Arch ToxicolDOI 10.1007/s00204-010-0567-z G E N O T O X I C I T Y A N D CA R C I N O G E N I CI T YEvaluation of the cytot...
Arch Toxicol                      O                                          USA). Methyl methanesulfonate (MMS), hydrogen...
Arch ToxicolSubsequently, 2 mL of soft agar (0.6% agar, 0.5% NaCl,          mutagenic evaluation under growth conditions, ...
Arch ToxicolOne day prior to treatment, cells were harvested by treat-      10% DMSO, pH 10.0) at 4°C for at least 1 h in ...
Arch Toxicolseparation module and UV detector 2487 (Waters). The                      assay. The test-substance was consid...
Arch ToxicolTable 2 Induction of his+ revertants in TA100 and TA102 S. typhimurium base-substitution strains by biXorin wi...
Arch ToxicolTable 3 Reversion of point mutation for (his1-7), ochre allele (lys1-1), and frameshift mutations (hom3-10) in...
Arch ToxicolTable 4 Reversion of point mutation for (his1-7), ochre allele (lys1-1), and frameshift mutations (hom3-10) in...
Arch Toxicol                                     100                                       75                      Surviva...
Arch Toxicol A 350                                                                                       A 100            ...
Arch Toxicol              100                                                   2002). As biXorin does not induce frameshi...
Arch ToxicolLemos TLG, Monte FJO, Santos AKL, Fonseca AM, Santos HS,                  of hydroxyl radical formation by det...
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Evaluation of the cytotoxic and antimutagenic e effects

  1. 1. Arch ToxicolDOI 10.1007/s00204-010-0567-z G E N O T O X I C I T Y A N D CA R C I N O G E N I CI T YEvaluation of the cytotoxic and antimutagenic eVectsof biXorin, an antitumor 1,4 o-naphthoquinone isolatedfrom Capraria biXora LMarne C. Vasconcellos · Dinara J. Moura · Renato M. Rosa · Miriana S. Machado · Temenouga N. Guecheva ·Izabel Villela · Bruna F. Immich · Raquel C. Montenegro · Aluísio M. Fonseca · Telma L. G. Lemos ·Maria Elisabete A. Moraes · Jenifer SaY · Letícia V. Costa-Lotufo · Manoel O. Moraes · João A. P. HenriquesReceived: 19 April 2010 / Accepted: 9 June 2010© Springer-Verlag 2010Abstract BiXorin is a natural quinone isolated from assay, biXorin was not mutagenic to TA97a TA98, TA100,Capraria biXora L. Previous studies demonstrated that biXorin and TA102 strains. However, biXorin was able to induceinhibits in vitro and in vivo tumor cell growth and presents cytotoxicity in haploid S. cerevisiae cells in stationary andpotent antioxidant activity. In this paper, we report concen- exponential phase growth. In diploid yeast cells, biXorintration-dependent cytotoxic, genotoxic, antimutagenic, and did not induce signiWcant mutagenic and recombinogenicprotective eVects of biXorin on Salmonella tiphymurium, eVects at the employed concentration range. In addition, theyeast Saccharomyces cerevisiae, and V79 mammalian cells, pre-treatment with biXorin prevented the mutagenic andusing diVerent approaches. In the Salmonella/microsome recombinogenic events induced by hydrogen peroxide (H2O2) in S. cerevisiae. In V79 mammalian cells, biXorin was cytotoxic at higher concentrations. Moreover, at low concentrations biXorin pre-treatment protected againstM. C. Vasconcellos · R. C. Montenegro · M. E. A. Moraes · H2O2-induced oxidative damage by reducing lipid per-L. V. Costa-Lotufo · M. O. MoraesDepartamento de Fisiologia e Farmacologia, oxidation and DNA damage as evaluated by normalFaculdade de Medicina, Universidade Federal do Ceará, and modiWed comet assay using DNA glycosylases. OurFortaleza, Ceará, Brazil results suggest that biXorin cellular eVects are concentration dependent. At lower concentrations, biXorin has signiWcantD. J. Moura · M. S. Machado · T. N. Guecheva · I. Villela ·B. F. Immich · J. SaY · J. A. P. Henriques (&) antioxidant and protective eVects against the cytotoxicity,Centro de Biotecnologia e Departamento de Biofísica, genotoxicity, mutagenicity, and intracellular lipid peroxida-Prédio 43422, Laboratório 210, Universidade Federal do Rio tion induced by H2O2 in yeast and mammalian cells, whichGrande do Sul, Campus do Vale, Av. Bento Gonçalves 9500, can be attributed to its hydroxyl radical-scavenging property.Bairro Agronomia, CEP 91501-970 Porto Alegre, RS, Brazile-mail: However, at higher concentrations, biXorin is cytotoxic and genotoxic.R. M. RosaLaboratório de Genética Toxicológica, Keywords BiXorin · Naphthoquinone · Yeast · V79 cells ·Universidade Luterana do Brasil, Canoas, RS, Brazil Antimutagenic activity · Salmonella/microsome assayA. M. Fonseca · T. L. G. LemosDepartamento de Química Orgânica e Inorgânica,Universidade Federal do Ceará, Fortaleza, Ceará, Brazil IntroductionJ. SaYDepartamento de Ciências Básicas da Saúde, Mass screening programs of natural products have identi-Universidade Federal de Ciências da Saúde de Porto Alegre, Wed the quinone moiety as an important pharmacophoricPorto Alegre, RS, Brazil unit for biological activity (Liu et al. 2004). Quinones areJ. A. P. Henriques secondary metabolic products of several microorganismsInstituto de Biotecnologia, and plants playing a pivotal role in energy metabolism. TheUniversidade de Caxias do Sul, Caxias do Sul, RS, Brazil wide spectrum of the biological activity of many quinones 123
