In vivo effects of α- dl-Difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei

Abstract

The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor α- dl-difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72–80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels (< 1 nmol mg −1 protein) after 36 h treatment. α- dl-Difluoromethylornithine-treated cells were unable to synthesize putrescine from [ 3H]ornithine but were able to convert [ 3H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S-adenosylmethionine was also altered in treated parasites: decarboxylated S-adenosylmethionine increased more than 1000-fold over untreated cells while S-adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50–100%, while protein synthesis increased up to 4-fold in 36-h treated cells. α- dl-Difluoromethylornithine treatment progressively limits the parasites' ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender → short stumpy transformation.