DNA Repair BER Pathway Inhibition Increases Cell Death Caused by Oxidative DNA Damage in Trypanosoma cruzi

Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Journal of Cellular Biochemistry (Impact Factor: 3.26). 08/2011; 112(8):2189-99. DOI: 10.1002/jcb.23138
Source: PubMed


Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas disease, an endemic and neglected pathology in Latin America. It presents a life cycle that involves a hematophagous insect and man as well as domestic and wild mammals. The parasitic infection is not eliminated by the immune system of mammals; thus, the vertebrate host serves as a parasite reservoir. Additionally, chronic processes leading to dysfunction of the cardiac and digestive systems are observed. To establish a chronic infection some parasites should resist the oxidative damage to its DNA exerted by oxygen and nitrogen free radicals (ROS/RNS) generated in host cells. Till date there are no reports directly showing oxidative DNA damage and repair in T. cruzi. We establish that ROS/RNS generate nuclear and kinetoplastid DNA damage in T. cruzi that may be partially repaired by the parasite. Furthermore, we determined that both oxidative agents diminish T. cruzi cell viability. This effect is significantly augmented in parasites subsequently incubated with methoxyamine, a DNA base excision repair (BER) pathway inhibitor, strongly suggesting that the maintenance of T. cruzi viability is a consequence of DNA repair mechanisms.

1 Follower
21 Reads
  • Source
    • "The QPCR results obtained herein are in agreement with a recently published article that investigated T. cruzi BER pathway [53]. In that work, the levels of nuclear and mitochondrial DNA damage generated by H2O2 were similar to the DNA damage profile verified by our group. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-/-) (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2)O(2)). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2)O(2) of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.
    Full-text · Article · Aug 2012 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Soon after discovery of the polymerase chain reaction (PCR), various laboratories have attempted to use quantitative PCR (QPCR) to detect DNA damage in specific gene segments. The development of techniques that facilitate long PCR increased the sensitivity of the assay so that biologically relevant doses of DNA-damaging agents could be assessed. QPCR has been used to survey DNA damage induced by different genotoxicants and to establish the repair kinetics of numerous genes. Current work seeks to analyze damage and repair in specific genes from animals exposed to specific DNA-damaging agents such as oxidative stress.
    Full-text · Article · Nov 2000 · Methods
  • [Show abstract] [Hide abstract]
    ABSTRACT: This chapter was written as a guide to using the long-amplicon quantitative PCR (QPCR) assay for the measurement of DNA damage in mammalian as well as nonmammalian species such as Caenorhabditis elegans (nematodes), Drosophila melanogaster (fruit flies), and two species of fish (Fundulus heteroclitus and Danio rerio). Since its development in the early 1990s (Kalinowski et al., Nucleic Acids Res 20:3485-3494, 1992; Salazar and Van Houten, Mutat Res 385:139-149, 1997; Yakes and Van Houten, Proc Natl Acad Sci USA 94:514-519, 1997), the QPCR assay has been widely used to measure DNA damage and repair kinetics in nuclear and mitochondrial genomes after genotoxin exposure (Yakes and Van Houten, Proc Natl Acad Sci USA 94:514-519, 1997; Santos et al., J Biol Chem 278:1728-1734, 2003; Mandavilli et al., Mol Brain Res 133:215-223, 2005). One of the main strengths of the assay is that the labor-intensive and artifact-generating step of mitochondrial isolation is not needed for the accurate measurement of mitochondrial DNA copy number and damage. Below we present the advantages and limitations of using QPCR to assay DNA damage in animal cells and provide a detailed protocol of the QPCR assay that integrates its usage in newly developed animal systems.
    No preview · Article · Sep 2012 · Methods in molecular biology (Clifton, N.J.)
Show more