RESISTANCE TO DL-ALPHA-DIFLUOROMETHYLORNITHINE BY CLINICAL ISOLATES OF TRYPANOSOMA-BRUCEI-RHODESIENSE - ROLE OF S-ADENOSYLMETHIONINE

The ornithine decarboxylase (ODC) inhibitor DL-alpha-difluoromethylorn ithine (DFMO) has emerged as a new treatment for West African sleeping sickness but is less effective against East African sleeping sickness . We examined uncloned clinical isolates of Trypanosoma brucei rhodesi ense, agent of the disease in East Africa, which were refractory to DF MO in laboratory infections, for characteristics that would explain th eir resistance. None of the isolates were from patients treated with D FMO. Two isolates took up [H-3]DFMO at 50-70% lower rates than drug-se nsitive strains but ODC activities, K(i) values for DFMO, spermidine a nd spermine uptake rates, polyamine content and inhibition of polymami ne metabolism by DFMO were statistically (P < 0.05) similar between se nsitive and refractory isolates. One cloned strain, continuously passa ged in vivo under DFMO pressure and included for comparison, had >85% lower ODC activity and up to 14-fold higher putrescine uptake rates th an sensitive controls. A statistically important trend was the metabol ism of S-adenosylmethionine (AdoMet): activities of AdoMet synthetase and AdoMet decarboxylase were 2- to 5-fold and 3- to 40-fold lower in resistant strains, respectively, while intracellular AdoMet pools (Ado Met + decarboxylated AdoMet) that were >60-fold elevated in sensitive strains during DFMO treatment, increased only 9-fold in refractory iso lates. The extreme elevation of the AdoMet pool in sensitive isolates from 0.7 to 44 nmol/mg protein and an intracellular pool concentration of approximately 5 mM may lead to an imbalance in methylation of prot eins or other cell constituents as a consequence of DFMO action. These studies indicate that the metabolism of AdoMet is altered significant ly in DFMO refractory isolates and suggest that differences in AdoMet metabolism may be responsible for increased tolerance to DFMO.