Research Article

Unveiling Benznidazole’s Mechanism of Action ThroughOverexpression of DNA Repair Proteins in Trypanosoma

cruzi

Matheus Andrade Raj~ao,1Carolina Furtado,1Ceres Luciana Alves,1

Danielle Gomes Passos-Silva,1 Michelle Barbi de Moura,1

Bruno Luiz Schamber-Reis,1 Marianna Kunrath-Lima,1 Aline Ara�ujo Zuma,2

Jo~ao PedroVieira-da-Rocha,1 Juliana Borio Ferreira Garcia,3

Isabela Cec�|lia Mendes,1 S�ergio Danilo Junho Pena,1

AndreaMaraMacedo,1Gl�oria Regina Franco,1

Nadja Cristhina de Souza-Pinto,4 Marisa Helena Gennari de Medeiros,4

Angela Kaysel Cruz,3 Maria Cristina MachadoMotta,2 Santuza MariaRibeiro Teixeira,1 and Carlos Renato Machado1*

1Departamento de Bioqu�ımica e Imunologia, Instituto de Ciencias Biol�ogicas,UFMG, Belo Horizonte, Minas Gerais

2Laborat�orio de Ultraestrutura Celular Hertha Meyer, Instituto de Biof�ısicaCarlos Chagas Filho, Centro de Ciencias da Sa�ude, Universidade Federal do

Rio de Janeiro, UFRJ, Cidade Universit�aria, Ilha do Fund~ao, Rio de Janeiro, Riode Janeiro

3Departamento de Biologia Celular e Molecular e Bioagentes Patogenicos,Faculdade de Medicina de Ribeir~ao Preto, Universidade de S~ao Paulo,

Ribeir~ao Preto, S~ao Paulo4Departamento de Bioqu�ımica, Instituto de Qu�ımica, Universidade de S~ao

Paulo, S~ao Paulo, S~ao Paulo

Benznidazole (BZ) is the most commonly used drugfor the treatment of Chagas disease. Although BZ isknown to induce the formation of free radicals andelectrophilic metabolites within the parasite Trypa-nosoma cruzi, its precise mechanisms of action arestill elusive. Here, we analyzed the survival of T.cruzi exposed to BZ using genetically modified par-asites overexpressing different DNA repair pro-teins. Our results indicate that BZ induces oxidationmainly in the nucleotide pool, as heterologousexpression of the nucleotide pyrophosphohydro-lase MutT (but not overexpression of the glycosy-lase TcOgg1) increased drug resistance in theparasite. In addition, electron microscopy indi-cated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic

DNA undergoes extensive heterochromatinunpacking following exposure to the drug. Further-more, the overexpression of proteins involved inthe recombination-mediated DNA repair increasedresistance to BZ, reinforcing the idea that the drugcauses double-stranded breaks. Our results alsoshow that the overexpression of mitochondrialDNA repair proteins increase parasite survivalupon BZ exposure, indicating that the drug induceslesions in the mitochondrial DNA as well. Thesefindings suggest that BZ preferentially oxidizes thenucleotide pool, and the extensive incorporation ofoxidized nucleotides during DNA replication leadsto potentially lethal double-stranded DNA breaks inT. cruzi DNA. Environ. Mol. Mutagen. 55:309–321, 2014. VC 2013 Wiley Periodicals, Inc.

Key words: Trypanosoma cruzi; oxidative stress; Benznidazole; DNA repair; drug resistance

*Correspondence to: Carlos Renato Machado, Departamento de Bioqu�ı-

mica e Imunologia, Instituto de Ciencias Biol�ogicas, UFMG, Belo Hori-

zonte, Minas Gerais, Brazil. E-mail: crmachad@icb.ufmg.br.

Matheus Andrade Raj~ao and Carolina Furtado contributed equally to this

work.

Received 19 October 2012; provisionally accepted 22 November 2013;

and in final form 24 November 2013

DOI 10.1002/em.21839

Published online 18 December 2013 in

Wiley Online Library (wileyonlinelibrary.com).

VC 2013Wiley Periodicals, Inc.

Environmental andMolecular Mutagenesis 55:309^321 (2014)

INTRODUCTION

The flagellate protozoan Trypanosoma cruzi is the

causative agent of Chagas disease, a debilitating illness

that affects approximately 10 million people, mainly in

Latin America. Although more than 25 million people are

at risk of infection, there is still no vaccine or effective

cure (Hotez et al., 2012). Chagas disease displays symp-

tomatic and pathologic variation among infected patients.

It is characterized by the presence of a short-term acute

phase, which in some patients can progress to a chronic

phase. The acute phase presents symptoms, such as fever

and local swelling at the site of infection, while the

chronic phase can lead to cardiomyopathy or gastrointes-

tinal disorders (Rassi et al., 2010).

The drugs currently used for the specific treatment of

T. cruzi infection are nifurtimox (4[(5-nitrofurfurylide-

ne)amino]-3-methylthiomorpholine-1,1-dioxide), derived

from nitrofuran, and benznidazole (BZ) (N-benzyl-2-nitro-

imidazole-1-acetamide), a nitroimidazole. Nifurtimox has

been used since 1967, commercialized by Bayer under

the brand name LampitVR

, and BZ was launched by Roche

in 1972 under the brand name RochaganVR

(Coura, 2002).

Clinical trials report that treatment based on these two

compounds (either simultaneously or separately) cures up

to 80% of patients in the acute phase (Bahia-Oliveira

et al., 2000), whereas in the chronic phase, the cure rate

is only 5–20% (Cancado, 2002). BZ is currently the main

drug used to treat Chagas disease because it is better tol-

erated by infected patients than nifurtimox. BZ is admin-

istered in the acute phase at 5 mg/kg/day in adults for 90

days. It is rapidly absorbed by the gastrointestinal tract,

and it is mainly metabolized in the liver by the cyto-

chrome P450 system (Maya et al., 2007).

