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      Alterations in lymphocyte signalling produced by exposure to mercury

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      Date
      2007-07-03
      Author
      Yole, Margaret Jane
      Type
      Thesis
      Degree Level
      Doctoral
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      Abstract
      The effects of 1 min – 4 hr exposures to mercuric chloride (HgCl2), methyl mercuric chloride (CH3HgCl), p-chloromercuribenzoate (p-CMB) and ethylmercurithiosalicylate (TMS) on cell viability and kinetics of cell death, microtubules, F-actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P), intracellular calcium [Ca2+]i and responses to polarized signals in YAC-1 lymphoma cells were investigated. We hypothesized that immunotoxic effects of HgCl2 (Hg2+) are initiated by global receptor triggering, accompanied by increased protein tyrosine phosphorylation (PTyr-P) and down-regulation of the T-cell receptor (TCR). As a polychloride anion with poor lipid solubility, inorganic Hg2+ may produce effects at the outer cell membrane before significant intracellular accumulation, loss of microtubule integrity (a sensitive target) and activation of cell death through apoptotic pathways. The organomercurial compound p-CMB is likewise thought to penetrate membranes slowly as a result of ionization. In contrast, the highly lipid-soluble organomercurial compounds CH3HgCl and TMS were expected to reduce responses to polarized stimuli only in conjunction with – and not prior to – loss of microtubule integrity and the onset of necrotic cell death. Two general patterns of effects were observed. In HgCl2-treated YAC-1 cells, inhibition of responses to polarized stimuli preceded loss of microtubules and onset of cell death. Effects on polarized stimuli were preceded by a transient Ca2+ signal; however, this Ca2+ signal appeared abortive, accompanied by a paradoxic decrease in PTyr-P and partial down-regulation of CD3 receptors. Responses to polarised stimuli were inhibited prior to extensive loss of microtubule staining, indicating effects preceded cytosolic Hg2+ accumulation. HgCl2 exposure was followed rapidly by necrotic cell death. Similarly, p-CMB-treated YAC-1 cells failed to respond to polarized stimuli before effects on microtubules or loss of viability, and proceeded rapidly to late apoptosis; however, a transient Ca2+ signal and progressive loss of F-actin preceded effects in all other assays and may account for loss of polarized responses. In CH3HgCl- and TMS-treated YAC-1 cells, CD3 receptor expression, [Ca2+] and PTyr-P were increased immediately, along with loss of microtubules. These reductions preceded inhibition of polarized signaling responses and seemed to indicate a general loss of cellular homeostasis not seen in HgCl2- and p-CMB-treated cells; loss of homeostasis did not necessarily produce simultaneous loss of viability, as TMS-treated cells remained viable for 30 min while CH3HgCl-treated cells became apoptotic within 1 min. Nonetheless, the YAC-1 cells proceeded to cell death more slowly, remaining early apoptotic after 4 hr, when almost all HgCl2- and p-CMB-treated cells were necrotic. These findings indicate the two groups of mercury compounds may alter responses to polarized stimuli and induce cell death by distinct pathways, one involving an apparently abortive signal and the other mediated by much more profound disruption of cellular homeostasis. Within the larger patterns there are further differences between the effects produced by each Hg compound, likely reflecting the combined influence of pharmaco–kinetic and –dynamic factors governing access to and interactions with different cellular targets leading to cell death. These distinct targets may in turn be reflected in the different immune effects produced by these compounds in vivo.
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Veterinary Biomedical Sciences
      Program
      Veterinary Biomedical Sciences
      Supervisor
      Blakley, Barry R.; Wickstrom, Mark
      Committee
      Singh, Baljit; Sharma, Raghubir P.; Rosenberg, Alan; Hamilton, Donald L.; Forsyth, George W.
      Copyright Date
      July 2007
      URI
      http://hdl.handle.net/10388/etd-07022007-140223
      Subject
      methyl mercuric chloride
      mercuric chloride
      phosphotyrosine
      CD3 receptor
      intracellular calcium
      microtubles
      actin
      immunological synapse
      organic mercury
      inorganic mercury
      p-chloromercuribenzoate
      thimerosal
      YAC-1 lymphoma
      cytotoxicity
      apoptosis
      viability
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