Role of SOD Peroxidase Activity in Oxidizing Drugs: Potential Modulation of Drug Toxicity

  • Author / Creator
    Aljuhani, Naif S
  • There is a close relationship between inflammation and cancer. At site of inflammation, reactive oxygen species (ROS) are generated, including hydrogen peroxide (H2O2). The generation of ROS could, in turn, activate transcription factors that are involved in inflammation, cellular transformation, tumor cell death, proliferation, angiogenesis, and metastasis. However, redox-buffering systems work as a cellular defense against ROS. Exhausting ROS-buffering capacity generates oxidative stress that are involved in many diseases, including cancer. The activities of two major antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), are decreased in cancer tissues. The potential accumulation of H2O2 is known to inactivate SOD but with the presence of certain intermediates, such as bicarbonate (HCO3-), the induction of oxidative stress can be propagated through carbonate radicals (CO3-); this is called SOD peroxidase activity. It is not known, however, if SOD peroxidase-derived CO3- can oxidize the drugs and consequently modulate their metabolism and cytotoxicity. We investigated the oxidation of phenylbutazone (PBZ) and 6-mercaptopurine (6-MP) by SOD peroxidase-derived CO3- through utilizing biochemical assays including, electron paramagnetic resonance (EPR)-spin trapping, oxygen analysis, UV-Vis and LC/MS measurements. Monitoring SOD protein and its activity and demonstrating the cytotoxic effect of oxidizing PBZ and 6-MP on cancer cells was also investigated. We found that SOD peroxidase activity was significantly attenuated by increased concentrations of PBZ that culminated in PBZ carbon-centered radicals as judged by computer simulation of hyperfine splitting constants. An identical PBZ carbon-centered radical was detected by reaction of PBZ with other peroxidases such as myeloperoxidase (MPO/H2O2) and horseradish peroxidase (HRP/H2O2). The oxygen uptake was significantly increased when PBZ was oxidized by SOD peroxidase activity, further confirming PBZ carbon radical formation. Both 4-hydroperoxyphenylbutazone (4-hydroxy-PBZ) and 4-hydroxyphenylbutazone (4-OH-PBZ) were detected after oxidizing PBZ by SOD peroxidase activity as evidenced by both UV-Vis and LC/MS analysis. By doing further investigations, we found that diethyldithiocarbamate (DDC), SOD inhibitor, inhibited SOD peroxidase activity. Also, the PBZ carbon-centered radical was significantly attenuated by glutathione (GSH) that resulted in thiyl radical formation. Sorbic acid decreased PBZ carbon-centered radicals in concentration-dependent manner. GSH, but not sorbic acid, protected SOD protein from oxidation by CO3- radicals. The cytotoxicity of PBZ was synergistically enhanced with the presence of H2O2 compared with either H2O2 or PBZ used alone. However, the synergistic cytotoxic effect of combined treatment on HepG2 cells was significantly attenuated by the presence of DDC. And extracellular addition of both human and bovine SOD significantly catalyzed the cytotoxic effect of combined PBZ with H2O2 on HepG2 cells. We also found that SOD peroxidase-derived CO3- radicals catalyzed the oxidation of 6-MP by bicarbonate-activated peroxidase system (H2O2/HCO3-). The oxidation of 6-MP by SOD peroxidase activity resulted in forming a major peak product (C5H4N4O2S; 182.9981 m/z), which corresponded to 6-sulfoxide-mercaptopurine (6-sulfoxide-MP) as indicated by using LC/MS analysis. 6-MP, but not azathioprine (AZA), significantly decreased CO3- radicals as evidenced by using EPR, further indicating that SOD peroxidase activity oxidized 6-MP. Interestingly, SOD activity was protected by 6-MP but not AZA. Lastly, pre-incubation of 6-MP with H2O2/HCO3- resulted in significantly decreased the cytotoxic effect of 6-MP as judged by the attenuated metabolic activity of a normal (HEK293) cell line and HepG2 cells. Our overall findings indicate that the combination of drugs with SOD peroxidase-derived CO3- radicals appeared to modulate their metabolism and consequently their cytotoxic effect. These findings may have relevance for drugs administered to patients who have chronic inflammation.

  • 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
  • Specialization
    • Pharmaceutical Sciences
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Wuest, Frank (Department of Oncology, University of Alberta)
    • Bandy, Brian ( College of Pharmacy and Nutrition, University of Saskatchewan)
    • Jurasz, Paul (Faculty of Pharmacy and Pharmaceutical Science, University of Alberta)
    • Ussher, John (Faculty of Pharmacy and Pharmaceutical Science, University of Alberta)