Genotoxicity of 15-deoxygoyazensolide in bacteria and yeast

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 , Antˆonio Eduardo Miller Crotti c , Jo˜ao Luis Callegari Lopes c , Cl´audia Pessoa a , Manoel Odorico de Moraes a , Norberto Peporine Lopes c , Let´ıcia V. Costa-Lotufo a , Jenifer Saffi d , Jo˜ao Antˆonio Pegas Henriques b,e,∗ a

c

Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Cear´a, Caixa Postal-3157, 60430-270 Fortaleza, Cear´a, Brazil b Centro de Biotecnologia e Departamento de Biof´ısica, Universidade Federal do Rio Grande do Sul, Caixa Postal 15005, 91501-970 Porto Alegre, Rio Grande do Sul, Brazil Faculdade de Ciˆencias Farmacˆeuticas de Ribeir˜ao Preto, Universidade de S˜ao Paulo, 14040-903 Ribeir˜ao Preto, S˜ao Paulo, Brazil d Laborat´ orio de Gen´etica Toxicol´ogica, 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 2007 Available online 7 April 2007

Abstract Sesquiterpene lactones (SLs) present a wide range of pharmacological activities. The aim of our study was to investigate the genotoxicity of 15-deoxygoyazensolide using the Salmonella/microsome assay and the yeast Saccharomyces cerevisiae. We also investigated the nature of induced DNA damage using yeast strains defective in DNA repair pathways, such as nucleotide excision repair (RAD3), error prone repair (RAD6), and recombinational repair (RAD52), and in DNA metabolism, such as topoisomerase 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 from 15-deoxygoyazensolide-induced DNA damage, whereas excision repair and mutagenic systems does not appear to be primarily involved. Top 1 defective yeast strain was highly sensitive to the cytotoxic activity of 15-deoxygoyazensolide, suggesting a possible involvement of this enzyme in the reversion of the putative complex formation between DNA and this SL, possibly due to intercalation. Moreover, the treatment with this lactone caused dose-dependent glutathione depletion, generating pro-oxidant status which 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 oxidative damage, which explains the high sensitivity to top1 strain. In summary, the present study shows that 15-deoxygoyazensolide is mutagenic 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 Biof´ısica, Pr´edio 43422, Laborat´orio 210, Campus do Vale, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonc¸alves 9500, Bairro Agronomia, CEP 91501-970 Porto Alegre, RS, Brazil. Tel.: +55 51 33086069; fax: +55 51 33087003. E-mail address: [email protected] (J.A.P. Henriques). 1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2007.04.002

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

17

1. Introduction Sesquiterpene lactones (SLs) are substances presenting different pharmacological activities, with anti-microbial, anti-viral, anti-inflammatory, and antitumor effects [1,2]. For instance, costunolide and dehydrocostus lactone inhibit the killing activity of cytotoxic T lymphocytes, nitric oxide (NO) production, and the expression of hepatitis B virus surface antigen [3–5]. Helenalin alleviated carrageen-induced edema of rat hindfeet, and suppressed cancer cell growth [6,7]. Parthenolide and encelin showed potent inhibitory effects on the expression of cyclooxygenase and tumor necrosis factor (TNF)-␣ [8]. This wide range of biological activities is mainly caused by the inactivation of the NF-␬B nuclear factor [8–10]. Cynaropicrin derived from Saussurea lappa suppresses the production of cytokines, such as TNF-␣ and cytokine-induced neutrophil chemoattractant-1 (interleukin-8), as well as NO release. It also strongly attenuates mitogenic stimulation of CD4+ and CD8+ lymphocyte proliferation [11,12]. Most of the observed biological effects of these compounds are attributed to their ability to inhibit enzymes and other essential proteins by covalently binding to free cysteine sulfhydryl groups of polypeptides [13] or by conjugation with glutathione (GSH), leading to thiol depletion and an increase in the susceptibility to oxidative damage [14]. Severe oxidative stress conferred by SLs-induced thiol depletion results in a disruption of the integrity of mitochondria, triggering mitochondrial permeability transition and release of mitochondrial pro-apoptotic proteins. SLs contain an ␣methylene-␥-lactone moiety, which is highly reactive with cellular thiols, leading to alkylation of sulphydryl residues through Michael-type addition [14–16]. NF␬B, an ubiquitous transcription factor that regulates inflammatory responses, cell growth/differentiation and apoptosis, seems to be the main target for many SLs [15,16]. According to R¨ungeler et al. [15], NF-␬B inhibition by SLs is caused by alkylation at the mentioned cysteine moieties in the subunit p65. In addition, some SL has also remarkable cytotoxic effects. Indeed, costunolide, helenalin and parthenolide are known to be strong inducers of cytotoxicity by triggering apoptosis through reactive oxygen speciesmediated oxidative stress, However, its genotoxic potential has not been thoroughly documented [17–20]. The most studied lactone in relation to genotoxicity is the parthenin, a sesquiterpene lactone from Parthenium hysterophorus L., which induces chromosomal aberrations, mainly chromatid breaks, in blood lymphocytes; its did not influence sister chromatid exchange and show

Fig. 1. Chemical structure of 15-deoxygoyazensolide.

