Hepatobiliary Transport of Arsenic

  • Author / Creator
    Roggenbeck, Barbara A
  • Arsenic is a proven human carcinogen and associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMAV), monomethylarsonous acid (MMAIII), dimethylarsinic acid (DMAV), and dimethylarsinous acid (DMAIII) and eliminated predominantly in urine. Hepatic basolateral transport of As species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. Based on rodent studies it appears that the ATP binding-cassette (ABC) transporters in subfamily, multidrug resistance proteins (MRPs/ABCCs), are likely to play a role in efflux of As out of the hepatocyte. The current studies use several techniques to identify MRPs that may be important for hepatobiliary transport of As. Sandwich cultured human hepatocytes (SCHH) were used as a physiological model to characterize hepatic efflux of As. Cytotoxicity assays were used to screen the basolateral MRP candidate proteins MRP3 (ABCC3) and MRP5 (ABCC5) for their ability to transport AsIII, AsV, MMAIII, MMAV, DMAIII, and DMAV. The data obtained from the SCHH studies, the cytotoxicity studies, and studies done with MRP4 (ABCC4) by other members of the lab led us to use RNA silencing to further investigate the role of MRP4 in the basolateral efflux of As. The SCHH model showed that basolateral transport of As was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2 (ABCC2)-dependence. Treatment of SCHH with AsIII (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1 (ABCC1), MRP6 (ABCC6), or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of As. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest As basolateral transport prevails over biliary excretion and is mediated at least in part by an MRP. Candidate MRP proteins at the basolateral surface of the hepatocytes include MRP3, MRP4, MRP5, and MRP6. HEK293 cells overexpressing MRP3 or MRP5 did not confer resistance to AsIII, AsV, MMAIII, MMAV, DMAIII, or DMAV in cytotoxicity assays and provided evidence that MRP3 and MRP5 do not provide cellular protection against arsenicals. The role of MRP6 is under investigation. MRP4/ABCC4 is a high affinity transporter of DMAV and the diglutathione conjugate of MMAIII (MMA(GS)2), therefore, its role in As efflux was further investigated using RNA silencing in SCHH. Knockdown of MRP4 using shRNA containing lentiviral particles did not decrease the levels of any arsenicals in the efflux media of two SCHH preparations under the conditions tested. Non-specific effects of the lentivirus made interpretation of the RNA silencing experiments difficult however, we conclude that other transport proteins localized to the basolateral membrane of hepatocytes should be considered for their contribution to the efflux of As into blood for eventual urinary elimination.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries 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.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Physiology
  • Supervisor / co-supervisor and their department(s)
    • Leslie, Elaine (Physiology)
  • Examining committee members and their departments
    • Young, James (Physiology)
    • Siraki, Arno (Pharmacy and Pharmaceutical Sciences)
    • Mao, Qingcheng (Pharmaceutics)
    • Li, Xing-Fang (Laboratory Medicine and Pathology)