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Toxicology Letters 190 (2009) 140–149

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

ytotoxic, mutagenic and genotoxic effects of new anti-T. cruzi-phenylethenylbenzofuroxans. Contribution ofhase I metabolites on the mutagenicity induction

auricio Cabrera a,1, María Laura Lavaggi a,1, Paola Hernández a, Alicia Merlino a,lejandra Gerpe a, Williams Porcal a, Mariana Boiani a, Ana Ferreira b, Antonio Monge c,dela López de Cerain c, Mercedes González a,∗, Hugo Cerecetto a,∗

Departamento de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, 11400 Montevideo, UruguayCátedra de Inmunología, Facultad de Ciencias-Facultad de Química, Universidad de la República, 11400 Montevideo, UruguayCentro de Investigaciones en Farmacobiología Aplicada, Universidad de Navarra, Pamplona, Spain

r t i c l e i n f o

rticle history:eceived 1 June 2009eceived in revised form 1 July 2009ccepted 2 July 2009vailable online 10 July 2009

eywords:hagas’ diseaseenzofuroxan

a b s t r a c t

5-Phenylethenylbenzofuroxans have displayed in vitro and in vivo activity against Trypanosoma cruzi,the etiologic agent of American Trypanosomiasis. On the basis of benzofuroxans pre-clinical studies weevaluated the potential of six 5-phenylethenyl derivatives to induce cytotoxicity, mutagenicity and geno-toxicity using different in vitro models. Cytotoxic effects were evaluated using a set of cells, mammalpre-monocytic macrophages, V-79 lung fibroblast from Chinese hamster, and colorectal adenocarcinomaCaco-2 cells, in the MTT viability assay. Mutagenicity was tested in the Ames assay using Salmonellatyphimurium TA98 strain with and without metabolic activation by S9-rat liver homogenate. The geno-toxic potentials were evaluated with the alkaline single cell gel electrophoresis (comet assay) in V-79

utagenicityenotoxicityetabolism

cells. In view of the Ames test results we study whether the main mammals’ phase I metabolites, thecorresponding o-nitroanilines, are involved in the mechanism of mutagenicity. These metabolites areproduced by NADPH-dependent enzymes in cytosol and by xanthine oxidase and cytochrome P450 inmicrosomes from rat liver. Among them, the electronic property of phenyl substituent seems to be respon-sible for this effect. It could be pointed out that the equimolecular mixture of compounds 1 and 2 (5E-and 5Z-(2-phenylethenyl)benzofuroxan, respectively) could be used in further clinical studies as anti-T.

Crow

cruzi drug.

. Introduction

Chagas’ disease or American Trypanosomiasis, one of the mostmportant public health problems in many South American coun-ries, is caused by the protozoan Trypanosoma cruzi (WHO/TDR,005; Moncayo, 1999). This parasitic disease, that affects more than8 million people in Latin America, has only two drugs as therapeu-ic alternatives, nifurtimox (Nfx, Lampit®, recently discontinued

y Bayer, Fig. 1) and benznidazole (Bnz, Rochagan®, Roche, Fig. 1)Cerecetto and González, 2002). These drugs have demonstratedeveral limitations in their use in part due to their low bioavailabil-ty, their limited efficacy in the different stages of the disease as

∗ Corresponding authors at: Iguá 4225, Montevideo, Uruguay.el.: +598 2 5258618x216; fax: +598 2 5250749.

E-mail addresses: megonzal@fq.edu.uy (M. González), hcerecet@fq.edu.uyH. Cerecetto).

1 These authors contributed equally to this article.

378-4274/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Ireland Ltoi:10.1016/j.toxlet.2009.07.006

n Copyright © 2009 Published by Elsevier Ireland Ltd. All rights reserved.

well as the development of parasite resistance (Castro Silva et al.,1989; De Andrade et al., 1996; González-Martin et al., 1998; Murtaet al., 1998; Nozaki et al., 1996). The other main contraindication ofboth drugs is the relevant toxic effects that they display.

Most frequent side effects of these drugs include anorexia, vom-iting, peripheral polyneuropathy and allergic dermopathy. The invivo toxic effects and mutagenicity of Nfx have been clearly proved(Bartel et al., 2007; Castro and Díaz de Toranzo, 1988; Gorla et al.,1989). Also Bnz has demonstrated genotoxic effects in vitro (Da SilvaMelo et al., 2000; Nagel, 1987; Souza et al., 1991) and relevant invivo alterations (Montalto de Mecca et al., 2008; Teixeira et al., 1990,1994). For this reason, the development of safer and more effectivedrugs for Chagas’ disease is an urgent priority.

The search for new trypanocidal compounds is currently

being done through the development of in vitro screening assays(Cerecetto and González, 2008). In this sense, our group has inves-tigated and developed new agents derived from the benzofuroxan(benzo[1,2-c]1,2,5-oxadiazole N-oxide) heterocycle (Aguirre et al.,2002, 2005a,b; Cerecetto et al., 1999; Olea-Azar et al., 2003a,b;

d. All rights reserved.

M. Cabrera et al. / Toxicology Letters 190 (2009) 140–149 141

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ig. 1. (a) Chemical structures of the anti-chagasic drugs used in therapy. (b) Chemictructure of one of the sub-product from the PhEBfx synthetic procedures (7). (d) Lefight: chemical structures of new identified mammal metabolites (14–19).

lea-Azar et al., 2005; Porcal et al., 2007) identifying excellentead compounds, 5-(2-phenylethenyl)benzofuroxan derivativesPhEBfx, cis and trans isomers, 1–6, Fig. 1). These compounds haveeen the subject of the following preclinical studies: pharmaceu-ical feasibility, in vivo efficacy, in vitro metabolism and in vitrooxicity. These studies were performed as part of a research projectupported by DNDi (Drugs for Neglected Disease initiative) orga-ization, which has as the primary goal the development of newnd more effective drugs for people suffering from neglected dis-ases in developing countries (http://www.dndi.org). In this sense,e have described a safe multigram procedure to prepare theesired PhEBfx, to analyse them chromatographically (Gerpe etl., 2008), to evaluate its in vivo efficacy and their metabolic pro-le in hepatic rat microsomes and cytosol and by the parasite T.ruzi. In the multigram synthetic procedures the corresponding de-xygenated analogues, phenylethenylbenzofurazans (PhEBfz, i.e., Fig. 1), were obtained in very low yields (Porcal et al., 2008)hat were eliminated after the purification processes. The bio-ransformations of the PhEBfx in mammal and parasitic systemsenerate the corresponding o-nitroanilines (PhENA, i.e. 8–13, Fig. 1)s mixtures of positional isomers with a higher proportion of 4-henylethenyl-2-nitroaniline ones (i.e. 8, 10, and 12, Fig. 1) (Boianit al., 2009). The PhEBfxs in vivo anti-T. cruzi efficacies were assessedn mice (Boiani et al., 2008), where compound 1 (Fig. 1) and thequimolecular mixture (1 + 2, as they were obtained in the syn-hetic procedure) were able to reduce the parasite loads of animalsith fully established T. cruzi infections and to prevent cardiac

