Abstract
Cadmium has recently been shown to induce apoptosis in C6 glioma cells via disruption of the mitochondrial membrane potential and subsequent caspase 9-activation. Here we show that both H2O2 and CdCl2 induced apoptotic DNA fragmentation in C6 cells. The employment of glutathione as an antioxidant prevented the induction of apoptotic DNA fragmentation by cadmium completely and catalase strongly reduced cadmium-induced DNA fragmentation suggesting that cadmium exerts its apoptotic effects at least partly via the production of H2O2. Apoptosis may be induced by cadmium indirectly through formation of oxidative stress, e.g., by inhibition of antioxidant enzymes. After incubation of C6 cells with cadmium for short times (up to 4 h), we analyzed the formation of intracellular reactive oxygen species and cellular lipid peroxidation. After 1 h of incubation with inreasing concentrations of CdCl2 (1–500 μM), no increase in dichlorofluorescein fluorescence was found. At variance, lipid peroxidation was slightly elevated after 2 h incubation with cadmium (50–100 μM). Furthermore, we analyzed the modulation of markers for oxidative stress after prolonged (24 h) exposure to cadmium. The intracellular glutathione content as measured using the fluorescent probe monobromobimane was decreased after incubation with CdCl2 (0.5–10 μM) for 24 h. Furthermore, we measured the effect of cadmium on the level of oxidized DNA lesions (predominantly 8-hydroxyguanine) using the bacterial Fpg-DNA-repair protein. After 24 h of incubation with 5 μM CdCl2 we found a sixfold increase in Fpg-sensitive DNA-lesions. We conclude that short time incubations with cadmium (up to 4 h) caused only slight or insignificant effects on the generation of reactive oxygen species (formation of thiobarbituric acid reactive substances, fluorescence of dichlorofluorescein), whereas incubation with this heavy metal for 24 h lead to a decrease in intracellular glutathione concentration and an increase in oxidative DNA-lesions. Our data demonstrate that cadmium as similar to H2O2 is a potent inducer of apoptosis in C6 cells. Even if cadmium unlike Fenton-type metals can not produce reactive oxygen species directly, the apoptotic effects of cadmium at least in part are mediated via induction of oxidative stress. Because both apoptosis and oxidative stress are thought to play important roles in neurodegenerative diseases, low concentrations of cadmium that initiate programmed cell death may lead to a selective cell death in distinct brain regions via generation of oxidative stress.
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References
Amoruso MA, Witz G, Goldstein BD. 1982 Enhancement of rat and human phagocyte superoxide anion radical production by cadmium in vitro. Toxicol Lett 10, 133–138.
Asmuss M, Mullenders LH, Eker A, Hartwig A. 2000 Differential effects of toxic metal compounds on the activities of Fpg and XPA, two zinc finger proteins involved in DNA repair. Carcinogenesis 21, 2097–2104.
Babich H, Shopsis C, Borenfreund E. 1986 In vitro cytotoxicity testing of aquatic pollutants (cadmium, copper, zinc, nickel) using established fish cell lines. Ecotox Environ Safety 11, 91–99.
Bagchi D, Joshi SS, Bagchi M, Balmoori J, Benner EJ, Kuszynski CA, Stohs SJ. 2000 Cadmium-and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. J Biochem Mol Toxicol 14, 33–41
Benda P, Lightbody J, Sato G, Levine L, Sweet W. 1968 Differentiated rat glial cell strain in tissue culture. Science 161, 370–371.
Beyersmann D, Hechtenberg S. 1997 Cadmium, gene regulation, and cellular signalling in mammalian cells. Toxicol Appl Pharmacol 144, 247–261.
Buttke TM, Sandstrom PA. 1994 Oxidative stress as a mediator of apoptosis. Immunol Today 15, 7-10.
Chin TA, Templeton DM. 1993 Protective elevations of glutathione and metallothionein in cadmium-exposed mesangial cells. Toxicology 77, 145–56.
Chrestensen CA, Starke DW, Mieyal JJ. 2000 Acute cadmium exposure inactivates thioltransferases (glutaredoxin), inhibits intracellular reduction of protein-glutathionyl-mixed disulfides, and initiates apoptosis. J Biol Chem 275, 26556–26565.
Chuang SM, Wang IC. Yang JL. 2000 Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis 21, 1423–1432.
Coogan TP, Bare RM, Bjornson EJ, Waalkes MP. 1994 Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells. J Toxicol Environ Health 41, 233–245.
Dally H, Hartwig A. 1997 Induction and repair inhibition of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis 18, 1021–1026.
Draper HH, Hadley M. 1990 Malondialdehyde determination as a index of lipid peroxidation. Meth Enzym 186, 421–431.
El-Maraghy SA, Gad MZ, Fahim AT, Hamdy MA. 2001 Effect of cadmium and aluminum intake on the antioxidant status and lipid peroxidation in rat tissues. J Biochem Mol Toxicol 15, 207–214.
Eneman JD, Potts RJ, Osier M, Shukla GS, Lee CH, Chiu JF, Hart BA. 2000 Suppressed oxidant-induced apoptosis in cadmium adapted alveolar epithelial cells and its potential involvement in cadmium carcinogenesis. Toxicology 147, 215–228.
Gaubin Y, Vaissade F, Croute F, Beau B, Soleilhavoup JP, Murat JC. 2000 Implication of free radicals and glutathione in the mech-anism of cadmium-induced expression of stress proteins in the A549 human lung cell line. Biochim Biophys Acta 1495, 4–13.
