Effect of dyskinetoplastic agents on ultrastructure and oxidative phosphorylation in Crithidia fasciculata

Ethidium bromide (EB) is an intercalating agent which binds specifically to the kinetoplast (mitochondrial) DNA (kDNA) of trypanosomatids. Accordingly , EB inhibits DNA replication, thus inducing dyskinetoplasty. Since in euka riotic organisms mitochondrial DNA encodes the genetic information for cyto chromes 6, aa(3) and F0F1 ATPase, it seemed of interest to establish whethe r a similar effect occurs in Crithidia fasciculata, a trypanosomatid used f or assay of potential trypanocidal drugs. Culturing of C. fasciculata in th e presence of EB inhibited growth and induced dyskinetoplasty, as confirmed by electron microscopy. The kinetoplast of EB-cultured crithidia lost its characteristic are shape, it was misplaced in the cell cytoplasm its matrix structure and membrane differentiation were specifically modified. Dyskine toplasty decreased crithidia respiration and oxidative phosphorylation, as indicated by the lower ATP level, ATP/ADP ratio and adenylate energy charge . The interference of EB with kinetoplastic constituents synthesis was conf irmed by the lack of action of EB on crithidia in the stationary phase of g rowth, that ruled out direct inhibition of oxidative phosphorylation enzyme s. The lipophilic o-naphthoquinone beta -lapachone produced structural alte rations in kinetoplast membranes, that correlated with inhibition of oxidat ive phosphorylation. These latter effects involved free radicals since they were prevented by free radical scavengers.