  2. 2. Arch Toxicol O USA). Methyl methanesulfonate (MMS), hydrogen perox- ide (H2O2), thiobarbituric acid (TBA), trichloroacetic acid O (TCA), cycloheximide, xanthine oxidase, hypoxanthine, salicylic acid, amino acids (L-histidine, L-threonine, L-methionine, L-tryptophan, L-leucine, L-lysine), and nitrogen bases (adenine and uracil) were purchased from O Sigma (St. Louis, MO, USA). Yeast extract, yeast nitrogen base, Bacto-peptone, and Bacto-agar were obtained fromFig. 1 Chemical structure of biXorin Difco Laboratories (Detroit, MI, USA). Oxoid nutrient broth No. 2 was obtained from Oxoid USA, Inc. (Maryland,is frequently related to their ability to generate reactive USA). Low-melting point agarose and normal agarose wereoxygen species (ROS) via redox cycling mechanism (Kim obtained from Invitrogen (Carlsbad, CA, USA). Giemsaet al. 2008). stain was from Merck (Darmstadt, Germany). Formamido- Many eYcient antineoplastic drugs are either quinones, pyrimidine DNA-glycosylase (FPG, also known as MutM)quinonoid derivatives (quinolones, genistein), or drugs that and endonuclease III (EndoIII, also known as Nth) werecan easily be converted to quinones by in vivo oxidation, obtained from BioLabs (New England, USA). The S9such as etoposide (Asche 2005). These anticancer agents fraction, prepared from the livers of Sprague–Dawley ratsare eVective inhibitors of DNA topoisomerase, contributing pre-treated with the polychlorinated biphenyl mixturefor their anticancer activity (Asche 2005). In addition to Aroclor 1254, was purchased from Moltox Inc. (Boone,their antitumor activities, quinones have also been studied NC, USA). All other reagents were of analytical to their wound-healing, anti-inXammatory, antiparasitic,and cytotoxic activities (Kim et al. 2008; Lemos et al. BiXorin isolation and identiWcation2007; Vasconcellos et al. 2005; Vasconcellos et al. 2007). Literature studies mention that the biological activities BiXorin was puriWed from the roots of Capraria biXora col-of quinones are centered in their ortho or para-quinonoid lected in a plantation in Fortaleza, Ceará, Brazil. The air-moiety (Monks and Jones 2002). BiXorin (Fig. 1) is a dried and powdered roots (450 g) were extracted twice with1,4 o-naphthoquinone isolated from Capraria biXora L., a petroleum ether and vacuum-Wltered, yielding a purple-redperennial herb widely distributed in several countries of precipitate (100 mg). The purple-red solid presented a melt-Tropical America (Fonseca et al. 2003). Previous studies ing point at 167–168°C, and was soluble in chloroform,demonstrated that biXorin inhibits both in vitro and in vivo ethyl acetate, and acetone. The chemical structure wastumor cell growth but diVerently from other cytotoxic determined by spectroscopic analyses (IR, UV, and 1NMR)naphthoquinones, biXorin does not induce oxidative stress and assignments of 1H and 13C NMR spectra, as described(Vasconcellos et al. 2005, 2007). In fact, it has potent anti- (Fonseca et al. 2003). The biXorin puriWcation was evalu-oxidant activity against the autoxidation of oleic acid in a ated through silica gel column chromatography methodwater/alcohol system assay (Vasconcellos et al. 2005). using gradient mixtures of 0–100% ethyl acetate—light The present study investigated the cytotoxic, genotoxic, petroleum as eluent, and was always above 95%.mutagenic, and protective eVects of biXorin in a prokaryotic For all treatments, dimethylsulfoxide (DMSO) stockmodel, Salmonella tiphymurium, and in two eukaryotic solutions of biXorin were prepared immediately prior to usemodels, yeast Saccharomyces cerevisiae and V79 cells, a so that the Wnal DMSO concentration in the medium neverpermanent cell line derived from Chinese hamster lung exceeded 0.2%. The negative control was exposed to anWbroblast. Furthermore, the antioxidant and antigenotoxic equivalent concentration of this solvent.potential of this natural quinone was studied using in vitrochemical systems, as well as biological systems, which Salmonella/microsome mutagenicity assayincluded yeasts and cultured mammalian cells. Salmonella typhimurium TA98, TA97a, TA100, and TA102 were kindly provided by B. M. Ames (University ofMaterials and methods California, Berkeley, CA, USA). Mutagenicity was assayed by the preincubation procedure. The S9 metabolic activa-Chemicals tion mixture (S9 mix) was prepared according to Maron and Ames (1983). BrieXy, 100 L of test bacterial cul-Dulbecco’s modiWed Eagle medium (DMEM), fetal bovine tures (1–2 £ 109 cells/mL) were incubated at 37°C withserum (FBS), trypsin–EDTA, L-glutamine, and antibiotics diVerent amounts of biXorin dissolved in DMSO in thewere purchased from Gibco BRL (Grand Island, NY, presence or absence of S9 mix for 20 min, without shaking.123
  3. 3. Arch ToxicolSubsequently, 2 mL of soft agar (0.6% agar, 0.5% NaCl, mutagenic evaluation under growth conditions, with diVer-50 M histidine, 50 M biotin, pH 7.4, 42°C) was added to ent concentrations of biXorin at 30°C for 3 h. In order tothe test tube and poured immediately onto a plate of mini- verify biXorin antimutagenic activity, cells were pre-treatedmal agar (1.5% agar, Vogel-Bonner E medium, containing with biXorin at 30°C for 3 h. Cells were then washed and2% glucose). AXatoxin B1 (1 g/plate) was used as positive treated with H2O2 (4 mM) at 30°C in the dark with agitationcontrol for all strains (in the presence of metabolic for 1 h. After treatment, appropriate cell dilutions wereactivation with S9 mix), 4-nitroquinoline-oxide (4-NQO, plated onto SC plates to determine cell survival, and cell0.5 g/plate) for TA97a, TA98, and TA102, and sodium suspension aliquots were plated on the appropriate omis-azide (1 g/plate) for TA100 (absence of S9 mix). Plates sion media, lacking lysine (SC-lys), histidine (SC-his), orwere incubated in the dark at 37°C for 48 h before counting homoserine (SC-hom). While his1-7 is a non-suppressiblethe revertant colonies. missense allele, and reversions result from mutation at the locus itself, lys1-1 is a suppressible ochre nonsense mutantSaccharomyces cerevisiae: strains, media, and growth