a positive increase in the micronucleated reticulocyte frequency in high doses [21]. Hymenoxon, helenalin, and tenulin were also tested for genotoxicity using six strains of Bacillus subtilis and hymenoxon and helenalin were found to produce lethal DNA damage in this model while tenulin did not produce lethal DNA damage in any of the strains tested [22]. Since that DNA damage has a central role in cytotoxic properties of several natural and synthetic molecules, the evaluation of genotoxicity of these molecules is very important [23]. The furanoheliangolide 15-deoxygoyazensolide (Fig. 1) is a SL isolated from several species of the sub-tribe Lychnophorinae (tribe Vernonieae, family Asteraceae), and possesses two functionalities in the form of an ␣-methylene-␥-lactone and an ␣,␤,␥,␦unsaturated carbonyl group [24,25], which could be considered as essential features for NF-␬B inhibition. Santos et al. [26] demonstrated that goyazensolide, which structure is similar to 15-deoxygoyazensolide, but for the oxidation at carbon 15, was strongly cytotoxic to tumor cell lines. In order to further understand the biological properties of SLs, this study aimed at investigating the genotoxicity of 15-deoxygoyazensolide in bacteria using the Salmonella/microsome assay, and at evaluating its genotoxic potential to the simple eukaryote S. cerevisiae, which mutagenesis and DNA repair mechanisms were assessed using haploid strains. This information is very important to evaluate the safety of possible future pharmacological applications of this compound, and to explore its potential pharmacological properties, such as antiproliferative activity. 2. Materials and methods 2.1. Chemicals All solvents were bi-distilled, and stored in dark flasks. Hydrogen peroxide (H2 O2 ), D-biotin, aflatoxin B1,

18

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

4-nitroquinoleine oxide (4-NQO), sodium azide, reduced glutathione (GSH), oxidized glutathione (GSSG), 1-chloro2,4-dinitrobenzene (CDNB), NADPH, glutathione reductase, aminoacids (l-histidine, l-threonine, l-methionine, l-tryptophan, l-leucine, l-lysine), l-canavanine, nitrogen bases (adenine and uracil), and dimethylsulfoxide (DMSO) were purchased from Sigma (St. Louis, USA). The S9 fraction, prepared with the polychlorinated biphenyl mixture Aroclor 1254, was purchased from Moltox (Annapolis, MD, USA), and glucose-6-phosphate and NADP were obtained from Sigma (St. Louis, USA). Oxoid nutrient broth No. 2 was obtained from Oxoid USA Inc. (Maryland, USA). Yeast extract, Bacto-peptone, and Bacto-agar were obtained from Difco Laboratories (Detroit, MI, USA). 2.2. 15-Deoxygoyazensolide isolation and identification Our previous phytochemical investigation of Minasia alpestris dried leaves, employing the five main fractions of a dichloromethane extract, showed that 15-deoxygoyazensolide is the main SL of this plant species [8]. Minor sub-fractions were analyzed using comparative HPLC, as previously described for seasonal variations of this class of secondary metabolites in Lychnophora ericoides [24]. Sub-fractions rich in this lactone were pooled together, and the resulting fraction was chromatographed in preparative HPLC (ODS-Shimadzu, 5.0 mm × 250 mm column, MeOH–H2 O, λ = 260 nm, flown 8 mL/min), yielding 30 mg of 15-deoxygoyazensolide. The structure of the isolated molecule was compared to the standard 15-deoxygoyazensolide structure obtained by authentic NMR spectrum and high-resolution mass spectrometry in a previous study [27].

[28], were kindly provided by B. M. Ames (University of California, Berkeley, CA, USA). Bacterial media were prepared according to Mortelmans and Zeiger [28]. Complete medium for growing strains (NB) contained 2.5% oxoid nutrient broth #2. Solidified medium with 1.5% bacto-agar, supplemented with 1× Vogel-Bonner salts and 2% glucose, was used for plates. The relevant genotypes of S. cerevisiae strains used in this work are given in Table 1. Haploid strain XV18514c 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 rad1rad6 double mutant were kindly provided by M. Grey (Frankfurt, Germany). The mutants rad3-e5 and rad52-1, defective in excision-resynthesis repair [29] and in DNA strand-breaks repair [31], respectively, were obtained by sporulation and tetrad analysis from the diploid of the genotype described in Table 1 [32]. Media, solutions, and buffers were prepared according to Burke et al. [33]. Complete YPD medium, containing 0.5% yeast extract, 2% bacto-peptone, and 2% glucose was used for routine growth of yeast cells. For plates, the medium was solidified with 2% bactoagar. Minimal medium (MM) contained 0.67% yeast nitrogen base with no amino acids, 2% glucose, and 2% bacto-agar supplemented with 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 mL of MM. For XV-185-14c strain mutagenesis, the omission media lacking lysine (SC-lys), histidine (SC-his), or homoserine (SC-hom) were used. Synthetic medium without arginine, 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 frameshift mutation 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 of leucine [GAG] by proline [GGG]), and TA102 (TAA (ochre: transversions/transitions) detects oxidative, alkylating mutagens and reactive oxygen species [ROS]), described in Ref.

Mutagenicity was assayed by the pre-incubation procedure proposed Maron and Ames [34], and revised by Mortelmans and Zeiger [28]. The S9 metabolic activation mixture (S9 mix) was prepared according to Maron and Ames [34]. 15Deoxygoyazensolide was dissolved in DMSO immediately before use. One hundred microliters of test bacterial cultures

Table 1 Saccharomyces cerevisiae strains used in this study Strains

Relevant genotypes

Source

Haploid: XV185-14c Y202 rad1Δ rad6Δ rad1Δ rad6Δ N123 Diploid: JH500a

MATα ade2-2 arg4-17 his1-7 lys1-1 trp5-48 hom3-10 MATa ura3-Δ100 ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 MATa ura3-Δ100 ade2-1 his3-11,15 trp1-1 can1-100 rad1::LEU2 MATa ade2-1 his3-11,15 leu2-3,112 trp1-1 can1-100 rad6::URA3 MATa ade2-1 his 3-11,15 trp1-1 can1-100 rad1::LEU2 rad6::URA3 MATa his1-7 gsh1 leak MATa/MATα rad3-e5/RAD3 RAD52/rad52-1 ADE2/ade2-1 ARG4/arg4-17 HIS1/his1-7 HIS5/his5-2 LEU1/leu1-12 LYS1/lys1-1 TRP5/trp5-48 HOM3/hom3-10

R.C. von Borstel M. Grey M. Grey M. Grey M. Grey J.A.P. Henriques R.C. von Borstel

a

Genotypes of zygotes from which haploid segregants were derived.

