Malondialdehyde-protein adducts in the spleens of aniline-treated rats: Immunochemical detection and localization

Previously it was reported that aniline exposure in rats induces increased lipid peroxidation and formation of malondialdehyde (MDA)-protein adducts in the spleen. In order to further elucidate the role of MDA-protein adducts in the splenic toxicity of aniline, studies were conducted to detect and localize these adducts in the spleen. Rabbit polyclonal antisera to MDA-keyhole limpet hemocyanin were employed for immunohistochemical localization and Western blot analyses of MDA-protein adducts in the spleens of aniline-treated (65 mg/kg/d aniline in the drinking water for 30 d) and control rats. For immunohistochemical localization of MDA-protein adducts in the spleen, a new approach using alkaline phosphatase-fast red (red color) to demonstrate bound primary antibodies was adopted, providing a sharper and increased contrast compared to horseradish peroxidase-diaminobenzidine (brown color) methodology. This new approach allowed us to differentiate the changes in aniline-treated spleens, which had extensive brownish deposits of iron proteins. Spleens from aniline-treated rats showed intense staining for these adducts in the red pulp areas (where iron was also localized), especially within the sinusoidal macrophages. Spleens from control rats showed only mild staining for adducts and only traces of iron. Western blot analyses of splenic microsomal proteins from aniline-treated and control rats showed the presence of 13 different MDA-modified proteins. However, 26-, 32-, and 44-kD proteins were more prominent in the aniline-treated rats. The colocalization of MDA-protein adducts with iron in the red pulp of the spleen suggests that iron-catalyzed lipid peroxidation leading to formation of MDA-protein adducts could be a potential mechanism for splenic toxicity of aniline.