Mutation Research 631 (2007) 16–25

Genotoxicity of 15-deoxygoyazensolide in bacteria and yeast

Marne C. Vasconcellos a, Renato M. Rosa b, Miriana S. Machado b, Izabel V. Villela b,Antonio Eduardo Miller Crotti c, Joao Luis Callegari Lopes c, Claudia Pessoa a,

Manoel Odorico de Moraes a, Norberto Peporine Lopes c, Letıcia V. Costa-Lotufo a,Jenifer Saffi d, Joao Antonio Pegas Henriques b,e,∗

a Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceara,Caixa Postal-3157, 60430-270 Fortaleza, Ceara, Brazil

b Centro de Biotecnologia e Departamento de Biofısica, Universidade Federal do Rio Grande do Sul,Caixa Postal 15005, 91501-970 Porto Alegre, Rio Grande do Sul, Brazil

c Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, 14040-903 Ribeirao Preto, Sao Paulo, Brazild Laboratorio de Genetica Toxicologica, Universidade Luterana do Brasil, 92425-900 Canoas, Rio Grande do Sul, Brazil

e

Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, Rio Grande do Sul, Brazil

Received 5 February 2007; received in revised form 2 April 2007; accepted 3 April 2007Available online 7 April 2007

Abstract

Sesquiterpene lactones (SLs) present a wide range of pharmacological activities. The aim of our study was to investigatethe genotoxicity of 15-deoxygoyazensolide using the Salmonella/microsome assay and the yeast Saccharomyces cerevisiae. Wealso investigated the nature of induced DNA damage using yeast strains defective in DNA repair pathways, such as nucleotideexcision repair (RAD3), error prone repair (RAD6), and recombinational repair (RAD52), and in DNA metabolism, such as topoi-somerase mutants. 15-deoxygoyasenzolide was not mutagenic in Salmonella typhimurium, but it was mutagenic in S. cerevisiae.The hypersensitivity of the rad52 mutant suggests that recombinational repair is critical for processing lesions resulting from15-deoxygoyazensolide-induced DNA damage, whereas excision repair and mutagenic systems does not appear to be primarilyinvolved. Top 1 defective yeast strain was highly sensitive to the cytotoxic activity of 15-deoxygoyazensolide, suggesting a pos-sible involvement of this enzyme in the reversion of the putative complex formation between DNA and this SL, possibly due tointercalation. Moreover, the treatment with this lactone caused dose-dependent glutathione depletion, generating pro-oxidant statuswhich facilitates oxidative DNA damage, particularly DNA breaks repaired by the recombinational system ruled by RAD52 in yeast.Consistent with this finding, the absence of Top1 directly affects chromatin remodeling, allowing repair factors to access oxidativedamage, which explains the high sensitivity to top1 strain. In summary, the present study shows that 15-deoxygoyazensolide ismutagenic in yeast due to the possible intercalation effect, in addition to the pro-oxidant status that exacerbates oxidative DNA

damage.© 2007 Elsevier B.V. All rights reserved.

Keywords: 15-Deoxygoyazensolide; Salmonella/microsome assay; Saccharomyces cerevisiae; Glutathione; Sesquiterpene lactones

∗ Corresponding author at: Departamento de Biofısica, Predio 43422, Laboratorio 210, Campus do Vale, Universidade Federal do Rio Grandedo Sul, Avenida Bento Goncalves 9500, Bairro Agronomia, CEP 91501-970 Porto Alegre, RS, Brazil. Tel.: +55 51 33086069;fax: +55 51 33087003.

E-mail address: pegas@cbiot.ufrgs.br (J.A.P. Henriques).

1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.mrgentox.2007.04.002

utation Research 631 (2007) 16–25 17

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M.C. Vasconcellos et al. / M

. Introduction

Sesquiterpene lactones (SLs) are substances pre-enting different pharmacological activities, withnti-microbial, anti-viral, anti-inflammatory, and anti-umor effects [1,2]. For instance, costunolide andehydrocostus lactone inhibit the killing activity ofytotoxic T lymphocytes, nitric oxide (NO) produc-ion, and the expression of hepatitis B virus surfacentigen [3–5]. Helenalin alleviated carrageen-induceddema of rat hindfeet, and suppressed cancer cell growth6,7]. Parthenolide and encelin showed potent inhibitoryffects on the expression of cyclooxygenase and tumorecrosis factor (TNF)-� [8]. This wide range of biolog-cal activities is mainly caused by the inactivation ofhe NF-�B nuclear factor [8–10]. Cynaropicrin derivedrom Saussurea lappa suppresses the production ofytokines, such as TNF-� and cytokine-induced neu-rophil chemoattractant-1 (interleukin-8), as well as NOelease. It also strongly attenuates mitogenic stimulationf CD4+ and CD8+ lymphocyte proliferation [11,12].