allele, which can be reverted either by locus-speciWc or by forward mutation in a suppressor gene. True reversions andThe following S. cerevisiae strains were used: XV185-14c forward (suppressor) mutations at the lys1-1 locus were(MAT ade2-2 arg4-17 his1-7 lys1-1 trp5-48 hom3-10) diVerentiated according to Schuller and von Borstel (1974),(von Borstel et al. 1971) in the mutagenicity assay, and where the reduced adenine content of the SC-lys mediumXS2316 (MATa/ his1-1/his1-1 leu1-1/leu1-12+/cyh2 shows locus reversions as red colonies, and suppressortrp5-48+/met13) in the recombinogenicity assay (Machida mutations as white colonies. It is believed that hom3-10and Nakai 1980). Media, solutions, and buVers were pre- contains a frameshift mutation due to its response to apared according to Burke et al. (2000). Complete YPD series of diagnostic mutagens.medium, containing 0.5% yeast extract, 2% bacto-peptone,and 2% glucose, was used for routine growth of the yeast Detection of induced mitotic recombination in XS2316cells. For plates, the medium was solidiWed with 2% bacto- diploid strainagar. The minimal medium (MM) contained 0.67% yeastnitrogen base with no amino acids, 2% glucose, and 2% Suspensions of exponential cells of diploid strainbacto-agar was supplemented with the appropriate amino (2 £ 107 cells/mL) were incubated for 3 h at 30°C in PBS,acids. The synthetic complete medium (SC) was MM sup- which contained diVerent biXorin concentrations. When theplemented with 2 mg adenine, 2 mg arginine, 5 mg lysine, protective eVect was investigated, cells were incubated with1 mg histidine, 2 mg leucine, 2 mg methionine, 2 mg uracil, biXorin for 3 h, washed, and exposed to H2O2 (4 mM) at2 mg tryptophan, and 24 mg threonine per 100 mL MM. 30°C in the dark with agitation for 1 h. After treatment,Stationary-phase cultures were obtained by inoculation of cells were diluted, plated on three diVerent media (SC,an isolated colony into liquid YPD for 48 h. Exponential SC-leu and SC+cycloheximide 0.2%), and incubated at 30°Cphase cultures were obtained by inoculating 5 £ 106 cells/mL for 5–7 days. Colonies grown in SC medium indicated cellof a stationary-phase YPD culture into fresh YPD survival and colonies grown in SC-leu and SC+cyh weremedium for 3 h. Before biXorin treatment, cells were har- scored for intragenic mitotic recombination (gene conver-vested, washed twice with phosphate-buVered saline solu- sion) and intergenic recombination (crossing-over), respec-tion (PBS; Na2HPO4, and NaH2PO4; 20 mM; pH 7.4), and tively. In order to measure the exact frequency of reciprocalbudding cell percentage in each culture were determined. crossing-over, it was necessary to eliminate the possibilityAll yeast assays were repeated at least four times, and plat- that some cycloheximide-resistant colonies were produceding was performed in triplicate for each dose. The 4-NQO by reversion at the CYH2 locus, as well as by chromosomeand H2O2 was used as positive control. The appropriate VII monosomy. Therefore, cycloheximide-resistant colo-concentration of H2O2 was determined by survival assay, nies were replica-plated on a series of SC-lys, SC-met andaccording to the diVerential sensitivity of used strain, and SC-ade media plates, with subsequent screening of thesesub-lethal concentration of the oxidant was used for all sub- markers for cyh2.sequent experiments. V79 mammalian cell culture and treatmentsDetection of reverse and frameshift mutationin XV185-14c haploid strain V79 cells were grown as monolayers under standard condi- tions in DMEM supplemented with 10% heat-inactivatedA suspension of 2 £ 108 cells/mL in stationary or exponen- FBS, 0.2 mg/mL L-glutamine, 100 IU/mL penicillin/tial growth phase was incubated in PBS for mutagenic eval- 100 g/mL streptomycin; and kept in tissue-culture Xasksuation under non-growth conditions, or in liquid YPD for at 37°C in a humidiWed atmosphere containing 5% CO2. 123
  4. 4. Arch ToxicolOne day prior to treatment, cells were harvested by treat- 10% DMSO, pH 10.0) at 4°C for at least 1 h in order toment with 0.15% trypsin-0.08% EDTA in PBS, and seeded remove cell membranes, leaving DNA as “nucleoids”. Ininto 5 mL DMEM complete medium in a 25-cm2 Xask. the modiWed Comet assay, slides were removed from theBiXorin was added to the FBS-free medium to achieve the lysis solution, washed three times in enzyme buVer (40 mMdiVerent designed concentrations, and the cells were incu- Hepes, 100 mM KCl, 0.5 Mm Na2EDTA, 0.2 mg/mL BSA,bated at 37°C for 3 h in a humidiWed atmosphere containing pH 8.0), and incubated with FPG (30 min 37°C) or EndoIII5% CO2. Oxidative challenge with 150 M H2O2 was car- (30 min 37°C). Slides were placed in a horizontal electro-ried out for 1 h in the dark in FBS-free medium. All experi- phoresis unit and incubated with fresh buVer solutionments with V79 cells were carried out at least three times. (300 mM NaOH, 1 mM EDTA, pH 13.0) at 4°C for 20 minTreatments of cells with MMS or H2O2 were used as posi- in order to allow DNA unwinding and the expression oftive control. alkali-labile sites. Electrophoresis was conducted for 20 min at 25 V (94 V/cm). All the above steps were per-Cytotoxicity evaluation by colony-forming ability formed under yellow light or in the dark in order to prevent(clonogenic assay) in V79 cells additional DNA damage. Slides were then neutralized (0.4 M Tris, pH 7.5) and stained using the silver stainingAfter biXorin treatment, cells (500 cells/per Xask) were protocol described by Nadin et al. (2001). After the stainingwashed and incubated in complete medium at 37°C in a step, gels were left to dry at room temperature overnighthumidiWed atmosphere containing 5% CO2 for 7 days. Col- and analyzed