Haase H, WÄtjen W, Beyersmann D. 2001 Zinc induces apoptosis that can be suppressed by lanthanum in C6 rat glioma cells. Biol Chem 382, 1227–1234.
Habeebu SS, Liu J, Klaassen CD. 1998 Cadmium-induced apoptosis in mouse liver. Toxicol Appl Pharmacol 149, 203–209.
Hamada T, Tanimoto A, Sasaguri Y. 1997 Apoptosis induced by cadmium. Apoptosis 2, 359–367.
Hart BA, Lee CH, Shukla A, Osier m, Eneman JD and Chiu JF 1999 Characterization of cadmium-induced apoptosis in rat lung epithelial cells: evidence for the participation of oxidative stress. Toxicology 133, 43–58.
Hartwig A, Klyszcz-Nasko H, Schlepegrell R, Beyersmann D. 1993 Cellular damage by ferric nitrilotriacetate and ferric citrate in V79 cells: Interrelationship between lipid peroxidation, DNA strand breaks and sister chromatid exchanges. Carcinogenesis 14, 107–112
Hartwig A, Dally H, Schlepegrell R. 1996 Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding. Toxicology 110, 1–6.
Hassoun EA, Stohs SJ. 1996 Cadmium-induced production of superoxide anion and nitic oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures. Toxicology 112, 219–226.
Hatcher EL, Chen Y, Kang YJ. 1995 Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities. Free Radic Biol Med 19, 805–812.
Kerr JF, Wyllie AH, Currie AR. 1972 Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26, 239–257.
Kim MS, Kim BJ, Woo HN, Kim KW, Kim KB, Kim IK, Jung YK. 2000 Cadmium induces caspase-mediated cell death: suppression by Bcl-2. Toxicology 145, 27–37.
Kondoh M, Araragi S, Sato K, Higashimoto M, Takiguchi M, Sato M. 2002 Cadmium induces apoptosis partly via caspase-9 activation in HL-60 cells. Toxicology 170, 111–117.
Kumar R, Agarwal AK, Seth PK. 1996 Oxidative stress-mediated neurotoxicity of cadmium. Toxicol Lett 89, 65–69.
Mosmann T. 1983 Rapid colorimetric assay for growth and survival: Application to proliferation and cytotoxicity assay. J Immunol Meth 65, 55–63.
Neuhoff V, Philipp K, Zimmer HG, Mesecke S. 1979 A simple, sensitive and volume-independent method for quantitative protein determination which is independent of other external influences. Hoppe-Seyler's Z Physiol Chem 360, 1657–1670.
Nigam D, Shukla GS, Agarwal AK. 1999 Glutathione depletion and oxidative damage in mitochondria following exposure to cadmium in rat liver and kidney. Toxicol Lett 106, 151–157.
Ochi T, Takahashi K, Ohsawa M. 1987 Indirect evidence for the induction of a prooxidant state by cadmium chloride in cultured mammalian cells and a possible mechanism for the induction. Mutat Res 180, 257–266.
Pourahmad J, O'Brien PJ. 2000 A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+. Toxicology 143, 263–273.
Shaikh ZA, Vu TT, Zaman K. 1999 Oxidative stress as a mechanism of chronic cadmium-induced hepatotoxicity and renal toxicity and protection by antioxidants. Toxicol Appl Pharmacol 154, 256–263.
Stacey NH, Cantilena LR, Klaassen CD. 1980 Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes. Toxicol Appl Pharmacol 53, 470–480.
Stohs SJ, Bagchi D. 1995 Oxidative mechanisms in the toxicity of metal ions. Free Rad Biol Med 18, 321–336.
Stohs SJ, Bagchi D, Hassoun E, Bagchi M. 2000 Oxidative mechanisms in the toxicity of chromium and cadmium ions. J Environ Pathol Toxicol Oncol 19, 201–213.
Stohs SJ, Bagchi D, Hassoun E, Bagchi M. 2001 Oxidative mechanisms in the toxicity of chromium and cadmium ions. J Environ Pathol Toxicol Oncol 20, 77–88.
Szuster-Ciesielska A, Stachura A, Slotwinska M, Kaminska T, Sniezko R, Paduch R, Abramczyk D, Filar J, Kandefer-Szerszen M. 2000 The inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in cell cultures. Toxicology 145, 159–171.
Tatrai E, Kovacikova Z, Hudak A, Adamis Z, Ungvary G. 2001 Comparative in vitro toxicity of cadmium and lead on redox cycling in type II pneumocytes. J Appl Toxicol 21, 479–483.
Xu C, Johnson JE, Singh PK, Jones MM, Yan H, Carter CE. 1996 In vivo studies of cadmium-induced apoptosis in testicular tissue of the rat and its modulation by a chelating agent. Toxicology 107, 1–8.
WÄtjen W, Cox M, Biagioli M, Beyersmann D. 2002 Cadmiuminduced apoptosis in C6 glioma cells: Mediation by caspase 9-activation. BioMetals 15, 15–25.
Yang CF, Shen HM, Shen Y, Zhuang ZX, Ong CN. 1997 Cadmiuminduced oxidative cellular damage in human fetal lung fibroblasts (MRC-5-cells). Environ Health Perspect 105, 712–716.
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Wätjen, W., Beyersmann, D. Cadmium-induced apoptosis in C6 glioma cells: Influence of oxidative stress. Biometals 17, 65–78 (2004). https://doi.org/10.1023/A:1024405119018
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DOI: https://doi.org/10.1023/A:1024405119018