M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25 171. Introduction Sesquiterpene lactones (SLs) are substances pre-senting different pharmacological activities, withanti-microbial, anti-viral, anti-inﬂammatory, and anti-tumor effects [1,2]. For instance, costunolide anddehydrocostus lactone inhibit the killing activity ofcytotoxic T lymphocytes, nitric oxide (NO) produc-tion, and the expression of hepatitis B virus surfaceantigen [3–5]. Helenalin alleviated carrageen-inducededema of rat hindfeet, and suppressed cancer cell growth[6,7]. Parthenolide and encelin showed potent inhibitoryeffects on the expression of cyclooxygenase and tumornecrosis factor (TNF)-␣ . This wide range of biolog- Fig. 1. Chemical structure of 15-deoxygoyazensolide.ical activities is mainly caused by the inactivation ofthe NF-B nuclear factor [8–10]. Cynaropicrin derived a positive increase in the micronucleated reticulocyte fre-from Saussurea lappa suppresses the production of quency in high doses . Hymenoxon, helenalin, andcytokines, such as TNF-␣ and cytokine-induced neu- tenulin were also tested for genotoxicity using six strainstrophil chemoattractant-1 (interleukin-8), as well as NO of Bacillus subtilis and hymenoxon and helenalin wererelease. It also strongly attenuates mitogenic stimulation found to produce lethal DNA damage in this model whileof CD4+ and CD8+ lymphocyte proliferation [11,12]. tenulin did not produce lethal DNA damage in any of the Most of the observed biological effects of these com- strains tested . Since that DNA damage has a centralpounds are attributed to their ability to inhibit enzymes role in cytotoxic properties of several natural and syn-and other essential proteins by covalently binding to thetic molecules, the evaluation of genotoxicity of thesefree cysteine sulfhydryl groups of polypeptides  molecules is very important .or by conjugation with glutathione (GSH), leading The furanoheliangolide 15-deoxygoyazensolideto thiol depletion and an increase in the susceptibil- (Fig. 1) is a SL isolated from several species of theity to oxidative damage . Severe oxidative stress sub-tribe Lychnophorinae (tribe Vernonieae, familyconferred by SLs-induced thiol depletion results in a Asteraceae), and possesses two functionalities in thedisruption of the integrity of mitochondria, triggering form of an ␣-methylene-␥-lactone and an ␣,␤,␥,␦-mitochondrial permeability transition and release of unsaturated carbonyl group [24,25], which could bemitochondrial pro-apoptotic proteins. SLs contain an ␣- considered as essential features for NF-B inhibition.methylene-␥-lactone moiety, which is highly reactive Santos et al.  demonstrated that goyazensolide,with cellular thiols, leading to alkylation of sulphydryl which structure is similar to 15-deoxygoyazensolide, butresidues through Michael-type addition [14–16]. NF- for the oxidation at carbon 15, was strongly cytotoxicB, an ubiquitous transcription factor that regulates to tumor cell lines. In order to further understand theinﬂammatory responses, cell growth/differentiation and biological properties of SLs, this study aimed at inves-apoptosis, seems to be the main target for many SLs tigating the genotoxicity of 15-deoxygoyazensolide[15,16]. According to R¨ ngeler et al. , NF-B inhi- u in bacteria using the Salmonella/microsome assay,bition by SLs is caused by alkylation at the mentioned and at evaluating its genotoxic potential to the simplecysteine moieties in the subunit p65. eukaryote S. cerevisiae, which mutagenesis and DNA In addition, some SL has also remarkable cytotoxic repair mechanisms were assessed using haploid strains.effects. Indeed, costunolide, helenalin and parthenolide This information is very important to