Most of the observed biological effects of these com-ounds are attributed to their ability to inhibit enzymesnd other essential proteins by covalently binding toree cysteine sulfhydryl groups of polypeptides [13]r by conjugation with glutathione (GSH), leadingo thiol depletion and an increase in the susceptibil-ty to oxidative damage [14]. Severe oxidative stressonferred by SLs-induced thiol depletion results in aisruption of the integrity of mitochondria, triggeringitochondrial permeability transition and release ofitochondrial pro-apoptotic proteins. SLs contain an �-ethylene-�-lactone moiety, which is highly reactiveith cellular thiols, leading to alkylation of sulphydryl

esidues through Michael-type addition [14–16]. NF-B, an ubiquitous transcription factor that regulates

nflammatory responses, cell growth/differentiation andpoptosis, seems to be the main target for many SLs15,16]. According to Rungeler et al. [15], NF-�B inhi-ition by SLs is caused by alkylation at the mentionedysteine moieties in the subunit p65.

In addition, some SL has also remarkable cytotoxicffects. Indeed, costunolide, helenalin and parthenolidere known to be strong inducers of cytotoxicity byriggering apoptosis through reactive oxygen species-

ediated oxidative stress, However, its genotoxicotential has not been thoroughly documented [17–20].he most studied lactone in relation to genotoxicity is

he parthenin, a sesquiterpene lactone from Partheniumysterophorus L., which induces chromosomal aberra-ions, mainly chromatid breaks, in blood lymphocytes;ts did not influence sister chromatid exchange and show

Fig. 1. Chemical structure of 15-deoxygoyazensolide.

a positive increase in the micronucleated reticulocyte fre-quency in high doses [21]. Hymenoxon, helenalin, andtenulin were also tested for genotoxicity using six strainsof Bacillus subtilis and hymenoxon and helenalin werefound to produce lethal DNA damage in this model whiletenulin did not produce lethal DNA damage in any of thestrains tested [22]. Since that DNA damage has a centralrole in cytotoxic properties of several natural and syn-thetic molecules, the evaluation of genotoxicity of thesemolecules is very important [23].

The furanoheliangolide 15-deoxygoyazensolide(Fig. 1) is a SL isolated from several species of thesub-tribe Lychnophorinae (tribe Vernonieae, familyAsteraceae), and possesses two functionalities in theform of an �-methylene-�-lactone and an �,�,�,�-unsaturated carbonyl group [24,25], which could beconsidered as essential features for NF-�B inhibition.Santos et al. [26] demonstrated that goyazensolide,which structure is similar to 15-deoxygoyazensolide, butfor the oxidation at carbon 15, was strongly cytotoxicto tumor cell lines. In order to further understand thebiological properties of SLs, this study aimed at inves-tigating the genotoxicity of 15-deoxygoyazensolidein bacteria using the Salmonella/microsome assay,and at evaluating its genotoxic potential to the simpleeukaryote S. cerevisiae, which mutagenesis and DNArepair mechanisms were assessed using haploid strains.This information is very important to evaluate the safetyof possible future pharmacological applications of thiscompound, and to explore its potential pharmacologicalproperties, such as antiproliferative activity.

2. Materials and methods

2.1. Chemicals

All solvents were bi-distilled, and stored in darkflasks. Hydrogen peroxide (H2O2), D-biotin, aflatoxin B1,

utation

18 M.C. Vasconcellos et al. / M

4-nitroquinoleine oxide (4-NQO), sodium azide, reducedglutathione (GSH), oxidized glutathione (GSSG), 1-chloro-2,4-dinitrobenzene (CDNB), NADPH, glutathione reduc-tase, aminoacids (l-histidine, l-threonine, l-methionine,l-tryptophan, l-leucine, l-lysine), l-canavanine, nitrogenbases (adenine and uracil), and dimethylsulfoxide (DMSO)were purchased from Sigma (St. Louis, USA). The S9 frac-tion, prepared with the polychlorinated biphenyl mixtureAroclor 1254, was purchased from Moltox (Annapolis, MD,USA), and glucose-6-phosphate and NADP were obtainedfrom Sigma (St. Louis, USA). Oxoid nutrient broth No. 2was obtained from Oxoid USA Inc. (Maryland, USA). Yeastextract, Bacto-peptone, and Bacto-agar were obtained fromDifco Laboratories (Detroit, MI, USA).