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

((1–2) × 109 cells/mL) were incubated in the dark at 37 ◦ C with different concentrations of 15-deoxygoyazensolide (8, 16, 24, 32, and 40 ␮g/plate) in the presence or absence of the S9 mix for 20 min, without shaking. Subsequently, 2 mL soft agar (0.6% agar, 0.5% NaCl, 50 ␮M histidine, 50 ␮M biotin, pH 7.4, 45 ◦ C) was added to the test tube, and immediately poured onto a minimal agar plate (1.5% agar, Vogel-Bonner E medium containing 2% glucose). Aflatoxin B1 (0.5 ␮g per plate) was used as positive control for all strains in the metabolic assay with the S9 mix. In the absence of the S9 mix, positive controls were 4NQO (0.5 ␮g/plate) for TA98, TA97a, and TA102, whereas sodium azide (5 ␮g/plate) was used for TA100. Plates were incubated in the dark at 37 ◦ C for 48 h before revertant colonies were counted. 2.5. Yeast growth Stationary-phase cultures were obtained by inoculation of an isolated colony into liquid YPD and, after 48 h incubation at 30 ◦ C with aeration by shaking, cultures were grown to (1–2) × 108 cells/mL. Cells were harvested, and washed twice with saline solution. Cell concentration and percentage of budding cells in each culture were determined in a Neubauer chamber by microscope counts. 2.6. Survival assays in S. cerevisiae strains The sensitivity to 15-deoxygoyazensolide was assayed by incubation of 2 × 108 cells/mL of stationary cultures in phosphate-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 compound in rotary shaker at 30 ◦ C for 3 h. After treatment, cells were harvested by centrifugation at 12,000 × g for 2 min, washed twice in PBS, counted, diluted, and plated on solid YPD. Plates were incubated at 30 ◦ C for 3–5 days before counting.

19

2.8. Detection of 15-deoxygoyazensolide-induced reverse and frameshift mutation in S. cerevisiae Mutagenesis was measured in S. cerevisiae XV185-14c strain. A suspension of 2 × 108 stationary-phase cells/mL was incubated for 3 h at 30 ◦ C with different concentrations of 15deoxygoyazensolide in PBS. Cell survival was determined in SC (3–5 days, 30 ◦ C), and mutation induction (LYS, HIS, or HOM revertants) in the appropriate omission media (7–10 days, 30 ◦ C). Whereas his1-7 is a non-suppressible missense allele, 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 mutation 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 [40], where the reduced adenine content of the SC-lys medium shows locus reversions as red and suppressor mutations as white colonies. It is believed that hom3-10 contains a frameshift mutation due to its response to a range of diagnostic mutagens [39]. Assays were repeated at least four times, and plating was performed in triplicate for each dose. 2.9. Detection of cytotoxic effects in mutants defective in DNA repair and topoisomerases The sensitivity of mutants defective in DNA repair pathways 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 inhibition assay, and total glutathione content was determined according to Akerboom and Sies [41]. Protein was measured by the Bradford method [42] using bovine serum albumin as standard.

2.7. Detection of 15-deoxygoyazensolide-induced forward mutation in S. cerevisiae

2.11. Data analysis

N123 strain was used for this analysis [35]. Yeast cells were cultured overnight in YPD medium at 30 ◦ C in an orbital shaker until cell suspension reached a density of (1–2) × 108 cells/mL. Cells were harvested, washed twice by centrifugation with PBS, and submitted to 15-deoxygoyazensolide (0.025, 0.05, 0.1, 0.25 and 0.5 mg/mL) treatment at 30 ◦ C for 3 h in the dark with shaking. Cell concentration and percentage of budding cells in each culture were determined by microscope counts using a Neubauer chamber. After treatment, appropriate dilutions of cells were plated onto SC plates to determine cell survival, and 100 ␮L aliquots of cell suspension (2 × 108 cells/mL) were plated onto SC media supplemented with 60 ␮g/mL canavanine in order to determine forward mutation in CAN1 locus. Mutants were counted after 4–5 days incubation at 30 ◦ C.

Mutagenicity data were analyzed using Salmonel software [43]. A compound was considered positive for mutagenicity only when: (a) the number of revertants was at least twice the spontaneous yield (MI ≥ 2; MI = mutagenic index: number of induced colonies in the sample/number of spontaneous colonies in the negative control samples); (b) a significant response 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 was considered as cytotoxic when MI ≤ 0.6. Data from mutagenesis and survival assays in S. cerevisiae were expressed as means and standard deviation from three independent experiments, and statistically analyzed using the Student’s ttest. Differences were considered significant when p < 0.05 [46].

20

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

Table 2 Induction 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

PCc NCd 15-Deoxygoyazensolide 8 16 24 32 40 a b c d

MIb

284.33 ± 38.04 15.67 ± 8.96 15.67 25.33 22.33 23.50 21.67

−S9

+S9

rev/platea

± ± ± ± ±

1.53 9.29 4.04 6.36 3.21

rev/plate

MI

469.33 ± 1.74 22.33 ± 6.03 1.00 1.61 1.42 1.50 1.38

± ± ± ± ±

18.00 23.67 22.33 17.00 20.33

+S9

rev/plate

MI

658.00 ± 85.15 119.33 ± 11.55

5.57 6.03 4.93 6.08 2.52

0.80 1.06 1.00 0.76 0.91

117.67 117.67 146.00 128.67 125.33

± ± ± ± ±

10.97 5.77 14.93 30.86 22.23

rev/plate

MI

734.66 ± 107.5 163.33 ± 65.19 0.98 0.98 1.22 1.07 1.05

176.00 156.67 186.67 155.67 212.67

± ± ± ± ±

39.40 28.31 6.11 35.44 46.32

1.07 0.96 1.14 0.95 1.30

Number of revertant per plate: mean of three independent experiments ±S.D. MI: mutagenic index (no. of his+ induced in the sample/no. of spontaneous his+ in the negative control). PC: positive control (−S9) 4-NQO (0.5 ␮g/plate); (+S9) aflatoxin B1 (0.5 ␮g/plate). NC: negative control dimethyl sulfoxide (10 ␮L) used as a solvent for 15-deoxygoyazensolide.