evaluate the safetyare known to be strong inducers of cytotoxicity by of possible future pharmacological applications of thistriggering apoptosis through reactive oxygen species- compound, and to explore its potential pharmacologicalmediated oxidative stress, However, its genotoxic properties, such as antiproliferative activity.potential has not been thoroughly documented [17–20].The most studied lactone in relation to genotoxicity is 2. Materials and methodsthe parthenin, a sesquiterpene lactone from Parthenium 2.1. Chemicalshysterophorus L., which induces chromosomal aberra-tions, mainly chromatid breaks, in blood lymphocytes; All solvents were bi-distilled, and stored in darkits did not inﬂuence sister chromatid exchange and show ﬂasks. Hydrogen peroxide (H2 O2 ), D-biotin, aﬂatoxin B1,
18 M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–254-nitroquinoleine oxide (4-NQO), sodium azide, reduced , were kindly provided by B. M. Ames (University of Cal-glutathione (GSH), oxidized glutathione (GSSG), 1-chloro- ifornia, Berkeley, CA, USA). Bacterial media were prepared2,4-dinitrobenzene (CDNB), NADPH, glutathione reduc- according to Mortelmans and Zeiger . Complete mediumtase, aminoacids (l-histidine, l-threonine, l-methionine, for growing strains (NB) contained 2.5% oxoid nutrient brothl-tryptophan, l-leucine, l-lysine), l-canavanine, nitrogen #2. Solidiﬁed medium with 1.5% bacto-agar, supplementedbases (adenine and uracil), and dimethylsulfoxide (DMSO) with 1× Vogel-Bonner salts and 2% glucose, was used forwere purchased from Sigma (St. Louis, USA). The S9 frac- plates. The relevant genotypes of S. cerevisiae strains usedtion, prepared with the polychlorinated biphenyl mixture in this work are given in Table 1. Haploid strain XV185-Aroclor 1254, was purchased from Moltox (Annapolis, MD, 14c was used in the mutagenicity assay (R.C. von Borstel,USA), and glucose-6-phosphate and NADP were obtained Edmonton, Canada). The isogenic strains containing rad1Δfrom Sigma (St. Louis, USA). Oxoid nutrient broth No. 2 mutant allele, defective in excision-resynthesis repair ,was obtained from Oxoid USA Inc. (Maryland, USA). Yeast rad6Δ mutant allele, blocked in the mutagenic repair pathwayextract, Bacto-peptone, and Bacto-agar were obtained from , and rad1 rad6 double mutant were kindly providedDifco Laboratories (Detroit, MI, USA). by M. Grey (Frankfurt, Germany). The mutants rad3-e5 and rad52-1, defective in excision-resynthesis repair  and in2.2. 15-Deoxygoyazensolide isolation and identiﬁcation DNA strand-breaks repair , respectively, were obtained by sporulation and tetrad analysis from the diploid of the geno- Our previous phytochemical investigation of Minasia type described in Table 1 . Media, solutions, and buffersalpestris dried leaves, employing the ﬁve main fractions of a were prepared according to Burke et al. . Complete YPDdichloromethane extract, showed that 15-deoxygoyazensolide medium, containing 0.5% yeast extract, 2% bacto-peptone, andis the main SL of this plant species . Minor sub-fractions 2% glucose was used for routine growth of yeast cells. Forwere analyzed using comparative HPLC, as previously plates, the medium was solidiﬁed with 2% bactoagar. Mini-described for seasonal variations of this class of secondary mal medium (MM) contained 0.67% yeast nitrogen base withmetabolites in Lychnophora ericoides . Sub-fractions rich no amino acids, 2% glucose, and 2% bacto-agar supplementedin this lactone were pooled together, and the resulting fraction with the appropriate amino acids. Synthetic complete mediumwas chromatographed in preparative HPLC (ODS-Shimadzu, (SC) was MM supplemented with 2 mg adenine, 2 mg arginine,5.0 mm × 250 mm column, MeOH–H2 O, λ = 260 