Detection of Efflux of Reactive Metabolites of Troglitazone from Primary Human Hepatocytes Using Stable Isotope Labeled Glutathione

Stable Isotope Labeled Glutathione I. Mezine, C. B. Bode, B. Raughley, S. Bhoopathy, A. Owen, I. J. Hidalgo Absorption Systems Purpose Drug-induced liver toxicity is one of the most common adverse drug effects, and formation of reactive metabolites (RM) could be a contributing factor. Troglitazone (TGZ), the model compound used in this study, was withdrawn from the market due to a high incidence of liver failure. The metabolism of TGZ is known to generate RM, which were detected as stable glutathione (GSH) conjugates in vivo and in vitro. We previously reported application of stable isotope-labeled (SIL) GSH methodology to trap and detect RM of TGZ generated in cryopreserved human hepatocytes in suspension. A major finding was different degrees of incorporation of exogenous GSH/SIL-GSH mixture into trapped RM. More recently, we have also reported evidence, obtained using oil-filtration methodology, in support of one of the hypothesized mechanisms: differential efflux of RM from cryopreserved human hepatocyte suspensions, followed in some cases by extracellular trapping as GSH conjugates. The aim of this work was to investigate if efflux of RM is also intrinsic to primary cultures of human hepatocytes. Methods Materials: TGZ and (C2, N-Gly)-GSH (isotopic purity>90%) were purchased from Toronto Research Chemicals (Toronto, ON, Canada) and Cambridge Isotope Labs (Cambridge, MA, USA), respectively. All other reagents were of analytical or HPLC grade (Sigma, St. Louis, MO, USA). Biology: Primary cultures of hepatocytes were purchased from Life Technologies (Grand Island, NY, USA), TRL (Research Triangle Park, NC, USA), and Bioreclamation IVT (Baltimore, MD, USA). Incubations with TGZ (5 and 50 μM), with and without addition of a mixture of GSH/SIL-GSH (ratio 1:1, total concentration 5 mM), were performed in duplicate. The medium was removed for analysis and replaced with fresh medium after each 24-hour interval. After 72 hours, cells were extracted for metabolite profiling using LC-HRAMS analysis. The concentrated medium (5-fold) and cell extracts were analyzed by reverse phase HPLC-HRAMS implemented on an LTQ Orbitrap XL equipped with HESI-II probe. The Orbitrap was operated at resolution 60,000 in survey scans (range 250-900 Th) and at resolution 15,000 in MS scans The acquired data files were processed manually for the presence of known GSH conjugates using Xcalibur software (Thermo). In addition, a comprehensive search for GSH conjugates was performed using MetWorks software (Thermo) via automatic detection of the monoisotopic pattern characteristic for the GSH/SIL-GSH mixture (split 3.0037 Da); a range of peak intensity ratios from 1:0.1 to 1:0.9 was evaluated.