nflammatory infiltrates with comparable results to that obtainedith both Nfx and Bnz. Furthermore, compound 1 (Fig. 1) did not

roduce remarkable damage on the animals in a performed acute-oxicity study (Boiani et al., 2008). In view of the aforementionedata, the determination of the complete cytotoxic, mutagenic andenotoxic potential of these compounds (1–6, Fig. 1) is neces-

ary in order to assess their safeness for possible use in humans.onsequently, in this study we evaluated the in vitro cytotoxic,utagenic and genotoxic potential of PhEBfxs 1–6, Fig. 1, and their

quimolecular mixtures, 1 + 2, 3 + 4, and 5 + 6, as they are syn-hetically obtained, including the PhEBfz 7 (Fig. 1). PhEBfz 7 is

ctures of the studied benzofuroxans as new anti-chagasic drugs (1–6). (c) Chemicalmical structures of the studied mammal and parasitic metabolites of PhEBfx (8–13);

the synthetic sub-product of the most mutagenic PhEBfx, deriva-tive 4. Cytotoxicity was tested using the MTT colorimetric viabilityassay, mutagenicity was examined using Ames assay, with andwithout metabolic activation, and induction of genotoxic effectswas evaluated in the Comet assay using the alkaline single cellgel electrophoresis version. Also, some aspects of the involvedmechanism of mutagenicity were studied based in the Ames testresults.

On the one hand, taking into account the mutagenic effect gen-erally was evidenced in the +S9-assays that phase I metabolites ofderivatives 1, 3 and 4 could be responsible of this effect. Accordingly,the pure metabolites (PhENA 8, and 9, Fig. 1) and its equimolecu-lar mixtures, as they are biologically generated (8 + 9, and 12 + 13),were studied on the mutagenicity assay. Furthermore the processesof 1–4 biotransformations were studied on terms of metabolizationrates and on term of the involved enzymes using enzyme selectiveinhibitors. Consequently, as during the enzymatic inhibition studies4-phenylethenyl-o-benzoquinone dioximes (PhEBDO, 14–19, Fig. 1)were identified as new metabolites, they were also submitted to theAmes test.

On the other hand, it can be considered that some chemicalstructural motives in the PhEBfx derivatives could play relevantrole in the mutagenic profiles. Thus to complete the study inthe electronic effect of phenyl-substitution, compounds with –H(in derivatives 1 and 2), electron-withdrawing-substitutions (–Cl,in derivatives 5 and 6) or with electron-donor substituents (3,4-methylendioxi, in derivatives 3 and 4); we also included on theAmes test another electron-donor-substituted E-PhEBfx previouslyobtained (derivative 20, Fig. 4, Boiani et al., 2009). We find thatelectron-donor chemical scaffolds could be the responsible of theenhanced mutagenic properties of PhEBfx more than the kind ofgeometric isomer.

2. Material and methods

2.1. Chemicals, media and cells

Compounds 1–20 were prepared according to the procedures reported ear-lier (Boiani et al., 2009; Porcal et al., 2008). Dimethyl sulfoxide (DMSO),

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-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), histine, bio-ine, streptomycin, penicillin, 2-aminofluorene (2-AF), 4-nitro-o-phenylendiamineNPD), NADP, d-glucose-6-phosphate, hydrogen peroxide, and the salts for mediand buffers were obtained from Sigma Chemical Company (St. Louis, MO). Nfx andnz were obtained from the corresponding commercial tablets extracting with ace-one, evaporating the solvent under reduced pressure and crystallizing successivelyntil adequate microanalysis. Compounds 1–20, its equimolecular mixtures, Nfx andnz in DMSO were freshly prepared at proper concentrations.

THP-1 human monocyte-like cells (ATCC, USA) were differentiated toacrophages by culture (5% CO2, 37 ◦C, 48 h) in the presence of 50 ng/mL phorbol-

2-myristate-13 acetate (Sigma) in RPMI 1640 medium supplemented with 10%eat inactivated foetal bovine serum (FBS). V-79 cell culture, assessing on a cell

ine derived from Chinese hamster lung fibroblasts, and Caco-2 cells (colorectaldenocarcinoma, ATCC/HTB-37), provided by the American Type Culture CollectionManassas, VA), were grown as monolayers in Dulbecco’s modified Eagle’s mediumDMEM) supplemented with 10% FBS (100 g/mL) and 1% of penicillin/streptomycinn a humidified atmosphere of 5% CO2 in air at 37 ◦C. The Salmonella typhimuriumis− , TA98 strain Moltox (NC, USA), was grown in Nutrient Broth No. 2 from Oxoid

25 g/L) (Wesel, Germany) for 12 h, at 37 ◦C. The S9 mix was obtained from MoltoxNC, USA).

.2. MTT viability assay

Cells (THP-1 differentiated to macrophages, V-79, or Caco-2) were seeded at aensity of 2.5–10.0 × 105 cells/well in 96-well plates flat bottom microplates (Nun-lon) and exposed, for 48 h in the case of macrophages and for 6 h in the rest of cells,o different doses of the studied compounds (1–14, 17, or the equimolecular mix-ures 1 + 2, 3 + 4, 5 + 6, 8 + 9, 10 + 11, 12 + 13, and 18 + 19), at 1000.0–12.5 �M doses,r vehicle for control. After treatment, the compounds were removed and the cellsashed once with PBS. Cell viability was then colorimetrically assessed by mea-

uring the mitochondrial-dependent reduction of MTT to formazan (Zhang et al.,007). For that purpose, the cells and MTT (0.4 mg/mL in the case of macrophagesnd 0.5 mg/mL in the rest of cells) were incubated in air at 37 ◦C for 3 h in the casef macrophages and for 4 h in the other cells. After the incubation period, the super-atant was removed and formazan crystals were dissolved with DMSO (180 �L). Thelates were shaken for 10 min and the optical densities were measured at 560 nm inmultiwall spectrophotometer. Each concentration was assayed three times and sixdditional controls (cells in medium) were used in each test. The data are presenteds the IC50, as the compound concentration required to reduce the cells by half.