nm, ﬂown 5 mg lysine, 1 mg histidine, 2 mg leucine, 2 mg methionine,8 mL/min), yielding 30 mg of 15-deoxygoyazensolide. The 2 mg uracil, 2 mg tryptophan, and 24 mg threonine per 100 mLstructure of the isolated molecule was compared to the standard of MM. For XV-185-14c strain mutagenesis, the omission15-deoxygoyazensolide structure obtained by authentic NMR media lacking lysine (SC-lys), histidine (SC-his), or homoser-spectrum and high-resolution mass spectrometry in a previous ine (SC-hom) were used. Synthetic medium without arginine,study . supplemented with 60 g/mL canavanine, was used for N123 strain assays.2.3. Strains and media 2.4. Salmonella/microsome mutagenicity assay Salmonella typhimurium strains TA98 (detects frameshiftmutation in DNA target -C-G-C-G-C-G-C-G), TA97a (detects Mutagenicity was assayed by the pre-incubation procedureframeshift mutation in -C-C-C-C-C-C-; +1 cytosine), TA100 proposed Maron and Ames , and revised by Mortelmans(base pair substitution mutation results from the substitution of and Zeiger . The S9 metabolic activation mixture (S9leucine [GAG] by proline [GGG]), and TA102 (TAA (ochre: mix) was prepared according to Maron and Ames . 15-transversions/transitions) detects oxidative, alkylating muta- Deoxygoyazensolide was dissolved in DMSO immediatelygens and reactive oxygen species [ROS]), described in Ref. before use. One hundred microliters of test bacterial culturesTable 1Saccharomyces cerevisiae strains used in this studyStrains Relevant genotypes SourceHaploid: XV185-14c MATα ade2-2 arg4-17 his1-7 lys1-1 trp5-48 hom3-10 R.C. von BorstelY202 MATa ura3-Δ100 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 M. Greyrad1Δ MATa ura3-Δ100 ade2-1 his3-11,15 trp1-1 can1-100 rad1::LEU2 M. Greyrad6Δ MATa ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 rad6::URA3 M. Greyrad1Δ rad6Δ MATa ade2-1 his 3-11,15 trp1-1 can1-100 rad1::LEU2 rad6::URA3 M. GreyN123 MATa his1-7 gsh1 leak J.A.P. HenriquesDiploid: JH500a MATa/MATα rad3-e5/RAD3 RAD52/rad52-1 ADE2/ade2-1 R.C. von Borstel ARG4/arg4-17 HIS1/his1-7 HIS5/his5-2 LEU1/leu1-12 LYS1/lys1-1 TRP5/trp5-48 HOM3/hom3-10 a Genotypes of zygotes from which haploid segregants were derived.
M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25 19((1–2) × 109 cells/mL) were incubated in the dark at 37 ◦ C with 2.8. Detection of 15-deoxygoyazensolide-induced reversedifferent concentrations of 15-deoxygoyazensolide (8, 16, 24, and frameshift mutation in S. cerevisiae32, and 40 g/plate) in the presence or absence of the S9 mix for20 min, without shaking. Subsequently, 2 mL soft agar (0.6% Mutagenesis was measured in S. cerevisiae XV185-14cagar, 0.5% NaCl, 50 M histidine, 50 M biotin, pH 7.4, 45 ◦ C) strain. A suspension of 2 × 108 stationary-phase cells/mL waswas added to the test tube, and immediately poured onto a mini- incubated for 3 h at 30 ◦ C with different concentrations of 15-mal agar plate (1.5% agar, Vogel-Bonner E medium containing deoxygoyazensolide in PBS. Cell survival was determined in2% glucose). Aﬂatoxin B1 (0.5 g per plate) was used as pos- SC (3–5 days, 30 ◦ C), and mutation induction (LYS, HIS, oritive control for all strains in the metabolic assay with the S9 HOM revertants) in the appropriate omission media (7–10mix. In the absence of the S9 mix, positive controls were 4- days, 30 ◦ C). Whereas his1-7 is a non-suppressible missenseNQO (0.5 g/plate) for TA98, TA97a, and TA102, whereas allele, and reversions result from mutation at the locus itselfsodium azide (5 g/plate) was used for TA100. Plates were , lys1-1 is a suppressible ochre nonsense mutant allele ,incubated in the dark at 37 ◦ C for 48 h before revertant colonies which can be reverted either by locus-speciﬁc or forward