BZ acts through the formation of free radicals and elec-

trophilic metabolites that are generated when its nitro

group is reduced to an amino group by the action of

nitroreductases (Maya et al., 2007; Wilkinson et al.,

2008). In contrast to nifurtimox, which also generates

O22 and H2O2 after the nitro group is reduced, BZ reduc-

tion does not produce reactive oxygen species (Docampo

and Moreno, 1984; Moreno et al., 1982). It is hypothe-

sized that the trypanocidal effect of BZ is caused by

covalent binding of its reduced metabolites to macromole-

cules of the parasite (Maya et al., 2004). Nevertheless, it

is also reported that BZ induces oxidative stress within

the parasite (Pedrosa et al., 2001).

Reactive oxygen species and electrophilic metabolites

can react with nucleic acids, which can lead to the gener-

ation of DNA lesions. To revert or even prevent the

resulting genomic damage, living organisms have devel-

oped biochemical pathways that promote the repair of dif-

ferent DNA lesions. These mechanisms ensure the

stability of the genome, protecting the DNA from poten-

tially mutagenic modifications and allowing its genetic

information to be transmitted accurately to further genera-

tions (Friedberg et al., 2006).

The main eukaryotic DNA repair mechanisms are i)

mismatch repair (MMR), which excises misincorporated

bases (Arczewska and Kusmierek, 2007), ii) nucleotide

excision repair, which detects and removes DNA sequen-

ces containing lesions that alter the DNA conformation

(Maddukuri et al., 2007), and iii) base excision repair

(BER), which removes oxidized, methylated, or deami-

nated bases through the action of glycosylases (Krwawicz

et al., 2007), and iv) recombination, which uses homology

regions of the sister chromatid to repair DNA double-

stranded breaks (Nowosielska, 2007). Despite the effi-

ciency of these DNA repair mechanisms, some lesions

might remain unrepaired during DNA replication. The

main strategy used by cells to replicate damaged DNA is

the recruitment of specialized DNA polymerases that are

able to bypass several types of DNA lesions, in a process

known as translesion synthesis (TLS) (Prakash et al.,

2005).

Our research group has been studying different T. cruziproteins that are involved in DNA metabolism (replica-

tion, repair, and recombination), describing its properties

using both in vitro and in vivo approaches (Aguiar et al.,

2013; Campos et al., 2011; Furtado et al., 2012; Lopes

et al., 2008; Moura et al., 2009; Raj~ao et al., 2009;

Regis-da-Silva et al., 2006; Schamber-Reis et al., 2012).

To investigate whether BZ efficacy against T. cruzi is due

to its action on DNA, we challenged our genetically

modified strains with BZ. Our results show that both

overexpression and reduced expression of proteins

involved in DNA metabolism alter T. cruzi resistance to

BZ.

MATERIALS ANDMETHODS

Parasite Growth

Epimastigote forms of the CL Brener strain of T. cruzi were grown at

28�C in liver infusion tryptose (LIT) medium (pH 7.3) supplemented

with 10% fetal calf serum, streptomycin sulfate (0.2 g/l), and penicillin

(200,000 U/l).

Genetically Modified Cell Lineages

T. cruzi cell lineages stably overexpressing the genes EcMUTT,

TcOGG1, TcMUTY, TcPOLB, TcPOLH, TcPOLK, or TcRAD51 (Aguiar

et al., 2013; Furtado et al., 2012; Lopes et al., 2008; Moura et al., 2009;

Raj~ao et al., 2009; Regis-da-Silva et al., 2006; Schamber-Reis et al.,

2012) were generated by cloning the respective genes into the pROCK

vector. This vector is capable of integrating into the b-TUBULIN locus

of the T. cruzi genome, and gene overexpression is achieved by the pres-

ence of a ribosomal promoter upstream the inserted gene (DaRocha

et al., 2004). The construction of these plasmids and generation of these

cell lines were previously described (Aguiar et al., 2013; Furtado et al.,

in press; Lopes et al., 2008; Moura et al., 2009; Raj~ao et al., 2009;

Regis-da-Silva et al., 2006; Schamber-Reis et al., 2012). TcMSH21/2

and TcRAD511/2 cells were generated by inserting a hygromycin

Environmental and Molecular Mutagenesis. DOI 10.1002/em

310 Raj~ao et al.

cassette into one of the TcMSH2 or TcRAD51 alleles, respectively (Cam-

pos et al., 2011). The hygromycin cassette was constructed by separately

subcloning the 50UTR region of TcRad51, the hygromycin resistance

gene, and the 30UTR region of TcRad51 into pGEM-T-easy (Promega).

The hygromycin gene was digested with XbaI and SalI and inserted into

pGEM-T-easy-50UTR, generating pGEM-T-easy-50UTR-hygromycin.

Next, the 30UTR region of TcRad51 was digested with SalI and SacI

and inserted into pGEM-T-easy-50UTR-hygromycin, generating the

pGEM-T-easy-50UTR-hygromycin-30UTR vector. The 50UTR-

hygromycin-30UTR cassette was liberated by digestion with ApaI and

SacI, and the purified fragment was used to transfect epimastigote cells,

following the protocol previously described (DaRocha et al., 2004). The

cassette insertion was confirmed by RT real-time PCR and DNA

sequencing.

Survival Curves

For testing resistance to BZ, parasite cultures containing 1 3 107

cells/ml were treated with 0, 60, 120, or 240 lM BZ for 48 hr. Cell

number was determined in a cytometry chamber using the erythrosine

vital stain to differentiate living and dead cells. The results were

expressed as the percentage of growth compared to untreated cultures.

Experiments were performed in triplicate.

