INDUCTION OF APOPTOSIS IN HUMAN T-CELLS BY ORGANOMERCURIC COMPOUNDS -A FLOW CYTOMETRIC ANALYSIS

Although several lines of investigation demonstrate that many heavy me tals are cytotoxic to host defense cells, the mechanism of killing is poorly understood. The major focus of this investigation was to determ ine if organic mercuric compounds kill human lymphocytes by inducing t he cells to undergo apoptosis and to evaluate possible flow cytometric systems for assessing cell death. T-cells were exposed to 0.6-5 mu M MeHgCl, EtHgCl, or PhHgCl for up to 24 hr and then analyzed by flow cy tometry. Mercury-treated cells exhibited increased Hoechst 33258 and 3 3342 fluorescence while maintaining their ability to exclude the vital stain 7-AAD. Furthermore, T-cells exposed to mercury exhibited change s in light scatter patterns that included decreased forward light scat ter and increased side scatter. The light scatter and fluorescent chan ges were consistent with changes that cells display during apoptosis. To further evaluate cell death and to distinguish between apoptosis an d necrosis, merocyanine 540 staining and annexin V binding to the plas ma membrane as well as DNA fragmentation were assessed. Mercury-treate d cells exhibited increased merocyanine 540 fluorescence and annexin V binding along with changes in nuclear morphology consistent with the notion of apoptosis. Conventional agarose gel electrophoresis failed t o demonstrate low-molecular-weight DNA bands; however, when probed by flow cytometry using both nick translation and a modified TUNEL assay, patterns consistent with nuclear fragmentation were evident. We noted that the percentage of T-cells undergoing apoptosis was dependent upo n the amount of serum present in the medium; as serum concentrations w ere increased from 0 to 10%, cell death declined. Apoptosis (33%) was observed within 1 hr of exposure to MeHgCl; maximum cell death (67%) o ccurred after 24 hr exposure. Induction of apoptosis was dependent on the mercury concentration and independent of the hydrophobicity of the mercury ligand. Finally, we assessed mercury-dependent apoptosis in a ctivated T-cells. When treated with mitogen, mercury failed to induce apoptosis in these cells. Indeed, there was no evidence of either apop tosis nor necrosis in these populations. It was concluded that the act ivation process prevented development of a metabolic state that was re quired for induction of apoptogenic genes. (C) 1997 Academic Press.