under a light microscope. One hundred cellsonies were Wxed with 3% formaldehyde, stained with 1% (50 cells from each of two replicate slides for each treat-crystal violet, counted, and the survival expressed as a per- ment) were selected and analyzed for DNA migration.centage relative to the negative control treatment. These cells were visually scored according to tail length into Wve classes: (1) class 0: undamaged, without a tail; (2)Measurement of lipid peroxidation in V79 cells class 1: with a tail shorter than the diameter of the head nucleus; (3) class 2: with a tail length 1–2£ the diameter ofThe extent of biXorin-induced lipid peroxidation was evalu- the head; (4) class 3: with a tail longer than 2£ the diameterated by the reaction of thiobarbituric acid (TBA) with mal- of the head; and (5) class 4: comets with no heads. Theondialdehyde (MDA), a product resulting from lipid value of damage index was assigned to each sample. Dam-peroxidation. The assays were carried out according to age index (DI) is an arbitrary score based on the number ofSalgo and Pryor (1996), with minor modiWcations. After cells in the diVerent damage classes, which are visuallytreatment, V79 cells (3 £ 106 cells) were lysed with Tris– scored by measuring DNA migration length and the amountHCl (15 mM for 1 h), 2 mL of 0.4 mg/mL TCA, and of DNA in the tail. DI ranges from 0 (no tail: 100 cells £ 0)0.25 M HCl, followed by incubation with 6.7 mg/mL TBA to 400 (with maximum migration: 100 cells £ 4) (Burlinsonat 100°C for 15 min. The mixture was centrifuged at 750£g et al. 2007). When selecting cells, edges and cells aroundfor 10 min and the supernatant was used for analyses. As air bubbles were disregarded.TBA reacts with products of lipid peroxidation other thanMDA, results were expressed in terms of thiobarbituric Hypoxanthine/xanthine oxidase assayreactive species (TBARS), as determined by their absor-bance at 532 nm. Hydrolyzed tetramethoxypropane (TMP) The in vitro assay based on a previously described methodwas used as standard. The results were normalized for pro- (Owen et al. 1996) was used to determine the biXorin anti-tein content (Lowry et al. 1951). oxidant potential. BrieXy, biXorin was dissolved in the assay buVer (1.0 mL hypoxanthine, Fe(III), EDTA, and sal-Comet assay in V79 cells icylic acid) at a concentration of 5.0 mg/mL and appropri- ately diluted (in triplicate) in assay buVer to a Wnal volumeAlkaline comet assay was performed as previously of 1.0 mL, resulting in a 0.025–2.0 mg/mL concentrationdescribed (Collins 2004). After treatment, cells were range. A 5 L aliquot of xanthine oxidase (18 mU) dis-washed with ice-cold PBS, trypsinized, and resuspended in solved in 3.2 mol/L (NH4)2SO4 was added to trigger thecomplete medium. Then, 20 L of cell suspension reaction. The sample tubes were incubated at 37°C for 3 h.(3 £ 106 cells/mL) was dissolved in 0.75% low-melting A 30 L aliquot of the reaction mixture was analyzed bypoint agarose, and immediately spread onto a glass micro- HPLC using chromatographic conditions, as described byscope slide pre-coated with a layer of 1% normal melting Owen et al. (1996). Chromatographic analysis was carriedpoint agarose. Agarose was allowed to set at 4°C for 5 min. out using a gradient based on methanol/water/acetic acidSlides were then incubated in ice-cold lysis solution (2.5 M with a BondaPak C18 reverse phase column (Waters), andNaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100, and detection at 325 nm. The HPLC equipment included a 2695123
  5. 5. Arch Toxicolseparation module and UV detector 2487 (Waters). The assay. The test-substance was considered toxic if the muta-amount of dihydroxyphenol (2,5-dihydroxybenzoic acid genic index (colony counts on the test plate/average countsand 2,3-dihydroxybenzoic acid) produced by the reaction of on the negative control plates; MI) value was lower thansalicylic acid with the produced hydroxyl radicals (OH·) 0.60 in at least two of the tested concentrations. The resultswas determined based on the standard curves of the respec- of the range-Wnder experiment were used to deWne the dosetive dihydroxyphenols. range to be applied in the mutagenicity test, which concen- tration range should be the highest allowed by the toxicityStatistical analysis or the solubility of the test substance. The range Wnding results indicate cytotoxicity in concentrations higher thanMutagenicity data in bacteria were analyzed using Salmo- 333.3 g/plate (data not shown), and therefore, the concen-nel software. A compound was considered positive for trations of 66.6, 133.5, 200.1, 266.7, and 333.3 g per platemutagenicity only when: (a) the number of revertants was were used in the mutagenicity assay. No biXorin mutage-at least the double of the spontaneous yield (MI ¸ 2— nicity eVect was detected, even at the highest concentration,mutagenic index (MI): number of induced colonies in the on TA97a (detects frameshift mutation in DNA targetsample/number of spontaneous colonies in the negative –C–C–C–C–C–C–; +1 cytosine), TA98 (detects frameshiftcontrol); (b) a signiWcant response was determined by anal- mutation in DNA target –C–G–C–G–C–G–C–G–), orysis of variance (P · 0.05); and (c) a reproducible positive TA100 (base-pair substitution mutation results from thedose–response (P · 0.01) was present. An eVect was con- substitution of a leucine [GAG] by a proline [GGG]), in thesidered cytotoxic when MI · 0.6. absence or presence of metabolic activation. Also, biXorin Data from assays with S. cerevisiae and in V79 cells did not show mutagenic eVect in TA102 cells that detectwere expressed as means and standard deviations and sta- oxidative and alkylating mutagens by an ochre mutationtistically