Assessment of the role of in situ generated (E)-2,4-diene-valproic acid in the toxicity of valproic acid and (E)-2-ene-valproic acid in sandwich-cultured rat hepatocytes

Evaluation of In Situ Generated Valproyl 1-O-β-Acyl Glucuronide in Valproic Acid Toxicity in Sandwich-Cultured Rat Hepatocytes

“…Adult male Sprague-Dawley rats (175-200 g) were obtained from Charles River Laboratories (Senneville, QC, Canada) and were housed and cared for in our animal care facility, as described previously (Surendradoss et al, 2012). All animal experiments were approved by the University of British Columbia Animal Care Committee and were conducted in accordance with the guidelines of the Canadian Council on Animal Care.…”

“…Rat hepatocytes were isolated by a two-step collagenase perfusion method (Seglen, 1993), as described previously (Tong et al, 2005a). Plating of hepatocytes (0.7 Â 10 6 cells/well), preparing the sandwich-culture configuration, and culturing of hepatocytes were performed, as reported earlier (Surendradoss et al, 2012). At 120 hours after plating, sandwich-cultured rat hepatocytes were treated with VPA, culture medium (vehicle for VPA), diclofenac, or DMSO (vehicle for diclofenac) for the next 24 hours at the concentrations indicated in each figure legend.…”

“…LDH release was used as a marker of cell necrosis (Jauregui et al, 1981). LDH activity in the culture supernatants and cell lysate samples was determined using the Cytotoxicity Detection Kit (Roche Diagnostics) (Surendradoss et al, 2012). LDH released into the culture supernatant was expressed as a percentage of the total cellular LDH activity (i.e., sum of the LDH activity in the culture supernatant and cell lysate).…”

“…Cellular accumulation of BODIPY 558/568 C 12 was used as an index of steatosis (Fujimura et al, 2009) and determined, as described previously (Surendradoss et al, 2012). Fluorescence was measured at a l ex of 484 nm and l em of 618 nm in a Biotek Synergy Mx microplate reader (Biotek Instruments, Winooski, VT).…”

“…Cellular content of total GSH was quantified using the Glutathione Assay Kit (Cayman Chemical), as described previously (Surendradoss et al, 2012). The rate of formation of the reaction product, 5-thio-2-nitrobenzoic acid, was determined spectrophotometrically in a kinetic mode at a wavelength of 405 nm in a Labsystems Multiskan Ascent multiwell plate reader (Thermo Electron, Burlington, ON, Canada).…”