Analysis of 8-OxoGAccumulation Induced by BZTreatment

Eight-oxodG accumulation was assessed by HPLC–electrochemical

detection. Cells (1 3 109 cells/ml) were treated with 120 mM BZ for 24

hr at 28�C and washed with PBS, and the DNA was isolated by the cha-

otropic NaI method (Nakae et al., 1995) in the presence of 0.1 mM des-

ferrioxamine. DNA samples were treated with nuclease P1 and alkaline

phosphatase and analyzed by HPLC. Samples (100 lg) of digested DNA

were injected into an HPLC/electrochemical detection system consisting

of a Shimadzu model LC-10AD pump connected to a Luna C18 (Phe-

nomenex, Torrance, CA, USA) reverse-phase column (250 mm 3 4.6

mm ID, particle size 5 lm). The flow rate of the isocratic eluent (50

mM potassium phosphate buffer, pH 5.5, and 8% methanol) was 1 ml/

min. Coulometric detection was obtained with a Coulochem II detector

(ESA, Chemsford, MA, USA). The potentials of the two electrodes were

set at 120 and 280 mV. Elution of unmodified nucleosides was moni-

tored simultaneously with a Shimadzu SPD-10A UV detector set at 254

nm. A Shimadzu Class-LC10 1.6 software was used to calculate the

peak areas. The molar ratio of 8-oxodG to dG in each DNA sample was

determined based on colorimetric detection at 280 mV for 8-oxodG and

on absorbance at 254 nm for dG in each injection.

Analysis of DNA Lesions After BZ Treatment UsingQuantitative PCR Assay

Parasite cultures at 1 3 107 cells/ml were harvested by centrifugation

at 3000g for 10 min. Cells were treated by incubating the parasites with

240 mM BZ for 24 hr. In parallel, other cells were treated with 2 mM

MMS (an alkylating agent) for 1 hr or with 200 mM camptothecin (a

topoisomerase I inhibitor) for 1 hr. After treatment, cells were harvested

immediately (DNA extraction, quantification, quantitative PCR assay

(QPCR) amplification, and data analysis were conducted as reported by

Santos et al. (2006). The large nuclear fragment was amplified using the

forward primer QPCRNuc2F (50-GCACACGGCTGCGAGTGACCATT

CAACTTT-30) and the reverse primer QPCRNuc2R (50-CCTCGCACA

TTTCTACCTTGTCCTTCAATGCCTGC-30). The small nuclear frag-

ment was amplified employing the internal primer QPCRNuc2Int

(50-TCGAGCAAGCTGACACTCGATGCAACCAAAG-30) and the

reverse primer QPCRNuc2R. The large mitochondrial fragment was

amplified using the forward primer QPCRMitF (50-TTTTATTTGGGGG

AGAACGGAGCG-30) and the reverse primer QPCRMitR (50-TTGAAA

CTGCTTTCCCCAAACGCC-30). The small mitochondrial fragment was

amplified with the internal primer QPCRMitInt (50-CGCTCTGCCCCC

ATAAAAAACCTT-30). The small fragment (250 bp) is used to normal-

ize the amplification of the large fragments (10 kb), which eliminates

the bias introduced by the different copy numbers of the nuclear and

mitochondrial genomes. The normalized amplification of treated samples

was then compared with untreated cells, and the relative amplification

was calculated. These values were next used to estimate the average

number of lesions per 10 kb of the genome, using a Poisson distribution.

The final results are the mean of two sets of PCR amplification for each

target gene from at least two biological experiments.

Transmission ElectronMicroscopy

Parasites were fixed in 2.5% glutaraldehyde diluted in 0.1 M cacodyl-

ate buffer, pH 7.2, at room temperature for 1 hr and were washed at the

same buffer. Next, cells were postfixed in 0.1 M cacodylate buffer con-

taining 1% OsO4 and 0.8% potassium ferricyanide for 1 hr. Cells were

washed with the same buffer and were dehydrated in a graded series of

acetone (50, 70, 90%, and 2 3 100%) and embedded in Epon resin

(Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were

stained with uranyl acetate and lead citrate and were observed in a Zeiss

900 transmission electron microscope (Zeiss, Oberkochen, Germany).

Pulse-Field Gel Electrophoresis

Parasites were treated with 0 mM BZ, 120 mM BZ for 24 hr, 240 mM

BZ for 48 hr, or 240 mM BZ for 72 hr. Samples for pulsed-field electro-

phoresis were prepared as described (Beverley, 1988). Each sample con-

tained 4 3 108 cells were separated by pulsed-field gel electrophoresis

(PFGE) in agarose gel 1% solution in TBE buffer 0.53 (Tris–borate

[4.5 mM] and EDTA [0.5 mM]) to 14�C. For the fractionation of the

chromosomes were employed pulses with increasing growth of 50 to

120 sec for 48 hr at 4.5 V/cm. The angle on the apparatus CHEF DRII

(BioRad) is fixed at 120�. The gel was stained with ethidium bromide

(0.5 mg/ml).

RESULTS

Heterologous Expression of MutT, but not Overexpressionof TcOgg1or TcMutY, Increases T. cruzi Resistance to BZ

The mode of action for BZ involves the generation of

free radicals and electrophilic metabolites, which can

oxidize certain molecules (Maya et al., 2007). Cellular

targets of free radicals include the 20-deoxyribonucleo-

side-50-triphosphates (dNTPs) that are substrates for DNA

synthesis, and oxidation of dNTPs frequently leads to

mutagenesis. The most abundant oxidized dNTP is oxi-

dized deoxyguanosine triphosphate (8-oxodGTP), which

can misincorporate into DNA by forming mispairs with

adenine, thereby causing AT to CG transversion muta-

tions (Shibutani et al., 1991). At high frequencies, this

event can lead to cell death (Ward et al., 1987). In bacte-

ria, 8-oxodGTP incorporation is prevented by the MutT

enzyme, which dephosphorylates 8-oxodGTP to 8-

oxodGMP (Maki and Sekiguchi, 1992). We decided to

investigate the action of BZ on free bases. Previous data

from our group have shown that MutT from Escherichiacoli retains its function when expressed in T. cruzi

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Unveiling Benznidazole’s Mechanism 311

(Aguiar et al., 2013). Therefore, we performed a

BZ-survival experiment using a genetically modified T.cruzi lineage that expresses E. coli MutT. Cells trans-

fected with pROCK-MutT or the empty pROCK vector

were challenged with 0, 60, 120, and 240 mM BZ, and

the number of living cells was evaluated after 48 hr of

drug treatment. The heterologous expression of MutT

increased T. cruzi survival upon BZ treatment at all tested

drug doses (Fig. 1A). At the highest tested concentration

(240 mM BZ), MutT expression increased the number of

living cells by threefold as compared to the control strain.