3. Results 3.1. Salmonella/microsome assay When dose range of 15-deoxygoyazensolide was evaluated with the TA100 strain, cytotoxicity was observed in concentrations higher than 40 ␮g/mL (data not shown). No mutagenicity of 15-deoxygoyazensolide was seen at concentrations of 8–40 ␮g/mL in strains TA98, TA97a, TA100, and TA102 in the absence or presence of metabolic activation. 3.2. Cytotoxic and mutagenic effects in S. cerevisiae Results of the mutagenicity tests are shown in Tables 4 and 5. 15-Deoxygoyazensolide induced mod-

erate dose-dependent cytotoxicity in haploid wild-type yeast cultures. The frequency of point (HIS1+, LYS1+), frameshift (HOM3+) (Table 4), and forward (Table 5) mutations during treatment of stationary-phase cells indicates a clear mutagenic effect at doses higher than 0.25 mg/mL (Tables 2 and 3). 3.3. Cytotoxic effects in S. cerevisiae DNA repair-defective strains and topoisomerase-defective strains The cytotoxic effects of 15-deoxygoyazensolide are shown in Fig. 2. The sensitivity to this SL depends on the repairing capacity of the yeast cell. The single mutants rad3-e5, rad1, and mutant rad6 (Fig. 2) exhibited the

Table 3 Induction of his+ revertants in S. typhimurium base pair substitution strains by 15-deoxygoyazensolide with and without metabolic activation (S9 mix) Substance: dose (␮g/plate)

TA100

TA102

−S9 rev/platea PCc NCd 15-Deoxygoyazensolide 8 16 24 32 40 a b c d

MIb

1247.33 ± 196.26 129.33 ± 7.09 126.67 149.00 147.00 128.67 118.33

−S9

+S9

± ± ± ± ±

30.73 2.65 14.73 12.01 25.70

rev/plate

MI

537.33 ± 105.78 117.33 ± 22.03 0.98 1.15 1.13 0.98 0.91

156.00 136.00 122.67 171.33 153.33

± ± ± ± ±

35.55 6.93 16.17 6.43 26.63

+S9

rev/plate

MI

3251.00 ± 259.75 548.00 ± 11.31 1.33 1.16 1.04 1.46 1.30

581.33 574.67 402.00 414.67 456.67

± ± ± ± ±

49.69 62.27 23.58 57.18 48.01

rev/plate

MI

781.00 ± 180.28 241.33 ± 16.17 1.06 1.04 0.73 0.75 0.83

230.67 312.67 293.33 252.00 260.00

± ± ± ± ±

24.44 21.39 38.02 31.75 24.98

Number of revertant per plate: mean of three independent experiments ±S.D. MI: mutagenic index (no. of his+ induced in the sample/no. of spontaneous his+ in the negative control). 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). NC: negative control dimethyl sulfoxide (10 ␮L) used as a solvent for 15-deoxygoyazensolide.

0.95 1.29 1.21 1.04 1.07

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

21

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

NCc PCd

Survival (%)

HIS1 (107 survivors)a

LYS1 (107 survivors)b

HOM3 (107 survivors)a

100.0 55.0

11.1 ± 5.0 83.0 ± 1.6*

1..3 ± 0.6 18.9 ± 7.0*

2.2 ± 1.3 43.0 ± 8.6*

15-Deoxygoyazensolide 0.025 mg/mL 0.05 mg/mL 0.1 mg/mL 0.25 mg/mL 0.5 mg/mL

90.4 73.2 60.1 35.1 20.2

14.4 23.7 30.6 49.0 32.0

± ± ± ± ±

0.7 1.0* 2.2* 3.7* 6.5*

2.4 5.1 15.4 24.9 30.8

± ± ± ± ±

0.35 2.5 2.7* 3.4* 1.2*

6.4 8.9 12.8 31.6 52.9

± ± ± ± ±

2.2 0.7* 4.0* 0.1* 0.8*

a

Locus-specific revertants. 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. b

same sensitivity to the wild type strain. Both the single mutant rad52-1 and the double mutant rad3-e5 rad521 showed extreme sensitivity to 15-deoxygoyazensolide (Fig. 2). Fig. 3 shows that topoisomerase I (Top1) enzymatic action has a crucial role in the resistance to the cytotoxicity of this SL. As the top1 strain was very sensitive to the SL treatment—at 0.1 mg/mL, survival was minimal. The mutant top3 showed similar sensitivity to wild type strain in virtually all evaluated doses, indicating that Top3 was not involved in the repair of the lesions induced by 15-deoxygoyazensolide. 3.4. Determination of total glutathione content As shown in Fig. 4, when wild-type cells were incubated with 15-deoxygoyazensolide, GSH content

Fig. 2. Sensitivity to 15-deoxygoyazensolide of different haploid rad mutant strains. WT RAD+ (solid square) and its isogenics mutants: rad3-e5 (solid triangle), rad52-1 (solid inverted triangle), rad3-e5 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 5 Induction of forward mutation (CAN1) in N123 strain of S. cerevisiae after 15-deoxygoyazensolide treatment during 3 h in PBS Substance

Survival (%)

Mutants (107 survivors)

NCa PCb

100.00 61.73 ± 4.57

4.75 ± 1.30 132.25 ± 3.17**

15-Deoxygoyazensolide 0.025 mg/mL 96.95 0.05 mg/mL 93.55 0.1 mg/mL 86.01 0.25 mg/mL 51.33 0.5 mg/mL 36.01

± ± ± ± ±

10.56 14.01 8.59 5.22 8.59

5.06 8.86 8.72 14.72 68.53

± ± ± ± ±

0.10 3.55 3.16 3.80* 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 15deoxygoyazensolide. b PC: positive control, 4-NQO (0.5 ␮g/plate).

