Doktorarbeit
Der Einfluss der Schwefelkomplexierung auf Absorption und Toxizität von Metalloiden in Zellkulturen
Sinikka Hinrichsen (01/2011-12/2015)
Betreuer: Britta Planer-Friedrich
Arsenic is a common poison and is classified as human carcinogen. Selenium is an essential nutrient, but is highly toxic when applied in high concentrations. The cytotoxic potential of both metalloids is modified when they form sulfur-containing complexes. For arsenic compounds, only some data existed about the bioavailability and cytotoxicity of methylated thioarsenates. No data existed about inorganic thioarsenates, even though their formation during pre-systemic arsenic metabolism was already proven. For selenium compounds, a reported general higher cytotoxicity of selenosulfate compared to selenite for cancer cell lines led to the claim of replacing selenite by selenosulfate in anti-cancer therapies.
In the first two studies of the present thesis, intestinal transport (e.g. bioavailability), cellular retention, and cytotoxicity of thioarsenates were investigated. The cellular retention and the intestinal transport of synthesized standard solutions of methylated and inorganic thioarsenates were compared to those of their non-thiolated analogues by means of a model of the human small intestine (Caco-2 cell monolayer). Analyses with AEC-ICP-MS were conducted to monitor species stability during the transport experiments. Both the transcellular uptake route (by phosphate transporters) and the paracellular uptake route (through the tight junctions) were investigated for each arsenic species.
The influence of sulfide on arsenite was investigated concerning the formation of inorganic thioarsenates and an accordingly modified cytotoxicity, quantified by means of MTT assay. The cytotoxic effects of arsenite, arsenate, and inorganic thioarsenates were compared for human hepatocytes (HepG2) and urothelial cells (UROtsa). Concentrations of each arsenic species leading to 50 % cell viability (IC50 values) were calculated. Cellular uptake of the different inorganic arsenic compounds was quantified and linked to their cytotoxicity.
As expected, arsenite showed the highest cellular retention and intestinal transport of all tested arsenic compounds. The bioavailability of thioarsenates strongly differed from that of their non-thiolated analogues. For dimethylmonothioarsenate, the highest cellular retention and intestinal transport among all methylated arsenic compounds were measured, which is of special concern as this species is known to possess a considerably higher cytotoxicity than its non-thiolated analogue dimethylarsenate. Only low cellular retention – comparable to
that of arsenate - was detected for the inorganic thioarsenates mono- and trithioarsenate, but their intestinal transport was considerably higher than that of arsenate. For trithioarsenate, the intestinal transport was even comparable to that of arsenite. Mono- and trithioarsenate were transported intact through the cell monolayer, but partial intracellular reduction to arsenite could not be excluded. Both cellular retention and intestinal transport was negligibly low for mono- and dimethylarsenate and for monomethylmonothioarsenate. The absence of phosphate increased cellular retention of all arsenic compounds indicating the importance of apical phosphate transporters. No data could be presented to interpret the importance of the paracellular transport route as the cell monolayer was damaged during these experiments.
Addition of sulfide to arsenite-containing cell growth medium resulted in immediate formation of inorganic thioarsenates and in reduced cytotoxicity. The order of cytotoxicity of the individually applied inorganic arsenic compounds after 24 h exposure was determined as arsenite > trithioarsenate > monothioarsenate > arsenate and this corresponded to the order of cellular arsenic uptake. Considering arsenite as the original present arsenic substrate for the formation of inorganic thioarsenates, thiolation can be seen as a detoxification process due to decreased intestinal transport and cytotoxicity. In case of dimethylmonothioarsenate, which is known to form from dimethylarsenate, thiolation can be seen as an activation process due to increased intestinal transport and cytotoxicity.
In the third study, cytotoxic effects and cellular uptake of selenosulfate and selenite were compared for three different cancer cell lines (HepG2, A375, and T24) to reassess the claim of selenosulfate being generally more cytotoxic than selenite for cancer cells. Experiments in absence and presence of amino acids linked the influence of amino acids with the cytotoxicity of the selenium compounds.
Selenosulfate was comparably toxic to the three cell lines (IC50 6.6-7.1 µM) and hardly influenced by incubation time and presence or absence of amino acids. Though, selenite cytotoxicity considerably differed among the three cell lines with the result that selenosulfate was more toxic than selenite for HepG2 cells (IC50 > 15 µM), but similar toxic to and lower toxic than selenite for A375 (IC50 4.7 µM) and T24 cells (IC50 3.5 µM).
In contrast to T24 cells, HepG2 cells were “routinely” cultivated with amino acids. Addition of amino acids to T24 cell growth medium led to reduced selenite uptake and toxicity, rendering it less toxic than selenosulfate. The strong effect of amino acids on selenite toxicity for T24 cells could be explained by an inhibition of the xc- transport system which facilitates cellular selenium uptake by secretion of cysteine and reduction of selenium compounds. Selenosulfate is less affected by the addition of amino acids as it is already a reduced species. Whether selenosulfate or selenite is more cytotoxic, does not only depend on the selenium species itself, but also on the sensitivity of the used cell line, the supplements of the cell growth medium, and the reductive state of the extracellular environment. The general claim of selenosulfate being more toxic than selenite therefore has to be reconsidered.