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 eukariotic organisms mitochondrial DNA encodes the genetic information for cytochromes b, aa3 and F0F1 ATPase, it seemed of interest to establish whether a similar effect occurs in Crithidia fasciculata, a trypanosomatid used for assay of potential trypanocidal drugs. Culturing of C. fasciculata in the presence of EB inhibited growth and induced dyskinetoplasty, as confirmed by electron microscopy. The kinetoplast of EB-cultured crithidia lost its characteristic arc shape, it was misplaced in the cell cytoplasm its matrix structure and membrane differentiation were specifically modified. Dyskinetoplasty 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 confirmed by the lack of action of EB on crithidia in the stationary phase of growth, that ruled out direct inhibition of oxidative phosphorylation enzymes. The lipophilic o-naphthoquinone beta-lapachone produced structural alterations in kinetoplast membranes, that correlated with inhibition of oxidative phosphorylation. These latter effects involved free radicals since they were prevented by free radical scavengers.

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 eukariotic organisms mitochondrial DNA encodes the genetic information for cytochromes b, aa3 and F0F1 ATPase, it seemed of interest to establish whether a similar effect occurs in Crithidia fasciculata, a trypanosomatid used for assay of potential trypanocidal drugs. Culturing of C. fasciculata in the presence of EB inhibited growth and induced dyskinetoplasty, as confirmed by electron microscopy. The kinetoplast of EB-cultured crithidia lost its characteristic arc shape, it was misplaced in the cell cytoplasm its matrix structure and membrane differentiation were specifically modified. Dyskinetoplasty 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 confirmed by the lack of action of EB on crithidia in the stationary phase of growth, that ruled out direct inhibition of oxidative phosphorylation enzymes. The lipophilic o-naphthoquinone beta-lapachone produced structural alterations in kinetoplast membranes, that correlated with inhibition of oxidative phosphorylation. These latter effects involved free radicals since they were prevented by free radical scavengers.

[Effect of lipophilic ortho-naphthoquinones on the growth of and production of peroxides by Leptomonas seymouri and Crithidia fasciculata]. / Efecto de orto-naftoquinonas lipofilicas sobre el crecimiento y la producción de peróxidos por Leptomonas seymouri y Crithidia fasciculata.

The lipophilic o-naphthoquinones CG 8-935, CG 9-442, CG 10-248 and mansonones A, C, E and F inhibit growth of the trypanosomatids Leptomonas seymouri (LS) and Crithidia fasciculata (CF). The most active mansonones were E and F (I50, 0.1-0.4 microM with LS; 0.3-1.2 microM with CF) with cytotoxic activities equal to or higher than quinones CG, as reported previously. Incubation of LS or CF with the CG-quinones and mansonones E and F caused the release of H2O2 and O2-. from the whole cells to the suspending medium, as detected by the microperoxidase and adrenochrome assays, respectively. Lower O2-. and H2O2 production values were obtained with perezone and primine, two p-benzoquinones used as controls. Quinones effect on O2-. and H2O2 production was closely related to their concentration and with mansonones E and F, O2-. production was 4-5-fold higher than H2O2 production. Smaller differences were observed with the CG-quinones. Peroxide production in the assayed organisms was the result of quinone redox-cycling, which involved an anaerobic, reductive phase producing quinols, and an aerobic phase (II), in which quinol oxidation and peroxide production (O2-., H2O2) occurred. With mansonones E and F, and quinone CG, phase II rate was faster than or similar to phase I rate, but with mansonones A and C, the quinols oxidation rate was 8-10-fold slower than the quinones reduction rate. These differences fit in well with a) the quinol oxidation rates, measured in vitro; b) O2-. production by quinols oxidation and c) quinols capability for producing lucigenin chemiluminescence. These results support the assumption that oxyradicals play a relevant role in o-naphthoquinones cytotoxic action, despite the presence of catalase and other protective enzymes in CF and LS. Other effects are not, however, ruled out. CF and LS sensitivity towards the assayed o-quinones was similar or higher than that of Trypanosoma cruzi, thus allowing the use of those organisms for preliminary screening of antichagasic drugs.

Efecto de orto-naftoquinonas lipofilicas sobre el crecimiento y la producción de peróxidos por Leptomonas seymouri y Crithidia fasciculata. / [Effect of lipophilic ortho-naphthoquinones on the growth of and production of peroxides by Leptomonas seymouri and Crithidia fasciculata]

