Modeled speciation of Hg(II) and endogenous thiols; their comparative toxicity in LLC-PK(1) cells
AuthorDivine, Kevin K, 1961-
AdvisorCarter, Dean E.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractIn vivo Hg(II) exists as a complex with endogenous ligands, not as a free ion. Previous in vitro studies have demonstrated that metal ion form has a profound influence upon toxicity. We modeled Hg(II) ligand complexes using two independently developed computer programs. Models generated at physiological pH using published Hg(II) ligand K(f)'s, predict Hg(II) primarily will exist as a Hg(thiol)₂ complex. The Hg(thiol)₂ complexes, Hg(SG)₂, Hg(Cys)₂ and Hg(CysGly)₂, were synthesized, purified and identified. Hg(II) was administered to LLC-PK cells growing in defined media either with or without exogenous thiols. Hg(II) was administered as HgCl₂, Hg(SG)₂, Hg(Cys)₂, and HgCl₂ + either 1 or 2 moles of; GSH, L-Cys, D-Cys, CysGly and albumin. The LLC-PK₁ cells were incubated in Hg(II) concentrations ranging from 0-120 μM for 20 hrs. Toxicity was determined by the XTT assay. All forms of Hg(II) were more toxic when administered to LLC-PK₁ cells growing in defined media without exogenous thiols. Under all conditions, the least toxic form of Hg(II) was the Hg(thiol)₂ complexes and the most toxic form was HgCl₂. Addition of a single thiol equivalent to LLC-PK₁ cells in thiol containing media shifted the Hg(II) dose-response curves rightward ≈ 20μ M. Addition of Hg(II):2 albumin reduced Hg(II) toxicity in both experimental protocols. A self consistent set of relative competitive or conditional Kf's for Hg(II) with GSH, L-Cys, CysGly, DMPS, DMSA and EDTA was obtained using immobilized metal ion affinity chromatography (IMAC). Determining conditional Kf's using IMAC is advantageous in that the physical nature of water associated with the packed column is similar to that found in biological conditions and buffer composition can be manipulated to resemble biological fluids. Thiol sepharose 4B (Pharmacia LKB Biotechnology Inc., Piscataway NJ), GSH immobilized to sepharose, columns were loaded with HgCl₂, or Hg(SG)₂ and the affinity of Hg for the ligands was determined by competitive displacement chromatography. Our results demonstrate that the relative ranking of Hg-ligand Kf's is DMSA and DMPS > GSH, GSH ≈ L-Cys ≈ CysGly, > EDTA. The absolute values for the relative Hg-thiol complex Kf's must be ≥ 10²¹·⁵. (Martell and Smith, 1982).
Degree ProgramGraduate College
Pharmacology and Toxicology