muta-were counted. tion in a suppressor gene [38,39]. True reversions and forward (suppressor) mutations at the lys1-1 locus were differentiated according to Schuller and von Borstel , where the reduced2.5. Yeast growth adenine content of the SC-lys medium shows locus reversions as red and suppressor mutations as white colonies. It is believed Stationary-phase cultures were obtained by inoculation of that hom3-10 contains a frameshift mutation due to its responsean isolated colony into liquid YPD and, after 48 h incuba- to a range of diagnostic mutagens . Assays were repeatedtion at 30 ◦ C with aeration by shaking, cultures were grown at least four times, and plating was performed in triplicate forto (1–2) × 108 cells/mL. Cells were harvested, and washed each dose.twice with saline solution. Cell concentration and percentageof budding cells in each culture were determined in a Neubauerchamber by microscope counts. 2.9. Detection of cytotoxic effects in mutants defective in DNA repair and topoisomerases2.6. Survival assays in S. cerevisiae strains The sensitivity of mutants defective in DNA repair path- ways or in topoisomerases was determined in exponential cells, The sensitivity to 15-deoxygoyazensolide was assayed as described in the survival assay.by incubation of 2 × 108 cells/mL of stationary cultures inphosphate-buffered saline solution (PBS 0.067 mol/L, pH 7.0)with different concentrations (0.5, 1.0 and 2.5 mg/mL) of the 2.10. Determination of total glutathione contentcompound in rotary shaker at 30 ◦ C for 3 h. After treatment,cells were harvested by centrifugation at 12,000 × g for 2 min, Cells were grown as described in the growth inhibitionwashed twice in PBS, counted, diluted, and plated on solid assay, and total glutathione content was determined accord-YPD. Plates were incubated at 30 ◦ C for 3–5 days before count- ing to Akerboom and Sies . Protein was measured bying. the Bradford method  using bovine serum albumin as standard.2.7. Detection of 15-deoxygoyazensolide-inducedforward mutation in S. cerevisiae 2.11. Data analysis N123 strain was used for this analysis . Yeast cells were Mutagenicity data were analyzed using Salmonel softwarecultured overnight in YPD medium at 30 ◦ C in an orbital shaker . A compound was considered positive for mutagenicityuntil cell suspension reached a density of (1–2) × 108 cells/mL. only when: (a) the number of revertants was at least twiceCells were harvested, washed twice by centrifugation with the spontaneous yield (MI ≥ 2; MI = mutagenic index: num-PBS, and submitted to 15-deoxygoyazensolide (0.025, 0.05, ber of induced colonies in the sample/number of spontaneous0.1, 0.25 and 0.5 mg/mL) treatment at 30 ◦ C for 3 h in the colonies in the negative control samples); (b) a signiﬁcantdark with shaking. Cell concentration and percentage of response was obtained in the analysis of variance (p ≤ 0.05); (c)budding cells in each culture were determined by micro- a reproducible positive dose-response (p ≤ 0.01) was present,scope counts using a Neubauer chamber. After treatment, as evaluated by the Salmonel software [44,45]. Effect wasappropriate dilutions of cells were plated onto SC plates to considered as cytotoxic when MI ≤ 0.6. Data from mutage-determine cell survival, and 100 L aliquots of cell suspension nesis and survival assays in S. cerevisiae were expressed(2 × 108 cells/mL) were plated onto SC media supplemented as means and standard deviation from three independentwith 60 g/mL canavanine in order to determine forward muta- experiments, and statistically analyzed using the Student’s t-tion in CAN1 locus. Mutants were counted after 4–5 days test. Differences were considered signiﬁcant when p < 0.05incubation at 30 ◦ C. .