analyzed by one-way ANOVA with Tukey’s mul- TAA in the hisG gene, which can be reverted by all six pos-tiple comparison test, using GraphPad Prism 4.00 Software. sible base-pair substitutions (Tables 1, 2).DiVerences were considered signiWcant when P < 0.05. Evaluation of mutagenesis, antimutagenesis, and recombinogenesis in yeastResults BiXorin induced dose-dependent cytotoxic eVects both inEvaluation of mutagenesis in bacteria stationary and in exponential phase cultures of the haploid yeast S. cerevisiae (Fig. 2). Therefore, we used sub-cyto-BiXorin was Wrst tested for TA100 strain toxicity at concen- toxic concentrations of this o-naphthoquinone (rangingtrations of 8–2,000 g/plate in Salmonella/microsome from 31.25 to 500 g/mL) to verify its mutagenic eVect onTable 1 Induction of his+ revertants in TA98 and 97a S. typhimurium frameshift strains by biXorin with and without metabolic activation (S9 mix)S. typhimurium TA98 TA97a ¡S9 +S9 ¡S9 +S9Substance Dose Rev./platea MIb Rev./plate MI Rev./plate MI Rev./plate MI ( g/plate)DMSOc 22.00 § 2.65 – 32.33 § 3.06 – 44.33 § 11.24 – 66.00 § 10.58 –PCd 383.66 § 93.32*** 17.43 724.00 § 142.00*** 22.39 875.66 § 471.46*** 19.75 1,630.50 § 265.16*** 24.70BiXorin 66.60 21.33 § 4.16 0.96 46.67 § 2.31 1.44 50.00 § 5.66 1.13 102.33 § 10.79 1.55 133.50 21.67 § 7.37 0.98 34.33 § 6.03 1.06 39.00 § 1.41 0.88 82.33 § 8.62 1.25 200.10 25.00 § 6.08 1.13 32.67 § 5.03 1.01 21.00 § 14.80 0.47 79.33 § 17.90 1.20 266.70 21.00 § 2.00 0.95 27.67 § 1.53 0.85 28.00 § 1.00 0.63 71.50 § 2.12 1.08 333.30 23.00 § 6.08 1.04 41.67 § 12.50 1.28 48.00 § 8.49 1.08 87.00 § 33.05 1.32a Number of revertants/plate: mean of three independent experiments § SDb MI: mutagenic index (number of his+ induced colonies in the sample/number of spontaneous his+ colonies in the negative control)c NC negative control: dimethyl sulfoxide (DMSO 25 L) used as a solvent for biXorind PC positive control: (¡S9) 4-nitroquinoline 1-oxide (0.5 g/plate); (+S9) aXatoxin B1 (1 g/plate)*** SigniWcant diVerence when compared to the negative control (solvent) at P < 0.001 123
  6. 6. Arch ToxicolTable 2 Induction of his+ revertants in TA100 and TA102 S. typhimurium base-substitution strains by biXorin with and without metabolicactivation (S9 mix)S. typhimurium TA100 TA102 ¡S9 +S9 ¡S9 +S9Substance Dose Rev./platea MIb Rev./plate MI Rev./plate MI Rev./plate MI ( g/plate)DMSOc 116.33 § 19.09 – 136.67 § 5.03 – 368.67 § 72.92 – 460.00 § 52.92 –PCd 1,922.00 § 402.07*** 16.52 1,530.33 § 113.50*** 11.19 2,115.60 § 502.14*** 5.73 1,934.33 § 279.01*** 4.20BiXorin 66.60 123.33 § 8.50 1.06 144.00 § 17.09 1.05 331.33 § 38.70 0.90 493.33 § 77.18 1.07 133.50 98.00 § 20.00 0.84 138.00 § 22.54 1.01 329.33 § 63.79 0.89 565.33 § 74.22 1.23 200.10 104.67 § 8.08 0.90 127.33 § 20.82 0.93 425.33 § 16.17 1.15 516.67 § 122.84 1.12 266.70 106.00 § 10.82 0.91 166.33 § 12.66 1.22 384.00 § 24.98 1.04 484.00 § 52.46 1.05 333.30 131.33 § 29.14 1.13 130.67 § 37.17 0.96 378.67 § 59.37 1.02 429.33 § 84.13 0.93a Number of revertants/plate: mean of three independent experiments § SDb MI: mutagenic index (number of his+ induced colonies in the sample/number of spontaneous his+ colonies in the negative control)c NC negative control: dimethyl sulfoxide (DMSO 25 L) used as a solvent for biXorind PC positive control: (¡S9) 4-nitroquinoline 1-oxide (0.5 g/plate) for TA102 or sodium azide (1 g/plate) for TA100; (+S9) aXatoxin B1 (1 g/plate)for all strains*** SigniWcant diVerence when compared to the negative control (solvent) at P < 0.001 (one-way ANOVA with Tukey’s multiple comparison test) 100 of crossing-over and gene conversion at the concentration range employed. As observed in mutagenicity assay, the pre-treatment with biXorin at concentrations of 33.25– 250 g/mL also prevented the recombinogenic events % survival induced by H2O2 (Table 5, lower panel). 10 BiXorin cytotoxicity and protective eVects against Stationary phase hydrogen peroxide in V79 cells Exponential phase BiXorin did not show signiWcant cytotoxic eVects on V79 1 cells in the clonogenic survival assay at concentrations 0 125 250 375 500 1000 1500 2000 lower than 10 g/mL (Fig. 3). The pre-treatment at non- Biflorin (μg/mL) toxic concentrations of 5 and 10 g/mL biXorin preventsFig. 2 BiXorin-induced cytotoxicity in XV185-14c haploid strain H2O2 toxicity (Fig. 3).treated during stationary-growth phase (open square) and exponential- The degree of lipid peroxidation in V79 cells can begrowth phase (open circle) in PBS after 3 h of treatment observed in Fig. 4. BiXorin itself does not induce an increase in TBARS levels at concentration range ofthe XV-18514c strain of S. cerevisiae. BiXorin did not 1–10 g/mL. In addition, the pre-treatment with biXorininduce locus non-speciWc, locus-speciWc, or frameshift reduces the extent of H2O2-induced lipid peroxidation, sug-mutations in this strain in none of the concentrations and gesting that this molecule can present antioxidant activitygrowth phases employed (Table 3). Stationary cells were (Fig. 4).used to verify an intracellular protective eVect of biXorinagainst H2O2-induced oxidative stress. When compared to BiXorin genotoxicity and antigenotoxic potential againstthe H2O2 treatment, biXorin pre-treatment enhanced cell hydrogen peroxide in V79 cellssurvival and decreased mutation frequency at concentra-tions of 31.25, 62.5, and 125 g/mL (Table 4). A protective The in vitro alkaline (pH > 13) comet assay detects DNA-eVect of 250 g/mL biXorin on mutagenesis induction but strand breaks and alkali-labile sites and can be performednot on the H2O2-induced cytotoxicity was observed. with a variety of cell types, including V79 cells (Collins The recombinogenic eVects of biXorin were investigated 2004). Our results showed that biXorin does not generatein XS2316 diploid yeast strain (Table 5, upper panel). DNA-strand breaks at doses lower than 10 g/mL in V79BiXorin did not induce signiWcant recombinogenic events cells. At cytotoxic concentrations of 20 and 30 g/mL123