.3. Ames assay

The preliminary Salmonella mutagenicity assay (Ames assay) was performedccording to the method described earlier (Maron and Ames, 1983). Firstly, we deter-ined the minimum toxic doses (MTD, �g/plate) of the studied compounds (1–9,

4, 17, 20, the equimolecular mixtures 1 + 2, 3 + 4, 5 + 6, 8 + 9, 12 + 13, 18 + 19, Nfxnd Bnz) against the bacteria (S. typhimurium His− , TA98 strain). Nfx and Bnz werencluded in this assay as references trypanocidal drugs. The determined MTD wereelected as the maximum doses to be assayed in the Ames test. For the test, suspen-ion of 2 × 109 S. typhimurium/mL was prepared and the preincubation procedureas performed both with and without S9 mix as an external enzymatic metabolizing

ystem. Rat liver S9, from commercial origin, was prepared as it was described ear-ier (Ames et al., 1975). Assays without metabolic activation (no S9) were performed

ixing 50 �L of each test substance solution with 500 �L of phosphate buffer (0.1 M,H 7.4) and 100 �L of bacteria suspension. Four to six consecutive dose levels weretudied beginning with the corresponding MTD and decreasing one third succes-ively (MTD/3, MTD/9, MTD/27, etc.). After 60 min of incubation, 2 mL of moltenop agar supplemented with traces of histine and biotine (50 �M each, final con-entration) were added, rapidly vortexed and poured on agar plates. As the topgar hardened, plates were inverted and incubated for 48 h, at 37 ◦C. Assays withetabolic activation were similarly performed replacing phosphate buffer by an

qual volume of S9 mix (10%, v/v S9, 4.7 mM NADP, 6 mM d-glucose-6-phosphate,9 mM MgCl2, 36 mM KCl, phosphate buffer 0.1 M, pH 7.4). Controls were tested inuplicate plates in each assay. A solvent control treated with DMSO and two posi-ive controls, NPD (20 �g/plate) without metabolic activation and 2-AF (10 �g/plate)ith metabolic activation, were always included. After 48 h incubation at 37 ◦C the

evertant colonies were counted. Two independent assays were carried out for eachxperimental condition. For all the assays the data were analysed using the modi-ed 2-fold rule (Chu et al., 1981) in which a response is considered positive if theverage response for at least two consecutive dose levels was more than twice thepontaneous frequencies and were subjected to multifactor analysis of variance.

.4. Alkaline comet assay

Monolayer V-79 cells in exponential growth were trypsinized and cell culturesere prepared in a 6-well plate: 2.5 × 105 cells/mL in 2 mL of DMEM containing 10%

BS and 1% of penicillin/streptomycin. Just after the 6 h treatment with the stud-ed compounds (1–3, 5, 6, or the equimolecular mixtures 1 + 2, 5 + 6, 8 + 9, Nfx andnz), at 100.0–12.0 �M doses, comet assay was carried out (Collins et al., 1997). Nfxnd Bnz were included in this assay as references trypanocidal drugs. One hundred

ters 190 (2009) 140–149

and sixty microliters of 0.5% agarose-low melting point containing 4.5 × 104 cellswere distributed quickly on a slide prepared previously with agarose-normal melt-ing point. A cover slip was added and the agarose was allowed to set for 5 min on ice.Then the cover slip was removed and the cells were lysed immediately by immer-sion of the slide in a cold solution (pH 10.5) of 2.5 M NaCl, 100 mM Na2EDTA, 10 mMTrizma–HCl and 1% Triton X-100 for 2 h at 4 ◦C. The slides were placed on a hori-zontal gel electrophoresis platform and covered with an alkaline solution made upof 300 mM NaOH and 1 mM Na2EDTA (pH 13.0). The slides were left in the solutionfor 20 min to allow unwinding of the DNA and expression of alkali-labile sites. Thepower supply was set at 0.7 V/cm (300 mA). The DNA was electrophoresed for 15 minand the slides were rinsed gently three times with 400 mM Trizma (pH 7.5) to neu-tralize the alkali excess. Each slide was stained with 30 mL of DAPI, covered with acover slip and coded after microscopic analysis. DAPI-stained nuclei were evaluatedwith a Nikon Eclipse TE 300 fluorescence microscope. A total of 100 comets on eachcomet slide were visually scored and classified as belonging to one of five classesaccording to the tail length. Each comet class was given a value between 0 and 4,with 0 means no damage and 4 means maximum damage. The total score was calcu-lated by the following equation: (percentage of cells in class 0 × 0) + (percentage ofcells in class 1 × 1) + (percentage of cells in class 2 × 2) + (percentage of cells in class3 × 3) + (percentage of cells in class 4 × 4). Consequently, the total score was in therange from 0 to 400 (Collins et al., 1995). Three comet slides were used for each con-dition, which makes a total of 300 comets. To check the performance of the cometassay, a positive control was included in all the experiments, V-79 cells treated withhydrogen peroxide (200 �M) during 15 min on ice.

2.5. Study of derivatives 1–4 metabolism

Two different kinds of experiments were performed. On the one hand, the dif-ferential rate of metabolization was studied for the +S9-non-mutagenic PhEBfx 2and for the +S9-mutagenic PhEBfx 1 and 3–4. On the other hand, identification ofenzymatic system involved in the metabolic processes was assessed by selectiveinhibition assays.

2.5.1. Preparation of the rat liver microsomal and cytosolic proteins andincubation conditions

Livers were obtained from female Wistar rats (250–300 g) from “Centro de Inves-tigaciones Nucleares, UdelaR” (Montevideo, Uruguay). The animals were allowedfood and water ad libitum. The experimental protocols with animals were evalu-ated and supervised by the local Ethics Committee and the research adhered tothe Principles of Laboratory Animal Care (Morton and Griffiths, 1985). The animalswere sacrificed by cervical dislocation and the livers, maintained in an ice bath,were perfused in situ with an ice-cold NaCl (0.9%) solution and washed with 3volumes of Tris–HCl (0.05 M)–sucrose (0.25 M) pH 7.4, then they were sliced andhomogenised in a Potter–Elvehjem glass-Teflon homogeniser. The homogenateswere centrifuged for 30 min at 900 × g at 4 ◦C and the supernatant fraction wascentrifuged at 10,000 × g for 1 h at 4 ◦C. The pellet was discarded and the super-natant fraction was further centrifuged at 100,000 × g for 1 h at 4 ◦C. The cytosolicand microsomal fractions, supernatant and pellet, respectively, were recovered. Thepellet was washed twice by resuspension in the above Tris–HCl buffer solution,resedimented by centrifugation for 1 h at 100,000 × g at 4 ◦C and finally resuspendedin the Tris–HCl buffer solution. Metabolic assays were carried out with microsomesand cytosol either fresh or frozen in Tris–HCl buffer and stored at −80 ◦C. Protein con-tent of the microsomal and cytosolic fractions was determined by the bicinchoninicacid assay from Sigma as suggested by the manufacturer.

