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Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line

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Abstract

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 μM induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 μM of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (ΔΨm) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC6(3), a cationic lipophilic dye into mitochondria indicating the disruption of ΔΨm. This drop in ΔΨm was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation → excess ROS production → GSH depletion → oxidative stress → disruption of ΔΨm → release of cytochrome C and other apoptosis related proteins to cytosol → apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.

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References

  1. Gupta PK, Gupta RC: Pharmacology, toxicology and degradation of endosulfan. Toxicology 13: 115–130, 1979

    PubMed  Google Scholar 

  2. Chaturvedi AK: Toxicological evaluation of mixture of ten widely used pesticides. J Appl Toxicol 13: 183–188, 1993

    PubMed  Google Scholar 

  3. Holcombe GW, Phipps GL, Fiandt JT: Toxicity of selected priority pollutants to various aquatic organisms. Ecotoxicol Environ Safety 7: 400–409, 1983

    Article  PubMed  Google Scholar 

  4. FAO/WHO: Endosulfan. In: 1974 Evaluations of Some Pesticide Residues in Food. Rome Food and Agriculture Organization of the United Nations

  5. WHO: Endosulfan, Environmental Health Criteria 40. World Health Organization, Geneva

  6. Chugh SN, Dhawan R, Agrawal N, Mahajan SK: Endosulfan poisoning in Northern India: A report of 18 cases. Int J Clin Pharmacol Ther 36: 474–477, 1998

    PubMed  Google Scholar 

  7. Garnier R, Chataigner D: Acute tubular necrosis following endosulfan insecticide poisoning. J Toxicol Clin Toxicol 33: 375, 377-378, 1995

    PubMed  Google Scholar 

  8. Kelch WJ, Kerr LA: Acute toxicosis in cattle sprayed with endosulfan. Vet Hum Toxicol 39: 29–30, 1997

    PubMed  Google Scholar 

  9. Zahm SH, Blair A: Pesticides and non-Hodgkin's lymphoma. Cancer Res 52: 5485s–5488s, 1992

    PubMed  Google Scholar 

  10. Zahm SH, Weisenburger DD, Saal RC, Vaught JB, Babbitt PA, Blair A: The role of agricultural pesticide use in the development of non-Hodgkin's lymphoma in women. Arch Environ Health 48: 353–358, 1993

    PubMed  Google Scholar 

  11. Rupa DS, Reddy PP, Sreemannarayana K, Reddi OS: Frequency of sister chromatid exchange in peripheral lymphocytes of male pesticide applicators. Environ Mol Mutagen 18: 136–138, 1991

    PubMed  Google Scholar 

  12. Fransson-Steen R, Flodstrom S, Warngard L: The insecticide endosulfan and its two stereoisomers promote the growth of altered hepatic foci in rats. Carcinogenesis 13: 2299–2303, 1992

    PubMed  Google Scholar 

  13. Banerjee BD, Hussain, QZ: Effect of sub-chronic endosulfan exposure on humoral and cell-mediated immune responses in albino rats. Arch Toxicol 59: 279–284, 1986

    Article  PubMed  Google Scholar 

  14. Kannan K: Studies on the immunotoxicologic evaluation of endosulfan in rodents. Ph.D thesis, Jawaharlal Nehru University, New Delhi, India, 1983

    Google Scholar 

  15. Kannan K, Das B, Shanker R, Bhatia B: Effect of endosulfan on oxygen consumption of leukocytes. Int J Immunopharmacol 4: (abstr) 322, 1982

    Article  Google Scholar 

  16. Sobti RC, Krishan A, Davies J: Cytokinetic and cytogenetic effect of agricultural chemicals on human lymphoid cells in vitro. II. Organochlorine pesticides. Arch Toxicol 52: 221–231, 1983

    Article  PubMed  Google Scholar 

  17. Dubey RK, Beg MU, Singh J: Effects of endosulfan and its metabolites on rat liver mitochondrial respiration and enzyme activities in vitro. Biochem Phamacol 33: 3405–3410, 1984

