Cytotoxicity of mercury compounds in LLC-PK1, MDCK and human proximal tubular cells

Abstract

Six mercury compounds [HgCl2 (MC), Hg(CH3COO)2(MA), Hg(NO3)2 (MN), C2H5HgSC6H4COONa (EMT), C6H5HgOCOCH3 (PMA) and CH3CIHg (MMC)] were studied using two kidney cell lines (MDCK and LLC-PK1), primary cultures of human proximal tubular cells (hPTC) and nonrenal cell lines (SAOS and Hep G2). Cell damage was measured with four different tests: neutral red uptake, mitochondrial dehydrogenase activity (MTT conversion), thymidine incorporation and protein content. Relative toxicity was established by the determination of the concentration of test compound inducing a 50% reduction of the parameter considered (EC50 value). Two groups could be distinguished: PMA, EMT and MMC are one order of magnitude more toxic than MC, MN and MA. Cellular uptake was measured by the HPLC-hybrid generation AAS after 24 hours treatment with 1.5 μM MC, MMC, PMA or EMT in MDCK cells, revealing Hg concentrations of 42.8 ± 2.5 ng/mg protein for MC, 596.9 ± 87.8 ng/mg protein for MMC, 269.8 ± 75.7 ng/mg protein for pMA and of 115.9 ± 25.2 ng/mg protein for EMT. Cytotoxicity was positively correlated with cellular uptake. The effect of the cellular GSH content on the toxicity of mercury was studied using the GSH synthesis inhibitor L-buthionine sulfoximine (BSO). In all cases an enhanced cytotoxicity was observed after BSO treatment. 2-Oxo-4-thiazolidine carboxylic acid (OTC) was used as a substrate for the GSH synthesis. Although OTC did not enhance the GSH content, the cytotoxicity of MC, MN and MA decreased significantly, no changes were observed for the other mercurials. HPLC-hydrid generation AAS revealed that OTC is complexed in the growth medium with MC, but not with EMT, whereas no interactions were observed between BSO and the mercury compounds. From a toxicological point of view two groups can be considered: MMC, EMT and PMA are one order of magnitude more toxic than MC, MA and MN in all cell lines studied. This difference in toxicity was proportional with the uptake of those compounds. Extracellular complexation of mercury with OTC is correlated with decreasing cytotoxicity in the absence of enhanced GSH. GSH inhibition by BSO resulted in a higher toxicity, within comparable cellular uptake, confirming the function of GSH in the detoxification of mercury.