  7. 7. Arch ToxicolTable 3 Reversion of point mutation for (his1-7), ochre allele (lys1-1), and frameshift mutations (hom3-10) in haploid XV185-14c strain ofS. cerevisiae after biXorin treatment at 30°C for 3 h Concentration Survival HIS1 revertants/ LYS1 revertants/ HOM3 revertants/ ( g/mL) (%) 107 survivorsa 107 survivorsb 107 survivorsaTreatment of stationary-phase cells under non-growth conditionsDMSOd 100 12.1 § 2.0c 1.3 § 0.7c 2.2 § 0.7c e4NQO 0.5 52.0 163.0 § 11.6*** 15.1 § 2.1*** 24.7 § 0.6***BiXorin 0 100 10.0 § 0.0 3.0 § 0.9 2.5 § 0.1 31.25 98.6 7.4 § 1.2 1.3 § 0.4 1.3 § 0.7 62.5 95.8 13.7 § 2.5 1.7 § 0.2 1.7 § 0.4 125 88.5 14.1 § 1.9 2.5 § 0.1 1.2 § 0.0 250 71.0 18.7 § 0.9 1.8 § 0.6 1.6 § 1.0 500 50.2 9.7 § 3.2 2.4 § 0.9 2.7 § 1.0Treatment of exponential-phase cells under non-growth conditionsDMSOd 100 18.7 § 0.9 8.1 § 1.3 6.7 § 0.3 e4NQO 0.5 41.4 182.5 § 8.0*** 34.2 § 11.1*** 48.1 § 0.7***BiXorin 0 100 10.0 § 1.0 7.8 § 2.2 7.6 § 0.4 31.25 93.6 9.2 § 1.9 5.2 § 0.8 6.5 § 0.7 62.5 87.5 10.7 § 2.3 5.5 § 1.2 7.3 § 0.9 125 87.4 11.2 § 1.2 6.3 § 0.9 7.4 § 1.5 250 83.1 11.3 § 1.0 6.8 § 0.1 7.2 § 0.4 500 55.7 10.0 § 0.4 5.1 § 0.1 6.0 § 2.0Treatment under growth conditionsDMSOd 100 10.3 § 0.9 3.0 § 0.5 4.8 § 0.9 e4NQO 0.5 51.4 160.5 § 21.3*** 24.9 § 0.9*** 26.3 § 2.3***BiXorin 0 100 11.0 § 0.6 3.1 § 0.9 3.8 § 0.1 31.25 98.5 11.5 § 2.8 3.9 § 1.0 2.8 § 0.7 62.5 89.6 13.1 § 3.3 4.6 § 0.7 3.5 § 0.4 125 88.0 12.4 § 1.6 4.9 § 2.2 4.9 § 1.7 250 60.9 10.1 § 1.0 3.4 § 0.6 4.8 § 0.1 500 55.4 14.4 § 0.6 5.1 § 1.4 3.9 § 0.0a Locus-speciWc revertantsb Locus non-speciWc revertantc Mean and standard deviation of three independent experimentsd Negative control (dimethyl sulfoxide 0.2% used as solvent)e Positive control, 4-nitroquinoleine-N-oxide 0.5 g/mL*** SigniWcantly diVerent when compared to the negative control (solvent) at P < 0.001 (one-way ANOVA with Tukey’s multiple comparisontest)(Fig. 3), it induces signiWcant DNA damage (Fig. 5a). certain damaged bases and create breaks. In the presentBiXorin pre-treatment at concentrations below 10 g/mL study, we used FPG, that is speciWc for oxidized purines,signiWcantly reduced the H2O2-induced DNA damage including 8-oxo-7,8-dihydroguanine, 2,6-diamino-4-hydroxy-(Fig. 5b). The pre-treatment at concentration of 30 g/mL 5-formamidopyrimidine, and 4,6-diamino-5-formamidopy-biXorin (that induces DNA damage by itself, Fig. 5a) rimidine, and other ring-opened purines, as well as EndoIII,slightly enhances the DNA migration after H2O2 treatment that recognizes oxidized pyrimidines, including thymine(Fig. 5b). glycol and uracil glycol. The levels of EndoIII and FPG While the alkaline version of the comet assay detects sensitive sites were calculated as the score obtained withDNA single- and double-strand breaks and alkali-labile enzymes minus the score without enzymes (only withsites, the modiWed comet assay is more speciWc than this enzyme buVer) after treatment with biXorin or H2O2. Thestandard method. In the modiWed version, there is an incu- results indicate that biXorin does not induce signiWcantbation step with lesion-speciWc enzymes, which recognize oxidative damage at concentration 2.5–30 g/mL (Fig. 6a). 123
  8. 8. Arch ToxicolTable 4 Reversion of point mutation for (his1-7), ochre allele (lys1-1), and frameshift mutations (hom3-10) in haploid XV185-14c strain ofS. cerevisiae after hydrogen peroxide treatment in stationary-phase cells pre-incubated with biXorin for 3 h under non-growth conditions Concentration Survival HIS1 revertants/ LYS1 revertants/ HOM3 revertants/ (%) 107 survivors 107 survivorsb 107 survivorsaDMSOd 100 10.3 § 0.9e 3.0 § 0.5e 4.8 § 0.9e eH2O2 4 mM 41.0 41.4 § 1.3*** 37.7 § 1.8*** 36.0 § 0.9***BiXorin pre-treatment 31.25 g/mL 66.4 7.4 § 1.8*** 3.2 § 0.4*** 4.5 § 1.4*** plus H2O2 4 mM 62.5 g/mL 70.3 7.1 § 2.0*** 3.6 § 0.4*** 4.9 § 1.8*** 125 g/mL 65.4 9.2 § 0.5*** 4.1 § 0.4 *** 5.1 § 1.1*** 250 g/mL 40.8 14.1 § 1.6*** 5.5 § 3.6*** 7.9 § 1.0*** 500 g/mL 41.9 34.4 § 7.9 43.4 § 2.7 35.6 § 3.7a Locus-speciWc revertantsb Locus non-speciWc revertantc Mean and standard deviation of three independent experimentsd Negative control (dimethyl sulfoxide 0.2% used as solvent)e Positive control, challenge mutagen—H2O2*** SigniWcantly diVerent at P < 0.001 (one-way ANOVA with Tukey’s multiple comparison test); Hydrogen peroxide treatment was comparedto the negative control, pre-treatments with biXorin were compared to the positive control (H2O2)Table 5 Induction of crossing-over (cyh2) and gene conversion (leu1-1/leu1-12) in the diploid strain XS2316 of S. cerevisiae after biXorin treat-ment during exponential growth phase and the eVects of this pre-treatment on hydrogen peroxide induced recombinogenesis Concentration Survival Crossing over/ Gene conversion/ (%) 105 survivors 105 survivorsDMSOb 100 9.1 § 0.8a 1.5 § 0.2 c4NQO 0.5 g/mL 67.0 185.0 § 26.6*** 49.0 § 3.0***BiXorin 31.25 g/mL 98.5 9.6 § 3.3 1.7 § 0.4 62.5 g/mL 96.7 10.1 § 2.7 1.2 § 0.6 125 g/mL 94.0 10.8 § 1.0 1.5 § 0.6 250 g/mL 91.0 9.7 § 3.1 1.7 § 0.3 500 g/mL 89.2 8.5 § 3.8 1.9 § 0.8 1.0 mg/mL 86.5 12.4 § 0.4 1.3 § 0.6 2.0 mg/mL 57.1 10.1 § 1.3 1.8 § 0.7 dH2O2 4 mM 51.7 106.5 § 18.4*** 43.4 § 9.0***BiXorin pre-treatment plus H2O2 4 mM exposure 31.25 g/mL 76.8 21.5 § 3.9AAA 8.1 § 0.9AAA 62.5 g/mL 78.3 23.0 § 4.6AAA 10.4 § 1.1AAA AAA 125 g/mL 71.9 33.1 § 9.3 9.9 § 1.3AAA AAA 250 g/mL 63.0 55.0 § 6.5 12.0 § 3.3AAA 500 g/mL 49.3 82.0 § 8.4 45.1 § 4.1a Mean and standard deviation of three independent experimentsb Negative control (dimethyl sulfoxide 0.2% used as solvent)c Positive control, 4-nitroquinoleine-N-oxide (0.5 g/mL)*** SigniWcant diVerence when compared to the negative control (solvent) at P < 0.00,1AAA SigniWcant diVerence when compared to the challenge mutagen—H2O2 at P < 0.001, as determined by one-way ANOVA with Tukey’s mul-tiple comparison testThese results suggest that the elevated DNA-strand breaks more, the DNA migration was also evaluated after incuba-observed after treatment at 20 and 30 g/mL in the alkaline tion with these enzymes in cells pre-treated with biXorincomet assay (Fig. 5a) do not have oxidative origin. Further- and then exposed to H2O2. The extent of oxidative damage123