    Article  Google Scholar 

  18. da Silva EM, Soares AM, Moreno AJ: The use of the mitochondrial transmembrane electric potential as an effective biosensor in ecotoxicological research. Chemosphere 36: 2375–2390, 1998

    PubMed  Google Scholar 

  19. Nelson BD, Williams C: Action of cyclodiene pesticides on oxidative metabolism in the yeast Saccharomyces cerevisiae. J Agri Food Chem 19: 339–341, 1971

    Article  Google Scholar 

  20. Rosa R, Rodriguez-Farre E, Sanfeliu C: Cytotoxicity of hexachlorocyclohexane isomers and cyclodienes in primary cultures of cerebellar granule cells. J Pharmacol Exp Ther 278: 163–169, 1996

    PubMed  Google Scholar 

  21. Susin SA, Zamzami N, Kroemer G: Mitochondria as regulators of apoptosis: Doubt no more. Biochim et Biophys Acta 1366: 151–165, 1998

    Google Scholar 

  22. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B, Kroemer G: Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 182: 367–377, 1995

    Article  PubMed  Google Scholar 

  23. Zamzami N, Susin SA, Marchetti P, Hirsch T, Gomez-Monterrey I, Castedo M, Kroemer G: Mitochondrial control of nuclear apoptosis. J Exp Med 183: 1533–1544, 1996

    Article  Google Scholar 

  24. Leist M, Single B, Castoldi AF, Kuhnle S, Nicotera P: Intracellular adenosine triphosphate (ATP) concentration: A switch in the decision between apoptosis and necrosis. J Exp Med 185: 1481–1486, 1997

    Article  PubMed  Google Scholar 

  25. Richter C, Schweizer M, Cossarizza A, Franceschi C: Control of apoptosis by the cellular ATP level. FEBS Lett 378: 107–110, 1996

    Article  PubMed  Google Scholar 

  26. Kasai T, Ohguchi K, Nakashima S, Ito Y, Naganawa T, Kondo N, Nozawa Y: Increased activity of oleate-dependent type phospholipase D during actinomycin D-induced apoptosis in Jurkat T cells. J Immunol 161: 6469–6474, 1998

    PubMed  Google Scholar 

  27. Wirleitner B, Baier-Bitterlich G, Bock G, Widner B, Fuchs D: 7,8-Dihydroneopterin-induced apoptosis in Jurkat T lymphocytes: A comparison with anti-Fas-and hydrogen peroxide-mediated cell death. Biochem Pharmacol 56: 1181–1187, 1998

    Article  PubMed  Google Scholar 

  28. Ahmed SA, Gogal RMJ, Walsh JE: A new rapid and simple nonradioactive assay to monitor and determine the proliferation of lymphocytes: An alternative to [3H] thymidine incorporation assay. J Immunol Meth 142: 199–206, 1994

    Google Scholar 

  29. de Fries R, Mitsubishi: Quantification of mitogen induced human lymphocyte proliferation: Comparison of alamar-blue™ assay to 3Hthymidine incorporation assay. J Clin Lab Anal 9: 89–95, 1995

    PubMed  Google Scholar 

  30. Springer JE, Azbill RD, Carlson SL: A rapid and sensitive assay for measuring mitochondrial metabolic activity in isolated neural tissue. Brain Res Protoc 2: 259–263, 1998

    Article  Google Scholar 

  31. Darzynkiewicz Z, Li X, Gong J: Assays of cell viability: Discrimination of cells dying by apoptosis. In: Z. Darzynkiewicz, J.P. Robinson, H.A. Crissman (eds). Methods in Cell Biology. Academic Press, 1994, pp 15–38

  32. Telford WG, King LE, Fraker PJ: Comparative evaluation of several DNA-binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 13: 137–143, 1992

    Article  PubMed  Google Scholar 

  33. Martin SJ, Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR: Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J Exp Med 182: 1545–1556, 1995

    Article  PubMed  Google Scholar 

  34. Shenker BJ, Datar S, Mansfield K, Shapiro IM: Induction of apoptosis in human T-cells by organomercuric compounds: A flow cytometric analysis. Toxicol Appl Pharmacol 143: 397–406, 1997

