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Parasitology Research

, Volume 114, Issue 2, pp 419–430 | Cite as

Unveiling the effects of berenil, a DNA-binding drug, on Trypanosoma cruzi: implications for kDNA ultrastructure and replication

  • Aline Araujo Zuma
  • Danielle Pereira Cavalcanti
  • Marcelo Zogovich
  • Ana Carolina Loyola Machado
  • Isabela Cecília Mendes
  • Marc Thiry
  • Antonio Galina
  • Wanderley de Souza
  • Carlos Renato Machado
  • Maria Cristina Machado Motta
Original Paper

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.

Keywords

DNA-binding drugs kDNA topology and replication Kinetoplast Trypanosoma cruzi Ultrastructure 

Notes

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).

Ethical standards

Ethical approval was not required in this work.

Conflict of interest

We do not have conflict of interest to declare in this work.

Supplementary material

436_2014_4199_Fig9_ESM.gif (51 kb)
Online Resource 1s

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)

436_2014_4199_MOESM1_ESM.tif (5.2 mb)
(TIFF 5281 kb)

References

  1. Barrett MP, Gemmell CG, Suckling CJ (2013) Minor groove binders as anti-infective agents. Pharmacol Ther 139:12–23. doi: 10.1016/j.pharmthera.2013.03.002 PubMedCrossRefGoogle Scholar
  2. Brack CH, Delain E, Riou G (1972a) Replicating, covalently, closed, circular DNA from kinetoplasts of T. cruzi. Proc Natl Acad Sci 69:1642–1646PubMedCentralPubMedCrossRefGoogle Scholar
  3. Brack CH, Delain E, Riou G, Festy B (1972b) Molecular organization of the kinetoplast DNA of Trypanosoma cruzi treated with berenil, a DNA interacting drug. J Ultrastruct Res 39:568–579. doi: 10.1016/S0022-5320(72)90122-0 PubMedCrossRefGoogle Scholar
  4. Camargo EP (1964) Growth and differentiation in Trypanosoma cruzi I. Origin of metacyclic trypanosomes in liquid media. Rev Inst Med Trop 6:93–100Google Scholar
  5. Cavalcanti DP, Gonçalves DL, Costa LT, De Souza W (2011) The structure of the kinetoplast DNA network of Crithidia fasciculata revealed by atomic force microscopy. Micron 42:553–559. doi: 10.1016/j.micron.2011.01.009 PubMedCrossRefGoogle Scholar
  6. De Souza W (2002) Special organelles of some pathogenic protozoa. Parasitol Res 88:1013–1025. doi: 10.1007/s00136-002-0696-2 PubMedCrossRefGoogle Scholar
  7. De Souza EM, da Silva PB, Nefertiti ASG, Ismail MA, Arafa RK, Tao B, Nixon-Smith CK, Boykin DW, Soeiro MNC (2011) Trypanocidal activity and selectivity in vitro of aromatic amidine compounds upon bloodstream and intracellular forms of Trypanosoma cruzi. Exp Parasitol 127:429–435. doi: 10.1016/j.exppara.2010.10.010 PubMedCrossRefGoogle Scholar
  8. Egbe-Nwiyi TN, Igbokwe IO, Onyeyili PA (2003) The pathogenicity of diminazene aceturate-resistant T. brucei in rats after treatment with the drug. J Comp Pathol 128:188–191. doi: 10.1053/jcpa.2002.0599 PubMedCrossRefGoogle Scholar
  9. Egbe-Nwiyi TN, Igbokwe IO, Onyeyili PA (2005) Diminazene aceturate resistence on the virulence of T. brucei for rats. J Comp Pathol 133:286–288. doi: 10.1016/j.jcpa.2005.05.002 PubMedCrossRefGoogle Scholar
  10. Elias MCQB, Faria M, Mortara RA, Motta MCM, De Souza W, Thiry M, Schenkman S (2002) Chromosome localization changes in the Trypanosoma cruzi nucleus. Eukaryot Cell 1:944–953. doi: 10.1128/EC.1.6.944-953.2002 PubMedCentralPubMedCrossRefGoogle Scholar
  11. Hajduk SL (1979) Dyskinetoplasty in two species of trypanosomatids. J Cell Sci 35:185–202PubMedCrossRefGoogle Scholar
  12. Henriques C, Moreira TLB, Maia-Brigagão C, Henriques-Pons A, Carvalho TMU, De Souza W (2011) Tetrazolium salt based methods for high-throughput evaluation of anti-parasite chemotherapy. Anal Methods 3:2148–2155. doi: 10.1039/C1AY05219E CrossRefGoogle Scholar
