Mechanistic Studies of Arsenic and Selenium Detoxification

  • Author / Creator
    Kaur, Gurnit
  • Over 200 million people worldwide are exposed to the proven human carcinogen arsenic, due to contaminated drinking water. Animal studies have shown that arsenic and the essential trace element selenium can undergo mutual detoxification through the formation of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]- which undergoes biliary excretion, resulting in fecal elimination of both compounds. The ATP-binding cassette (ABC) transporter multidrug resistance protein 2 (MRP2/ABCC2), localized to the canalicular surface of hepatocytes, is a transporter of this conjugate. [(GS)2AsSe]- is also formed in animal red blood cells (RBCs), resulting in the sequestration of arsenic and selenium. In human cells, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The objectives of this thesis were (a) to characterize arsenite and selenite uptake and metabolism in human RBCs (hRBCs), (b) to characterize the effects of selenite on the hepatobiliary efflux of arsenic in sandwich-cultured human hepatocytes (SCHH) and (c) to characterize the effects of different selenium species on arsenic cytotoxicity and accumulation in HepG2 cells. The overarching hypothesis was that selenium would alter arsenic metabolism and transport through the formation of [(GS)2AsSe]-. 75Se-selenite uptake inhibition assays and experiments with heterologous overexpression systems showed selenite uptake in hRBCs to be mediated by the Cl−/bicarbonate (HCO3−) anion exchanger 1 (AE1). 73As-arsenite uptake inhibition assays showed arsenite uptake in hRBCs to be partially AQP3-mediated, while no evidence of GLUT1 involvement was found. Speciation analysis using X-ray absorption spectroscopy showed that 73% of the arsenic and selenium present in RBCs co-treated with arsenite and selenite was in the form of [(GS)2AsSe]−. The remaining 26% of As was present as As(GS)3, providing the first evidence of the formation of both As-glutathione conjugates in human cells. Consistent with published literature, all 73As biliary efflux in SCHH was glutathione-dependent. Selenite either reduced or did not alter 73As efflux across the sinusoidal and canalicular surfaces of SCHH. Preliminary data showed that selenide increases or does not change 73As biliary efflux, 75Se-selenide accumulation in hepatocytes was higher than 75Se-selenite; and selenide was able to protect SCHH against arsenite-induced toxicity while, selenite only conferred protection when the SCHH preparation displayed a potential for arsenic biliary efflux. Further characterization of selenite and selenide uptake and cytotoxicity differences in HepG2 cells also showed a higher IC50 value for cells treated with arsenite + selenide (138 ± 76 µM) than arsenite + selenite (50 ± 6 µM). Cytotoxicity assays of arsenite + selenite and arsenite + selenide at different treatment concentration ratios revealed higher overall mutual antagonism of arsenite + selenide toxicity than arsenite + selenite. In comparison to 75Se-selenite, HepG2 cells in suspension were at least 3-fold more efficient at accumulating selenium from reduced 75Se-selenide, and its accumulation was further increased by arsenite. These results were corroborated by X-ray fluorescence imaging of HepG2 cells which also showed increased arsenic accumulation in the presence of selenide. These results from studies in SCHH and HepG2 cells are consistent with a greater intracellular availability of selenide relative to selenite for protection against arsenite, and the formation and retention of a less toxic product, likely [(GS)2AsSe]−. Lastly, although arsenic-induced toxicities are known to display inter-individual variability, selenium supplementation trials are underway in arsenic-endemic regions in efforts to find cost-effective therapeutic solutions for arsenic-induced toxicities. As such, the focus of this thesis work was also to characterize single nucleotide polymorphic variants of MRP2 in order to gain a comprehensive understanding of the protective role of [(GS)2AsSe]− not only through its sequestration in RBCs, but also its efflux from hepatocytes. This work provides support in understanding the the mechanistic aspects of arsenic and selenium cellular handling in humans.

  • Subjects / Keywords
  • Graduation date
    Fall 2020
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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