Cell death and cytotoxic effects in YAC-1 lymphoma cells following exposure to various forms of mercury.
The effects of 1 min-4 h exposures to four Hg compounds (mercuric chloride [HgCl2], methyl mercuric chloride [CH3HgCl], p-chloromercuribenzoate [p-CMB] and thimerosal [TMS; ethylmercurithiosalicylate]) on cell death, microtubules, actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P) and intracellular calcium ([Ca2+]i) levels were investigated in YAC-1 lymphoma cells using flow cytometry. YOPRO-1 (YP) and propidium iodide (PI) dye uptake indicated all forms of Hg tested were toxic at concentrations ranging from 25.8-48.4 microM, with two distinct patterns of effects. Early apoptosis was prolonged for CH3HgCl- and TMS-treated cells, with more than 50% remaining YP+/PI- after 4h. Both CH3HgCl and TMS induced complete loss of beta-tubulin fluorescence, indicative of microtubule depolymerization and inhibition of tubulin synthesis and/or beta-tubulin degradation, while F-actin fluorescence diminished to a lesser degree and only after loss beta-tubulin. CH3HgCl and TMS induced an almost immediate two-fold increase in CD3 fluorescence, with levels returning to baseline within minutes. With continued exposure, CD3 fluorescence was reduced to approximately 50% of baseline values. Both compounds also increased PTyr-P two- to three-fold immediately, with levels returning to baseline at 4h. Similarly, two- to three-fold increases in [Ca2+]i were noted after 1 min exposure. [Ca2+]i increased progressively, reaching levels five- to eight-fold greater than control values. In contrast, dye uptake was delayed with HgCl2 and p-CMB, although cell death proceeded rapidly, with almost all non-viable cells being late apoptotic (YP+/PI+) by 4h. p-CMB produced early reductions in F-actin, and after 4h, complete loss of F-actin with only partial reduction of total beta-tubulin was seen with both p-CMB and HgCl2. HgCl2 reduced CD3 expression and PTyr-P slightly within minutes, while p-CMB produced similar effects on CD3 only at 4h, at which time PTyr-P was increased two- to three-fold. Both compounds increased [Ca2+]i within minutes, though levels remained under twice the baseline concentration after 15 min exposure. With continued exposure, [Ca2+]i increased to levels two- to five-fold greater than control values. These findings indicate the two groups of Hg compounds may induce cell death by distinct pathways, reflecting interactions with different cellular targets leading to cell death.
Yole
Margaret
M
Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, 52 Campus Drive, University of Saskatchewan, Saskatoon SK S7N 5B4, Canada. yole@sask.usask.ca
Wickstrom
Mark
M
Blakley
Barry
B
D000199:Actins / Q000378:metabolism; D000818:Animals; D017252:CD3 Complex / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D008627:Mercuric Chloride / Q000633:toxicity*; D008767:Methylmercury Compounds / Q000633:toxicity*; D051379:Mice; D019000:Phosphotyrosine / Q000378:metabolism; D013849:Thimerosal / Q000633:toxicity*; D014404:Tubulin / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid / Q000633:toxicity*
D000199:Actins; D017252:CD3 Complex; D008767:Methylmercury Compounds; D014404:Tubulin; D019000:Phosphotyrosine; D013849:Thimerosal; D008627:Mercuric Chloride; D020245:p-Chloromercuribenzoic Acid; C004925:methylmercuric chloride; D002118:Calcium