20 M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25Table 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 MIPCc 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 ± 65.1915-Deoxygoyazensolide 8 15.67 ± 1.53 1.00 18.00 ± 5.57 0.80 117.67 ± 10.97 0.98 176.00 ± 39.40 1.07 16 25.33 ± 9.29 1.61 23.67 ± 6.03 1.06 117.67 ± 5.77 0.98 156.67 ± 28.31 0.96 24 22.33 ± 4.04 1.42 22.33 ± 4.93 1.00 146.00 ± 14.93 1.22 186.67 ± 6.11 1.14 32 23.50 ± 6.36 1.50 17.00 ± 6.08 0.76 128.67 ± 30.86 1.07 155.67 ± 35.44 0.95 40 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. b MI: mutagenic index (no. of his+ induced in the sample/no. of spontaneous his+ in the negative control). c PC: positive control (−S9) 4-NQO (0.5 g/plate); (+S9) aﬂatoxin B1 (0.5 g/plate). d NC: negative control dimethyl sulfoxide (10 L) used as a solvent for 15-deoxygoyazensolide.3. Results erate dose-dependent cytotoxicity in haploid wild-type yeast cultures. The frequency of point (HIS1+, LYS1+),3.1. Salmonella/microsome assay frameshift (HOM3+) (Table 4), and forward (Table 5) mutations during treatment of stationary-phase cells When dose range of 15-deoxygoyazensolide was indicates a clear mutagenic effect at doses higher thanevaluated with the TA100 strain, cytotoxicity was 0.25 mg/mL (Tables 2 and 3).observed in concentrations higher than 40 g/mL (datanot shown). No mutagenicity of 15-deoxygoyazensolidewas seen at concentrations of 8–40 g/mL in strains 3.3. Cytotoxic effects in S. cerevisiae DNATA98, TA97a, TA100, and TA102 in the absence or repair-defective strains and topoisomerase-defectivepresence of metabolic activation. strains3.2. Cytotoxic and mutagenic effects in S. cerevisiae The cytotoxic effects of 15-deoxygoyazensolide are shown in Fig. 2. The sensitivity to this SL depends on the Results of the mutagenicity tests are shown in repairing capacity of the yeast cell. The single mutantsTables 4 and 5. 15-Deoxygoyazensolide induced mod- rad3-e5, rad1 , and mutant rad6 (Fig. 2) exhibited theTable 3Induction of his+ revertants in S. typhimurium base pair substitution strains by 15-deoxygoyazensolide with and without metabolic activation (S9mix)Substance: dose (g/plate) TA100 TA102 −S9 +S9 −S9 +S9 rev/platea MIb rev/plate MI rev/plate MI rev/plate MIPCc 1247.33 ± 196.26 537.33 ± 105.78 3251.00 ± 259.75 781.00 ± 180.28NCd 129.33 ± 7.09 117.33 ± 22.03 548.00 ± 11.31 241.33 ± 16.1715-Deoxygoyazensolide 8 126.67 ± 30.73 0.98 156.00 ± 35.55 1.33 581.33 ± 49.69 1.06 230.67 ± 24.44 0.95 16 149.00 ± 2.65 1.15 136.00 ± 6.93 1.16 574.67 ± 62.27 1.04 312.67 ± 21.39 1.29 24 147.00 ± 14.73 1.13 122.67 ± 16.17 1.04 402.00 ± 23.58 0.73 293.33 ± 38.02 1.21 32 128.67 ± 12.01 0.98 171.33 ± 6.43 1.46 414.67 ± 57.18 0.75 252.00 ± 31.75 1.04 40 118.33 ± 25.70 0.91 153.33 ± 26.63 1.30 456.67 ± 48.01 0.83 260.00 ± 24.98 1.07 a Number of revertant per plate: mean of three independent experiments ±S.D. b MI: mutagenic index (no. of his+ induced in the sample/no. of spontaneous his+ in the negative control). c PC: positive control (−S9) 4-NQO (0.5 g/plate) for TA102 e sodium azide (5 g/plate) for TA100; (+S9) aﬂatoxin B1 (0.5 g/plate). d NC: negative control dimethyl sulfoxide (10 L) used as a solvent for 15-deoxygoyazensolide.
