Mercuric Chloride Induces Apoptosis in Human T Lymphocytes: Evidence of Mitochondrial Dysfunction

• Published in: Toxicology and applied pharmacology 1998-12, Vol.153 (2), p.250-257
• Main Authors: Guo, Tai Liang, Miller, Mark A., Shapiro, Irving M., Shenker, Bruce J.
• Format: Article
• Language: English
• Subjects: Apoptosis - drug effects; Biological and medical sciences; Blotting, Western; Cells, Cultured; Chemical and industrial products toxicology. Toxic occupational diseases; DNA Fragmentation - drug effects; Flow Cytometry; Fluorescence; Humans; Hydrogen-Ion Concentration; Medical sciences; Membrane Potentials; Mercuric Chloride - toxicity; Metals and various inorganic compounds; Mitochondria - drug effects; Oxidative Stress - drug effects; Phosphatidylserines - pharmacokinetics; Poly(ADP-ribose) Polymerases - metabolism; T-Lymphocytes - drug effects; Toxicology
• ISSN: 0041-008X; 1096-0333
• DOI: 10.1006/taap.1998.8549

The major objective of our study was to define the mechanism by which mercuric chloride (HgCl2) induces human T-cell death. Human peripheral blood T-cells were exposed to 0–40 μmHgCl2and then analyzed for biochemical and molecular features of T-cell apoptosis. HgCl2-treated cells exhibited increased Hoechst 33258 fluorescence while maintaining their ability to exclude the vital stain 7-aminoactinomycin D. To further evaluate cell death and distinguish between apoptosis and necrosis, translocation of phosphatidylserine to the outer layer of the plasma membrane (annexin V binding), DNA fragmentation (TUNEL assay), and cleavage of poly (ADP-ribose) polymerase (PARP) were assessed. In the presence of 20–40 μmHgCl2, T-cells exhibited increased annexin V binding (28%) and DNA fragmentation (31%). HgCl2-dependent PARP cleavage was also observed by Western blot analysis. Because degradative changes associated with apoptosis are often preceded by disruption of mitochondrial function, HgCl2-treated cells were assessed for disruption of the mitochondrial transmembrane potential (ΔΨm) and development of the mitochondrial permeability transition state. Using DiOC6(3), we demonstrated that HgCl2exposure resulted in a decrease in the ΔΨm. Because a decline in ΔΨmcan disturb the intracellular pH (pHi), we used the fluorescent probe, SNARF-1, to assess intracellular acidification. Treatment of T-cells with HgCl2resulted in reduced pHifrom 7.0 to 6.7. Concomitant with these observations, the fluorescent probe, hydroethidine, was utilized to demonstrate that uncoupled mitochondrial electron transport resulted in increased reactive oxygen species (ROS) generation. Interestingly, in spite of these alterations to mitochondrial function, translocation of cytochrome c to the cytosol was not detected; this correlated with enhanced bcl-2 levels in HgCl2-treated cells. In conclusion, HgCl2exposure results in oxidative stress and activation of death signaling pathways leading to apoptosis. Collectively, our studies indicate that individual mercurial species are capable of inducing T-cell death by activating specific apoptotic cascades.