Master Thesis

In vitro toxicity of thioarsenates for human urothelial cells and hepatocytes

Regina Lohmayer (03/2010-11/2010)

Support: Britta Planer-Friedrich, Sasan Rabieh

Arsenic is known as human carcinogen with bladder and liver being two of the main target organs. In vitro toxicity tests have shown that arsenite is more toxic than arsenate, which is ascribed to the higher uptake of arsenite and the in vivo reactivity towards sulfur-containing compounds such as proteins. In contrast to oxyarsenicals, little is known about the cytotoxicity of inorganic thioarsenicals, which are the predominant arsenic species in sulfidic environments and potentially also form in vivo by intracellular reaction of arsenite with sulfur.

In the present study, human urothelial cells (UROtsa) and hepatocytes (HepG2) were exposed to arsenite, arsenate, mono- and dimethylarsenate, mono- and tetrathioarsenate, to mixtures thereof, and to arsenite-sulfide mixtures. The chosen exposure times were 1, 24, 48, and 72 h and the arsenic concentrations ranged from 0.1 µM to 10 mM. The cytotoxicity was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-test.

A significant reduction of arsenite toxicity was observed in the presence of a 4-fold excess of sulfide. Speciation analysis by anion exchange chromatography (AEC) coupled to an inductively coupled plasma mass spectrometer (ICP-MS) showed that thioarsenates – especially trithioarsenate – had formed in the arsenite-sulfide solutions. Investigating the cytotoxicity of the individual thioarsenates, monothioarsenate was found to be generally less toxic than arsenite. However, its toxicity increased significantly over time and was after 72 h comparable to that of arsenite after 24 h exposure. The experiments with mixtures of different arsenicals revealed an additive toxic effect of arsenite, arsenate, and monothioarsenate. This relationship was used to deduce the cytotoxicity of trithioarsenate, which was not available as pure standard. Speciation analysis of experiments with tetrathioarsenate, however, revealed that the tetrathioarsenate standard when dissolved in the cell medium dissociated to a 1:1 mixture of arsenite and trithioarsenate. Subtracting the toxicity of arsenite from that of the arsenite-trithioarsenate mixture, trithioarsenate cytotoxicity was calculated. The resulting order of toxicity was arsenite > arsenate > monothioarsenate > trithioarsenatecalculated > dimethylarsenate > monomethylarsenate for UROtsa cells and arsenite > trithioarsenatecalculated > monothioarsenate > arsenate > dimethylarsenate > monomethylarsenate for HepG2 cells. These results indicate that with an increasing number of thio-groups the toxicity of thioarsenates decreases for UROtsa cells but increases for HepG2 cells. Differences in arsenic sensitivity of the two cell types may be due to their differing methylation capacity, as HepG2 cells are able to methylate inorganic arsenicals whereas UROtsa cells are not. The lower toxicity of thioarsenates in comparison to arsenite may be explained by lower uptake as it is assumed that the uptake mechanisms of thioarsenates are similar to the ones of arsenate. Also the reactivity towards sulfur-containing compounds might decrease in comparison to arsenite if the arsenic species itself already contains thio-groups. In general, it can be concluded that the formation of thioarsenates – at least at short term exposure – can lead to a detoxification, whereas the role of the number of thio-groups needs further investigation.