M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25 21Table 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)aNCc 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-Deoxygoyazensolide 0.025 mg/mL 90.4 14.4 ± 0.7 2.4 ± 0.35 6.4 ± 2.2 0.05 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. d PC: positive control, 4-NQO (0.5 g/plate). * Mean and standard deviation per three independent experiments. Data signiﬁcant in relation to negative control group (solvent) at p < 0.05;ANOVA.same sensitivity to the wild type strain. Both the singlemutant rad52-1 and the double mutant rad3-e5 rad52-1 showed extreme sensitivity to 15-deoxygoyazensolide(Fig. 2). Fig. 3 shows that topoisomerase I (Top1) enzy-matic action has a crucial role in the resistance to thecytotoxicity of this SL. As the top1 strain was verysensitive to the SL treatment—at 0.1 mg/mL, survivalwas minimal. The mutant top3 showed similar sensi-tivity to wild type strain in virtually all evaluated doses,indicating that Top3 was not involved in the repair of thelesions induced by 15-deoxygoyazensolide.3.4. Determination of total glutathione content Fig. 2. Sensitivity to 15-deoxygoyazensolide of different haploid rad mutant strains. WT RAD+ (solid square) and its isogenics mutants: As shown in Fig. 4, when wild-type cells were rad3-e5 (solid triangle), rad52-1 (solid inverted triangle), rad3-e5incubated with 15-deoxygoyazensolide, GSH content rad52-1 (solid diamond); WT Y202 (open circle) and its isogenics mutants: rad1 (open square), rad6 (open triangle) and rad1 rad6 (open inverted triangle).Table 5Induction of forward mutation (CAN1) in N123 strain of S. cerevisiaeafter 15-deoxygoyazensolide treatment during 3 h in PBSSubstance Survival (%) Mutants (107 survivors)NCa 100.00 4.75 ± 1.30PCb 61.73 ± 4.57 132.25 ± 3.17**15-Deoxygoyazensolide 0.025 mg/mL 96.95 ± 10.56 5.06 ± 0.10 0.05 mg/mL 93.55 ± 14.01 8.86 ± 3.55 0.1 mg/mL 86.01 ± 8.59 8.72 ± 3.16 0.25 mg/mL 51.33 ± 5.22 14.72 ± 3.80* 0.5 mg/mL 36.01 ± 8.59 68.53 ± 16.68*Data 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- Fig. 3. Sensitivity to 15-deoxygoyazensolide of different topoiso-deoxygoyazensolide. b PC: positive control, 4-NQO (0.5 g/plate). merase mutants. Wild type strain (solid square), top1 (solid triangle), top3 (solid inverted triangle).
22 M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25 As consequence of this abnormal reduction of glu- tathione, free radicals generated during normal aerobic metabolism, such as the hydroxyl radical, can attack DNA, 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 forward mutations. In contrast, no mutagenic response was observed in bacteria in concentrations up to 40 g/mL. However, at higher concentrations, this SL is toxic to bacteria. The explanation for these ﬁndings can be due the differences in metabolism in bacteria and yeast, in membrane permeability and in detoxiﬁcation cellularFig. 4. Effect of 15-deoxygoyazensolide on the total GSH content in systems, which explains the different results obtained inwild type yeast during incubation in synthetic complete media dur- these biological models for several genotoxins [53–55].ing 18 h at 30 ◦ C. Values are means ± S.D. (n = 6). SL: treatmentwith 15-deoxygoyazensolide; formaldehyde 0.1%; CDNB: 1-chloro- Studies with other SLs showed that some furanohe-2,4-dinitrobenzene (1 mM); negative control: DMSO 10%. * Data liangolides have a direct impact on DNA, causing lethalsigniﬁcant in relation to negative control at p < 0.05 (ANOVA). cell damage in bacteria , as well as chromosomal aberrations [57,58] and DNA strand breaks in mam- malian cells . However, previous genotoxic researchdecreased in a dose-dependent manner. Positive control with 15-deoxygoyazensolide did not observe signiﬁcanttreatments for GSH depletion, using formaldehyde and increase in the frequency of sister chromatid exchangesCDNB, were used to validate the results. in human lymphocytes in vitro . In the present study we suggested that the mutagenic activity of 15-4. Discussion desoxygoyasenzolide in S. cerevisiae could be at least partially due to an indirect free radical-DNA damage SLs, as well as ﬂavonoids and lignans, are consid- generated by GSH depletion. SLs, such as costuno-ered as important classes of natural products with a lide , helenalin [17,18] and parthenolide , arewide spectrum of biological effects, including antitumor, known to be potent inducers of cytotoxicity