  9. 9. Arch Toxicol 100 75 Survival (%) # * # 50 25 *** 0 SO 0 .0 20 30 0 0 .0 0 μM 5. 1. 5. 1. 10 10 M 0 D 15 2 2O Biflorin ( μg/mL) Biflorin ( μg/mL) pre-treatment H plus H2O2 exposureFig. 3 Clonogenic survival of V79 cells after treatment with biXorin oxide, used as solvent) at p < 0.05; ***p < 0.001; #p < 0.05, whenfor 3 h or pre-treated with biXorin for 3 h and challenged with H2O2 biXorin pretreated cells were compared to cells exposed to challenge(150 M) for 1 h at 37°C in the dark, at the indicated concentrations. mutagen—H2O2 (150 M). Statistical analyses were carried out usingData are presented as mean § SD of three independent experiments. one-way ANOVA and Tukey’s multiple comparison test*SigniWcantly diVerent when compared to the control (dimethyl sulf- 60 (nmol MDA/mg protein) 50 MDA equivalents 40 * 30 # 20 # # 10 0 SO 0 .0 0 0 .0 0 5. 1. 5. μM 1. 10 10 M D 0 2 15 2O Biflorin ( μg/mL) Biflorin ( μg/mL) pre-treatment H plus H 2O2 exposureFig. 4 Determination of thiobarbituric acid reactive substances used as solvent) at p < 0.05; # p < 0.05, when biXorin pretreated cells(TBARS) in V79 cells pretreated with biXorin at the indicated concen- were compared to cells exposed to H2O2 (150 M). Statistical analysestrations for 3 h and subsequently exposed to H2O2 (150 M). Data are were carried out using one-way ANOVA and Tukey’s multiple com-presented as mean § SD of three independent experiments. *SigniW- parison testcantly diVerent when compared to the control (dimethyl sulfoxide,recognized by the lesion-speciWc enzymes signiWcantly hypoxanthine/xanthine oxidase assay. The decrease in totaldecreased after pre-treatment at low biXorin concentrations oxidation products as a function of the amount of biXorin(2.5, 5.0, and 10.0 g/mL), indicating that the antigenotoxic added to the assay is shown in Fig. 7, indicating that biXo-eVects of this compound can be related to its antioxidant rin presents signiWcant antioxidant capacity in a dose-properties (Fig. 6b). dependent manner.BiXorin free-radical scavenging ability DiscussionBiXorin antioxidant capacity was determined by monitoringthe production of hydroxylated benzoic acid (DHBA) Quinones are secondary metabolites found in several livingresulting from the attack of ROS on salicylic acid in the cells and are widely used as anticancer, antibacterial, or 123
  10. 10. Arch Toxicol A 350 A 100 DMSO biflorin 2.5 μg/mL 300 biflorin 5.0 μg/mL Oxidative damage score 80 biflorin 10 μg/mL biflorin 20 μg/mL Damage Index 250 biflorin 30 μg/mL 200 *** *** 60 150 *** 40 100 50 20 0 μM SO 5 0 .0 20 .0 0 2. 5. 10 30 M 40 EndoIII FPG D S M M Biflorin (μ g/mL) H2O 2 150 μM biflorin 2.5 μg/mL plus H2O 2 B 100 biflorin 5.0 μg/mL plus H2O 2 B 350 biflorin 10.0 μg/mL plus H2O 2 biflorin 20.0 μg/mL plus H2O 2 Oxidative damage score 300 80 biflorin 30.0 μg/mL plus H2O 2 250 Damage Index 60 200 ** 150 ** ** *** 40 *** 100 *** *** *** 20 50 *** 0 0 0 .0 20 .0 5 μM 5. 2. 10 30 EndoIII FPG 0 2 15 2O Fig. 6 DNA damage measured by comet assay in V79 cells exposed Biflorin( μg/mL) pre treatment H to biXorin at 37°C for 3 h with subsequent treatment with buVer, Endo- plus H2O2 150 μM III or FPG. The levels of EndoIII and FPG sensitive sites were calcu- lated as the score obtained with enzymes minus the score withoutFig. 5 a Evaluation of genotoxicity of biXorin treatment for 3 h using enzymes (buVered) after treatment with biXorin or H2O2. Means andcomet assay in V79 cells. b EVect of the pre-treatment with biXorin for standard deviation values were determined from an average of 1003 h on hydrogen peroxide (150 M) induced DNA damage in V79 cells per replicate, with three replicates per concentration. **SigniW-cells using comet assay. Bars represent the mean § SD of three inde- cantly diVerent when compared to the positive control (H2O2) atpendent experiments. ***DiVerences are signiWcant when compared to p < 0.01 and ***p < 0.001, as determined by one-way Anova andthe control in a (dimethyl sulfoxide, used as solvent) or in b to H2O2 at Tukey’s multiple comparison testp < 0.001; **p < 0.01 with Tukey’s multiple comparison test at one-way ANOVA–Tukey’s multiple comparison test has a dose-dependent cytotoxic eVect on diVerent eukary-antimalarial drugs. In fact, naphthoquinones, mainly repre- otic cells (Figs. 2, 3) but it is not mutagenic to bacteria andsented by -lapachone, are a promising group of com- yeast (Tables 1, 2, 3, 4 and 5). Employing TBARS determi-pounds with antitumor properties (Asche 2005). In nation and modiWed comet assay with ENDOIII and FPGaddition, quinone moieties are present in many drugs, such repair proteins, we found that biXorin did not induce oxida-as anthracyclines daunorubicin, doxorubicin, mitomycin, tive damage in V79 cells (Figs. 4, 6a). Moreover, biXorinmitoxantrones, and saintopin, which are clinically used in reduced DNA damage and mutation triggered by H2O2 intherapy of solid cancers (Verma 2006). In the present study, yeast (Tables 4, 5) and V79 cells (Figs. 5b, 6b), suggestingwe investigated the cytotoxic, genotoxic, mutagenic, and an antioxidant eVect. BiXorin protective capacity againstantimutagenic eVects of biXorin using diVerent approaches H2O2-induced oxidative stress was also conWrmed byon prokaryotic and eukaryotic cells systems. reductions in lipid peroxidation in V79 cells (Fig. 4). BiXorin is a 1,4-o-napthoquinone isolated from C. biX- Hydrogen peroxide is an important ROS compound that, inora that presents in vivo and in vitro antitumor eVects; combination with reduced trace metals such as iron or cop-however, its exact mechanism of action is still unknown per, is transformed via Fenton reaction into the highly reac-(Vasconcellos et al. 2007). We showed here that biXorin tive hydroxyl radicals (OH·), which causes damage to123
