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[Introduction of changes in the DNA of Trypanosoma cruzi by trypanocidal agents]
S G Goijman1, A C Frasch, A O Stoppani
1Centro de Investigaciones Bioenergéticas, Facultad de Medicina, Universidad de Buenos Aires, Argentina.
Abstract:
Incubation of Trypanosoma cruzi culture (epimastigote) forms with nifurtimox (10 or 100 microM), benznidazole (38 or 380 microM) and beta-lapachone (1.6 or 7.8 microM) produced damage of nuclear DNA, as shown by the increased rate of the "unscheduled DNA synthesis" in epimastigotes arrested at phase S (9-, 3-, and 6-fold, respectively). alpha-lapachone, a position isomer of beta-lapachone, was completely ineffective. In order to demonstrate the "unscheduled repair of DNA", the semiconservative replication was inhibited by preincubating the epimastigotes for 16 hours with 10 mM hydroxyurea and 0.3 mM cycloheximide. Kinetoplast DNA (kDNA) extracted from epimastigotes pretreated with the trypanocidal agents revealed an increased number of single-strand breaks. After alkaline agarose-gel electrophoresis, a fast moving DNA fraction was detected in the kDNA from nifurtimox, benznidazole and beta-lapachone-treated parasites, while trapping of alkali-denatured kDNA by nitrocellulose filters, was significantly increased after treating the epimastigotes with the same drugs. Reincubation of these epimastigotes in fresh medium for 24 h, reestablished kDNA electrophoretic and filtration patterns to normality, except with 7.8 microM beta-lapachone, thus proving the reversibility of DNA lesions. Redox-cycling of nifurtimox and beta-lapachone in T. cruzi generates oxygen radicals, and accordingly, the higher effectiveness of these drugs (as compared with benznidazole and alpha-lapachone) supports the role of oxygen radicals for the trypanocidal action.
Insights
Trypanosoma cruzi parasites treated with nifurtimox, benznidazole, and beta-lapachone showed DNA damage, indicated by increased DNA repair synthesis and strand breaks. These lesions were mostly reversible, suggesting oxygen radicals play a role in the drugs' trypanocidal effects.
Area of Science:
- Molecular Biology
- Parasitology
- Pharmacology
Background:
- Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical illness.
- Nifurtimox and benznidazole are current front-line treatments, but their efficacy and safety profiles are suboptimal.
- Understanding drug mechanisms is crucial for developing improved therapeutic strategies.
Purpose of the Study:
- To investigate the DNA-damaging effects of nifurtimox, benznidazole, and beta-lapachone on Trypanosoma cruzi epimastigotes.
- To elucidate the role of DNA repair and oxygen radicals in the trypanocidal activity of these compounds.
Main Methods:
- Epimastigote forms of Trypanosoma cruzi were incubated with varying concentrations of nifurtimox, benznidazole, and beta-lapachone.
- Unscheduled DNA synthesis was measured to assess DNA damage and repair.
- Kinetoplast DNA (kDNA) single-strand breaks were analyzed using alkaline agarose-gel electrophoresis and nitrocellulose filter trapping.
Main Results:
- Nifurtimox, benznidazole, and beta-lapachone significantly increased unscheduled DNA synthesis, indicating nuclear DNA damage.
- These drugs also induced single-strand breaks in kDNA, altering its electrophoretic and filtration properties.
- The DNA lesions were largely reversible upon drug removal, except for high-dose beta-lapachone treatment.
Conclusions:
- Nifurtimox, benznidazole, and beta-lapachone induce significant DNA damage in Trypanosoma cruzi.
- The generation of oxygen radicals via redox-cycling likely contributes to the trypanocidal action of nifurtimox and beta-lapachone.
- Understanding these mechanisms may guide the development of novel anti-parasitic agents.