throughanti-ulcer, anti-inﬂammatory, neurotoxic, cytotoxic, and ROS-mediated oxidative stress. Interestingly, mutagen-cardiotonic activities [3–15,47]. As previously men- esis was not observed in 15-deoxygoyazensolide-treatedtioned, SLs can covalently bind to molecules containing Salmonella TA102 strain, which has a proven abilitya sulphydryl group like GSH, leading to thiol depletion, to detect mutagens that generate ROS and oxidativeand affecting protein structure and functions [13,14]. DNA damage although this SL has showed inducesThis is consistent with our ﬁnding that the total cyto- GSH depletion and mutagenesis in yeast cells. Similarly,plasmatic GSH content in yeast cells treated with parthenin did not show mutagenesis in Salmonella but it15-deoxygoyazensolide decreased in a dose-response caused oxidative stress in a hepatoma cell line in culture,manner. Polygoidal, pungent sesquiterpenoid unsatu- showing that differents biological effects are detected inrated dialdehyde also depletes intracellular GSH in S. relation to genotoxicity of SL in different prokaryoticcerevisiae, thus promoting a pro-oxidant status in cells and eukaryotic models as above mentioned .. Furthermore, it was recently demonstrated that In order to understand the modes of genotoxic actionendogenous GSH level in leukocytes treated with fura- of 15-deoxygoyazensolide on yeast, we studied thenoheliangolide lychnopholide was drastically reduced, response of S. cerevisiae mutants defective in DNAwithout corresponding increases in the oxidized form. repair to the treatment with this compound. The hyper-Analysis of electrospray mass spectrometry led to the sensitivity of the rad52-1 mutant strain suggests that theidentiﬁcation of glutathionyl-lychnopholide adduct in recombinational repair is critical for processing poten-cellular extracts . Data obtained in the present study tially lethal genetic lesions caused by oxidative damage,show that 15-deoxygoyazensolide reduces intracellular such as DNA single-strand breaks and closely locatedGSH content, promoting DNA oxidative damage prob- single strand breaks. However, when the cell attempts toably through an adduct similar to that described for repair these single strand breaks, it may, due to exhaus-glutathionyl-lychnopholide. tive repairing, also causes double breaks. Indeed, in S.
M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25 23cerevisiae, members of the RAD52 epistasis group are exact mechanism of the interaction among this naturalessential for successful meiotic and mitotic recombina- molecule, DNA, and topoisomerases.tion and survival after treatment with ionizing radiation, Moreover, the intracellular glutathione depletionalkylating agents, some oxidative mutagens, and cross- induced by 15-deoxygoyazensolide treatment appearslinking agents [34,36,61]. The cytotoxic responses of have a central role in its mutagenic effects in yeast Insingle mutant rad52-1 and double mutant rad3-e5 rad52- addition, the action of Top1 also is essential to repair1 to 15-deoxygoyazensolide are identical, which is a the lesions induced by this natural molecule, indicatingconsequence of the absence of the RAD52 pathway. that possibly the topology of nucleic acids are disturbed The data obtained here suggest that 15- by the lesiosn generated by this SL, probably a putativedesoxygoyazensolide-induced DNA damage could be intercalation effect.a consequence of its pro-oxidant properties. Althoughoxidative damage can be the primary mechanism to Acknowledgmentsexplain the genotoxicity of this molecule in yeast, theresults of the top1 mutant indicate a possible effect The authors are grateful to Dr. Diego Bonatto for criti-of intercalation, which suggests a putative complex cal reading of the manuscript and the Brazilian Agenciesformation between DNA and this SL that may also be FINEP, CNPq, BNB/FUNDECI, PRONEX, FAPESP,involved in its genotoxic effect. Indeed, the absence of GENOTOX, Genotoxicity Laboratory, Royal InstituteTop1 increased the sensitivity to the drug, as shown in and CAPES for fellowships and ﬁnancial support.Fig. 3. Topoisomerase I (Top1) catalyzes two transester- Referencesiﬁcation reactions: single-strand cleavage and DNAreligation, which are normally coupled for the relaxation  A.K. Picman, Biological activities of sesquiterpene lactones,of DNA supercoiling during chromatin transcription and Biochem. Syst. Ecol. 14 (1986) 255–281.replication. 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