Fig. 3. Sensitivity to 15-deoxygoyazensolide of different topoisomerase 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

Fig. 4. Effect of 15-deoxygoyazensolide on the total GSH content in wild type yeast during incubation in synthetic complete media during 18 h at 30 ◦ C. Values are means ± S.D. (n = 6). SL: treatment with 15-deoxygoyazensolide; formaldehyde 0.1%; CDNB: 1-chloro2,4-dinitrobenzene (1 mM); negative control: DMSO 10%. * Data significant in relation to negative control at p < 0.05 (ANOVA).

decreased in a dose-dependent manner. Positive control treatments for GSH depletion, using formaldehyde and CDNB, were used to validate the results. 4. Discussion SLs, as well as flavonoids and lignans, are considered as important classes of natural products with a wide spectrum of biological effects, including antitumor, anti-ulcer, anti-inflammatory, neurotoxic, cytotoxic, and cardiotonic activities [3–15,47]. As previously mentioned, SLs can covalently bind to molecules containing a 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 cytoplasmatic GSH content in yeast cells treated with 15-deoxygoyazensolide decreased in a dose-response manner. Polygoidal, pungent sesquiterpenoid unsaturated dialdehyde also depletes intracellular GSH in S. cerevisiae, thus promoting a pro-oxidant status in cells [48]. Furthermore, it was recently demonstrated that endogenous GSH level in leukocytes treated with furanoheliangolide lychnopholide was drastically reduced, without corresponding increases in the oxidized form. Analysis of electrospray mass spectrometry led to the identification of glutathionyl-lychnopholide adduct in cellular extracts [49]. Data obtained in the present study show that 15-deoxygoyazensolide reduces intracellular GSH content, promoting DNA oxidative damage probably through an adduct similar to that described for glutathionyl-lychnopholide.

As consequence of this abnormal reduction of glutathione, 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 activity could be expected for this class of molecules. Indeed, our results show that 15-deoxygoyazensolide was mutagenic 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 cellular systems, which explains the different results obtained in these biological models for several genotoxins [53–55]. Studies with other SLs showed that some furanoheliangolides have a direct impact on DNA, causing lethal cell damage in bacteria [56], as well as chromosomal aberrations [57,58] and DNA strand breaks in mammalian cells [59]. However, previous genotoxic research with 15-deoxygoyazensolide did not observe significant increase in the frequency of sister chromatid exchanges in human lymphocytes in vitro [60]. In the present study we suggested that the mutagenic activity of 15desoxygoyasenzolide in S. cerevisiae could be at least partially due to an indirect free radical-DNA damage generated by GSH depletion. SLs, such as costunolide [19], helenalin [17,18] and parthenolide [20], are known to be potent inducers of cytotoxicity through ROS-mediated oxidative stress. Interestingly, mutagenesis was not observed in 15-deoxygoyazensolide-treated Salmonella TA102 strain, which has a proven ability to detect mutagens that generate ROS and oxidative DNA damage although this SL has showed induces GSH depletion and mutagenesis in yeast cells. Similarly, parthenin did not show mutagenesis in Salmonella but it caused oxidative stress in a hepatoma cell line in culture, showing that differents biological effects are detected in relation to genotoxicity of SL in different prokaryotic and eukaryotic models as above mentioned [20]. In order to understand the modes of genotoxic action of 15-deoxygoyazensolide on yeast, we studied the response of S. cerevisiae mutants defective in DNA repair to the treatment with this compound. The hypersensitivity of the rad52-1 mutant strain suggests that the recombinational repair is critical for processing potentially lethal genetic lesions caused by oxidative damage, such as DNA single-strand breaks and closely located single strand breaks. However, when the cell attempts to repair these single strand breaks, it may, due to exhaustive repairing, also causes double breaks. Indeed, in S.

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

cerevisiae, members of the RAD52 epistasis group are essential for successful meiotic and mitotic recombination and survival after treatment with ionizing radiation, alkylating agents, some oxidative mutagens, and crosslinking agents [34,36,61]. The cytotoxic responses of single mutant rad52-1 and double mutant rad3-e5 rad521 to 15-deoxygoyazensolide are identical, which is a consequence of the absence of the RAD52 pathway. The data obtained here suggest that 15desoxygoyazensolide-induced DNA damage could be a consequence of its pro-oxidant properties. Although oxidative damage can be the primary mechanism to explain the genotoxicity of this molecule in yeast, the results of the top1 mutant indicate a possible effect of intercalation, which suggests a putative complex formation between DNA and this SL that may also be involved in its genotoxic effect. Indeed, the absence of Top1 increased the sensitivity to the drug, as shown in Fig. 3. Topoisomerase I (Top1) catalyzes two transesterification reactions: single-strand cleavage and DNA religation, which are normally coupled for the relaxation of DNA supercoiling during chromatin transcription and replication. Therefore, it is involved in multiple DNA transactions, including DNA replication, transcription, chromosome condensation and decondensation, and, probably, DNA recombination [62]. Besides promoting DNA relaxation, necessary to eliminate torsional stresses associated to these processes, Top1 may have other functions related to its interaction with other cellular proteins. Top1 cleavage complexes are also produced by endogenous and exogenous DNA lesions, including UVinduced base modifications, guanine methylation and oxidation, polycyclic aromatic carcinogenic adducts, base mismatches, abasic sites, cytosine arabinoside or gemcitabine incorporation, and DNA nicks. Cleavage complexes can produce DNA damage after collisions of replication forks and transcription complexes [63]. These lesions, particularly replication fork collisions, need to be repaired for the cell to survive. We suppose that this SL is capable of intercalating into DNA, and the DNA-SL complex requires Top1 action for DNA resolution and repair. DNA topoisomerases are the targets of antimicrobial and anticancer drugs [62,63]. The precise role of Top1 in 15-deoxygoyazensolide cytotoxicity is still unclear [64,65]. As topoisomerase II inhibition induces DNA breaks, which are repaired almost exclusively by Rad52dependent homologous recombination in yeast, the high sensitivity of the rad52 mutant may involve, at least in part, the interference of this SL in Top2 activity [66]. Therefore, further studies are needed to clarify the

