Assessment of the covalent binding potential of 2,2,4-trimethylpentane to rat α2u-globulin

Abstract

Subchronic exposure of male rats to the nephrotoxin 2,2,4-trimethylpentane (TMP) causes an accumulation of protein droplets in the epithelial cells of the renal cortex. Experimental evidence suggests that these droplets contain α 2u-globulin, a low-molecular-weight protein found specifically in the urine of male rats. It has been proposed that aldehyde metabolites of TMP form Schiff base adducts with the lysine groups of α 2u-globulin and thereby inhibit renal lysosomal processing of the protein. Accordingly, the ability of TMP and its metabolites to covalently bind to α 2u-globulin was examined. As a model, a [ 14C]formaldehyde -α 2u - globulin Schiff base was formed. This protein adduct was stabilized by reduction with cyanoborohydride and could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein analysis by SDS-PAGE demonstrated that hepatocytes isolated from male Fischer-344 rats produced significant quantities of α 2u-globulin in culture, whereas hepatocytes from female rats did not. A 15-hr exposure of metabolically competent, primary cultures of male rat hepatocytes to [ 14C]TMP (0.1 and 0.5%, v/v), followed by reduction with cyanoborohydride, dialysis, and analysis with SDS-PAGE, revealed no evidence of radiolabeled α 2u-globulin. When [ 14C]TMP was administered to an adult male Fischer-344 rat (300 mg/kg, ig) 22, 16, and 10 hr before sacrifice, 16% of the administered radioactivity was eliminated in the urine as TMP metabolites. Analysis as above showed no TMP-derived radioactivity in fractions containing α 2u-globulin from liver, blood, kidney cortex, or urine. The absence of a detectable covalent interaction between TMP and α 2u-globulin following in vitro or in vivo exposure suggests that a TMP- α 2u-globulin adduct is not responsible for the excessive formation of protein droplets in the renal cortex of exposed male rats.