The standard incubation mixture (Boiani et al., 2009) contains MgCl2 (1.3 mM),NADP+ (0.4 mM), glucose 6-phosphate (3.5 mM), 0.5 U/mL glucose 6-phosphatedehydrogenase in a phosphate buffer (0.1 M, pH 7.4) containing EDTA (1.5 mM) andthe corresponding PhEBfx (400 �M, from stock solutions in DMSO) with 1 mL offinal volume. The final concentration of the DMSO in the incubation mixture wasbelow 1.0%. After pre-equilibration of the mixture at 37 ◦C, appropriate volumes ofmicrosomal or cytosolic fraction were added to give a final protein concentration of1 mg/mL. The mixtures were incubated between 0.5 and 4 h, in 24-well plates opento air, at 37 ◦C. The experiments were performed by triplicate. Two control incu-bations were done: (1) without �-nicotinamide adenine dinucleotide phosphate(NADPH)-generating system; (2) using the subcellular fractions inactivated by heat-ing for 5 min at 95 ◦C. At the end of the incubation, 400 �L of methanol were addedand the mixture was kept at 4 ◦C for protein precipitation. The incubated mixtureswere extracted with EtOAc (3 × 400 �L) and the organic layer was evaporated todryness under reduced pressure. The residue was treated twice with acetonitrile(AcCN), 500 �L each, the combined organic layers were filtered through RC regener-ated cellulose filters 0.45 �m pore size (Sartorius, Germany). The 100 �L aliquot ofthe obtained AcCN solution was analysed by a RP column, 25 cm × 0.46 cm, 10 �mparticle size, with a PerkinElmer LC-135C/LC-235C HPLC system equipped with adiode array detector, series 410 LC BIO PUMP. HPLC analyses were carried out at

a flow rate of 1 mL min−1 with AcCN:H2O (4:6 (v:v)) as mobile phase and roomtemperature as working temperature. Detection was at 300 nm (Gerpe et al., 2008).

2.5.2. Identification of the involved enzymesThe identification of the involved metabolic enzymes was made pre-incubating

during 30 min the corresponding fraction, microsomal or cytosolic hepatic fraction,

gy Letters 190 (2009) 140–149 143

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Table 2Cytotoxicity on THP-1 cells treated with mixture of geo-metric isomers of compounds 1–6 (as they are obtainedin the synthetic procedures), synthetic sub-product 7and metabolites 8–14, and 17–19.

Compound THP-1, IC50a (�M)

1 + 2b 149.00c

3 + 4b 81.0c

5 + 6b 66.7c

7 >1000.08 >1000.09 >400.0d

8 + 9e >800.0d

10 >400.0d

11 170.010 + 11e 128.012 180.013 192.012 + 13e 144.014 175.817 >400.018 + 19 84.6

a The results are the means of three independentexperiments with a SD less than 10% in all cases.

b Equimolecular mixture of each of the geometric iso-mer (E and Z).

c From Porcal et al. (2007).d Large amount could not be assayed for solubility

problems in the corresponding biological milieu.

M. Cabrera et al. / Toxicolo

ith previously described enzymatic inhibitors (400 �M). The employed inhibitorsere: dicoumarol for DT-diaphorase (DTD), menadione (Men) for aldehyde oxidase

AO), allopurinol for xanthine oxidase (XO), and ketoconazole (Ket) for cytochrome450 (CYP) (Lavaggi et al., 2008). An artifact found in the Men-inhibition assay (seeelow) conduct us to employ benzaldehyde as enhancer electron donor of AO orther NADPH-dependent enzyme (Sun et al., 2000). Consequently, the possible par-icipation of AO, or other NADPH-dependent enzymes, was studied enhancing itsctivity with benzaldehyde in absence of NADPH-generating system. Studied com-ounds were dissolved in DMSO (400 �M) and were incubated, during 30 min at7 ◦C in 0.1 M potassium phosphate buffer (pH 7.4) containing EDTA (1.5 mM) withhe corresponding enzymatic fraction (1 mg protein/mL) and benzaldehyde (40 �M,issolved in DMSO) as an electron donor. Taking into account that some inhibitionas observed with Ket, which is defined as general CYP inhibitor with preferen-

ial CYP3A4/5 isoforms inhibition capability, two others specific CYP inhibitors weretudied, ticlopidine for CYP2C19 isoforms (Ha-Duong et al., 2001) and furafylline forYP1A2 isoforms (Atherton et al., 2006).

.5.3. Chromatographic monitoring of metabolitesThe incubated mixtures were extracted with EtOAc (3 × 400 �L) and the organic

ayer was evaporated to dryness in vacuo. The residue was treated twice withtOAc, 500 �L each, the combined organic layer was filtered through RC regen-rated cellulose filters 0.45 �m pore size (Sartorius). The 100 �L aliquot of thebtained AcCN solution was analysed by a RP column, 25 cm × 0.46 cm, 10 �m par-icle size, with a PerkinElmer LC-135C/LC-235C HPLC system equipped with a dioderray detector, series 410 LC BIO PUMP. HPLC analyses were carried out at a flowate of 1 mL min−1 with AcCN:H2O (4:6 (v:v)) as mobile phase and room tem-erature as working temperature. Detection was at 300 nm (Gerpe et al., 2008).long with HPLC experiments, TLC was performed as qualitative study. These stud-

es were done in SiO2 as solid phase and EtOAc:hexane (80:20) (three times) asobile phase. The spots were visualised using UV-light (240 nm) and directly for its

haracteristic colours (PhEBfx, yellow; PhENA, orange-yellow and orange; PhEBDO,rown).

. Results and discussion

.1. MTT assays

The MTT assays demonstrated that compounds 1–6 differen-ially decreased the mitochondrial activities in the different cellularystems and studied experimental conditions (Table 1). Accordingo the decrease in the mitochondrial activity PhEBfx 3 and 5 werehe least cytotoxic against the three cellular systems. Except forompound 4, the percentages of survival with respect to the con-rols in V-79 cells after 6 h treatment with the studied compoundst 100.0 �M were more than 70%. According to previous descriptionHenderson et al., 1998) and taking into account that compound

was not included in the alkaline comet assay (see below), dosesower than 100.0 �M in V-79 cells are good to evaluate the potentialenotoxicity of these compounds in the alkaline comet assay.

