Abstract
Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a single mitochondrion with an enlarged portion termed kinetoplast. This unique structure harbors the mitochondrial DNA (kDNA), composed of interlocked molecules: minicircles and maxicircles. kDNA is a hallmark of kinetoplastids and for this reason constitutes a valuable target in chemotherapeutic and cell biology studies. In the present work, we analyzed the effects of berenil, a minor-groove-binding agent that acts preferentially at the kDNA, thereby affecting cell proliferation, ultrastructure, and mitochondrial activity of T. cruzi epimastigote form. Our results showed that berenil promoted a reduction on parasite growth when high concentrations were used; however, cell viability was not affected. This compound caused significant changes in kDNA arrangement, including the appearance of membrane profiles in the network and electron-lucent areas in the kinetoplast matrix, but nuclear ultrastructure was not modified. The use of the TdT technique, which specifically labels DNA, conjugated to atomic force microscopy analysis indicates that berenil prevents the minicircle decatenation of the network, thus impairing DNA replication and culminating in the appearance of dyskinetoplastic cells. Alterations in the kinetoplast network may be associated with kDNA lesions, as suggested by the quantitative PCR (qPCR) technique. Furthermore, parasites treated with berenil presented higher levels of reactive oxygen species and a slight decrease in the mitochondrial membrane potential and oxygen consumption. Taken together, our results reveal that this DNA-binding drug mainly affects kDNA topology and replication, reinforcing the idea that the kinetoplast represents a potential target for chemotherapy against trypanosomatids.
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Acknowledgments
The authors are grateful to Rachel Rachid, Camila Silva Gonçalves, and Daniela Leão Gonçalves for technical assistance. This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Programa de Apoios a Núcleos de Excelência (Pronex).
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Analysis of the ROS generation in T. cruzi treated with berenil for 24 and 48 hours. ROS production enhanced in a time and concentration dependent manner. The data are the average of three independent experiments (GIF 50 kb)
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Zuma, A.A., Cavalcanti, D.P., Zogovich, M. et al. Unveiling the effects of berenil, a DNA-binding drug, on Trypanosoma cruzi: implications for kDNA ultrastructure and replication. Parasitol Res 114, 419–430 (2015). https://doi.org/10.1007/s00436-014-4199-8
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DOI: https://doi.org/10.1007/s00436-014-4199-8