23

exact mechanism of the interaction among this natural molecule, DNA, and topoisomerases. Moreover, the intracellular glutathione depletion induced by 15-deoxygoyazensolide treatment appears have a central role in its mutagenic effects in yeast In addition, the action of Top1 also is essential to repair the lesions induced by this natural molecule, indicating that possibly the topology of nucleic acids are disturbed by the lesiosn generated by this SL, probably a putative intercalation effect. Acknowledgments The authors are grateful to Dr. Diego Bonatto for critical reading of the manuscript and the Brazilian Agencies FINEP, CNPq, BNB/FUNDECI, PRONEX, FAPESP, GENOTOX, Genotoxicity Laboratory, Royal Institute and CAPES for fellowships and financial support. References [1] A.K. Picman, Biological activities of sesquiterpene lactones, Biochem. Syst. Ecol. 14 (1986) 255–281. [2] S. Zang, Y.K. Won, C.N. Ong, H.M. Shen, Anti-cancer potential of sesquiterpene lactones: bioactivity and molecular mechanisms, Curr. Med. Chem. Anticancer Agents 5 (2005) 239–249. [3] M. Taniguchi, T. Kataoka, H. Suzuki, M. Uramoto, M. Ando, K. Arao, J. Magae, T. Nishimura, N. Otake, K. Nagai, Costunolide and dehydrocostus lactone as inhibitors of killing function of cytotoxic T lymphocytes, Biosci. Biotechnol. Biochem. 59 (1995) 2064–2067. [4] H.J. Lee, N.Y. Kim, H.J. Son, K.M. Kim, D.H. Sohn, S.H. Lee, J.H. Ryu, A sesquiterpene, dehydrocostus lactone, inhibits the expression of inducible nitric oxide synthase and TNF-alpha in LPS-activated macrophages, Planta Med. 65 (1999) 104–108. [5] H.C. Chen, C.K. Chou, S.D. Lee, J.C. Wang, S.F. Yeh, Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells, Antiviral Res. 27 (1995) 99–109. [6] I.H. Hall, K.H. Lee, C.O. Starnes, Y. Sumida, R.Y. Wu, T.G. Waddell, T.W. Cochran, K.G. Gerhart, Anti-inflammatory activity of sesquiterpene lactones and related compounds, J. Pharm. Sci. 68 (1979) (1979) 537–541. [7] I.H. Hall, K.H. Lee, E.C. Mar, C.O. Starnes, T.G. Wadell, Antitumor agents: 21. A proposed mechanism for inhibition of cancer growth by tenulin and helenalin and related cyclopentenones, J. Med. Chem. 20 (1977) 333–337. [8] D. Hwang, N.H. Fischer, B.C. Jang, H. Tak, J.K. Kim, W. Lee, Inhibition of the expression of inducible cyclooxygenase and proinflammatory cytokines by sesquiterpene lactones in macrophages correlates with the inhibition of MAP kinases, Biochem. Biophys. Res. Commun. 226 (1996) 810–818. [9] P.M. Bork, M.L. Schmitz, M. Kuhnt, C. Escher, M. Heinrich, Sesquiterpene lactone containing Mexican Indian medicinal plants and pure sesquiterpene lactones as potent inhibitors of transcription factor NF-kappaB, FEBS Lett. 402 (1997) 85–90. [10] S.P. Hehner, M. Heinrich, P.M. Bork, M. Vogt, F. Ratter, V. Lehmann, K. Schulze-Osthoff, W. Droge, M.L. Schmitz,

24

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24]

[25]

[26]