    Article  PubMed  Google Scholar 

  35. Wyllie AH, Kerr JF, Currie AR: Cell death: The significance of apoptosis. Int Rev Cytol 68: 251–306, 1980

    PubMed  Google Scholar 

  36. Osorio LM, De Santiago A, Aguilar-Santelises M, Mellstedt H, Jondal M: CD6 ligation modulates the Bcl-2/Bax ratio and protects chronic lymphocytic leukemia B cells from apoptosis induced by anti-IgM. Blood 89: 2833–2841, 1997

    PubMed  Google Scholar 

  37. Buetler E: Reduced glutathione. In: E. Buetler (ed). Red Cell Metabolism: A Manual of Biochemical Methods, 3rd edn. Grune & Stratton Inc., 1984, pp 131–136

  38. Hockenbery DM: Bcl-2, a novel regulator of cell death. Bioessays 7: 631–638, 1995 (review)

    Article  Google Scholar 

  39. Reed JC: Bcl-2 and regulation of programmed cell death. J Cell Biol 124: 1–6, 1994

    Article  PubMed  Google Scholar 

  40. Kroemer G: The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nature Med 6: 614–620, 1997

    Article  Google Scholar 

  41. Dean JH, Luster MI, Munson AE, Kimber I: Immunotoxicology and Immunopharmacology, 2nd edn. Raven Press, New York, 1994

    Google Scholar 

  42. Kerkvliet N, Burleson GR: Immunotoxicity of TCDD and related halogenated aromatic hydrocarbons. In: J.H. Dean, M.I. Luster, A.E. Munson, I. Kimber (eds). Immunotoxicology and Immunopharmacology, 2nd edn. Raven Press Ltd., New York, 1994, pp 97–122

    Google Scholar 

  43. Faith RE, Luster MI, Vos JG: Effect of immunocompetence by chemicals of environmental concern. Rev Biochem Toxicol 2: 173–211, 1980

    Google Scholar 

  44. Vos JG: Immune suppression as related to toxicology. CRC Crit Rev Toxicol 5: 67–101, 1977

    PubMed  Google Scholar 

  45. Barnett JB, Rodgers KE: Pesticides. In: J.H. Dean, M.I. Luster, A.E. Munson, I. Kimber (eds). Immunotoxicology and Immunopharmacology, 2nd edn. Raven Press Ltd., New York, 1994, 191–212

    Google Scholar 

  46. McConkey D, Jondal MB, Orrenius SG: Chemical-induced apoptosis in the immune system. In: J.H. Dean, M.I. Luster, A.E. Munson, I. Kimber (eds). Immunotoxicology and Immunopharmacology, 2nd edn. Raven Press Ltd., New York, 1994, pp 473–486

    Google Scholar 

  47. Aw TY, Nicotera N, Manzo L, Orrenius S: Tributyltin stimulates apoptosis in rat thymocytes. Arch Biochem Biophys 283: 46–50, 1990

    Article  PubMed  Google Scholar 

  48. Tebourbi O, Rhouma KB, Sakly M: DDT induces apoptosis in rat thymocytes. Bull Environ Contam Toxicol 61: 216–223, 1998

    Article  PubMed  Google Scholar 

  49. Kerr JFR, Wyllie AH, Currie AR: Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257, 1972

    PubMed  Google Scholar 

  50. Savill J, Fadok V, Henson P, Haslett C: Phagocyte recognition of cells undergoing apoptosis. Immunol Today 14: 131–136, 1993

    Article  PubMed  Google Scholar 

  51. McConkey DJ, Hartzell P, Nicotera P, Wyllie AH, Orrenius S: Stimulation of endogenous endonuclease activity in hepatocytes to oxidative stress. Toxicol Lett 42: 123–130, 1988

    Article  PubMed  Google Scholar 

  52. Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H, Tsujimoto Y: Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors. Oncogene 13: 21–29, 1996