2.2. 15-Deoxygoyazensolide isolation and identification

Our previous phytochemical investigation of Minasiaalpestris dried leaves, employing the five main fractions of adichloromethane extract, showed that 15-deoxygoyazensolideis the main SL of this plant species [8]. Minor sub-fractionswere analyzed using comparative HPLC, as previouslydescribed for seasonal variations of this class of secondarymetabolites in Lychnophora ericoides [24]. Sub-fractions richin this lactone were pooled together, and the resulting fractionwas chromatographed in preparative HPLC (ODS-Shimadzu,5.0 mm × 250 mm column, MeOH–H2O, λ = 260 nm, flown8 mL/min), yielding 30 mg of 15-deoxygoyazensolide. Thestructure of the isolated molecule was compared to the standard15-deoxygoyazensolide structure obtained by authentic NMRspectrum and high-resolution mass spectrometry in a previousstudy [27].

2.3. Strains and media

Salmonella typhimurium strains TA98 (detects frameshiftmutation in DNA target -C-G-C-G-C-G-C-G), TA97a (detects

frameshift mutation in -C-C-C-C-C-C-; +1 cytosine), TA100(base pair substitution mutation results from the substitution ofleucine [GAG] by proline [GGG]), and TA102 (TAA (ochre:transversions/transitions) detects oxidative, alkylating muta-gens and reactive oxygen species [ROS]), described in Ref.

Table 1Saccharomyces cerevisiae strains used in this study

Strains Relevant genotypes

Haploid: XV185-14c MATα ade2-2 arg4-17 his1-7 lys1-1Y202 MATa ura3-Δ100 ade2-1 his3-11,15rad1Δ MATa ura3-Δ100 ade2-1 his3-11,15rad6Δ MATa ade2-1 his3-11,15 leu2-3,112rad1Δ rad6Δ MATa ade2-1 his 3-11,15 trp1-1 canN123 MATa his1-7 gsh1 leakDiploid: JH500a MATa/MATα rad3-e5/RAD3 RAD52

ARG4/arg4-17 HIS1/his1-7 HIS5/hisTRP5/trp5-48 HOM3/hom3-10

a Genotypes of zygotes from which haploid segregants were derived.

Research 631 (2007) 16–25

[28], were kindly provided by B. M. Ames (University of Cal-ifornia, Berkeley, CA, USA). Bacterial media were preparedaccording to Mortelmans and Zeiger [28]. Complete mediumfor growing strains (NB) contained 2.5% oxoid nutrient broth#2. Solidified medium with 1.5% bacto-agar, supplementedwith 1× Vogel-Bonner salts and 2% glucose, was used forplates. The relevant genotypes of S. cerevisiae strains usedin this work are given in Table 1. Haploid strain XV185-14c was used in the mutagenicity assay (R.C. von Borstel,Edmonton, Canada). The isogenic strains containing rad1Δ

mutant allele, defective in excision-resynthesis repair [29],rad6Δ mutant allele, blocked in the mutagenic repair pathway[30], and rad1�rad6� double mutant were kindly providedby M. Grey (Frankfurt, Germany). The mutants rad3-e5 andrad52-1, defective in excision-resynthesis repair [29] and inDNA strand-breaks repair [31], respectively, were obtained bysporulation and tetrad analysis from the diploid of the geno-type described in Table 1 [32]. Media, solutions, and bufferswere prepared according to Burke et al. [33]. Complete YPDmedium, containing 0.5% yeast extract, 2% bacto-peptone, and2% glucose was used for routine growth of yeast cells. Forplates, the medium was solidified with 2% bactoagar. Mini-mal medium (MM) contained 0.67% yeast nitrogen base withno amino acids, 2% glucose, and 2% bacto-agar supplementedwith the appropriate amino acids. Synthetic complete medium(SC) was MM supplemented with 2 mg adenine, 2 mg arginine,5 mg lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine,2 mg uracil, 2 mg tryptophan, and 24 mg threonine per 100 mLof MM. For XV-185-14c strain mutagenesis, the omissionmedia lacking lysine (SC-lys), histidine (SC-his), or homoser-ine (SC-hom) were used. Synthetic medium without arginine,supplemented with 60 �g/mL canavanine, was used for N123strain assays.

