Mouse Monocytes (RAW CELLS) and the Handling of Cysteine and Homocysteine S-Conjugates of Inorganic Mercury and Methylmercury

Abstract

Although there is evidence indicating that mononuclear phagocytes can take up mercury by some forms of endocytosis, very little is known about the potential for the uptake of mercuric species by carrier-mediated processes. Thus, we hypothesized that monocytes also possess mechanisms allowing these cells to take up inorganic mercury (Hg²⁺) and/or methylmercury (CH₃Hg⁺) as cysteine (Cys) and/or homocysteine (Hcy) S-conjugates by certain membrane transport proteins. The specific thiol S-conjugates were chosen for study because our laboratory and those of some other investigators have demonstrated that these species of mercury are indeed transportable substrates for several membrane transport proteins in certain types of epithelial cells. We chose to use RAW 264.7 cells for our experiments. These cells represent an adherent line of mouse monocytes. Kinetic analyses for the uptake of Cys-Hg-Cys, CH₃Hg-Cys, Hcy-Hg-Hcy, and CH₃Hg-Hcy revealed that uptake occurred by a saturable, concentration-dependent mechanism, displaying Michaelis–Menten properties. Interestingly, in the cells exposed to the Cys or Hcy S-conjugate of Hg²⁺, significantly more Hg²⁺ was taken up in the presence of 140 mM sodium chloride (NaCl) than in the presence of 140 mM N-methyl-d-glucamine (NMDG), indicating that Na-dependent processes play more of a role in the uptake of these species of Hg²⁺ than sodium-independent ones. With respect to the uptake of CH₃Hg⁺, rates of uptake of the Cys and Hcy S-conjugates of CH₃Hg⁺ were similar under both Na-dependent and Na-independent conditions, although the levels of uptake of these mercuric species far exceeded the levels of uptake of the corresponding S-conjugate of Hg²⁺. Uptake of Hg²⁺ and CH₃Hg⁺, as the Cys or Hcy S-conjugates, was also time-dependent. We also showed that when the temperature in the bathing medium was reduced to 4°C, uptake of the Cys S-conjugates Hg²⁺ or CH₃Hg⁺ was for the most part reduced to negligible levels in the RAW cells; indicating that the preponderance of uptake at 37°C was not due primarily to simple diffusion and/or non-specific binding. Overall, the present findings strongly suggest that the uptake of the Cys and Hcy S-conjugates of Hg²⁺ and/or CH₃Hg⁺ occurs in monocytes by one or more mechanisms involving carrier proteins.

This project was supported, in part, by grants ES05157, ES05980, and ES11288 from the National Institute of Environmental Health Science and by grant MOP-49441 funded by the Canadian Institutes of Health Research. The authors thank Dr. Delon Barfuss, at Georgia State University, for his assistance in supplying radioactive mercury for the experiments described in this article. The authors also thank Dr. Christy C. Bridges for reading the article and providing editorial suggestions.