Differential immunotoxic effects of ethanol on murine EL-4 lymphoma and normal lymphocytes is mediated through increased ROS production and activation of p38MAPK.
Ethanol has been used to achieve thymic depletion in myasthenia gravis patients. Ethanol (95%) has also been used widely in the therapy of many tumors including hepatocellular carcinoma. In light of these findings, we delineated the differential immunotoxic behavior and mechanism of lower concentration of ethanol towards murine EL-4 lymphoma and its normal counterpart lymphocytes. EL-4 lymphoma and normal lymphocytes were cultured with ethanol (0%-5%) for 6 h and cytotoxicity was measured by various methods. EL-4 cells treated with ethanol showed concentration-dependent loss of viability at 2%-5% ethanol concentration and exhibit proliferative arrest at preG1 stage. Acridine-orange and ethidium-bromide staining indicated that ethanol induced death in EL-4 cells, by induction of both apoptosis and necrosis which was further supported by findings of DNA-fragmentation and trypan blue dye exclusion test. However, treatment of lymphocytes with similar concentration of ethanol did not show any death-associated parameters. Furthermore, ethanol induced significantly higher ROS generation in EL-4 cells as compared to lymphocytes and caused PARP cleavage and activation of apoptotic proteins like p53 and Bax, in EL-4 cells and not in normal lymphocytes. In addition, ethanol exposure to EL-4 cells led to phosphorylation of p38MAPK, and upregulation of death receptor Fas (CD95). Taken together, these results suggest that ethanol upto a concentration of 5% caused no significant immunotoxicity towards normal lymphocytes and induced cell death in EL-4 cells via phosphorylation of p38MAPK and regulation of p53 leading to further activation of both extrinsic (Fas) and intrinsic (Bax) apoptotic markers.
Immunology and Hyperthermia Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India.
Sudha
S
Premachandran
Nazir M
NM
Khan
Vikas S
VS
Thakur
Jyoti
J
Shukla
T B
TB
Poduval
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D003603:Cytotoxins / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D016193:G1 Phase / Q000187:drug effects; D008214:Lymphocytes / Q000378:metabolism* / Q000473:pathology; D008223:Lymphoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D009336:Necrosis; D010766:Phosphorylation / Q000187:drug effects; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012997:Solvents / Q000494:pharmacology; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
C494827:Bax protein, mouse; D003603:Cytotoxins; D017382:Reactive Oxygen Species; D012997:Solvents; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D000431:Ethanol; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D048051:p38 Mitogen-Activated Protein Kinases