2.4. Salmonella/microsome mutagenicity assay

Mutagenicity was assayed by the pre-incubation procedure

proposed Maron and Ames [34], and revised by Mortelmansand Zeiger [28]. The S9 metabolic activation mixture (S9mix) was prepared according to Maron and Ames [34]. 15-Deoxygoyazensolide was dissolved in DMSO immediatelybefore use. One hundred microliters of test bacterial cultures

Source

trp5-48 hom3-10 R.C. von Borstelleu2-3,112 trp1-1 can1-100 M. Greytrp1-1 can1-100 rad1::LEU2 M. Greytrp1-1 can1-100 rad6::URA3 M. Grey1-100 rad1::LEU2 rad6::URA3 M. Grey

J.A.P. Henriques/rad52-1 ADE2/ade2-15-2 LEU1/leu1-12 LYS1/lys1-1

R.C. von Borstel

utation

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M.C. Vasconcellos et al. / M

(1–2) × 109 cells/mL) were incubated in the dark at 37 ◦C withifferent concentrations of 15-deoxygoyazensolide (8, 16, 24,2, and 40 �g/plate) in the presence or absence of the S9 mix for0 min, without shaking. Subsequently, 2 mL soft agar (0.6%gar, 0.5% NaCl, 50 �M histidine, 50 �M biotin, pH 7.4, 45 ◦C)as added to the test tube, and immediately poured onto a mini-al agar plate (1.5% agar, Vogel-Bonner E medium containing

% glucose). Aflatoxin B1 (0.5 �g per plate) was used as pos-tive control for all strains in the metabolic assay with the S9

ix. In the absence of the S9 mix, positive controls were 4-QO (0.5 �g/plate) for TA98, TA97a, and TA102, whereas

odium azide (5 �g/plate) was used for TA100. Plates werencubated in the dark at 37 ◦C for 48 h before revertant coloniesere counted.

.5. Yeast growth

Stationary-phase cultures were obtained by inoculation ofn isolated colony into liquid YPD and, after 48 h incuba-ion at 30 ◦C with aeration by shaking, cultures were growno (1–2) × 108 cells/mL. Cells were harvested, and washedwice with saline solution. Cell concentration and percentagef budding cells in each culture were determined in a Neubauerhamber by microscope counts.

.6. Survival assays in S. cerevisiae strains

The sensitivity to 15-deoxygoyazensolide was assayedy incubation of 2 × 108 cells/mL of stationary cultures inhosphate-buffered saline solution (PBS 0.067 mol/L, pH 7.0)ith different concentrations (0.5, 1.0 and 2.5 mg/mL) of the

ompound in rotary shaker at 30 ◦C for 3 h. After treatment,ells were harvested by centrifugation at 12,000 × g for 2 min,ashed twice in PBS, counted, diluted, and plated on solidPD. Plates were incubated at 30 ◦C for 3–5 days before count-

ng.

.7. Detection of 15-deoxygoyazensolide-inducedorward mutation in S. cerevisiae

N123 strain was used for this analysis [35]. Yeast cells wereultured overnight in YPD medium at 30 ◦C in an orbital shakerntil cell suspension reached a density of (1–2) × 108 cells/mL.ells were harvested, washed twice by centrifugation withBS, and submitted to 15-deoxygoyazensolide (0.025, 0.05,.1, 0.25 and 0.5 mg/mL) treatment at 30 ◦C for 3 h in theark with shaking. Cell concentration and percentage ofudding cells in each culture were determined by micro-cope counts using a Neubauer chamber. After treatment,ppropriate dilutions of cells were plated onto SC plates to

etermine cell survival, and 100 �L aliquots of cell suspension2 × 108 cells/mL) were plated onto SC media supplementedith 60 �g/mL canavanine in order to determine forward muta-

ion in CAN1 locus. Mutants were counted after 4–5 daysncubation at 30 ◦C.

Research 631 (2007) 16–25 19

2.8. Detection of 15-deoxygoyazensolide-induced reverseand frameshift mutation in S. cerevisiae

Mutagenesis was measured in S. cerevisiae XV185-14cstrain. A suspension of 2 × 108 stationary-phase cells/mL wasincubated for 3 h at 30 ◦C with different concentrations of 15-deoxygoyazensolide in PBS. Cell survival was determined inSC (3–5 days, 30 ◦C), and mutation induction (LYS, HIS, orHOM revertants) in the appropriate omission media (7–10days, 30 ◦C). Whereas his1-7 is a non-suppressible missenseallele, and reversions result from mutation at the locus itself[36], lys1-1 is a suppressible ochre nonsense mutant allele [37],which can be reverted either by locus-specific or forward muta-tion in a suppressor gene [38,39]. True reversions and forward(suppressor) mutations at the lys1-1 locus were differentiatedaccording to Schuller and von Borstel [40], where the reducedadenine content of the SC-lys medium shows locus reversionsas red and suppressor mutations as white colonies. It is believedthat hom3-10 contains a frameshift mutation due to its responseto a range of diagnostic mutagens [39]. Assays were repeatedat least four times, and plating was performed in triplicate foreach dose.

