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Arch Toxicol
DOI 10.1007/s00204-010-0567-zGENOTOXICITY AND CARCINOGENICITY
Evaluation of the cytotoxic and antimutagenic eVects of biXorin, an antitumor 1,4 o-naphthoquinone isolated from Capraria biXora L
Marne 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. Henriques
Received: 19 April 2010 / Accepted: 9 June 2010© Springer-Verlag 2010
Abstract BiXorin is a natural quinone isolated fromCapraria biXora L. Previous studies demonstrated that biXorininhibits in vitro and in vivo tumor cell growth and presentspotent antioxidant activity. In this paper, we report concen-tration-dependent cytotoxic, genotoxic, antimutagenic, andprotective eVects of biXorin on Salmonella tiphymurium,yeast Saccharomyces cerevisiae, and V79 mammalian cells,using diVerent approaches. In the Salmonella/microsome
assay, biXorin was not mutagenic to TA97a TA98, TA100,and TA102 strains. However, biXorin was able to inducecytotoxicity in haploid S. cerevisiae cells in stationary andexponential phase growth. In diploid yeast cells, biXorindid not induce signiWcant mutagenic and recombinogeniceVects at the employed concentration range. In addition, thepre-treatment with biXorin prevented the mutagenic andrecombinogenic events induced by hydrogen peroxide(H2O2) in S. cerevisiae. In V79 mammalian cells, biXorinwas cytotoxic at higher concentrations. Moreover, at lowconcentrations biXorin pre-treatment protected againstH2O2-induced oxidative damage by reducing lipid per-oxidation and DNA damage as evaluated by normaland modiWed comet assay using DNA glycosylases. Ourresults suggest that biXorin cellular eVects are concentrationdependent. At lower concentrations, biXorin has signiWcantantioxidant and protective eVects against the cytotoxicity,genotoxicity, mutagenicity, and intracellular lipid peroxida-tion induced by H2O2 in yeast and mammalian cells, whichcan be attributed to its hydroxyl radical-scavenging property.However, at higher concentrations, biXorin is cytotoxic andgenotoxic.
Keywords BiXorin · Naphthoquinone · Yeast · V79 cells · Antimutagenic activity · Salmonella/microsome assay
Introduction
Mass screening programs of natural products have identi-Wed the quinone moiety as an important pharmacophoricunit for biological activity (Liu et al. 2004). Quinones aresecondary metabolic products of several microorganismsand plants playing a pivotal role in energy metabolism. Thewide spectrum of the biological activity of many quinones
M. C. Vasconcellos · R. C. Montenegro · M. E. A. Moraes · L. V. Costa-Lotufo · M. O. MoraesDepartamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil
D. J. Moura · M. S. Machado · T. N. Guecheva · I. Villela · B. F. Immich · J. SaY · J. A. P. Henriques (&)Centro de Biotecnologia e Departamento de Biofísica, Prédio 43422, Laboratório 210, Universidade Federal do Rio Grande do Sul, Campus do Vale, Av. Bento Gonçalves 9500, Bairro Agronomia, CEP 91501-970 Porto Alegre, RS, Brazile-mail: [email protected]
R. M. RosaLaboratório de Genética Toxicológica, Universidade Luterana do Brasil, Canoas, RS, Brazil
A. M. Fonseca · T. L. G. LemosDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil
J. SaYDepartamento de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brazil
J. A. P. HenriquesInstituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS, Brazil
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is frequently related to their ability to generate reactiveoxygen species (ROS) via redox cycling mechanism (Kimet al. 2008).
Many eYcient antineoplastic drugs are either quinones,quinonoid derivatives (quinolones, genistein), or drugs thatcan easily be converted to quinones by in vivo oxidation,such as etoposide (Asche 2005). These anticancer agentsare eVective inhibitors of DNA topoisomerase, contributingfor their anticancer activity (Asche 2005). In addition totheir antitumor activities, quinones have also been studiedas to their wound-healing, anti-inXammatory, antiparasitic,and cytotoxic activities (Kim et al. 2008; Lemos et al.2007; Vasconcellos et al. 2005; Vasconcellos et al. 2007).
Literature studies mention that the biological activitiesof quinones are centered in their ortho or para-quinonoidmoiety (Monks and Jones 2002). BiXorin (Fig. 1) is a1,4 o-naphthoquinone isolated from Capraria biXora L., aperennial herb widely distributed in several countries ofTropical America (Fonseca et al. 2003). Previous studiesdemonstrated that biXorin inhibits both in vitro and in vivotumor cell growth but diVerently from other cytotoxicnaphthoquinones, biXorin does not induce oxidative stress(Vasconcellos et al. 2005, 2007). In fact, it has potent anti-oxidant activity against the autoxidation of oleic acid in awater/alcohol system assay (Vasconcellos et al. 2005).
The present study investigated the cytotoxic, genotoxic,mutagenic, and protective eVects of biXorin in a prokaryoticmodel, Salmonella tiphymurium, and in two eukaryoticmodels, yeast Saccharomyces cerevisiae and V79 cells, apermanent cell line derived from Chinese hamster lungWbroblast. Furthermore, the antioxidant and antigenotoxicpotential of this natural quinone was studied using in vitrochemical systems, as well as biological systems, whichincluded yeasts and cultured mammalian cells.