The equimolecular mixture of PhEBfx 3 + 4 and 5 + 6, as they are

btained in the synthetic procedures, displayed higher toxicitiesgainst THP-1 cells (Table 2) than each of the individual isomer ofhe mixture. It shows some kind of toxic-synergism when both geo-

etric isomers are co-administered. Contrarily, the equimolecularixture 1 + 2 was less toxic than each of the mixture component

able 1ytotoxicity in the three studied biological systems treated with compounds 1–6.

ompound IC50a (�M)

THP-1b V-79 Caco-2

109.9 130.0 >200.0c

62.6 178.0 >400.0220.0 >400.0 >400.0120.0 44.0 >400.0

>200.0c 400.0 >400.097.0 140.0 >400.0

a The results are the means of three independent experiments with a SD less than0% in all cases.

b From Porcal et al. (2007).c Large amount could not be assayed for solubility problems in the corresponding

iological milieu.

e In equimolecular ratio, simulating the proportion ofthe o-nitroanilines generated in the metabolic condi-tions.

showing a favourable effect in the equimolecular mixture ofisomers. The studied sub-product from the synthetic procedure,PhEBfz 7, was completely non-toxic against THP-1 cells (Table 2).The main mammals’ metabolites of PhEBfx previously identified(Boiani et al., 2009), o-nitroanilines 8–13, showed different profilesof cytotocities against THP-1 cells (Table 2). Clearly, the metabolitesderived from PhEBfx 1, PhENA 8 and 9, did not affect mitochon-drial activities at the assayed doses. When they were studied inthe proportion that they are generated in mammal cells, 8 + 9,absence of cytotoxicity was also observed. The metabolites fromPhEBfx 2 and 4, o-nitroanilines 10–13, were more cytotoxic againstTHP-1 than PhEBfx 1’s metabolites. Interestingly, in both cases,the equimolecular mixture of metabolites (10 + 11 and 12 + 13,entries 7 and 10, Table 2) displayed more cytotoxicities againstTHP-1 than each of the independent metabolite. Again a negativetoxic-synergism was observed. The corresponding o-benzoquinonedioximes, 14–19 (Fig. 1), the new metabolites identified here whenCYP was inhibited, were evaluated against THP-1 cells. Dependingon its chemical structures they were more or less toxic than thecorresponding o-nitroaniline analogues, compare 14-cytotoxicitywith 8- or 9-cytotoxicity or compare 17-cytotoxicity with 12- or13-cytotoxicity correspondingly.

In summary, our findings indicated cytotoxic effects againstthe three studied cellular systems were so different. Cytotoxicitiesagainst THP-1 macrophages were wide-ranging while Caco-2 cellswere insensitive against PhEBfx. THP-1 cell line provides a valu-able model for studying the mechanisms involved in macrophagedifferentiation and for exploring the regulation of macrophage-specific genes (Auwerx, 1991) while the Caco-2 cells derived froma human colon adenocarcinoma, spontaneously differentiate afterreaching confluence in culture, exhibiting several morphological

and functional characteristics of mature enterocytes (Liu and Chen,2004). Then again, V-79 cell line, known as a well-established cel-lular model, was an adequate system to perform alkaline cometassay, with doses lower than 100 �M, where the PhEBfx, exceptcompound 4, posses IC50 higher than 100 �M.

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.2. Ames assay

The results on mutagenicity of compounds 1–6 examined bysing S. typhimurium test strain TA98, with and without S9 acti-ation, are gathered in Table 3. Unlike the trypanocidal referencerugs, Nfx and Bnz, the studied PhEBfx 1, 2, 3, 5, and 6 andhe corresponding equimolecular mixtures as they are obtainedn the synthetic procedures (1 + 2 and 5 + 6) were not mutagenic

ithout S9 activation. In both conditions, with and without S9ctivation, and in the range 3.0–83.0 �g/plate the Z-isomer 4 sig-ificantly increased colony formation in a dose-dependant manner.he equimolecular mixture of E and Z isomers 3 and 4 showed theame behaviour as compound 4 alone. In the presence of S9 mix theombination 1 + 2 was unable to produce a number of revertantsigher than the untreated system similar behaviour observed withHP-1 cellular system. On the other hand, in presence of S9 mix andt the highest doses, PhEBfx 1, 3, 5, and 6, and the equimolecularixtures 5 + 6 displayed significant increase of revertants indicat-

ng the S9 mix capability to promote the generation of mutagenicntities. Additionally, we studied the mutagenic potential of theecondary product obtained from the synthesis of 4, the mostutagenic studied compound, PhEBfz 7. The results summarized

n Table 3 demonstrate that PhEBfz 7 is mutagenic, according tohu et al. (1981) criteria, after S9 activation.

These results show that some chemical structural motives inhe PhEBfx derivatives could play relevant role in the mutagenicrofiles, as the electronic effect of phenyl-substitution (com-are −S9-mutagenic profile of un-substituted derivatives 1 andor electron-withdrawing-substituted derivatives 5 and 6 to theutagenic profile of electron-donor-substituted derivative 4) or

he geometric isomerism (compare the −S9-mutagenicity of Z-somerism 4 to the absence of mutagenicity of E-isomer 3). To gainnsight this fact others studies were performed (see below, Section.4).

able 3evertants in Salmonella typhimurium treated with different doses of compounds 1–6, equ

ompound Doses(�g/plate)

−S9 +S9 Compound Doses(�g/plate)

−

0.0 20 ± 5 15 ± 4 3 0.01.0 13 ± 3 16 ± 6 1.02.0 8 ± 2 18 ± 3 2.05.0 11 ± 5 25 ± 10 6.0

15.0 3 ± 1 54 ± 12a 17.045.0 4 ± 2 134 ± 6a,b 50.050.0 26 ± 1 152 ± 2a

0.0 21 ± 4 31 ± 10 4 0.00.4 12 ± 9 28 ± 4 3.01.0 19 ± 6 22 ± 6 9.03.0 21 ± 5 35 ± 5 28.0 1

10.0 24 ± 6 48 ± 2 83.0 130.0 41 ± 11 38 ± 1 250.0 3

+ 2c 0.0 19 ± 1 25 ± 1 3 + 4c 0.00.5 10 ± 1 24 ± 2 2.01.0 19 ± 8 23 ± 6 6.04.0 17 ± 4 21 ± 6 17.0

13.0 Toxicity 39 ± 13 50.0150.0 6

fx 0.0 21 ± 4 31 ± 10 Bnz 0.00.5 29 ± 6 37 ± 5 62.01.0 43 ± 17 39 ± 18 185.03.0 62 ± 2a 53 ± 9 556.0

10.0 144 ± 11a,b 64 ± 6a 1667.030.0 117 ± 17a 139 ± 11a,b 5000.0 25

a Revertants number was at least two times higher than DMSO (0.0 �g/plate of compoub Mutagenic, according to Chu et al. (1981): second consecutive dose levels twice the spc Equimolecular mixture of each of the geometric isomer (E and Z). Positive controls: N

evertants.

ters 190 (2009) 140–149

3.3. Alkaline comet assay

Induction of genotoxic effect was evaluated by measuring DNAdamage in the alkaline comet assay (Azqueta et al., 2007) in V-79cells. Fig. 2 illustrates the results of these experiments performedon compounds 1, 2, 3, 5, 6, the corresponding equimoleculargeometric–isomers mixtures (1 + 2, and 5 + 6) as they were obtainedin the synthetic procedures, the equimolecular mixture of themetabolites 8 and 9, and the trypanocidal reference-drugs, Nfx andBnz. Compound 4 were not included in this study due to its hightoxicity against V-79 cells (IC50 = 44.0 �M) and, on the other hand,its mutagenic profile in both conditions (−S9 and +S9) that trans-form PhEBfx 4 as a no adequate entity as potential drug for Chagasdisease.