  13. Hill GC, Anderson WA (1969) Effects of acriflavine on the mitochondria and kinetoplast of Crithidia fasciculata. J Cell Biol 41:547–561PubMedCentralPubMedCrossRefGoogle Scholar
  14. Jensen RE, Englund PT (2012) Network news: the replication of kinetoplast DNA. Annu Rev Microbiol 66:473–491. doi: 10.1146/annurev-micro-092611-150057 PubMedCrossRefGoogle Scholar
  15. Kennedy PGE (2013) Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol 12:186–194. doi: 10.1016/S1474-4422(12)70296-X PubMedCrossRefGoogle Scholar
  16. Korshunov SS, Skulachev VP, Starkov AA (1997) High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett 416:15–18. doi: 10.1016/S0014-5793(97)01159-9 PubMedCrossRefGoogle Scholar
  17. LIU B, LIU Y, MOTYKA SA, AGBO EEC, ENGLUND PT (2005) Fellowship of the rings: the replication of kinetoplast DNA. Trends Parasitol 21:363–369. doi: 10.1016/j.pt.2005.06.008 PubMedCrossRefGoogle Scholar
  18. Macadam RF, Williamson J (1972) Drug effects on the fine structure of Trypanosoma rhodesiense: diamidines. Trans R Soc Trop Med Hyg 66:897–904. doi: 10.1016/0035-9203(72)90125-3 PubMedCrossRefGoogle Scholar
  19. Macedo-Silva ST, Silva TLAO, Urbina JA, de Souza W, Rodrigues JCF (2011) Antiproliferative, ultrastructural and physiological effects of amiodarone on promastigote and amastigote forms of Leishmania amazonensis. Mol Biol Int 13:876021. doi: 10.4061/2011/876021 Google Scholar
  20. Manchester TM, Cavalcanti DP, Zogovich M, de Souza W, Motta MCM (2013) Acriflavine treatment promotes dyskinetoplasty in Trypanosoma cruzi as revealed by ultrastructural analysis. Parasitology 140:1422–1431. doi: 10.1017/S0031182013001029 PubMedCrossRefGoogle Scholar
  21. Motta MC, de Souza W, Thiry M (2003) Immunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids. FEMS Microbiol Lett 111:17–23CrossRefGoogle Scholar
  22. Palchaudhuri R, Hergenrother PJ (2007) DNA as a target for anticancer compounds: methods to determine the mode of binding and the mechanism of action. Curr Opin Biotechnol 18:497–503. doi: 10.1016/j.copbio.2007.09.006 PubMedCrossRefGoogle Scholar
  23. Peregrine AS, Mamman M (1993) Pharmacology of diminazene: a review. Acta Trop 54:185–203. doi: 10.1016/0001-706X(93)90092-P PubMedCrossRefGoogle Scholar
  24. Pérez-Morga DL, Englund PT (1993) The structure of replicating kinetoplast DNA networks. J Cell Biol 123:1069–1079PubMedCrossRefGoogle Scholar
  25. Portugal J (1994) Berenil acts as a poison of eukaryotic topoisomerase II. FEBS Lett 344:136–138. doi: 10.1016/0014-5793(94)00363-7 PubMedCrossRefGoogle Scholar
  26. Regis-da-Silva CG, Freitas JM, Passos-Silva DG, Furtado C, Augusto-Pinto L, Pereira MT et al (2006) Characterization of the Trypanosoma cruzi Rad51 gene and its role in recombination events associated with the parasite resistance to ionizing radiation. Mol Biochem Parasitol 2:191–200. doi: 10.1016/j.molbiopara.2006.05.012 CrossRefGoogle Scholar
  27. Shapiro TA, Englund PT (1990) Selective cleavage of kinetoplast DNA minicircles promoted by antitrypanosomal drugs. Proc Natl Acad Sci 87:950–954PubMedCentralPubMedCrossRefGoogle Scholar
  28. Silva CF, Meuser MB, De Souza EM, Meirelles MNL, Stephens CE, Som P, Boykin DW, Soeiro MNC (2007) Cellular effects of reversed amidines on Trypanosoma cruzi. Antimicrob Agents Chemother 51:3803–3809. doi: 10.1128/AAC. 00047-07 PubMedCentralPubMedCrossRefGoogle Scholar
  29. Silva TM, Peloso EF, Vitor SC, Ribeiro LH, Gadelha FR (2011) O2 consumption rates along the growth curve: new insights into Trypanosoma cruzi mitochondrial respiratory chain. J Bioenerg Biomembr 43:409–417. doi: 10.1007/s10863-011-9369-0 PubMedCrossRefGoogle Scholar