2.9. Detection of cytotoxic effects in mutants defective inDNA repair and topoisomerases

The sensitivity of mutants defective in DNA repair path-ways or in topoisomerases was determined in exponential cells,as described in the survival assay.

2.10. Determination of total glutathione content

Cells were grown as described in the growth inhibitionassay, and total glutathione content was determined accord-ing to Akerboom and Sies [41]. Protein was measured bythe Bradford method [42] using bovine serum albumin asstandard.

2.11. Data analysis

Mutagenicity data were analyzed using Salmonel software[43]. A compound was considered positive for mutagenicityonly when: (a) the number of revertants was at least twicethe spontaneous yield (MI ≥ 2; MI = mutagenic index: num-ber of induced colonies in the sample/number of spontaneouscolonies in the negative control samples); (b) a significantresponse was obtained in the analysis of variance (p ≤ 0.05); (c)a reproducible positive dose-response (p ≤ 0.01) was present,as evaluated by the Salmonel software [44,45]. Effect wasconsidered as cytotoxic when MI ≤ 0.6. Data from mutage-

nesis and survival assays in S. cerevisiae were expressedas means and standard deviation from three independentexperiments, and statistically analyzed using the Student’s t-test. Differences were considered significant when p < 0.05[46].

20 M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25

Table 2Induction of his+ revertants in S. typhimurium frameshift strains by 15-deoxygoyazensolide with and without metabolic activation (S9 mix)

Substance: dose (�g/plate) TA98 TA97a

−S9 +S9 −S9 +S9

rev/platea MIb rev/plate MI rev/plate MI rev/plate MI

PCc 284.33 ± 38.04 469.33 ± 1.74 658.00 ± 85.15 734.66 ± 107.5NCd 15.67 ± 8.96 22.33 ± 6.03 119.33 ± 11.55 163.33 ± 65.19

15-Deoxygoyazensolide8 15.67 ± 1.53 1.00 18.00 ± 5.57 0.80 117.67 ± 10.97 0.98 176.00 ± 39.40 1.0716 25.33 ± 9.29 1.61 23.67 ± 6.03 1.06 117.67 ± 5.77 0.98 156.67 ± 28.31 0.9624 22.33 ± 4.04 1.42 22.33 ± 4.93 1.00 146.00 ± 14.93 1.22 186.67 ± 6.11 1.1432 23.50 ± 6.36 1.50 17.00 ± 6.08 0.76 128.67 ± 30.86 1.07 155.67 ± 35.44 0.9540 21.67 ± 3.21 1.38 20.33 ± 2.52 0.91 125.33 ± 22.23 1.05 212.67 ± 46.32 1.30

a Number of revertant per plate: mean of three independent experiments ±S.D.ntaneouB1 (0.5for 15-

b MI: mutagenic index (no. of his+ induced in the sample/no. of spoc PC: positive control (−S9) 4-NQO (0.5 �g/plate); (+S9) aflatoxind NC: negative control dimethyl sulfoxide (10 �L) used as a solvent

3. Results

3.1. Salmonella/microsome assay

When dose range of 15-deoxygoyazensolide wasevaluated with the TA100 strain, cytotoxicity wasobserved in concentrations higher than 40 �g/mL (datanot shown). No mutagenicity of 15-deoxygoyazensolidewas seen at concentrations of 8–40 �g/mL in strainsTA98, TA97a, TA100, and TA102 in the absence orpresence of metabolic activation.

3.2. Cytotoxic and mutagenic effects in S. cerevisiae

Results of the mutagenicity tests are shown inTables 4 and 5. 15-Deoxygoyazensolide induced mod-

Table 3Induction of his+ revertants in S. typhimurium base pair substitution strains bmix)

Substance: dose (�g/plate) TA100

−S9 +S9

rev/platea MIb rev/plate

PCc 1247.33 ± 196.26 537.33 ± 105.7NCd 129.33 ± 7.09 117.33 ± 22.03

15-Deoxygoyazensolide8 126.67 ± 30.73 0.98 156.00 ± 35.5516 149.00 ± 2.65 1.15 136.00 ± 6.9324 147.00 ± 14.73 1.13 122.67 ± 16.1732 128.67 ± 12.01 0.98 171.33 ± 6.4340 118.33 ± 25.70 0.91 153.33 ± 26.63

a Number of revertant per plate: mean of three independent experiments ±Sb MI: mutagenic index (no. of his+ induced in the sample/no. of spontaneouc PC: positive control (−S9) 4-NQO (0.5 �g/plate) for TA102 e sodium azid NC: negative control dimethyl sulfoxide (10 �L) used as a solvent for 15-

s his+ in the negative control).�g/plate).deoxygoyazensolide.

erate dose-dependent cytotoxicity in haploid wild-typeyeast cultures. The frequency of point (HIS1+, LYS1+),frameshift (HOM3+) (Table 4), and forward (Table 5)mutations during treatment of stationary-phase cellsindicates a clear mutagenic effect at doses higher than0.25 mg/mL (Tables 2 and 3).

