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Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis inTrypanosoma cruzi and stimulated O2 uptake and $$O_2 ^{\bar .} $$ generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and $$O_2 ^{\bar .} $$ generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H2O2 production were obtained withCrithidia fasciculata despite the presence of catalase in this organism.

Activity of H(+)-ATPase of Trypanosoma cruzi (RA strain) submitochondrial particles is increased in parasites which have a low spermine content caused by growing in a polyamine free medium or in a medium containing inhibitors of polyamine synthesis. Under these conditions, the proliferation rate is markedly decreased. Kinetics of the enzyme inhibition by spermine indicates a non-competitive inhibition. Spermine, through its action on the H(+)-ATPase hydrophobic environment, affects the enzyme activity. Together with the ATPase protein inhibitor, this could be a mechanism regulating ATP levels needed for the parasite proliferation.

Growth and polyamine content of epimastigotes of Trypanosoma cruzi were studied in the presence of precursors or inhibitors of putrescine synthesis. Arginine and agmatine turned out to be better precursors than ornithine. alpha-D-difluoromethylarginine, an inhibitor of arginine decarboxylase, inhibited cellular proliferation and decreased putrescine, spermidine and spermine contents while that of cadaverine remained unchanged. These effects were reversed both by agmatine and putrescine. alpha-D-difluoromethylornithine, an inhibitor of ornithine decarboxylase did not inhibit growth of parasites grown in a polyamine free medium. These results suggest that epimastigotes need polyamines to grow, and that the parasite are able to synthesize them mainly through the arginine decarboxylase pathway.

Despite its epidemiological importance, chemotherapy of Chagas' disease is still an unsolved problem. Many drugs have been assayed for their trypanocidal action on T. cruzi, but, so far, naftoquinone-imines have not been included in drug screenings. Fernández A.E. et al., prepared a series of 4-(aminomethylisoxazolyl)-1,2-naftoquinones. Some of these, namely, IIIA (2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naftoquinone-4- imine), IIIC hydroxy-N-3,5-dimethyl-4-isoxazolyl)-1,4-naftoquinone-4-i min e, IIIE (2-hydroxy-N-(3-methyl-5-isoxazolyl)-1,4-naftoquinone-4-i min e), and IVD (5-methyl-3-isoxazolyl)-1,2-naftoquinone-4-imine) were assayed on T. cruzi epimastigotes. Parasite growth and DNA synthesis were inhibited 12-36% and 14-49%, respectively, by 18-19 microM quinones or 44-83% and 37-73%, respectively, by 60-78 microM quinones (Table 1). The assayed compounds (37-100 microM concentration) stimulated oxygen uptake (Figs. 2 and 3) and superoxide anion generation by epimastigotes to 0.20-0.70 nmol/min/mg of cell protein (O2- measured by the adrenochrome method, Table 2). In the presence of quinones, T. cruzi mitochondrial fraction supplemented with 0.42 mM NADH produced O2- at a rate of 1.10-1.45 nmol/min/mg of protein (31 and 75 microM quinone, respectively; Table 3) whereas, under the same experimental conditions, the microsomal fraction supplemented with NADPH produced O2- at rate of 0.85 and 1.03, respectively (same units). The kinetics of O2- generation by the mitochondrial fraction supplemented with quinone IIIC and NADH revealed two interacting sites (Fig. 5). The corresponding Km(s) were 5.2 and 17 microM and the Vm, 13 and 17 nmoles O2-/min/mg of protein, respectively. The quinone interaction with the mitochondrial membranes involved a "quinone reductase" located on the substrate site of the antimycin site, as shown by the 3.6-fold increase of the oxygen uptake rate induced by quinone IIIE in Fig. 2. Apparently, the quinone excess inhibited the reductase since by varying the quinone concentration in the 0-500 microM range, the oxygen uptake versus quinone concentration plots showed a maximum at 100-150 microM quinone (Fig. 3). O2- generation by quinones depended on the latter redox-cycling, as shown by a) the decrease of quinone absorption at 480-500 nm after incubation with T. cruzi epimastigotes for the time necessary to exhaust the reaction mixture oxygen, and b) the reappearance of the quinone absorption band after oxygenation of the anaerobic reaction mixture (Fig. 6).

Differential reduced minus oxidized (Red-Ox) or reduced. CO minus reduced (Red. CO-Red) spectra of Trypanosoma cruzi metacyclic trypomastigotes (Tulahuen strain), revealed the presence of cytochromes aa3, b, c5 5 8, o and possibly d (Fig. 1). Mitochondrial membranes from the epimastigote form of the parasite showed similar cytochrome spectra (Fig. 2A). Extraction of the mitochondrial membranes with guanidine and cholate, and spectroscopy of the cytochrome o -cyanide derivative proved that this cytochrome is an integral constituent of the mitochondrial membranes (Figs. 2B and 4, and Table 1). Contamination of the investigated samples with hemoglobin, peroxidase, catalase, cytochrome P-420, cytochrome P-450 or culture medium hemoproteins was ruled out by filtering the cells and mitochondrial membranes on Sephadex G-50, or by differential spectroscopy of pigments, or by differential centrifugations of samples. Investigation of pyridine-hemochromes revealed hemes A, B, C and D (Fig. 3), thus confirming the presence of the postulated cytochromes. Comparative spectroscopy of a series of T. cruzi stocks (including Tulahuen and Y strains), many of them obtained from acute or chronic forms of Chagas disease, revealed significant variability in the cytochrome content (Table 2). Taking cytochromes o and b as standard for comparison, the epimastigotes samples could be grouped as follows (in parenthesis number of passages through the culture medium): 1) stocks with a relatively high content of cytochromes b and o, prevailing the former (stocks Y (116), RA (114), AF, FN, TN and MG (14 y 16); 2) stocks with a relatively low content of both cytochromes: Y (119), AWP and UP; 3) stocks with a low content of cytochrome b, without cytochrome o: CA-I and CA-I (V); 4) stocks without cytochromes: Y(117 and 118) and RA(113). In some strains (e.g. Y and RA), significant variation of cytochrome content in different stocks of the same isolate was observed (Table 2), but the Tulahuen strain proved to be less variable. Comparison of cytochrome distribution and other properties of parasites, namely, lethality for mice and morphology, did not allow to establish positive correlations.

Isolated mitochondria from widely different biological sources are capable of synthesizing protein in vitro (1,2). Extensive studies have proved that the mitochondrial synthesizing apparatus resembles the better-known cytoplasmic and bacterial systems, especially the latter one.