    PubMed  Google Scholar 

  53. Bindoli A: Lipid peroxidation in mitochondria. Free Rad Biol Med 5: 247–261, 1996

    Article  Google Scholar 

  54. Yu BP, Chen JJ, Kang CM, Choe M, Maeng YS, Kristal BS: Mitochondrial aging and lipoperoxidative products. Ann NY Acad Sci 786: 44–56, 1996

    PubMed  Google Scholar 

  55. Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H: Oxidative damage, mitochondrial oxidant generation and anti-oxidant defenses during aging and in response to food restriction in the mouse. Mech Age Dev 74: 121–133, 1994

    Article  Google Scholar 

  56. Zhang Y, Marcillat O, Giulivi C, Ernster L, Davies KJ: The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J Biol Med 265: 16330–16336, 1990

    Google Scholar 

  57. Kristal BS, Park BK, Yu BP: 4-Hydroyhexanal is a potent inducer of the mitochondrial permeability transition. J Biol Chem 271: 6033–6038, 1966

    Google Scholar 

  58. Hincal F Gurbay A, Giray B: Induction of lipid peroxidation and alteration of glutathione redox status by endosulfan. Biol Trace Elem Res 47: 321–326, 1995

    PubMed  Google Scholar 

  59. Agarwal DK, Seth PK, Gupta PK: Effect of endosulfan on drug metabolizing enzymes and lipid peroxidation in rat. J Environ Sci Health [C] 13: 49–62, 1978

    Google Scholar 

  60. Singh SK, Pandey RS: Toxicity of endosulfan on kidney of male rats in relation to drug metabolizing enzymes and microsomal lipid peroxidation. Indian J Exp Biol 27: 725–728, 1989

    PubMed  Google Scholar 

  61. Kazzaz JA, Xu J, Palaia TA, Mantell L, Fein AM, Horowitz S: Cellular oxygen toxicity. Oxidant injury without apoptosis. J Biol Chem 271: 15182–15186, 1996

    Article  PubMed  Google Scholar 

  62. McConkey DJ: Biochemical determinants of apoptosis and necrosis (mini-review). Toxicol Lett 99: 157–168, 1988

    Article  Google Scholar 

  63. Macho A, Hirsch T, Marzo I, Marchetti P, Dallaporta B, Susin SA, Zamzami N, Kroemer G: Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J Immunol 158: 4612–4619, 1997

    PubMed  Google Scholar 

  64. Messmer UK, Brune B: Nitric oxide (NO) in apoptotic vs. necrotic RAW 264.7 macrophage cell death: The role of NO-donor exposure. NAD+ content, and p53 accumulation. Arch Biochem Biophys 327: 1–10, 1996

    Article  PubMed  Google Scholar 

  65. Hampton MB, Orrenius S: Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett 414: 552–556, 1997

    Article  PubMed  Google Scholar 

  66. Boise LH, Gottschalk AR, Quintans J, Thompson CB: Bcl-2 and Bcl-2 related proteins in apoptosis regulation. Curr Top Microbiol Immunol 200: 107–121, 1995

    PubMed  Google Scholar 

  67. Guo TL, Miller MA, Shapiro IM, Shenker BJ: Mercuric chloride induces apoptosis in human T-lymphocytes: Evidence of mitochondrial dysfunction. Toxicol Appl Pharmacol 153: 250–257, 1998

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, Louisiana, USA

    Krishnaswamy Kannan, Xavier Alvarez-Hernandez & Jonathan Glass

  2. Division of Hematology/Oncology, University of California, Irvine, California, USA

    Randall F. Holcombe

  3. Department of Pediatrics, LSU Health Sciences Center, Shreveport, Louisiana, USA

    Sushil K Jain

  4. Department of Microbiology and Immunology, LSU Medical Center, Shreveport, Louisiana, USA

    Robert Chervenak

  5. Arthritis Center, LSU Health Sciences Center, Shreveport, Louisiana, USA

    Krishnaswamy Kannan & Robert E Wolf

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  1. Krishnaswamy Kannan
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  2. Randall F. Holcombe
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Kannan, K., Holcombe, R.F., Jain, S.K. et al. Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line. Mol Cell Biochem 205, 53–66 (2000). https://doi.org/10.1023/A:1007080910396

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