The Influence of Selenium on Arsenic Hepatobiliary Transport

• Author / Creator

  Zhou, Janet R

• Chronic exposure to arsenic causes lung, skin, and bladder cancer in humans. Conservative estimates suggest at least 92-220 million people worldwide are exposed to arsenic through consumption of contaminated water. Unfortunately, removal of arsenic from contaminated water sources is not economically feasible. Selenium is an essential trace nutrient but is toxic in excess. Interestingly, arsenic and selenium are mutually protective. Simultaneous exposure to toxic doses of arsenic and selenium in laboratory animals result in reduced accumulation of both compounds through the formation and biliary excretion of the seleno-bis (S-glutathionyl) arsinium ion [(GS)2AsSe]-. Completed selenium-supplementation trials in arsenic-endemic regions have used various chemical species of selenium including selenite and selenomethionine. Methylselenocysteine has been shown to have anti-cancer effects in vitro. Despite completed selenium-supplementation trials, the influence of selenium on human arsenic hepatobiliary transport has not been studied using optimal human models. There is also likely a genetic component to susceptibility to arsenic-induced diseases as different outcomes between individuals with similar exposure histories are observed. In human hepatocytes, the ATP-binding cassette transporter, multidrug resistance protein 2 (MRP2, gene symbol ABCC2) is localized to the canalicular/apical membranes for biliary excretion of arsenic-glutathione conjugates, and the related multidrug resistance protein 4 (MRP4/ABCC4) is localized to the sinusoidal/basolateral membranes for arsenic transport into systemic circulation.
  The objectives of this thesis were to: (i) investigate the influence of different selenium chemical forms on arsenic hepatobiliary transport, (ii) investigate the stimulatory effect of methylselenocysteine on arsenic sinusoidal efflux, and lastly, (iii) investigate the effect of naturally occurring MRP2/ABCC2 variants on arsenic transport.
  Human HepaRG cells, a surrogate for primary human hepatocytes were established as a model for studying arsenic hepatobiliary transport. Arsenite + selenite and arsenite + selenide at different molar ratios revealed mutual toxicity antagonisms, with the latter being higher. Significant arsenic biliary excretion was detected with a biliary excretion index (BEI) of 14 ± 8%, which was stimulated to 32 ± 7% by selenide. Consistent with the formation and biliary efflux of [(GS)2AsSe]-, arsenite increased the BEI of selenide from 0% to 24 ± 5%. Arsenic biliary excretion was lost in the presence of selenite, selenomethionine, and methylselenocysteine. Sinusoidal arsenic efflux was stimulated by ~1.6-fold by methylselenocysteine, but unchanged by other selenium forms. Arsenic efflux across canalicular and sinusoidal membranes (± selenide) was temperature- and glutathione-dependent and inhibited by MK571, an MRP inhibitor.
  Since MRP2/ABCC2 and MRP4/ABCC4 mediate biliary and sinusoidal efflux of arsenic, respectively, ABCC2 and ABCC4 were knocked down (individually) in HepaRG cells, resulting in barely detectable levels of MRP2 or MRP4. Experiments using ABCC2-knockdown HepaRG cells revealed that MRP2 accounted for all detectable biliary efflux of arsenic (± selenide). Similarly, arsenic sinusoidal efflux decreased by 27%, 57%, and 31% after glutathione depletion, temperature reduction and addition of MK571, respectively, which also resulted in a loss of methylselenocysteine stimulation. Further strategies were employed to determine if this process is MRP4-mediated. Arsenic sinusoidal efflux decreased by 35%, and methylselenocysteine-stimulated efflux was lost in the presence of ceefourin-1, an MRP4 inhibitor. Arsenic efflux from ABCC4-knockdown HepaRG cells decreased by 30%, and methylselenocysteine-stimulated arsenic export was completely lost relative to control HepaRG cells with parental levels of MRP4. Overall, the chemical form of selenium and human MRP2 and MRP4 strongly influenced arsenic hepatobiliary transport.
  Lastly, 13 naturally occurring MRP2 variants with a single nucleotide substitution resulting in an amino acid change were investigated for their influence on MRP2 level, plasma membrane localization, MRP2-enriched membrane vesicle transport of As(GS)3 and [(GS)2AsSe]−, and cellular accumulation of arsenic ± selenium (selenide and selenite). The variants R412G-, V1188E-, C1515Y- and V1188E/C1515Y-MRP2 were similar to WT-MRP2 for protein levels, cell surface localization, and transport activity while S789F- and A1450T-MRP2 were undetected at the cell surface. As(GS)3 transport activity of T1477M-MRP2 was 60% of WT-MRP2. R353H-, R1181L-, and P1291L-MRP2-enriched vesicles had [(GS)2AsSe]− transport activity increased by 160%, 145% and 170%, respectively, compared to WT-MRP2. In contrast, V417I- and R1150H- had [(GS)2AsSe]− transport that was 59% and 37%, respectively, of WT-MRP2. This thesis advances knowledge about the influence of selenium on human hepatic handling of arsenic and our understanding about inter-individual differences in arsenic transport pathways. Future studies could use our findings to help design efficacious selenium-supplementation trials and predict responses to selenium-supplementation.

• Subjects / Keywords

  • arsenic
  • selenium
  • multidrug resistance protein 2
  • multidrug resistance protein 4

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-7368-x016

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.