  11. 11. Arch Toxicol 100 2002). As biXorin does not induce frameshift mutation in bacteria and yeast in our study, we supposed that it is not intercalating agent as well. Another mechanism leading to 75 DNA-strand breaks induction is the interaction with DNA topoisomerase. It has been shown that -lapachone and derivatives can stimulate DNA double-strand breaks forma- % DHBA tion in the presence of topoisomerase II (Frydman et al. 50 1997). Similar mechanism of action can explain the strand breaks induction in V79 cells observed in comet assay after treatment at higher biXorin concentrations (Fig. 5a). The 25 topoisomerase II poisoning could be responsible for the cytotoxic eVect of biXorin, contributing for the anticancer activity. 0 In summary, our Wndings indicate that the cellular eVects 0.0 0.5 1.0 1.5 2.0 of biXorin are concentration dependent. At a range of low Biflorin (mg/mL) doses, the hydroxyl radical-scavenging property of biXorinFig. 7 Inhibition of the generation of reactive oxygen species by can contribute to the signiWcant antioxidant and protectivebiXorin in a hypoxanthine-xanthine oxidase system. Solvent (open eVects against the cytotoxicity, genotoxicity, mutagenicity,square) and biXorin (open circle). Mean § SD of four independent and intracellular lipid peroxidation induced by H2O2. Onexperiments the other hand, at higher concentrations biXorin is cyto- toxic, possibly due to its ability to induce DNA-strandvirtually all macromolecules (Halliwell and Gutteridge breaks.2000). Semiquinones, which often act reducing O2 to O2·¡(Fenton intermediary), in aqueous solution can favor O2·¡ Acknowledgments We are grateful to Dr. Marc François Richter for assistance with HPLC analysis. This research was supported by thedismutation reaction (Halliwell and Gutteridge 2000). The Brazilian Agencies FINEP, CNPq, BNB/FUNDECI, PRONEX,in vitro hypoxanthine-xanthine oxidase assay in this study GENOTOX-Royal Institute—Genotoxicity Laboratory, Universidadeshowed that biXorin can interfere with the Fenton reaction, Federal do Rio Grande do Sul and CAPES.acting as O2·¡ and/or OH· scavenger, suggesting that theprotection against H2O2–induced damage provided by ConXict of interest statement The authors declare that they have no conXicts of interest.biXorin is probably due to its ability to quench free radicals(Fig. 7). Interestingly, the protective eVects of biXorin againstoxidative mutagenesis in yeast and in V79 cultured cells Referencesoccurred only in the lower concentrations tested whereas at Asche C (2005) Antitumour quinones. Mini Rev Med Chem 5:449–467high concentration biXorin was cytotoxic and induced Burke D, Dawson D, Stearns T (2000) Methods in yeast genetics. ColdDNA-strand breaks as evidenced by the comet assay. The Spring Harbor Laboratory Press, OxfordDNA damage induction by biXorin could justify its Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY,anti-proliferative potential (Vasconcellos et al. 2007). Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A (2007)Therefore, it is possible that the toxicity observed in con- Fourth international workgroup on genotoxicity testing: results ofcentrations higher than 250 g/mL in haploid yeast strain the in vivo Comet assay workgroup. Mutat Res 627:31–35and 10 g/mL in V79 cells is a result of the DNA damage Collins AR (2004) The comet assay for DNA damage and repair: prin-induced by this 1,4-naphthoquinone. The biXorin-induced ciples, applications, and limitations. Mol Biotechnol 26:249–261 Fonseca AM, Pessoa ODL, Silveira ER, Monte FJQ, Braz-Filho R,DNA lesions in V79 mammalian cells, at concentrations Lemos TLG (2003) Total assignments of 1H and 13C spectra ofabove 10 g/mL (Fig. 5a), do not include oxidized bases biXorin and bis-biXorin from Capraria biXora. Magn Resonthat are substrate of the ENDOIII and FPG enzymes Chem 41:1038–1040(Fig. 6a). Also the absence of point mutation induction in Frydman B, Marton LJ, Sun JS, Neder K, Witiak DT, Liu AA, Wang HM, Mao Y, Wu HY, Sanders MM, Liu LF (1997) Induction ofbacteria and yeast reinforced our results obtained in V79 DNA topoisomerase II-mediated DNA cleavage by beta-lapach-cells, indicating that biXorin does not induce oxidative one and related naphthoquinones. Cancer Res 57:620–627damage. Most of the antitumour quinones belong to the Halliwell B, Gutteridge JMC (2000) Free radicals in biology and med-groups of DNA intercalating and/or alkylating agents and icine, 3rd edn. Oxford University Press, New York Kim JS, Rhee HK, Park HJ, Lee SK, Lee CO, Park Choo HY (2008)also can be eVective inhibitors of DNA topoisomerase Synthesis of 1-/2-substituted-[1, 2, 3]triazolo[4, 5-g]phthalazine-(Asche 2005). Previous study has reported that the 4, 9-diones and evaluation of their cytotoxicity and topoisomerase -lapachone does not intercalate into DNA (Pardee et al. II inhibition. Bioorg Med Chem 16:4545–4550 123
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