These results suggest that BZ intermediates attack free

dGTP, producing 8-oxodGTP.

In addition to dNTP oxidation, free radicals can also

oxidize the DNA molecule itself, generating oxidized

bases and DNA strand breaks. 8-oxoguanine (8-oxoG) is

one of the most common oxidative lesions, and it is

repaired mainly by BER (Barnes and Lindahl, 2004).

This multistep process is initiated by a specific DNA gly-

cosylase that recognizes and removes the modified base,

leaving an abasic site (AP site). Subsequently, the DNA

backbone is cleaved by an AP endonuclease, and repair is

completed by the activity of a phosphodiesterase, a DNA

polymerase and a DNA ligase (Robertson et al., 2009). 8-

Oxoguanine glycosylase 1 (Ogg-1) acts specifically in

8-oxoG removal (Michaels and Miller, 1992). To verify

whether BZ treatment could cause the generation of 8-

oxoG in the parasite DNA, we performed a BZ-survival

experiment using a genetically modified T. cruzi lineage

overexpressing T. cruzi Ogg1 (TcOgg1) (Furtado et al.,

2012). In contrast to what is observed with MutT-

expressing parasites, the overexpression of TcOgg1 did

Fig. 1. (A) Survival analysis of MutT-expressing parasites following

treatment with BZ. Control parasites (empty vector, open diamond);

MutT-expressing parasites (MutT, filled square). (B) Survival analysis of

TcOgg1-overexpressing parasites following treatment with BZ. Control

parasites (empty vector, open diamond); TcOgg1-overexpressing parasites

(TcOgg1, filled square). (C) Survival analysis of TcMutY-overexpressing

parasites following treatment with BZ. Control parasites (empty vector,

open diamond); TcMutY-overexpressing parasites (TcMutY, filled

square). Parasites were treated with 0, 60, 120, or 240 mM BZ. Cells

were counted in a cytometric chamber 48 hr after treatment, using the

erythrosine vital stain to differentiate living and dead cells. All experi-

ments were performed in triplicate. The error bars indicate standard devi-

ations of the mean. The statistical analysis used was an unpaired t test.

*P< 0.05; **P< 0.005; ***P< 0.0005.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

312 Raj~ao et al.

not modify the response of T. cruzi to BZ treatment at

any tested dose (Fig. 1B). In addition, we also tested if

the overexpression of TcMutY could alter the parasite’s

susceptibility to BZ. This glycosylase has the ability to

remove adenines misincorporated opposite 8-oxoG lesions

(Lu and Fawcett, 1998). As for TcOgg1-overexpressing

parasites, overexpression of TcMutY did not modify the

survival of T. cruzi against BZ treatment. These results

suggest that either BZ does not generate large amounts of

8-oxoG in the DNA of T. cruzi or, if it does generate 8-

oxoG, the lesion is preferentially removed by another

mechanism in this context.

Quantification of 8-oxoG in Parasites Treated with BZ

To address the question of whether BZ generates 8-

oxoG in T. cruzi, we assessed 8-oxoG accumulation in

parasites treated with this drug. Parasites were treated

with 120 mM BZ for 24 hr, after which the DNA samples

were isolated, processed, and analyzed by HPLC–electro-

chemical detection. As shown in Figure 2A, nontreated

empty vector parasites contained 3.08 8-oxoG/1 3 106

dG. The expression of MutT reduced 8-oxoG accumula-

tion to 1.58 8-oxoG/1 3 106 dG, showing that the activity

of MutT against 8-oxodGTP decreases the amount of 8-

oxoG in T. cruzi (Fig. 2A). When empty vector parasites

were treated with BZ, 8-oxoG levels rose to 8.33 8-oxoG/

1 3 106 dG (Fig. 2A). The expression of MutT again

reduced the level of 8-oxoG (5.60 8-oxoG/1 3 106 dG),

but in this case, the reduction occurred to a greater extent

than observed in nontreated parasites (Fig. 2A). These

results confirm that BZ induces 8-oxoG formation in

T. cruzi and indicate that at least part of this BZ-induced

DNA damage is caused by incorporation of nucleotides

that are oxidized by the drug.

Single-Allele Knockout of T. cruzi MSH2 ImprovesResistance Against BZ

Although the expression of MutT decreased the accumu-

lation of 8-oxoG in T. cruzi, it did not completely prevent

its incorporation into DNA. However, TcOgg1 and

TcMutY overexpression did not alter the resistance of T.cruzi against BZ, as would be expected in such a scenario.

The postreplicative DNA MMR system has a known func-

tion in the recognition and removal of 8-oxoG that is incor-

porated during replication (Colussi et al., 2002). The lesion

recognition and binding steps are carried out by the protein

MSH2. Following these initial steps, MMR components

drive the downstream events of strand discrimination,

lesion excision, and DNA resynthesis. We next investigated

whether the repair of BZ-induced 8-oxoG lesions is per-

formed by MMR. To do so, we performed a BZ-survival

experiment using a T. cruzi cell-line containing a single

allele deletion of T. cruzi MSH2 gene (TcMSH2) (Campos

et al., 2011). Our results show that the single knockout of

TcMSH2 enhanced resistance to BZ (Fig. 2B). The higher

survival rate observed in TcMSH21/2 cells indicates that

the MMR pathway is responsible for correcting BZ-

induced 8-oxoG lesions. Together with the fact that

Fig. 2. (A) Accumulation of 8-oxoG in T. cruzi cells treated with BZ.

