the rapid decline induced by necrotic death. These chelating agents-Cu2+ complexes also produced the same decline pattern of ATP content. Following the increased oxidative stress, the chelating agents-Cu2+ complexes persistently activated the JNKs, but only transiently increased p38, and ERK activity. All of these effects were consistent with the increased intracellular copper contents. The non-permeable copper-specific chelator bathocuproine disulfonate (BCPS) but not the permeable Cu2+ chelator neocuproine abolished all the observed effects. Antioxidants (N-acetyl-cysteine, vitamin C), Cu2+ binding proteins (albumin, hemoglobin and higher serum) reduced the cytotoxic effects of chelating agents-Cu2+ complexes. Therefore, we concluded that the death signaling pathways induced by these chelating agents-Cu2+ complexes were mediated by the decrease of mitochondria membrane potential, increase of oxidative stress, depletion of GSH, ATP and subsequent JNK, NF-kB and AP-1 activation. These findings imply that it should be cautious to use these chelating agents, a widely used in pesticides or medicines by antioxidant or chelating effects which are capable of penetrating the blood brain barrier. Furthermore we need to prevent the food- and environmental- contamination by appropriated control to keep from their direct neurotoxic effects or through the dysfunction of neuroprotective astrocytes.