Induction of apoptosis in human T-cells by organomercuric compounds: a flow cytometric analysis.
Although several lines of investigation demonstrate that many heavy metals are cytotoxic to host defense cells, the mechanism of killing is poorly understood. The major focus of this investigation was to determine if organic mercuric compounds kill human lymphocytes by inducing the cells to undergo apoptosis and to evaluate possible flow cytometric systems for assessing cell death. T-cells were exposed to 0.6-5 microM MeHgCl, EtHgCl, or PhHgCl for up to 24 hr and then analyzed by flow cytometry. Mercury-treated cells exhibited increased Hoechst 33258 and 33342 fluorescence while maintaining their ability to exclude the vital stain 7-AAD. Furthermore, T-cells exposed to mercury exhibited changes in light scatter patterns that included decreased forward light scatter and increased side scatter. The light scatter and fluorescent changes were consistent with changes that cells display during apoptosis. To further evaluate cell death and to distinguish between apoptosis and necrosis, merocyanine 540 staining and annexin V binding to the plasma membrane as well as DNA fragmentation were assessed. Mercury-treated 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 to 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 upon the amount of serum present in the medium; as serum concentrations were increased from 0 to 10%, cell death declined. Apoptosis (33%) was observed within 1 hr of exposure to MeHgCl; maximum cell death (67%) occurred 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 activated T-cells. When treated with mitogen, mercury failed to induce apoptosis in these cells. Indeed, there was no evidence of either apoptosis nor necrosis in these populations. It was concluded that the activation process prevented development of a metabolic state that was required for induction of apoptogenic genes.
Shenker
B J
BJ
Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Datar
S
S
Mansfield
K
K
Shapiro
I M
IM
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D016923:Cell Death; D002462:Cell Membrane / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008213:Lymphocyte Activation / Q000502:physiology; D008563:Membrane Lipids / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009941:Organomercury Compounds / Q000633:toxicity*; D010835:Phytohemagglutinins / Q000494:pharmacology; D011744:Pyrimidinones / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000502:physiology*
D017304:Annexin A5; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D009941:Organomercury Compounds; D010835:Phytohemagglutinins; D011744:Pyrimidinones; C003954:merocyanine dye; D004247:DNA