Ethanol Increases Apoptotic Cell Death of Thymocytes in Vitro

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Abstract
Exposure of animals to ethanol causes thymic atrophy in adults and fetuses. Whether direct effects of ethanol contribute to thymic atrophy or whether indirect effects are entirely responsible is at present unknown. In the normal animal, large numbers of thymocytes undergo a physiological form of cell death referred to as “apoptosis.” To determine if ethanol affects the process of apoptosis, studies were undertaken in which mouse thymocytes were cultured overnight in the presence or absence of ethanol. Apoptotic cell death was analyzed by flow cytometric quantitation of apoptotic nuclei, by fluorometric measurement of DNA fragments, and by gel electrophoretic analysis of DNA fragments. Ethanol in concentrations of 0.2% to 0.8% produced significantly higher levels of apoptosis than were seen in control cultures. The DNA fragmentation was characterized as apoptotic on the basis of inhibition by aurintricarboxylic acid (an inhibitor of nucleases) and by the presence of characteristic oligonucleosomal‐sized fragments of DNA. The effect of ethanol on apoptosis was additive to that induced by immobilized anti‐CD3 monoclonal antibody. CD4+CD8+ cells underwent apoptosis as indicated by reduction in CD4 and CD8 surface antigen expression. An inhibitor of protein kinases (H‐7) reduced the DNA degradation induced by ethanol and by anti‐CD3. These results suggest that direct effects of ethanol contribute to thymic atrophy in alcohol‐consuming animals.