Leakage of incorporated radiolabelled adenine--a marker for drug-induced damage of macrofilariae in vitro.

An objective in vitro assay has been developed for quantifying drug-induced damage in Dipetalonema viteae macrofilariae. The method involves radiolabelling the worms ATP pool by incubating macrofilariae with [U-14C]-adenine. As determined by HPLC 72% of the incorporated label was in ATP, 15% in ADP and about 4% in each of NAD and AMP. Macrofilariae labelled with [U-14C]-adenine show a linear efflux of [14C]-label amounting to 21.3% of the total incorporation (mainly as uncharged catabolites) over a time course of 120 h in vitro incubation. When prelabelled worms were exposed to compounds exerting macrofilaricidal effects in vitro a marked stimulation in the leakage of [14C]-label from the worms was noted. The [14C]-label leakage appears to be linked with membrane (or cuticle) damage and the reduction of macrofilarial ATP levels. Determination of the amount of [14C]-label remaining in drug-treated worms relative to appropriate control provides a simple, sensitive and quantitative measure of drug-induced damage in macrofilariae (including Onchocerca). The method has been used to describe the macrofilaricidal activity of a wide range of antifilarial standards, membrane disruptive agents, respiratory inhibitors, fasciolicides and anti-cestode compounds.