3.3. Cytotoxic effects in S. cerevisiae DNArepair-defective strains and topoisomerase-defectivestrains

The cytotoxic effects of 15-deoxygoyazensolide areshown in Fig. 2. The sensitivity to this SL depends on therepairing capacity of the yeast cell. The single mutantsrad3-e5, rad1�, and mutant rad6� (Fig. 2) exhibited the

y 15-deoxygoyazensolide with and without metabolic activation (S9

TA102

−S9 +S9

MI rev/plate MI rev/plate MI

8 3251.00 ± 259.75 781.00 ± 180.28548.00 ± 11.31 241.33 ± 16.17

1.33 581.33 ± 49.69 1.06 230.67 ± 24.44 0.951.16 574.67 ± 62.27 1.04 312.67 ± 21.39 1.291.04 402.00 ± 23.58 0.73 293.33 ± 38.02 1.211.46 414.67 ± 57.18 0.75 252.00 ± 31.75 1.041.30 456.67 ± 48.01 0.83 260.00 ± 24.98 1.07

.D.s his+ in the negative control).

de (5 �g/plate) for TA100; (+S9) aflatoxin B1 (0.5 �g/plate).deoxygoyazensolide.

M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25 21

Table 4Reversion of point mutation for (his1-7), ochre allele (lys1-1) and frameshift mutations (hom3-10) in haploid XV185-14c strain of S. cerevisiaeafter 15-deoxygoyazensolide treatment during 3 h in PBS

Survival (%) HIS1 (107 survivors)a LYS1 (107 survivors)b HOM3 (107 survivors)a

NCc 100.0 11.1 ± 5.0 1..3 ± 0.6 2.2 ± 1.3PCd 55.0 83.0 ± 1.6* 18.9 ± 7.0* 43.0 ± 8.6*

15-Deoxygoyazensolide0.025 mg/mL 90.4 14.4 ± 0.7 2.4 ± 0.35 6.4 ± 2.20.05 mg/mL 73.2 23.7 ± 1.0* 5.1 ± 2.5 8.9 ± 0.7*

0.1 mg/mL 60.1 30.6 ± 2.2* 15.4 ± 2.7* 12.8 ± 4.0*

0.25 mg/mL 35.1 49.0 ± 3.7* 24.9 ± 3.4* 31.6 ± 0.1*

0.5 mg/mL 20.2 32.0 ± 6.5* 30.8 ± 1.2* 52.9 ± 0.8*

a Locus-specific revertants.b Locus non-specific revertants (forward mutation).c NC: negative control, dimethyl sulfoxide used as a solvent for 15-deoxygoyazensolide.

Data significant in relation to negative control group (solvent) at p < 0.05;A

sm1(mcswtil

3

i

TIa

S

NP

1

D*

d

d PC: positive control, 4-NQO (0.5 �g/plate).* Mean and standard deviation per three independent experiments.NOVA.

ame sensitivity to the wild type strain. Both the singleutant rad52-1 and the double mutant rad3-e5 rad52-showed extreme sensitivity to 15-deoxygoyazensolide

Fig. 2). Fig. 3 shows that topoisomerase I (Top1) enzy-atic action has a crucial role in the resistance to the

ytotoxicity of this SL. As the top1� strain was veryensitive to the SL treatment—at 0.1 mg/mL, survivalas minimal. The mutant top3� showed similar sensi-

ivity to wild type strain in virtually all evaluated doses,ndicating that Top3 was not involved in the repair of theesions induced by 15-deoxygoyazensolide.