  30. Simpson L (1968) Effect of acriflavine on the kinetoplast of L. tarentolae. Mode of action and physiological correlates of the loss of kinetoplast DNA. J Cell Biol 37:660–682PubMedCentralPubMedCrossRefGoogle Scholar
  31. Storr SJ, Woolston CM, Zhang Y, Martin SG (2013) Redox environment, free radical, and oxidative DNA damage. Antioxid Redox Signal 20:2399–2408. doi: 10.1089/ars.2012.4920 CrossRefGoogle Scholar
  32. Stuart KD (1971) Evidence for the retention of kinetoplast DNA in acriflavine-induced dyskinetoplastic strain of Trypanosoma brucei which replicates the altered central element of the kinetoplast. J Cell Biol 49(189–195):1971Google Scholar
  33. Trager W, Rudzinska MA (1964) The riboflavin requirement and the effects of acriflavin on the fine structure of the kinetoplast of Leishmania tarentolae. J Protozool 11:133–145. doi: 10.1111/j.1550-7408.1964.tb01734.x PubMedCrossRefGoogle Scholar
  34. Wang MZ, Zhu X, Srivastava A, Liu Q, Sweat JM, Pandharkar T, Stephens CE, Riccio E, Parman T, Munde M, Mandal S, Madhubala R, Tidwell RR, Wilson WD, Boykin DW, Hall JE, Kyle DE, Werbovetz KA (2010) Novel arylimidamides for treatment of visceral leishmaniasis. Antimicrob Agents Chemother 54:2507–2516. doi: 10.1128/AAC. 00250-10 PubMedCentralPubMedCrossRefGoogle Scholar
  35. Wilson WD, Nguyen B, Tanious FA, Mathis A, Hall JE, Stephens CE, Boykin DW (2005) Dications that target the DNA minor groove: compound design and preparation, DNA interactions, cellular distribution and biological activity. Curr Med Chem 5:389–408. doi: 10.2174/1568011054222319 Google Scholar
  36. Witola WH, Atsuda A, Inoue N, Ohashi K, Onuma M (2005) Acquired resistance to berenil in a cloned isolate of T. evansi is associated with upregulation of a novel gene, TeDR40. Parasitology 131:635–646. doi: 10.1017/S003118200500836X PubMedCrossRefGoogle Scholar
  37. Zuma AA, Cavalcanti DP, Maia MC, de Souza W, Motta MCM (2011) Effect of topoisomerase inhibitors and DNA-binding drugs on the cell proliferation and ultrastructure of Trypanosoma cruzi. Int J Antimicrob Agents 37:449–456. doi: 10.1016/j.ijantimicag.2010.11.031 PubMedCrossRefGoogle Scholar
  38. Zuma AA, Mendes IC, Reignault LC, Elias MC, de Souza W, Maçado CR, Motta MCM (2014) How Trypanosoma cruzi handles cell cycle arrest promoted by camptothecin, a topoisomerase I inhibitor. Mol Biochem Parasitol 193:93–100. doi: 10.1016/j.molbiopara.2014.02.001 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Aline Araujo Zuma
    • 1
    • 2
  • Danielle Pereira Cavalcanti
    • 2
  • Marcelo Zogovich
    • 2
  • Ana Carolina Loyola Machado
    • 1
    • 2
  • Isabela Cecília Mendes
    • 3
  • Marc Thiry
    • 4
  • Antonio Galina
    • 5
  • Wanderley de Souza
    • 1
    • 2
  • Carlos Renato Machado
    • 3
  • Maria Cristina Machado Motta
    • 1
    • 2
  1. 1.Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas FilhoUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil
  2. 2.Instituto Nacional de MetrologiaQualidade e Tecnologia-InmetroDuque de CaxiasBrazil
  3. 3.Laboratório de Genética Bioquímica, Departamento de Bioquímica e Imunologia, Instituto de Ciências BiológicasUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  4. 4.Department of life Sciences, GIGA-Neurosciences, Unit of Cell and Tissue BiologyUniversity of LiegeLiegeBelgium
  5. 5.Laboratório de Bioenergética e Fisiologia Mitocondrial, Programa de Biofísica e Bioquímica Celular, Instituto de Bioquímica Médica Leopoldo de MeisUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil

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