Binding of methylmercury(II) by HeLa S3 suspension-culture cells: Intracellular methylmercury levels and their effect on DNA replication and protein synthesis

Abstract

HeLa S3 cells were exposed to varied concentrations of methylmercury over varied periods of time and its binding by the cells was studied using 203Hg-labeled methylmercuric chloride as radioactive marker. Also studied was the effect of cell-bound methylmercury on DNA replication and protein synthesis and on the growth rate of the cells. The results show that methylmercury binding is a rapid process, with much of the organomercurial bound within the the first 60 min of incubation, and that considerable quantities of organic mercury become affixed to the cells. The amounts of bound methylmercury, [CH 3Hg(II)] bound , given in mol/cell, range from 2 × 10 −16 (at 1 h of incubation and at 1 μM CH 3Hg(II) in the medium) to almost 4 × 10 −14 (at 24 h of incubation and at 100 μM CH 3Hg(II) in the medium). A [CH 3Hg(II)] bound value of about 30 × 10 −16 mol/cell appears to be the threshold below which cells display a normal growth pattern and below which metabolic events such as DNA replication or protein synthesis are affected only to a minor degree but above which major changes in cell metabolism and cell growth take place. Methylmercury binding by the cells is tight so that only 20% of the bound material is released from the cells over a 3-h incubation period when the cells are placed into fresh, methylmercury-free growth medium. Analysis of the binding data in terms of binding to identical and completely independent sites yields an association constant K of 7.92 × 10 4 l/mol and for the maximum concentration of cellular binding sites the value 2.40 × 10 −14 mol/cell or 1.45 × 10 10 sites/cell. Evidence is presented which shows that cellular sulfhydryl groups do not suffice to provide all the sites taken up by methylmercury and that binding, in all likelihood, involves basic nitrogen, too. The levels of cell-bound methylmercury are such that binding to HeLa DNA and HeLa chromatin, for instance, can readily take place. Methylmercury binding data obtained by using the technique of particle-induced X-ray emission (PIXE) are in good agreement with the data obtained via isotope dilution.