.4. Determination of total glutathione content

As shown in Fig. 4, when wild-type cells werencubated with 15-deoxygoyazensolide, GSH content

able 5nduction of forward mutation (CAN1) in N123 strain of S. cerevisiaefter 15-deoxygoyazensolide treatment during 3 h in PBS

ubstance Survival (%) Mutants (107 survivors)

Ca 100.00 4.75 ± 1.30Cb 61.73 ± 4.57 132.25 ± 3.17**

5-Deoxygoyazensolide0.025 mg/mL 96.95 ± 10.56 5.06 ± 0.100.05 mg/mL 93.55 ± 14.01 8.86 ± 3.550.1 mg/mL 86.01 ± 8.59 8.72 ± 3.160.25 mg/mL 51.33 ± 5.22 14.72 ± 3.80*0.5 mg/mL 36.01 ± 8.59 68.53 ± 16.68*

ata significant in relation to negative control group (solvent):p < 0.05; **p < 0.01 (ANOVA).a NC: negative control, dimethyl sulfoxide used as a solvent for 15-eoxygoyazensolide.b PC: positive control, 4-NQO (0.5 �g/plate).

Fig. 2. Sensitivity to 15-deoxygoyazensolide of different haploid radmutant strains. WT RAD+ (solid square) and its isogenics mutants:rad3-e5 (solid triangle), rad52-1 (solid inverted triangle), rad3-e5rad52-1 (solid diamond); WT Y202 (open circle) and its isogenicsmutants: rad1� (open square), rad6� (open triangle) and rad1�

rad6� (open inverted triangle).

Fig. 3. Sensitivity to 15-deoxygoyazensolide of different topoiso-merase mutants. Wild type strain (solid square), top1� (solid triangle),top3� (solid inverted triangle).

22 M.C. Vasconcellos et al. / Mutation

Fig. 4. Effect of 15-deoxygoyazensolide on the total GSH content inwild type yeast during incubation in synthetic complete media dur-ing 18 h at 30 ◦C. Values are means ± S.D. (n = 6). SL: treatment

with 15-deoxygoyazensolide; formaldehyde 0.1%; CDNB: 1-chloro-2,4-dinitrobenzene (1 mM); negative control: DMSO 10%. * Datasignificant in relation to negative control at p < 0.05 (ANOVA).

decreased in a dose-dependent manner. Positive controltreatments for GSH depletion, using formaldehyde andCDNB, were used to validate the results.

4. Discussion

SLs, as well as flavonoids and lignans, are consid-ered as important classes of natural products with awide spectrum of biological effects, including antitumor,anti-ulcer, anti-inflammatory, neurotoxic, cytotoxic, andcardiotonic activities [3–15,47]. As previously men-tioned, SLs can covalently bind to molecules containinga sulphydryl group like GSH, leading to thiol depletion,and affecting protein structure and functions [13,14].This is consistent with our finding that the total cyto-plasmatic GSH content in yeast cells treated with15-deoxygoyazensolide decreased in a dose-responsemanner. Polygoidal, pungent sesquiterpenoid unsatu-rated dialdehyde also depletes intracellular GSH in S.cerevisiae, thus promoting a pro-oxidant status in cells[48]. Furthermore, it was recently demonstrated thatendogenous GSH level in leukocytes treated with fura-noheliangolide lychnopholide was drastically reduced,without corresponding increases in the oxidized form.Analysis of electrospray mass spectrometry led to theidentification of glutathionyl-lychnopholide adduct incellular extracts [49]. Data obtained in the present study

show that 15-deoxygoyazensolide reduces intracellularGSH content, promoting DNA oxidative damage prob-ably through an adduct similar to that described forglutathionyl-lychnopholide.

Research 631 (2007) 16–25

As consequence of this abnormal reduction of glu-tathione, free radicals generated during normal aerobicmetabolism, such as the hydroxyl radical, can attackDNA, generating strand breaks and base oxidation[50–52]. Considering these properties, a genotoxic activ-ity could be expected for this class of molecules. Indeed,our results show that 15-deoxygoyazensolide was muta-genic in yeast, inducing point, frameshift, and forwardmutations. In contrast, no mutagenic response wasobserved in bacteria in concentrations up to 40 �g/mL.However, at higher concentrations, this SL is toxic tobacteria. The explanation for these findings can be duethe differences in metabolism in bacteria and yeast, inmembrane permeability and in detoxification cellularsystems, which explains the different results obtained inthese biological models for several genotoxins [53–55].