Nontreated empty vector parasites (empty vector, white column); non-

treated MutT-expressing parasites (MutT, light gray column); BZ-treated

empty vector parasites (empty vector 1 BZ, dark gray column); BZ-

treated MutT-expressing parasites (MutT 1 BZ, black column). Parasites

were treated with 120 mM BZ for 24 hr and 8-oxoG accumulation was

assessed by HPLC–electrochemical detection. (B) Survival analysis of

TcMSH21/2 parasites following treatment with BZ. Control parasites

(empty vector, open diamond); TcMSH21/2 parasites (TcMSH21/2, filled

square). Parasites were treated with 0, 60, 120, or 240 mM BZ. Cells

were counted in a cytometric chamber 48 hr after treatment, using the

erythrosine vital stain to differentiate living and dead cells. Experiments

were performed in triplicate. The error bars indicate standard deviations

of the mean. The statistical analysis used was an unpaired t test.

*P< 0.05; **P< 0.005; ***P< 0.0005.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Unveiling Benznidazole’s Mechanism 313

TcOgg1 overexpression did not alter the survival of T. cruziexposed to BZ, the results above suggest that most of the 8-

oxoG induced by this drug is removed by MMR during

DNA replication.

Overexpression of T. cruzi Pol b, g, and j IncreasesResistance to BZ

DNA polymerization step of BER is responsible for

filling the AP sites generated by the glycosylase, thus

allowing the further steps of the pathway to take place. In

several organisms, DNA polymerase beta (Polb) is the

main DNA polymerase involved in this step (Robertson

et al., 2009). In T. cruzi, however, this DNA polymerase

is localized exclusively in the mitochondria, and it is also

involved in the gap-filling events that occur at the end of

mitochondrial DNA replication (Jensen and Englund,

2012; Lopes et al., 2008). We decided to investigate

whether overexpression of TcPolb would confer any addi-

tional resistance to BZ. Our results show that TcPolb

overexpression increased the BZ resistance of T. cruziwhen the parasite was treated with the highest drug dose

(Fig. 3A). This result suggests that BZ also induces dam-

age to mitochondrial DNA.

In addition to DNA repair, another strategy used by

cells to respond to DNA damage is TLS, the process that

allows the DNA replication machinery to bypass DNA

lesions (Prakash et al., 2005). Among the DNA polymer-

ases that are able to perform TLS, DNA polymerase eta

(Polh) is one of the most versatile, capable of bypassing

several different types of DNA lesions (Prakash et al.,

2005). Previous work from our group showed that

T. cruzi Polh (TcPolh) is localized to the nucleus and

has the ability to incorporate nucleotides opposite to

Fig. 3. (A) Survival analysis of TcPolb-overexpressing parasites follow-

ing treatment with BZ. Control parasites (empty vector, open diamond);

TcPolb-overexpressing parasites (TcPol beta, filled square). (B) Survival

analysis of TcPolh-overexpressing parasites following treatment with BZ.

Control parasites (empty vector, open diamond); TcPolh-overexpressing

parasites (TcPol eta, filled square). (C) Survival analysis of TcPolj-

overexpressing parasites following treatment with BZ. Control parasites

(empty vector, open diamond); TcPolj-overexpressing parasites (TcPol

kappa, filled square). Parasites were treated with 0, 60, 120, or 240 mM

BZ. Cells were counted in a cytometric chamber 48 hr after treatment,

using the erythrosine vital stain to differentiate living and dead cells. All

experiments were performed in triplicate. The error bars indicate standard

deviations of the mean. Statistical analysis performed using the unpaired ttest. *P< 0.05; **P< 0.005; ***P< 0.0005.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

314 Raj~ao et al.

an 8-oxoG lesion (Moura et al., 2009). In addition, its over-

expression increases T. cruzi resistance to H2O2 (Moura

et al., 2009). To investigate whether BZ treatment generates

lesions that can be bypassed through TLS, we assessed the

resistance of TcPolh-overexpressing parasites to this drug.

Our results show that TcPolh improves the resistance of T.cruzi to BZ, especially when the parasite was treated with

120 and 240 mM BZ (Fig. 3B). This finding indicates that at

least a fraction of the BZ-induced DNA lesions are

bypassed through TLS mechanisms. Another DNA poly-

merase involved in TLS is DNA polymerase kappa (Polj),

which acts mainly towards oxidative lesions (Bavoux et al.,

2005). Previous work from our group characterized the

mitochondrial copy of T. cruzi Polk (TcPolj) and reported

that this DNA polymerase is able to efficiently bypass 8-

oxoG lesions in vitro (Raj~ao et al., 2009). Accordingly, its

overexpression also enhanced T. cruzi resistance to H2O2

(Raj~ao et al., 2009). We used this TcPolj-overexpressing

lineage to test whether this DNA polymerase could also

increase T. cruzi resistance to BZ. As observed for TcPolh,

TcPolj overexpression augmented the parasite’s tolerance

to BZ, and this effect was also more evident after treatment

with the higher dose (Fig. 3C). In addition to lesion bypass,

mitochondrial TcPolj is also involved in DNA recombina-

tion (Raj~ao et al., 2009). Therefore, the higher resistance of

TcPolj overexpressors to BZ could indicate the action of

TcPolj towards mitochondrial DNA lesions through either

TLS or recombination repair.