The lipophilic o-naphthoquinones CG 8-935, CG 9-442, CG 10-248 and mansonones A, C, E and F inhibit growth of the trypanosomatids Leptomonas seymouri (LS) and Crithidia fasciculata (CF). The most active mansonones were E and F (I50, 0.1-0.4 microM with LS; 0.3-1.2 microM with CF) with cytotoxic activities equal to or higher than quinones CG, as reported previously. Incubation of LS or CF with the CG-quinones and mansonones E and F caused the release of H2O2 and O2-. from the whole cells to the suspending medium, as detected by the microperoxidase and adrenochrome assays, respectively. Lower O2-. and H2O2 production values were obtained with perezone and primine, two p-benzoquinones used as controls. Quinones effect on O2-. and H2O2 production was closely related to their concentration and with mansonones E and F, O2-. production was 4-5-fold higher than H2O2 production. Smaller differences were observed with the CG-quinones. Peroxide production in the assayed organisms was the result of quinone redox-cycling, which involved an anaerobic, reductive phase producing quinols, and an aerobic phase (II), in which quinol oxidation and peroxide production (O2-., H2O2) occurred. With mansonones E and F, and quinone CG, phase II rate was faster than or similar to phase I rate, but with mansonones A and C, the quinols oxidation rate was 8-10-fold slower than the quinones reduction rate. These differences fit in well with a) the quinol oxidation rates, measured in vitro; b) O2-. production by quinols oxidation and c) quinols capability for producing lucigenin chemiluminescence. These results support the assumption that oxyradicals play a relevant role in o-naphthoquinones cytotoxic action, despite the presence of catalase and other protective enzymes in CF and LS. Other effects are not, however, ruled out. CF and LS sensitivity towards the assayed o-quinones was similar or higher than that of Trypanosoma cruzi, thus allowing the use of those organisms for preliminary screening of antichagasic drugs.

[Effect of quinones and nitrofurans on Trypanosoma mega and Crithidia fasciculata]. / Efecto de quinonas y nitrofuranos sobre Trypanosoma mega y Crithidia fasciculata.

Demonstration of trypanocidal effects in vitro is a first step for the development of new antichagasic drugs. In order to obtain an experimental model allowing the pre-screening of potential trypanocides for Trypanosoma cruzi in a short time and under safe conditions, the trypanosomatids T. mega and C. fasciculata were assayed for their response to a) compounds known for their action on T. cruzi, and b) compounds not tested before on the latter. The drugs were assayed on the organisms growth in a liquid culture medium, cell multiplication being measured by the medium turbidity increase, using a photoelectric colorimeter previously calibrated with cell suspensions of known concentration. A series of quinones (Lapachones and related compounds), naftoquinone-imines, benzoquinones (perezone and dihydroperezone), a quinol (miconidine) and several nitrofurans, including nifurtimox and (5-nitro-2-furfurylidene)-amino (NF-group) derivatives, inhibited the flagellates growth, specially T. mega, with half-maximal inhibitory concentrations lesser than 5.0 microM, for the most active compounds. T. mega response to nifurtimox, NF-derivatives and beta-lapachone was in close agreement with that of T. cruzi. Cultures of T. mega in the presence of NF-pyrazole, NF-indazoles and NF-imidazole but not nifurtimox, showed irreversible damage since, after re-incubation in fresh medium without inhibitor, these cells grew significantly less than their corresponding controls. Similar effects were observed in C. fasciculata, with beta-lapachone and one naftoquinone-imine. Our results qualify T. mega as an adequate experimental model for the assay of antichagasic agents, as C. fasciculata and T. brucei brucei do for the african trypanosomes.

Efecto de quinonas y nitrofuranos sobre Trypanosoma mega y Crithidia fasciculata. / [Effect of quinones and nitrofurans on Trypanosoma mega and Crithidia fasciculata]

Demonstration of trypanocidal effects in vitro is a first step for the development of new antichagasic drugs. In order to obtain an experimental model allowing the pre-screening of potential trypanocides for Trypanosoma cruzi in a short time and under safe conditions, the trypanosomatids T. mega and C. fasciculata were assayed for their response to a) compounds known for their action on T. cruzi, and b) compounds not tested before on the latter. The drugs were assayed on the organisms growth in a liquid culture medium, cell multiplication being measured by the medium turbidity increase, using a photoelectric colorimeter previously calibrated with cell suspensions of known concentration. A series of quinones (Lapachones and related compounds), naftoquinone-imines, benzoquinones (perezone and dihydroperezone), a quinol (miconidine) and several nitrofurans, including nifurtimox and (5-nitro-2-furfurylidene)-amino (NF-group) derivatives, inhibited the flagellates growth, specially T. mega, with half-maximal inhibitory concentrations lesser than 5.0 microM, for the most active compounds. T. mega response to nifurtimox, NF-derivatives and beta-lapachone was in close agreement with that of T. cruzi. Cultures of T. mega in the presence of NF-pyrazole, NF-indazoles and NF-imidazole but not nifurtimox, showed irreversible damage since, after re-incubation in fresh medium without inhibitor, these cells grew significantly less than their corresponding controls. Similar effects were observed in C. fasciculata, with beta-lapachone and one naftoquinone-imine. Our results qualify T. mega as an adequate experimental model for the assay of antichagasic agents, as C. fasciculata and T. brucei brucei do for the african trypanosomes.