Organ Distribution of Multidrug Resistance Proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and Hepatic Induction of Mrp3 by Constitutive Androstane Receptor Activators in Rats

Abstract

Thalidomide produces numerous birth defects, the most notable being phocomelia. Mechanisms behind thalidomide-induced malformations have not been fully elucidated, although recent evidence suggests a role for reactive oxygen species. A thalidomide-resistant (rat) and -sensitive (rabbit) species were used to compare potential inherent differences related to oxidative stress that may provide a more definitive understanding of mechanisms of thalidomide embryopathy. Limb bud cells (LBCs) were removed from the rat and rabbit embryo, dissociated, and plated in culture for 24 h. A fluorescence (6-carboxy-2′,7′-dichlorofluorescin diacetate; DCF) assay for oxidative stress was used with varying concentrations of thalidomide (5–100 μM). Thalidomide (100 μM) showed a 6-fold greater production of oxidative stress in rabbit cultures than in rat. Lower concentrations (50 and 25 μM) also showed a significant increase in reactive oxygen species. Confocal microscopy revealed DCF fluorescence preferentially in rabbit LBC nuclei compared with the uniform distribution of DCF fluorescence in rat LBC. Localization of glutathione (GSH) was determined using 5-chloromethylfluorescein diacetate fluorescent confocal microscopy. In rat cultures, significant thalidomide-induced GSH depletion was detected in the cytosol but the nuclei maintained its GSH content, but rabbit LBC showed significant GSH depletion in both compartments. GSH depletion was confirmed by high-performance liquid chromatography analysis. These observations provide evidence that thalidomide preferentially produces oxidative stress in the thalidomide-sensitive species but not the thalidomide-resistant species. Nuclear GSH content in the rabbit LBC is selectively modified and indicates a shift in the nuclear redox environment. Redox shifts in the nucleus may result in the misregulation of transcription factor/DNA interactions and cause defective growth and development.