Overexpression of TcRad51Improves T. cruzi Resistanceto BZ

The main protein involved in DNA recombination is

the recombinase Rad51. This protein acts by forming a

nucleoprotein filament on the 30-end of ssDNA, which is

necessary for the search for homologous repair templates

and catalysis of the subsequent strand invasion (Li and

Heyer, 2008). Rad51 in T. cruzi (TcRad51) is localized in

the nucleus, and its overexpression increases the para-

site’s resistance to genotoxic agents that are repaired by

recombination, such as gamma radiation and zeocin

(Regis-da-Silva et al., 2006). We evaluated the participa-

tion of TcRad51 in the response to BZ-induced DNA

lesions. Parasites overexpressing TcRad51 were more

resistant to BZ, and as observed in the previous experi-

ments, this difference was more pronounced when the

parasite was treated with the higher BZ dose (Fig. 4). In

addition, TcRAD511/2 parasites were more susceptible to

BZ. Together with the fact that a TcMSH2 single knock-

out increased T. cruzi resistance to BZ, these data support

the idea that this drug might induce DNA double-

stranded breaks.

Analysis of BZ-Induced DNA Lesions in T. cruziNuclear andMitochondrial DNA

As shown in Fig. 2A, we confirmed that BZ is able to

induce 8-oxoG formation within T. cruzi. An 8-oxoG

lesion per se is not cytotoxic to cells because it only

slightly slows the replication fork. Nevertheless, the proc-

essing and repair of this lesion can lead to abasic sites

and DNA strand breaks. We investigated the number of

cytotoxic DNA lesions that are induced by BZ using a

quantitative PCR (QPCR) assay described by Santos et al.

(2006). Wild-type parasites were treated with 240 mM BZ

for 24 hr, a period of time in which we still do not

observe cell death (data not shown). After treatment, the

number of nuclear and mitochondrial DNA lesions was

analyzed. As shown in Fig. 5A, the number of BZ-

induced lesions that are able to block DNA polymerase is

low in both nuclear and mitochondrial DNA (approxi-

mately 0.26 and 0.12 lesions per 10 kb, respectively). As

controls, we also performed the QPCR assay on parasites

treated with MMS and camptothecin. MMS generates

alkylating lesions in DNA, such as N7-methylguanine and

N3-methyladenine, and camptothecin is a topoisomerase I

inhibitor that induces double-stranded breaks (DSBs) in

cells that are in S-phase (Krwawicz et al., 2007; Pom-

mier, 2009). Cell death was not observed after these treat-

ments too. The treatment with MMS produced a

significant number of DNA lesions in T. cruzi DNA (Fig.

5B). On the other hand, the number of DNA lesions

caused by camptothecin was low in both the nuclear and

mitochondrial DNA (Fig. 5C), similar to what was

observed when treating parasites with BZ (Fig. 5A). Pre-

vious results from our group have shown that treatment

with the oxidative agent H2O2 induces high numbers of

QPCR-detectable DNA lesions (Furtado et al., 2012),

which differs from what we observed with BZ treatment.

Fig. 4. Survival analysis of TcRad51-overexpressing and TcRad511/2

parasites following treatment with BZ. Control parasites (empty vector,

open diamond, dashed line), TcRad51-overexpressing parasites (TcRad51,

�, solid line) and TcRad511/2 parasites (TcRad511/2, �, dashed line)

were treated with 0, 60, 120, or 240 mM BZ. Cells were counted in a

cytometric chamber 48 hr after treatment, using the erythrosine vital stain

to differentiate living and dead cells. The experiment was performed in

triplicate. The error bars indicate standard deviations of the mean. Statisti-

cal analysis was performed using the unpaired t test. *P< 0.05;

**P< 0.005; ***P< 0.0005.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Unveiling Benznidazole’s Mechanism 315

Thus, the results obtained here reinforce the hypothesis

that BZ-induced DNA lesions are mostly formed and

repaired during DNA replication.

BZ Induces DNA Breaks into T. cruziGenome

Because we could not detect a high amount of BZ-

induced DNA damage using the QPCR assays, we analyzed

the nuclear and mitochondrial ultrastructure by transmission

electron microscopy (TEM). After treatment of wild-type

parasites with 60 or 120 mM BZ for 24 hr, we observed that

in contrast to nontreated cells (Fig. 6A), the nuclear DNA of

T. cruzi underwent heterochromatin unpacking (Fig. 6B and

6C), similar to what is observed when the parasite is exposed

to high doses of gamma irradiation (Nardelli et al., 2009).

This result indicates that BZ induces the formation of

double-stranded breaks in T. cruzi nuclear DNA. We also

observed accumulation of electron dense vesicles in

response to BZ treatment, which may correspond to lipid

inclusions in the cytoplasm (Fig. 6C and 6D). Mitochondrial

swelling with a loss of matrix was observed in response to

treatment with 120 mM and 240 mM BZ for 24 or 48 hr (Fig.

6D and 6E). Protozoan ultrastructure was more affected after

treatment with 240 mM, for which we observed a more pro-

nounced mitochondrial swelling (Fig. 6E) and loss the

nuclear envelope (Fig. 6F). To confirm that BZ induces

DNA breaks into T. cruzi genome, we examined the DNA

integrity of parasites treated with BZ, by performing a PFGE

assay. As seen in Fig. 7, BZ leads to DNA fragmentation in

T. cruzi, which is showed by the smear that increases along

with the period of incubation with the drug (Fig. 7). Taken

together, the findings obtained in this work suggest that BZ

oxidizes the dNTP pool, which is subsequently incorporated

into T. cruzi genome. The high influx of oxidized dNTPs

may lead to DSBs, causing cytotoxicity to the parasite.

