Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine.
Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.
Ahmed
Tanzeel
T
Environmental Biochemistry and Immunology Laboratory, Department of Biochemistry, University College of Medical Sciences. G. T. B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, India.
Tripathi
Ashok K
AK
Ahmed
Rafat S
RS
Das
Shukla
S
Suke
Sanvidhan G
SG
Pathak
Rahul
R
Chakraboti
Ayanabha
A
Banerjee
Basu Dev
BD
D000111:Acetylcysteine / Q000494:pharmacology*; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D053938:DNA Fragmentation / Q000187:drug effects; D004726:Endosulfan / Q000633:toxicity*; D005978:Glutathione / Q000172:deficiency*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000187:drug effects*; D008856:Microscopy, Fluorescence; D009336:Necrosis
D017304:Annexin A5; D005978:Glutathione; D004726:Endosulfan; D000111:Acetylcysteine