Inductions of DNA strand breaks lower than positive control(H2O2, 200 �M, 15 min treatment) were observed at every one ofthe studied doses (100.0–12.0 �M, 6 h treatment) for all the stud-ied compounds and equimolecular mixtures of compounds (1, 2,1 + 2, 3, 5, 6, 5 + 6, Nfx and Bnz) and metabolites from compound1 (8 + 9). Contrarily to in vivo data for Nfx (Gorla et al., 1989), thisassay could not categorize it as genotoxic. Conversely, PhEBfx Z geo-metric isomers, 2 and 6, showed the highest DNA damage at thetwo highest studied doses (100.0 and 50.0 �M) with some degreesof cells belonging to category 4. On the contrary, the equimolec-ular mixture of geometric isomers, 1 + 2 and 5 + 6, did not displayDNA strand breaks at any of the studied doses being, together withNfx and Bnz, the entities with lowest total scores in these assays.Again, like in the THP-1 viability test and in the Ames assay, afavourable effect in the behaviour of the equimolecular mixture ofgeometric isomers 1 + 2 was observed. For compounds 1, Nfx, Bnz

and the equimolecular mixture 8 + 9 were not evidenced cometsbelonging to category 4 at any of the assayed doses. No com-pounds or mixtures reached total scores near to 400 at the assayeddoses.

imolecular mixtures of them, Nfx and Bnz.

S9 +S9 Compound Doses(�g/plate)

−S9 +S9

21 ± 4 31 ± 10 5 0.0 21 ± 4 31 ± 1017 ± 2 46 ± 0 1.0 18 ± 3 32 ± 217 ± 1 39 ± 6 2.0 20 ± 1 35 ± 426 ± 8 66 ± 1a 6.0 25 ± 2 86 ± 7a

27 ± 3 262 ± 5a 17.0 23 ± 4 102 ± 20a

21 ± 1 225 ± 25a,b 50.0 26 ± 0 118 ± 17a,b

21 ± 4 31 ± 10 6 0.0 19 ± 1 25 ± 153 ± 6a 40 ± 7 1.0 17 ± 4 30 ± 678 ± 4a 76 ± 5a 3.0 20 ± 4 49 ± 0

14 ± 6a,b 350 ± 25a 8.0 16 ± 1 79 ± 1a

83 ± 31a 382 ± 24a,b 25.0 45 ± 3a 59 ± 9a,b

93 ± 39a 226 ± 3a

19 ± 1 25 ± 1 5 + 6c 0.0 20 ± 0.5 11 ± 722 ± 13 22 ± 1 1.0 24 ± 5 16 ± 241 ± 5a 31 ± 1 2.0 28 ± 2 22 ± 241 ± 4a 47 ± 4 5.0 17 ± 5 27 ± 9

50 ± 14a 373 ± 0a 15.0 14 ± 5 71 ± 13a

6 ± 24a,b 545 ± 32a,b 45.0 27 ± 2 81 ± 9a,b

21 ± 4 31 ± 10 7 0.0 19 ± 3 25 ± 156 ± 1a 65 ± 9a 2.8 20 ± 1 21 ± 0

77 ± 6a,b 83 ± 5a 8.3 22 ± 3 34 ± 0115 ± 8a 151 ± 2a 25.0 27 ± 7 35 ± 0232 ± 2a 192 ± 10a,b 75.0 37 ± 17 68 ± 0a

4 ± 103a 309 ± 3a,b 225.0 41 ± 22 292 ± 0a,b

nd).ontaneous frequencies of revertants.PD (20.0 �g/plate, −S9) 1223 ± 237 revertants, 2-AF (10.0 �g/plate, +S9) 801 ± 82

M. Cabrera et al. / Toxicology Letters 190 (2009) 140–149 145

Fig. 2. Alkaline comet assay analysis in V-79 cells incubated with the studied compounds for 6 h, expressed as cells in five different categories (0–5) and quantified as Collinset al. (1997). Cells in category zero are not showed because they are not counted in the total score. Percentages of cells in category zero: (1) Negative control: 57. (2) Positivecontrol: 14. (3) Derivative 1: 22 (100 �M), 19 (50 �M), 19 (25 �M), 26 (12 �M). (4) Derivative 2: 1 (100 �M), 0 (50 �M), 21 (25 �M), 43 (12 �M). (5) Derivative 3: 50 (100 �M),50 (25 �M), 46 (12 �M). (6) Derivative 5: 28 (100 �M), 22 (50 �M), 34 (25 �M), 25 (12 �M). (7) Derivative 6: 19 (100 �M), 1 (50 �M), 35 (25 �M), 19 (12 �M). (8) Nfx: 62(100 �M), 60 (50 �M), 48 (25 �M), 44 (12 �M). (9) Bnz: 47 (100 �M), 58 (50 �M), 50 (25 �M), 46 (12 �M). (10) Equimolecular mixture 1 + 2: 46 (100 �M), 56 (50 �M), 89(25 �M), 97 (12 �M). (11) Equimolecular mixture 5 + 6: 54 (100 �M), 53 (50 �M), 96 (25 �M), 86 (12 �M). (12) Equimolecular mixture 8 + 9: 24 (100 �M), 15 (50 �M), 16(25 �M), 17 (12 �M).

Fig. 3. Relationship between PhEBfx V-79 cytotoxicities, expressed as log10(IC50), and DNA damages, expressed as sum of the comets in categories 1–4 (a) at 25 �M; (b) at100 �M. For derivative 3 the IC50.V-79 was estimated.

146 M. Cabrera et al. / Toxicology Letters 190 (2009) 140–149

Table 4Revertants in Salmonella typhimurium treated with different doses of metabolites 8, 9, 8 + 9, 12 + 13.