DISCUSSION

BZ is the drug of first choice for the treatment of Cha-

gas disease, but despite being intensely used for several

years, the precise molecular basis for its mode of action

is still unclear. It is well documented that BZ is reduced

within the cell, leading to the formation of various free

radical intermediates and electrophilic metabolites, but lit-

tle is known about its physiological consequences (Maya

et al., 2007). In this respect, one of the first mechanistic

studies reported that BZ inhibits both protein and RNA

synthesis in T. cruzi. This same study also found that

DNA synthesis is slightly decreased, and no alterations in

aerobic respiration were detected (Polak and Richle,

1978). Other studies have confirmed that BZ induces

DNA damage, and this damaging ability relies on the pro-

duction of nitro derivatives (Zahoor et al., 1987) and this

drug induces oxidative stress within the parasite (Pedrosa

et al., 2001). T. cruzi does not have catalase or glutathi-

one peroxidase activities, and its superoxide dismutase

activity is very low (Turrens, 2004; Wilkinson and Kelly,

2003). Its main defenses against free radicals are reduced

glutathione and a glutathione–spermidine conjugate called

trypanothione (Ariyanayagam and Fairlamb, 2001). The

sequestration of the thiols mentioned above by BZ elec-

trophilic metabolites reduces the parasite’s defenses

against free radicals even further, which is thought to be

the cause of BZ-induced oxidative stress within T. cruzi(Maya et al., 1997).

In the present work, we found that the heterologous

expression of E. coli MutT increased the parasite’s resist-

ance to BZ treatment. MutT specifically degrades 8-

oxoG-containing nucleoside triphosphates to correspond-

ing nucleoside monophosphates (Maki and Sekiguchi,

1992). Thus, our results indicate that BZ treatment leads

to oxidation of free guanine (and most likely other free

bases as well), although we cannot affirm whether this

oxidation is performed directly by BZ intermediates or

whether it is a collateral effect of the oxidative stress gen-

erated by the drug. The contrasting results obtained with

the expression of EcMutT in comparison to the overex-

pression of glycosylases TcOgg1 and TcMutY suggest

that BZ oxidizes the nucleotide pool, but it does not

Fig. 5. (A) Quantitative QPCR-based measurement of BZ-induced

lesions. Cells were treated with 240 mM BZ for 24 hr. (B) Quantitative

QPCR-based measurement of MMS-induced lesions. Cells were treated

with 2 mM MMS for 1 hr. (C) Quantitative QPCR-based measurement of

camptothecin-induced lesions. Cells were treated with 200 mM camptothe-

cin for 1 hr. All results are expressed as the mean of two biological

experiments. The error bars indicate standard deviations of the mean.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

316 Raj~ao et al.

Fig. 6. Ultrastructural analysis of T. cruzi epimastigotes by transmission

electron microscopy. (A) An untreated parasite presents typical features

of the nucleus, containing the nucleolus (nu), the peripheral heterochro-

matin (ht), the kinetoplast (k), the flagellum (f), the Golgi complex (gc),

and reservosomes (r). Parasites treated with benznidazole (B) 60 lM for

24 hr, (C) 120 lM for 24 hr, (D) 120 lM for 48 hr, (E) 240 lM for 24

hr, and (F) 240 lM for 48 hr. Ultrastructural alterations observed after

drug treatment included unpacking of nuclear heterochromatin (B, C; E,

F), mitochondrial swelling (D, E asterisk), membrane profiles in the mito-

chondrial matrix (B black arrows) and the presence of electron-dense

vesicles (C, D white arrows). After treatment with the highest drug con-

centration (240 lM) the mitochondrial swelling was more intense (E

asterisks), the nuclear envelope was disrupted (F arrowhead), and several

reservosomes were observed in the cytosol. Bars 5 2 mm (A, E, F) and 1

mm (B–D).

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Unveiling Benznidazole’s Mechanism 317

generate high levels of 8-oxoG in the genomic DNA

itself. This unexpected result could be explained by the

fact that free bases can act, to some extent, as a protec-

tive barrier to DNA oxidation. The majority of oxidative

DNA lesions occur as a consequence of the direct interac-

tion between reactive oxygen species (ROS) and nucleic

acids, which makes the nucleotide pool more vulnerable

to oxidation than the genomic DNA, which is packed into

chromatin (Rai, 2010). Studies have reported that the rel-

ative amounts of 8-oxoG and 2-hydroxyadenine produced

by ROS are higher in free dNTPs compared to genomic

DNA (Kamiya and Kasai, 1995; Mo et al., 1992).

Accordingly, it has been recently shown that elevated lev-

els of ROS within bacteria promote cell death predomi-

nantly by specific oxidation of the guanine nucleotide

pool (Foti et al., 2012).

The images obtained by TEM revealed that BZ induces

heterochromatin unpacking within the nucleus, suggesting

that the nuclear DNA contained DSBs. In addition, the

increased resistance to BZ observed in parasites overex-

pressing TcRad51 also reinforces the idea that BZ pro-

duces DSBs, given that T. cruzi primarily repairs DSBs

through recombination, with Rad51 being the major

player for this process (Regis-da-Silva et al., 2005). It has

been reported that DSBs can arise as a consequence of

free guanine oxidation. In this case, the extensive incor-

poration of 8-oxodGTP into the genome during S-phase

may cause high levels of closely spaced DNA lesions that

are inefficiently repaired and subsequently result in lethal

DSBs (Foti et al., 2012). In fact, it has been reported that

the cell death induced by bactericidal antibiotics is pre-

dominantly caused by this specific oxidation of the gua-

nine nucleotide pool and its subsequent use in DNA

replication (Foti et al., 2012). We were not able to detect

high numbers of DNA lesions induced by BZ using

QPCR. DSBs are among the most harmful types of DNA

damage, as they are injurious to the cell even at low

amounts. In bacteria, even a single DSB within the entire

genome is potentially lethal (Bonura et al., 1975). Our

results suggest that BZ induces DSBs at a frequency that

is relatively low, but sufficient to induce chromatin

unpacking and cell death. Similar results have been

observed when treating the parasite with drugs that inhibit

mitochondrial (unpublished data) and nuclear topoisomer-

ases (Zuma et al., 2011). These drugs are capable of

inducing extensive heterochromatin unpacking due to the

formation of DSBs, and in these experiments, we could

not detect DNA lesions through the QPCR technique.

However, we could verify DNA fragmentation when

examining the total DNA integrity of the parasite by per-

forming a PFGE assay, confirming that BZ is causing

DNA breaks in T. cruzi genome.

