title: Elucidation of the Mechanisms through Which the Reactive Metabolite Diclofenac Acyl Glucuronide Can Mediate Toxicity.
abstract: We have previously reported that mice lacking the efflux transporter Mrp3 had significant intestinal injury after toxic diclofenac (DCF) challenge, and proposed that diclofenac acyl glucuronide (DCF-AG), as a substrate of Mrp3, played a part in mediating injury. Since both humans and mice express the uptake transporter OATP2B1 in the intestines, OATP2B1 was characterized for DCF-AG uptake. In vitro assays using human embryonic kidney (HEK)-OATP2B1 cells demonstrated that DCF-AG was a substrate with a maximal velocity (Vmax) and Km of 17.6 ± 1.5 pmol/min per milligram and 14.3 ± 0.1 μM, respectively. Another key finding from our in vitro assays was that DCF-AG was more cytotoxic compared with DCF, and toxicity occurred within 1-3 hours of exposure. We also report that 1 mM DCF-AG caused a 6-fold increase in reactive oxygen species (ROS) by 3 hours. Investigation of oxidative stress through inhibition of superoxide dismutase (SOD) revealed that DCF-AG had 100% inhibition of SOD at the highest tested dose of 1 mM. The SOD and ROS results strongly suggest DCF-AG induced oxidative stress in vitro. Lastly, DCF-AG was screened for pharmacologic activity against COX-1 and COX-2 and was found to have IC50 values of 0.620 ± 0.105 and 2.91 ± 0.36 μM, respectively, which represents a novel finding. Since cyclooxygenase (COX) inhibition can lead to intestinal ulceration, it is plausible that DCF-AG can also contribute to enteropathy via COX inhibition. Taken in context, the work presented herein demonstrated the multifactorial pathways by which DCF-AG can act as a direct contributor to toxicity following DCF administration.
authors: Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut.
authors: Renato J
authors: RJ
authors: Scialis
authors: Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut jose.manautou@uconn.edu.
authors: José E
authors: JE
authors: Manautou
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D051545:Cyclooxygenase 1 / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004008:Diclofenac / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D005767:Gastrointestinal Diseases / Q000139:chemically induced / Q000473:pathology; D020719:Glucuronides / Q000494:pharmacology / Q000633:toxicity*; D006801:Humans; D007700:Kinetics; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D027361:Organic Anion Transporters / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D051545:Cyclooxygenase 1; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D004008:Diclofenac; D020719:Glucuronides; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D027361:Organic Anion Transporters; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; C487366:1-O-(2-((2',6'-dichlorophenyl)amino)phenylacetyl)glucopyranuronic acid; C450859:SLCO2B1 protein, human; C114462:multidrug resistance-associated protein 3