Compound Doses (�g/plate) −S9 +S9 Compound Doses (�g/plate) −S9 +S9

8 0.0 24 ± 7 32 ± 4 8 + 9c 0.0 24 ± 7 32 ± 42.8 11 ± 0 25 ± 1 2.8 16 ± 5 41 ± 78.3 28 ±0 36 ± 4 8.3 15 ± 0 216 ± 0a

25.0 22 ±6 62 ± 2 25.0 15 ± 1 249 ± 25a,b

75.0 26 ± 5 54 ± 9 75.0 19 ± 1 169 ± 18a

225.0 53 ± 4 53 ± 5 225.0 33 ± 2 161 ± 11a

9 0.0 24 ± 7 32 ± 4 12 + 13c 0.0 19 ± 1 32 ± 42.8 16 ± 0 111 ± 9a 6.2 44 ± 5 64 ± 08.3 20 ± 0 305 ± 113a,b 18.5 27 ± 0 179 ± 0a

25.0 20 ± 0 360 ± 35a 55.6 38 ± 7 738 ± 0a,b

75.0 25 ± 4 342 ± 37a 166.7 73 ± 12a 674 ± 0a

225.0 Toxicity 336 ± 0a 500.0 95 ± 0a,b 681 ± 0a

mpouthe sptroani

bvbsrBcgsttdih

3

Soofcfeea2mtgomehtc

+m

msoo

s

a Revertants number was at least two times higher than DMSO (0.0 �g/plate of cob Mutagenic, according to Chu et al. (1981): second consecutive dose levels twicec Equimolecular mixture of each of the positional isomer (4-aryl- and 5-aryl-2-ni

While the neutral comet assay is known to detect primarily dou-le strand breaks (dsb) there is general agreement that the alkalineersion (pH ≥13), performed here, is most appropriate to detect aroad spectrum of primary DNA lesions with high sensitivity, i.e.ingle strand breaks (ssb) and dsb, alkali-labile sites, incompleteepair sites and crosslinks (Tice et al., 2000; Hartmann et al., 2003;rendler-Schwaab et al., 2005; Burlinson et al., 2007). However,ytotoxicity only correlates to dsb and in our case we could observeood fitting between the viability on V-79 cells (and THP-1 cells,ee Fig. S1, Supplementary content), expressed as log10(IC50), andhe comet numbers (obtained in the alkaline conditions at 25 �M)hat allow us to infer the compounds are able to produce mainlysb at this sub-toxic dose (Fig. 3a). Due to derivative 2 is an outlier

n this conditions this could shows different kind of DNA lesions atigher doses (i.e. ssb) operating in this compound.

.4. Study of the mechanism of mutagenicity of PhEBfx in S9 mix

In order to confirm and identify the mutagenic entities after the9 metabolization we studied the mutagenic potential of the previ-usly characterized (Boiani et al., 2009) main mammal metabolites,-nitroanilines (i.e. 8–13, Fig. 1). We chose the main metabolitesrom the +S9-mutagenic PhEBfx 1, PhENA 8 and 9 and the biologi-al generated equimolecular mixture 8 + 9 (Boiani et al., 2009), androm the both −S9- and +S9-mutagenic PhEBfx 4, the biological gen-rated equimolecular mixture 12 + 13. The results, shown in Table 4,videnced a clear dependence of the o-nitroanilines mutagenicitiesnd the chemical structure, i.e. nitro position in the phenyl group.-Nitro-4-phenylethenylaniline 8 is not mutagenic in both experi-ental conditions, with and without metabolic fraction, however

he positional isomer 2-nitro-5-phenylethenylaniline, 9, is muta-enic after metabolization. Probably, the biological transformationf 9 into the 1,2-phenylenediamine could be the responsible of theutagenicity in presence of S9 fraction (Chung et al., 1995). The

quimolecular mixture of 8 + 9, as they are obtained biologically,as the same behaviour as derivative 9 alone. On the other hand,he equimolecular mixture of 12 + 13 is mutagenic in both studiedonditions (−S9 and +S9 fraction).

Consequently, these experiments could be demonstrating thatS9-mutagenicity of PhEBfx 1, and 3–6 is due to the positional iso-ers 5-aryl-2-nitroaniline mutagenicities after its metabolizations.

In order to gain insight the participation of derivatives 1, 3–6etabolites on the PhEBfx mutagenicities, after its metabolizations,

ome others experiments were performed. For that, the processesf 1–4 biotransformations were studied in terms of rates of metab-lization and in term of involved metabolic enzymes.

In the first experiment the PhEBfx 1–4 were incubated in micro-omal and cytosolic rat-hepatic fractions during 4 h following the

nd).ontaneous frequencies of revertants.line).

disappearance of the PhEBfx via HPLC analysis (see Fig. S2, Supple-mentary content) (Boiani et al., 2009). Neither time nor origins ofthe enzymes were identified as responsible of the different muta-genic properties between 1, 3–6 and 2. Therefore compound 2,the non-mutagenic derivative in both conditions, has a similarbehaviour on the metabolization rates of compound 4, the mostmutagenic PhEBfx, large metabolism in the citosolic fraction andmoderate in the microsomal. On the other hand, as we previouslyobserved (Boiani et al., 2009) the E-isomers are less metabolizedthan the Z-ones in both conditions.

Secondly, from the identification of the involved enzymesassays using specific enzymatic inhibitors some relevant factswere achieved (see Table S1, Supplementary content). In the o-nitroanilines 8, 9, 12, and 13 biological production is not involvedDTD in cytosol. However, from the study with benzaldehydeas enhancer electron donor of AO, or other NADPH-dependentenzyme, it was proved that this enzyme is involved in the reduc-tion of PhEBfx to PhENA in the cytosol. The study with Menas inhibitor was not able to probe the inhibition of AO becausethe mixture Men + NADPH, without enzymatic fraction, is capa-ble to reduce chemically the parent PhEBfx to the correspondingo-nitroaniline derivatives (see Supplementary content). On theother hand, XO and CYP are comprised partially in the PhENA pro-duction in microsomes. According to the microsomal inhibitiondata, using Ket, furafilline and ticlopidine as inhibitors, CYP3A4/5but not CYP2C19 nor CYP1A2 isoforms are involved. No differ-ences on the involved metabolic enzymes of PhEBfx 1 and 4were observed. However, a very interesting fact occurred duringthe inhibition assays with dicoumarol in cytosolic fraction andallopurinol, Ket, and ticlopidine in microsomal fraction, a newmetabolite was observed. The corresponding 4-phenylethenyl-o-benzoquinone dioxime (PhEBDO, 14–19, Fig. 1) was identified.This kind of metabolite was previously described for benzofuroxan(Grosa et al., 2004) however in our experimental conditions wewere unable to detect the dioxime biological intermediate untilnow (Boiani et al., 2009). Due to this generation, the new observedmetabolites, 14, 17 and the equimolecular mixture 18 + 19, wereevaluated in the Ames test in order to know its mutagenic properties(Table 5).

As it could be seen, the PhEBDO derived from compounds 1, 5and 6, dioximes 14 and equimolecular mixture of C C geometricisomers 18 + 19 are not mutagenic in the assayed conditions how-ever the dioxime 17, that is generated biologically from compound

4, is mutagenic like the parent compound.