M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25 Sesquiterpene lactones specifically inhibit activation of NF-kappa B by preventing the degradation of I kappa B-alpha and I kappa B-beta, J. Biol. Chem. 272 (1998) 135–143. J.Y. Cho, K.U. Baik, J.H. Jung, M.H. Park, In vitro antiinflammatory effects of cynaropicrin, a sesquiterpene lactone, from Saussurea lappa, Eur. J. Pharmacol. 398 (2000) 399–407. J.Y. Cho, A.R. Kimb, J.H. Jungb, T. Chunc, M.H. Rheed, E.S. Yooe, Cytotoxic pro-apoptotic activities of cynaropicrin, a sesquiterpene lactone, on the viability of leukocyte cancer cell lines, Eur. J. Pharmacol. 492 (2004) 85–94. T.J. Schimidt, Helenanolide-type sesquiterpene lactones. III. Rates and stereochemistry in the reaction of helenalin and releated helenanolides with sulfhydryl containing biomolecules, Bioorg. Med. Chem. 5 (1997) 645–653. F.F. Tukov, S. Anand, R.S. Gadepalli, A.A. Gunatilaka, J.C. Matthews, J. Rimoldi, Inactivation of the cytotoxic activity of repin, a sesquiterpene lactone from Centaurea repen, Chem. Res. Toxicol. 17 (2004) 1170–1176. P. R¨ungeler, V. Castro, G. Mora, N. Goren, W. Vichenewski, H.L. Pahl, L. Melfort, T.J. Schimidt, Inhibition of transcription factor NF-kappaB by sesquiterpene lactones: a proposed molecular mechanism of action, Bioorg. Med. Chem. 7 (1999) 2343–2352. B. Siedle, A.J. Garc´ıa-Pi˜neres, R. Murillo, J. Schulte-Monting, V. Castro, P. R¨ungeler, C.A. Klaas, F.B. Da Costa, W. Kisiel, I. Merfort, Quantitative structure-activity relationship of sesquiterpene lactones as inhibitors of the transcription factor NF-␬B, J. Med. Chem. 47 (2004) 6042–6054. V.M. Dirsch, H. Stuppner, A.M. Vollmar, Cytotoxic sesquiterpene lactones mediate their death-inducing effect in leukemia T cells by triggering apoptosis, Planta Med. 67 (2001) 557–559. V.M. Dirsch, H. Stuppner, A.M. Vollmar, The sesquiterpene lactone helenalin triggers apoptosis in leukemia Jurkat T cells independently of the CD95 receptor and even in cells overexpressing Bcl-xL or Bcl-2, Cancer Res. 61 (2001) 5817–5823. H.J. Park, S.H. Kwon, Y.N. Han, J.W. Choi, K. Miyamoto, S.H. Lee, K.T. Lee, Apoptosis-inducing costunolide and a novel acyclic monoterpene from the stem bark of Magnolia sieboldii, Arch. Pharm. Res. 24 (2001) 342–348. J. Wen, K.R. You, S.Y. Lee, C.H. Song, D.G. Kim, Oxidative stress-mediated apoptosis: the anticancer effect of the sesquiterpene lactone parthenolide, J. Biol. Chem. 277 (2002) 38954–38964. A. Ramos, R. Rivero, A. Visozo, J. Piloto, A. Garc´ıa, Parthenin, a sesquiterpene lactone of Parthenium hysterophorus L. is a high toxicity clastogen, Mutat. Res. 514 (2002) 19–27. D.H. Jones, H.L. Kim, K.C. Donnelly, DNA damaging effects of three sesquiterpene lactones in repair-deficient mutants of Bacillus subtilis, Res. Commun. Chem. Pathol. Pharmacol. 34 (1981) 161–164. W.P. Roos, B. Kaina, DNA damage-induced cell death by apoptosis, Trends Mol. Med. 12 (2006) 440–450. H.T. Sakamoto, D. Flausino, E.E. Castellano, B.W.C. Stark, J.P. Gates, N.P. Lopes, Sesquiterpene lactones from Lychnophora ericoides, J. Nat. Prod. 66 (2003) 693–695. A.E.M. Crotti, W.R. Cunha, N.P. Lopes, J.L.C. Lopes, Sesquiterpene lactones from Minasia alpestris, J. Br. Chem. Soc. 16 (2005) 677–680. P.A. Santos, M.F.C. Amarante, A.M.S. Pereira, B. Bertoni, S. Castro Franca, C. Pessoa, M.O. Moraes, L.V. Costa-Lotufo, M.R.P. Pereira, N.P. Lopes, Production of an furanoheliangolide by Lychnophora ericoides cell culture, Chem. Pharm. Bull. 12 (2004) 1433–1435.

[27] A.E.M. Crotti, J.L.C. Lopes, N.P. Lopes, Triple quadrupole tandem mass spectrometry of sesquiterpene lactones: a study of goyazensolide and its congeners, J. Mass Spectrom. 40 (2005) 1030–1034. [28] K. Mortelmans, E. Zeiger, The Ames Salmonella/microsome mutagenicity assay, Mutat. Res. 455 (2000) 29–60. [29] L. Prakash, S. Prakash, Nucleotide excision repair in yeast, Mutat. Res. 451 (2000) 13–24. [30] J.C. Game, The Saccharomyces cerevisiae DNA repair genes at the end of the century, Mutat. Res. 451 (2000) 277–293. [31] M. Kupiec, Damage-induced recombination in the yeast Saccharomyces cerevisiae, Mutat. Res. 451 (2000) 91–105. [32] J.A.P. Henriques, K.V.C.L. Da Silva, E. Moustacchi, Interaction between genes controlling sensitivity to psoralen (pso) and to radiation (rad) after 3carbethoxypsoralen plus 365 nm UV light treatment in yeast, Mol. Gen. Genet. 201 (1985) 415–420. [33] D. Burke, D. Dawson, T. Stearns, Methods in Yeast Genetics, Cold Spring Harbour Laboratory Course Manual, CSH Laboratory Press, Cold Spring Harbour, 2000. [34] D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113 (1983) 173–215. [35] M. Brendel, M. Grey, A. Maris, J. Hietkamp, Z. Fesus, C. Pich, L. Dafr´e, M. Schmidt, F. Eckardt-Schupp, J. Henriques, Low glutathione pools in the original pso3 mutant of Saccharomyces cerevisiae are responsible for its pleiotropic sensitivity phenotype, Curr. Genet. 33 (1998) 4–9. [36] S. Snow, Absence of suppressible alleles at the his 1 locus of yeast, Mol. Gen. Genet. 164 (1978) 341–342. [37] D.C. Hawthorne, Identification of nonsense codons in yeast, J. Mol. Biol. 43 (1969) 71–75. [38] D.C. Hawthorne, R.K. Mortimer, Suppressors in yeast, Genetics 48 (1963) 617–620. [39] R.C. Von Borstel, K.T. Cain, C.M. Steinberg, Inheritance of spontaneous mutability in yeast, Genetics 69 (1971) 17–27. [40] R.C. Schuller, R.C. von Borstel, Spontaneous mutability in yeast. I. Stability of lysine reversion rates to variation of adenine concentration, Mutat. Res. 24 (1974) 17–23. [41] T.P. Akerboom, H. Sies, Assay of glutathione, glutathione disulfide and glutathione mixed disulfides in biological samples, Meth. Enzymol. 77 (1981) 373–382. [42] M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72 (1976) 248–254. [43] L. Myers, N. Adams, L. Kier, T.K. Rao, B. Shaw, L. Williams, Microcomputer software for data management and statistical analysis of the Ames/Salmonella test, in: D. Krewski, C.A. Franklin (Eds.), Statistics in Toxicology, Gordon and Breach, New York, 1991, pp. 265–279. [44] A.A.M. Cavalcante, G. Rubensam, J.N. Picada, E.G. da Silva, J.C.F. Moreira, J.A.P. Henriques, Mutagenicity, antioxidant potential, and antimutagenic activity against hydrogen peroxide of cashew (Anacardium occidentale) apple juice and cajuina, Environ. Mol. Mutagen. 41 (2003) 360–369. [45] J.N. Picada, A.F. Maris, K. Ckless, M. Salvador, N.N. KhromovBorisov, J.A.P. Henriques, Differential mutagenic, antimutagenic and cytotoxic responses induced by apomorphine and its oxidation product, 8-oxo-apomorphine-semiquinone, in bacterial and yeast, Mutat. Res. 539 (2003) 29–41. [46] A. Camussi, F. Moller, M. Ottavianio, S. Gorla, Distribuizone campionarie e stima dei parametri in una popolazione Metodi statistici per la sperimentazione biologica, vol. 3, Zanichelli, Bologna, 1986, pp. 68–70.