Studies with other SLs showed that some furanohe-liangolides have a direct impact on DNA, causing lethalcell damage in bacteria [56], as well as chromosomalaberrations [57,58] and DNA strand breaks in mam-malian cells [59]. However, previous genotoxic researchwith 15-deoxygoyazensolide did not observe significantincrease in the frequency of sister chromatid exchangesin human lymphocytes in vitro [60]. In the presentstudy we suggested that the mutagenic activity of 15-desoxygoyasenzolide in S. cerevisiae could be at leastpartially due to an indirect free radical-DNA damagegenerated by GSH depletion. SLs, such as costuno-lide [19], helenalin [17,18] and parthenolide [20], areknown to be potent inducers of cytotoxicity throughROS-mediated oxidative stress. Interestingly, mutagen-esis was not observed in 15-deoxygoyazensolide-treatedSalmonella TA102 strain, which has a proven abilityto detect mutagens that generate ROS and oxidativeDNA damage although this SL has showed inducesGSH depletion and mutagenesis in yeast cells. Similarly,parthenin did not show mutagenesis in Salmonella but itcaused oxidative stress in a hepatoma cell line in culture,showing that differents biological effects are detected inrelation to genotoxicity of SL in different prokaryoticand eukaryotic models as above mentioned [20].

In order to understand the modes of genotoxic actionof 15-deoxygoyazensolide on yeast, we studied theresponse of S. cerevisiae mutants defective in DNArepair to the treatment with this compound. The hyper-sensitivity of the rad52-1 mutant strain suggests that therecombinational repair is critical for processing poten-tially lethal genetic lesions caused by oxidative damage,

such as DNA single-strand breaks and closely locatedsingle strand breaks. However, when the cell attempts torepair these single strand breaks, it may, due to exhaus-tive repairing, also causes double breaks. Indeed, in S.

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M.C. Vasconcellos et al. / M

erevisiae, members of the RAD52 epistasis group aressential for successful meiotic and mitotic recombina-ion and survival after treatment with ionizing radiation,lkylating agents, some oxidative mutagens, and cross-inking agents [34,36,61]. The cytotoxic responses ofingle mutant rad52-1 and double mutant rad3-e5 rad52-

to 15-deoxygoyazensolide are identical, which is aonsequence of the absence of the RAD52 pathway.

The data obtained here suggest that 15-esoxygoyazensolide-induced DNA damage could beconsequence of its pro-oxidant properties. Although

xidative damage can be the primary mechanism toxplain the genotoxicity of this molecule in yeast, theesults of the top1� mutant indicate a possible effectf intercalation, which suggests a putative complexormation between DNA and this SL that may also benvolved in its genotoxic effect. Indeed, the absence ofop1 increased the sensitivity to the drug, as shown inig. 3.

Topoisomerase I (Top1) catalyzes two transester-fication reactions: single-strand cleavage and DNAeligation, which are normally coupled for the relaxationf DNA supercoiling during chromatin transcription andeplication. Therefore, it is involved in multiple DNAransactions, including DNA replication, transcription,hromosome condensation and decondensation, and,robably, DNA recombination [62]. Besides promotingNA relaxation, necessary to eliminate torsional stresses

ssociated to these processes, Top1 may have otherunctions related to its interaction with other cellularroteins. Top1 cleavage complexes are also produced byndogenous and exogenous DNA lesions, including UV-nduced base modifications, guanine methylation andxidation, polycyclic aromatic carcinogenic adducts,ase mismatches, abasic sites, cytosine arabinoside oremcitabine incorporation, and DNA nicks. Cleavageomplexes can produce DNA damage after collisionsf replication forks and transcription complexes [63].hese lesions, particularly replication fork collisions,eed to be repaired for the cell to survive. We supposehat this SL is capable of intercalating into DNA, andhe DNA-SL complex requires Top1 action for DNAesolution and repair.

DNA topoisomerases are the targets of antimicrobialnd anticancer drugs [62,63]. The precise role of Top1n 15-deoxygoyazensolide cytotoxicity is still unclear64,65]. As topoisomerase II inhibition induces DNAreaks, which are repaired almost exclusively by Rad52-

ependent homologous recombination in yeast, the highensitivity of the rad52 mutant may involve, at leastn part, the interference of this SL in Top2 activity66]. Therefore, further studies are needed to clarify the

[

Research 631 (2007) 16–25 23

exact mechanism of the interaction among this naturalmolecule, DNA, and topoisomerases.

Moreover, the intracellular glutathione depletioninduced by 15-deoxygoyazensolide treatment appearshave a central role in its mutagenic effects in yeast Inaddition, the action of Top1 also is essential to repairthe lesions induced by this natural molecule, indicatingthat possibly the topology of nucleic acids are disturbedby the lesiosn generated by this SL, probably a putativeintercalation effect.

Acknowledgments

The authors are grateful to Dr. Diego Bonatto for criti-cal reading of the manuscript and the Brazilian AgenciesFINEP, CNPq, BNB/FUNDECI, PRONEX, FAPESP,GENOTOX, Genotoxicity Laboratory, Royal Instituteand CAPES for fellowships and financial support.

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