In summary, it is clear that the mutagenic activities after expo-sition to S9-mix of PhEBfx 1, 3–6 are the consequence of the5-aryl-2-nitroanilines and also of the o-benzoquinone dioximein the case of derivative 4. However, observing the displayed

M. Cabrera et al. / Toxicology Letters 190 (2009) 140–149 147

ns in t

mrtwstCidcabeekP

TRm

C

1

1

c

t

C

Fig. 4. Chemical structural consideratio

utagenicities without metabolization it could be consider someelevant chemical-structural features that probably explain theoxic differential mechanisms. On the one hand, mutagenic activityithout S9-mix could be affected by the electronic effect of phenyl-

ubstituent (compare results of PhEBfx 1 and 4, Table 3, Fig. 4) or, onhe other hand, by the geometric isomerism around double bond

C (compare results of 3 and 4, Table 3, Fig. 4). In order to gainnsight this fact, we included in the Ames test another previouseveloped PhEBfx (derivative 20, Fig. 4) (Boiani et al., 2009) thatonjugates in the same structure the electron-donor substituentnd the E-geometric isomerism. As the results revealed (Table 5),eing compound 20 also mutagenic in −S9-mix conditions, the

lectron-donor chemical scaffolds could be the responsible of thenhanced −S9 mutagenic properties of the PhEBfx more than theind of geometric isomer. The lack of −S9 mutagenic effects ofhEBfx 3 could be result of the lower solubility of this derivative

able 5evertants in Salmonella typhimurium treated with different doses of new identifiedetabolites 14, 17, 18 + 19, and the new PhEBfx 20 included in this study.

ompound Doses(�g/plate)

−S9 Compound Doses(�g/plate)

−S9

4 0 19 ± 3 18 + 19c 0.0 19 ± 30.4 21 ± 3 0.4 18 ± 41.1 20 ± 2 1.1 21 ± 13.3 27 ± 2 3.3 21 ± 810.0 26 ± 6 10.0 Toxicity30.0 Toxicity

7 0.0 19 ± 3 20 0.0 19 ± 30.4 25 ± 1 0.4 11 ± 31.1 34 ± 6 1.1 25 ± 43.3 49 ± 11a 3.3 62 ± 8a

10.0 44 ± 1a,b 10.0 80 ± 7a,b

30.0 38 ± 5a 30.0 Toxicity

a Revertants number was at least two times higher than DMSO (0.0 �g/plate ofompound).

b Mutagenic, according to Chu et al. (1981): second consecutive dose levels twicehe spontaneous frequencies of revertants.

c Equimolecular mixture of each of the geometric isomer around the double bondC.

he −S9 mutagenicity of studied PhEBfx.

in the biological milieu, this kind of phenomena was not deepeststudied here.

4. Conclusions

The mutagenic and/or genotoxic effects of the two drugs used inChagas’ disease, Nfx and Bnz, have been previously demonstrated(Bartel et al., 2007; Castro and Díaz de Toranzo, 1988; Da Silva Meloet al., 2000; Gorla et al., 1989; Nagel, 1987; Souza et al., 1991).Besides, it is interesting to note that some research groups areworking in the chemical modulation of the nitroimidazole systemin order to generate antiparasite agents without mutagenic effects(Crozet et al., 2009). In this sense, we are looking for new anti-Chagas’ disease drugs with reduced toxic effects and we found thatPhEBfx system could be adequate pharmacological agents. For thisreason, PhEBfx were submitted to an initial in vitro toxicologicalstudy. From the Ames assays, it is possible to evidence that PhEBfx4 is mutagenic with and without S9 activation like the trypanoci-dal drugs, Nfx and Bnz. Except compound 2, the rest of the PhEBfx,1, 3, 5, and 6, were mutagenic only with S9 activation. Genotoxiceffects evaluated with the Comet assay were moderately observedat the highest doses only for compounds 2 and 6. According to theobserved relationship to cellular cytotoxicity, the DNA-damage atsub-toxic dose is probably due to double strand break. All of theresults allow us to identify compounds to endorse clinical stud-ies, especially the synthetic equimolecular mixture of compounds1 and 2 (in this study 1 + 2). This mixture has an especial behaviourin all the performed tests when it is compared with the parent com-pounds separately, 1 or 2, finding a positive effect lacking mutagenicand genotoxic effects.

In reference to the mechanism of mutagenicity and accord-ing to the results with compound 1, the +S9-mutagenic effectsseem to be the result of the 5-aryethenyl-2-nitroaniline metabolites(exemplified with metabolite 9 in Fig. S3) that are mutagen-

ics after biotransformations with S9 mix. Interesting biologicalresult was observed with 4-arylethenyl-2-nitroaniline metabolitethat results non-mutagenic (see example with metabolite 8 inFig. S3) with and without metabolic mixture showing the rele-vance of the structure in the mutagenicity mechanism. The main

1 gy Let

maaPdPaaeipteroiosPimogLuc

sld

C

A

DCa

A

t

R

A

A

A

A

A

A

48 M. Cabrera et al. / Toxicolo

etabolites of compound 4, compounds 12 and 13 (evaluateds the equimolecular mixture of 12 + 13), are mutagenic withnd without S9 mix explaining the mutagenicity of the parenthEBfx with metabolic activation (Fig. S3). We were unable to findifferences on the metabolic processes of the different studiedhEBfxs. The metabolic rates in hepatic microsomes and cytosolnd the involved metabolic enzymes were similar for mutagenicnd non-mutagenic derivatives being the responsible metabolicnzymes, at least in part, AO or other NADPH-dependent enzymen cytosol and XO and CYP3A4/5 in microsomes. The kind ofhenyl-substitution among the different studied PhEBfx could behe key for the −S9 mutagenicity. Mesomeric-electron-donor moi-ties, 2-(3,4-methylenedioxy) or 4-hydroxy, could play a relevantole in the mechanism of mutagenesis of these compounds with-ut metabolic mix. Profoundly observing the chemical structure

nvolve in the studied compounds other conclusion could be takenut. Being compound 4, the mixture of metabolites, 12 + 13, theynthetic by-product, PhEBfz 7, and the new identified metabolitehEBDO 17 mutagenics in all the studied conditions it could also be

ndicating that the shared chemical structural feature, the 2-(3,4-ethylenedioxyphenyl)ethenyl substituent, could be responsible

f these effects as it was previously described in several muta-enic/genotoxic products (Chen et al., 2006; Chung et al., 2008;ee et al., 2005; Yun et al., 2003) (Fig. 4). All of these results allows to speculate on the mechanism of mutagenicity of the studiedompounds.

The data obtained here point to some structural motives thathould be used for further chemical modifications in order to obtainess toxic new anti-T. cruzi agents able to be used as drugs for Chagas’isease.

onflict of interest statement

The authors declare that they have no conflict of interest.

cknowledgement

This investigation was done as part of the project “Clinicalevelopment of Arylethenylbenzofuroxan Derivatives as Drugs forhagas Disease” supported by Drugs for Neglected Diseases initiativend supported in part by RIDIMEDCHAG-CYTED.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.toxlet.2009.07.006.

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