Selective protein adducts to membrane proteins in cultured rat hepatocytes exposed to diclofenac: radiochemical and immunochemical analysis.

The nonsteroidal anti-inflammatory drug diclofenac can be bioactivated to the reactive acyl glucuronide, which covalently binds to hepatocellular proteins in rat hepatocytes. Short term cultured rat hepatocytes were used to further study the formation and nature of protein adducts after exposure to diclofenac. Incubation of cells with [14C]diclofenac (30 microM) for up to 24 hr was associated with a time-dependent increase in radioactivity bound to proteins. Upon subcellular fractionation of hepatocytes exposed to diclofenac for 2 hr, the majority of the radiolabel appeared in the microsomal fraction. By 24 hr, the specific binding had decreased by 50% in this cell compartment. In contrast, the hepatocellular plasma membrane fraction, which also was associated with high specific binding of diclofenac-derived radioactivity by 2 hr, exhibited a approximately 3-fold increase in adduct formation by 24 hr. Lesser amounts of radioactivity were associated with cytosolic proteins. After resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, the radioactivity was associated with a major protein band with an apparent molecular mass of 60 kDa that was present in both microsomes and plasma membranes. Further, we developed an antidiclofenac antibody against diclofenac-protein adducts by Protein A chromatography of a polyclonal antiserum raised in rabbits against a diclofenac-keyhole limpet hemocyanin adduct. The antidiclofenac antibody did recognize diclofenac-protein adducts on Western blots of homogenates of cultured rat hepatocytes exposed to diclofenac. The major detected adducts included the 60-kDa protein, which was present at all diclofenac concentrations used. In addition, the antibody recognized proteins with apparent molecular masses of 50, 80, and 126 kDa that were not evident in the radiochemical assay. There were no detectable cross-reactive epitopes of proteins recognized by the antibody on Western blots of cultured hepatocytes not treated with diclofenac. Moreover, immunoblots of liver homogenates from rats treated with diclofenac (30 mg/kg/day, intraperitoneally, for 4 days) also exhibited adducts with the 60- and 80-kDa proteins. Collectively, these results suggest that binding of diclofenac to rat hepatocyte proteins is selective and that a 60-kDa microsomal membrane protein (or protein subunit) that accumulates in the plasma membrane fraction appears to be the major target for alkylation both in cultured hepatocytes exposed to diclofenac and in vivo.