The mitochondrial DNA from T. cruzi consists of thou-

sands of minicircles and a dozen maxicircles that are con-

catenated in a condensed network of DNA and proteins.

This network is present in an enlarged portion of the

mitochondrion called the kinetoplast (Jensen and Englund,

2012). Electron microscopy analysis did not show altera-

tions in the topology of T. cruzi mitochondrial DNA, usu-

ally called kinetoplast DNA (kDNA), but only revealed

swelling in the mitochondrial branches, suggesting that

kDNA did not suffer extensive DSBs. Nevertheless, the

results obtained through overexpression of the mitochon-

drial proteins TcPolb and TcPolj indicate that the kDNA

of T. cruzi is also damaged by BZ. A previous paper

from our group showed that TcPolj functions in recombi-

nation repair, as it augments T. cruzi resistance to both

gamma radiation and zeocin and participates in DNA syn-

thesis in recombination intermediates (Raj~ao et al., 2009).

Fig. 7. Analysis of DNA integrity by pulse field gel electrophoresis. Par-

asites were treated with 0 mM BZ, 240 mM BZ for 24 hr, 240 mM BZ for

48 hr, or 240 mM BZ for 72 hr. Samples were prepared and subjected to

a pulsed-field gel electrophoresis. The agarose gel was stained with ethi-

dium bromide.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

318 Raj~ao et al.

Therefore, if BZ causes DSBs in the kDNA, TcPoljcould act in DNA polymerization during the recombina-

tion process. The kDNA structure might help maintain its

topology despite the strand breaks. In fact, the replication

of kDNA requires the generation of several strand breaks

in other to separate the concatenated minicircles, and the

DNA network does not lose its structure during this pro-

cess (Jensen and Englund, 2012). TcPolb is known to be

involved in kDNA replication during the process of filling

in the gaps between Okazaki fragments (Saxowsky et al.,

2003; Torri and Englund, 1995). The higher resistance of

TcPolb-overexpressing parasites to BZ suggests that

TcPolb is possibly employed in the final stages of the

recombination-mediated repair of BZ-induced DSBs, act-

ing in the gap-filling steps. On the other hand, if the

incorporation of oxidized nucleotides in kDNA does not

occur at a rate that is high enough to induce DSBs,

TcPolb could also be acting in mitochondrial BER.

The overexpression of TcPolh also increased the sur-

vival of T. cruzi exposed to BZ. Although the mammalian

Polh has been shown to participate in recombination, our

previous results suggest that the T. cruzi counterpart does

not play a role in this process (Moura et al., 2009). That

previous study showed that the overexpression of TcPolh

in T. cruzi does not confer any additional resistance to

gamma radiation (Moura et al., 2009), in contrast to what

is observed with the overexpression of TcRad51 (Regis-

da-Silva et al., 2006). Because previous results indicate

that TcPolh functions exclusively in TLS mechanisms,

the increase in the resistance to BZ provided by TcPolh

overexpression suggests that a small fraction of incorpo-

rated oxidized nucleotides does not cause DSBs and also

escapes DNA repair.

BZ is best known for interfering with the T. cruzi para-

site’s RNA and protein synthesis (Polak and Richle,

1978). This work provides new insights concerning how

BZ can assault the integrity of T. cruzi DNA, which our

results indicate to be mainly through formation of DSBs.

T. cruzi has considerable resistance to DSBs due to its

efficient recombination repair. Therefore, improving

understanding of how T. cruzi repairs BZ-induced DNA

lesions may enable new pharmacological approaches that

could enhance the effectiveness of BZ treatment.

ACKNOWLEDGMENTS

This work was supported by the following Brazilianresearch funding institutions: FAPEMIG (Fundac~ao deAmparo �a Pesquisa do Estado de Minas Gerais), FAPESP(Fundac~ao de Amparo �a Pesquisa do Estado de S~aoPaulo), CNPq (Conselho Nacional para o Desenvolvi-mento Cient�ıfico e Tecnol�ogico), CAPES (Coordenac~aode Aperfeicoamento de Pessoal de N�ıvel Superior), INCTde Processos Redox em Biomedicina—Redoxoma, andPr�o-Reitoria de Pesquisa da USP. We would like to thank

Thais Pereira Lopes and Neuza Antunes Rodrigues fortheir excellent assistance.

AUTHORCONTRIBUTIONS

Drs Carlos Renato Machado, Gl�oria Regina Franco and

Andrea Mara Macedo designed the study. Drs Matheus

Andrade Raj~ao, Carolina Furtado, Ceres Luciana Alves,

Danielle Gomes Passos-Silva, Michelle Barbi de Moura,

Bruno Luiz Schamber-Reis, Marianna Kunrath-Lima,

Aline Ara�ujo Zuma, Jo~ao Pedro Vieira-da-Rocha, Juliana

Borio Ferreira Garcia and Isabela Cec�ılia Mendes col-

lected the data. Drs Carlos Renato Machado, Santuza

Maria Ribeiro Teixeira, Angela Kaysel Cruz, Maria Cris-

tina Machado Motta, Matheus Andrade Raj~ao, Carolina

Furtado, Ceres Luciana Alves, Danielle Gomes Passos-

Silva, Michelle Barbi de Moura, Bruno Luiz Schamber-

Reis, Marianna Kunrath-Lima, Aline Ara�ujo Zuma, Jo~ao

Pedro Vieira-da-Rocha, Juliana Borio Ferreira Garcia, Isa-

bela Cec�ılia Mendes, Nadja Cristhina de Souza-Pinto,

Marisa Helena Gennari de Medeiros, Maria Cristina

Machado Motta and S�ergio Danilo Junho Pena analyzed

the data and prepared draft figures and tables. Drs Carlos

Renato Machado prepared the manuscript draft with

important intellectual input from Drs Matheus Andrade

Raj~ao and Carolina Furtado.

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