Defluorination of fluoroacetate in vitro by rat liver subcellular fractions

Abstract

An earlier report showing enhanced accumulation of ionic fluoride in bones of rats given fluoroacetate (FAc) suggested an in vivo defluorination of fluoroacetate. Rat liver an organ which shows minimal pathological and biochemical effects in FAc intoxication was found to possess defluorination activity in vitro. Subcellular fractionation of livers from male Sprague-Dawley rats was performed in 0.25 m sucrose and yielded the following fractions: whole homogenate, nuclear, mitochondrial, microsomal and 105,000 g supernatant. Defluorination activity was measured by incubating subcellular fractions and their boiled controls with FAc (1 hr, 37°C, pH 7.4, in 0.1 m Tris-HCl or 0.1% Triton X-100) and comparing the difference in ionic fluoride content at the end of the incubation. Defluorination activity based on protein content was consistently the highest in the 105,000 g supernatant fraction, the only fraction showing an activity increase over the original homogenate. The microsomal fraction had minimal activity. Defluorination activity in the 105,000 g supernatant as a function of time after fraction isolation at 4°C revealed a time-dependent reduction in activity. After 24 hr at 4°C, activity was almost completely lost. The time-dependent loss of activity at 4°C could be regained when glutathione (GSH) was added to this fraction at a final concentration of 5 m m. Furthermore, GSH significantly increased the defluorination activity in all subcellular fractions. A study of the optimum pH for defluorination activity in the 105,000 g supernatant fraction was performed in 0.1 m Tris-maleate buffer. Surprisingly, a complete loss of activity with this buffer resulted throughout the pH range studied (pH 5.6–8.0). Furthermore, defluorination activity of all subcellular rat liver fractions was completely inhibited by maleate and stimulated by glutathione. These results are consistent with the involvement of a sulfhydryl group in the defluorination of FAc in rat liver.