M.C. Vasconcellos et al. / Mutation Research 631 (2007) 16–25 [47] M. Robles, M. Aregullin, J. West, E. Rodriguez, Recent studies on the zoopharmacognosy, pharmacology, and neurotoxicology of sesquiterpene lactone, Planta Med. 61 (1995) 199–203. [48] K. Machida, T. Tanaka, M. Taniguchi, Depletion of glutathione as a cause of the promotive effects of polygodial, a sesquiterpene on the production of reactive oxygen species in Saccharomyces cerevisiae, J. Biosci. Bioeng. 88 (1999) 526–530. [49] M.R.P.L. Brigad˜ao, N.P. Lopes, P. Colep´ıcolo, Inhibitory mechanism of leukocyte respiratory burst by the sesquiterpene lactone Lychnopholide through protein glutathionylation decrease, Free Radic. Biol. Med. 36 (2004) S57–S58. [50] M. Cunningham, J. Peak, M. Peak, Single-strand DNA breaks in rodent and human cells produced by superoxide anion or its reduction products, Mutat. Res. 184 (1987) 217–222. [51] R. Meneghini, Iron homeostasis, oxidative stress and DNA damage, Free Radic. Biol. Med. 23 (1997) 783–792. [52] A. Barbouti, D. Paschalis-Thomas, L. Nousis, M. Tenopoulou, D. Galares, DNA damage and apoptosis in hydrogen peroxideexposed Jurkat cells: bolus addition versus continuous generation of hydrogen peroxide, Free Radic. Biol. Med. 33 (2002) 691–702. [53] Z. Kirpnick, M. Homiski, E. Rubitski, M. Repnevskaya, N. Howlett, J. Aubrecht, R.H. Schiestl, Yeast DEL assay detects clastogens, Mutat. Res. 582 (2005) 116–134. [54] R.J. Brennan, R.H. Schiestl, Free radicals generated in yeast by the Salmonella test-negative carcinogens benzene, urethane, thiourea and auramine O, Mutat. Res. 403 (1998) 65–73. [55] T. Nunoshiba, E. Watanabe, T. Takahashi, Y. Daigaku, S. Ishikawa, M. Mochiziki, A. Ui, T. Enomoto, K. Yakamoto, Ames test-negative carcinogen, ortho-phenyl phenol, binds tubulin and causes aneuploidy in budding yeast, Mutat. Res. 617 (2007) 90–97. [56] D.H. Jones, H.L. Kim, H.C. Donnelly, DNA damaging effects of three sesquiterpene lactones in repair deficient mutants of Bacillus subtilis, Res. Commun. Chem. Pathol. Pharmacol. 34 (1981) 161–164.

25

[57] R.V. Burim, R. Canalle, J.L.C. Lopes, C.S. Takahashi, Genotoxic Action of the sesquiterpene lactone Glaucolide B on mammalian in vitro and in vivo, Genet. Mol. Biol. 22 (1999) 401–406. [58] M.S. Mantovani, C.S. Takahashi, W. Vichnewski, Evaluation of the genotoxic activity of the sesquiterpene lactone goyazensolide in mammalian systems in vitro and in vivo, Rev. Br. Gen´et. 16 (1993) 967–975. [59] V.G. Vaidya, I. Kulkarni, B.A. Nagasampagi, In vitro and in vivo cytogenetic effects of sesquiterpene lactone parthenin derived from Parthenium hysterophorus Linn, Indian J. Exp. Biol. 16 (1978) 1117–1118. [60] V.E.P. Vicentini-Dias, Estudo da ac¸a˜ o citot´oxica das lactonas sesquiterpˆenicas eremantolido C e 15-desoxigoyazensolido, sobre medula o´ ssea de ratos e linf´ocitos humanos, Ph.D. thesis, Faculdade de Medicina de Ribeir˜ao Preto, Universidade de S˜ao Paulo, Ribeir˜ao Preto, S˜ao Paulo, 1992. [61] A. Abdulovic, N. Kim, S. Jinks-Robertaon, Mutagenesis and the three R’s in yeast, DNA Repair (Amst) 8 (5) (2006) 409– 421. [62] Y. Pommier, C. Redon, V.A. Rao, J.A. Seiler, O. Sordet, H. Takemura, S. Antony, L.H. Meng, Z.Y. Liao, G. Kohlhagen, H.L. Zhang, K.W. Kohn, Repair of and checkpoint to topoisomerase I-mediated DNA damage, Mutat. Res. 532 (2003) 173–203. [63] P. Pourquier, Y. Pommier, Topoiserase I—mediated DNA damage, Adv. Cancer Res. 80 (2001) 189–216. [64] O. Sordet, Q.A. Khan, Y. Pommier, Apoptotic topoisomerase I–DNA complex induced by oxygen radicals and mitochondrial dysfunction, Cell Cycle 3 (2004) 1095–1097. [65] P. Daroui, S.D. Desai, T.K. Lin, A.A. Liu, L.F. Liu, Hydrogen peroxide induces topoisomerase I-mediated DNA damage and cell death, J. Biol. Chem. 15 (2004) 14587–14594. [66] M. Sabourin, J.L. Nitiss, K.C. Nitiss, K. Tatebayashi, H. Ikeda, N. Osheroff, Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks, Nucl. Acids Res. 31 (2003) 4373–4384.