In VitroUptake of Methyltert-Butyl Ether in Male Rat Kidney: Use of a Two-Compartment Model to Describe Protein Interactions

Abstract

Methyltert-butyl ether (MTBE) is a gasoline additive that causes renal tumors in male rats. In the process of measuring chemical specific parameters necessary to develop a quantitative dosimetry model of MTBE in rats, the uptake of MTBE was found to be 5.5 times greater in male than in female F-344 rat kidney homogenate. The objectives of this study were to characterize the factor(s) that influences the high uptake of MTBE into male rat kidneyin vitroand to develop a system to evaluate the interaction of MTBE with the male rat-specific protein, α2u-globulin (α2u). The uptake of MTBE in male, but not female, rat kidney homogenate was found to be dependent on protein and chemical concentrations. When [14C]MTBE was incubated with male rat kidney homogenate, radioactivity coeluted with the total protein fraction on a gel filtration column. An interaction between [14C]MTBE and male rat kidney proteins was not found under conditions of dialysis or anion exchange chromatography. A two-compartment vial equilibration model was used to assess the interaction between MTBE and α2u. Using this system, the dissociation constant for MTBE and α2u was estimated to be 2.15 × 10−4m, which is in the range of other chemicals known to bind to α2u and cause α2u-mediated nephropathy.d-Limonene oxide was used to validate this two-compartment vial equilibration system. These findings illustrate a technique useful in estimating the dissociation constant for a volatile chemical and a protein, as well as explain the process that contributes to the uptake of MTBE into male rat kidney homogenatein vitro.A description of the weak interaction between MTBE and α2u will be used to refine a physiologically based pharmacokinetic model to describe the target tissue (kidney) concentrations of MTBE.