Materials and methods
Chemicals
Dulbecco’s modiWed Eagle medium (DMEM), fetal bovineserum (FBS), trypsin–EDTA, L-glutamine, and antibioticswere purchased from Gibco BRL (Grand Island, NY,
USA). Methyl methanesulfonate (MMS), hydrogen perox-ide (H2O2), thiobarbituric acid (TBA), trichloroacetic acid(TCA), cycloheximide, xanthine oxidase, hypoxanthine,salicylic acid, amino acids (L-histidine, L-threonine,L-methionine, L-tryptophan, L-leucine, L-lysine), andnitrogen bases (adenine and uracil) were purchased fromSigma (St. Louis, MO, USA). Yeast extract, yeast nitrogenbase, Bacto-peptone, and Bacto-agar were obtained fromDifco Laboratories (Detroit, MI, USA). Oxoid nutrientbroth No. 2 was obtained from Oxoid USA, Inc. (Maryland,USA). Low-melting point agarose and normal agarose wereobtained from Invitrogen (Carlsbad, CA, USA). Giemsastain was from Merck (Darmstadt, Germany). Formamido-pyrimidine DNA-glycosylase (FPG, also known as MutM)and endonuclease III (EndoIII, also known as Nth) wereobtained from BioLabs (New England, USA). The S9fraction, prepared from the livers of Sprague–Dawley ratspre-treated with the polychlorinated biphenyl mixtureAroclor 1254, was purchased from Moltox Inc. (Boone,NC, USA). All other reagents were of analytical grade.
BiXorin isolation and identiWcation
BiXorin was puriWed from the roots of Capraria biXora col-lected in a plantation in Fortaleza, Ceará, Brazil. The air-dried and powdered roots (450 g) were extracted twice withpetroleum ether and vacuum-Wltered, yielding a purple-redprecipitate (100 mg). The purple-red solid presented a melt-ing point at 167–168°C, and was soluble in chloroform,ethyl acetate, and acetone. The chemical structure wasdetermined by spectroscopic analyses (IR, UV, and 1NMR)and assignments of 1H and 13C NMR spectra, as described(Fonseca et al. 2003). The biXorin puriWcation was evalu-ated through silica gel column chromatography methodusing gradient mixtures of 0–100% ethyl acetate—lightpetroleum as eluent, and was always above 95%.
For all treatments, dimethylsulfoxide (DMSO) stocksolutions of biXorin were prepared immediately prior to useso that the Wnal DMSO concentration in the medium neverexceeded 0.2%. The negative control was exposed to anequivalent concentration of this solvent.
Salmonella/microsome mutagenicity assay
Salmonella typhimurium TA98, TA97a, TA100, andTA102 were kindly provided by B. M. Ames (University ofCalifornia, Berkeley, CA, USA). Mutagenicity was assayedby the preincubation procedure. The S9 metabolic activa-tion mixture (S9 mix) was prepared according to Maronand Ames (1983). BrieXy, 100 �L of test bacterial cul-tures (1–2 £ 109 cells/mL) were incubated at 37°C withdiVerent amounts of biXorin dissolved in DMSO in thepresence or absence of S9 mix for 20 min, without shaking.
Fig. 1 Chemical structure of biXorin
O
O
O
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Arch Toxicol
Subsequently, 2 mL of soft agar (0.6% agar, 0.5% NaCl,50 �M histidine, 50 �M biotin, pH 7.4, 42°C) was added tothe test tube and poured immediately onto a plate of mini-mal agar (1.5% agar, Vogel-Bonner E medium, containing2% glucose). AXatoxin B1 (1 �g/plate) was used as positivecontrol for all strains (in the presence of metabolicactivation with S9 mix), 4-nitroquinoline-oxide (4-NQO,0.5 �g/plate) for TA97a, TA98, and TA102, and sodiumazide (1 �g/plate) for TA100 (absence of S9 mix). Plateswere incubated in the dark at 37°C for 48 h before countingthe revertant colonies.
Saccharomyces cerevisiae: strains, media, and growth
The following S. cerevisiae strains were used: XV185-14c(MAT� ade2-2 arg4-17 his1-7 lys1-1 trp5-48 hom3-10)(von Borstel et al. 1971) in the mutagenicity assay, andXS2316 (MATa/� his1-1/his1-1 leu1-1/leu1-12+/cyh2trp5-48+/met13) in the recombinogenicity assay (Machidaand Nakai 1980). Media, solutions, and buVers were pre-pared according to Burke et al. (2000). Complete YPDmedium, containing 0.5% yeast extract, 2% bacto-peptone,and 2% glucose, was used for routine growth of the yeastcells. For plates, the medium was solidiWed with 2% bacto-agar. The minimal medium (MM) contained 0.67% yeastnitrogen base with no amino acids, 2% glucose, and 2%bacto-agar was supplemented with the appropriate aminoacids. The synthetic complete medium (SC) was MM sup-plemented 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 MM.Stationary-phase cultures were obtained by inoculation ofan isolated colony into liquid YPD for 48 h. Exponentialphase cultures were obtained by inoculating 5 £ 106 cells/mLof a stationary-phase YPD culture into fresh YPDmedium for 3 h. Before biXorin treatment, cells were har-vested, washed twice with phosphate-buVered saline solu-tion (PBS; Na2HPO4, and NaH2PO4; 20 mM; pH 7.4), andbudding cell percentage in each culture were determined.All yeast assays were repeated at least four times, and plat-ing was performed in triplicate for each dose. The 4-NQOand H2O2 was used as positive control. The appropriateconcentration of H2O2 was determined by survival assay,according to the diVerential sensitivity of used strain, andsub-lethal concentration of the oxidant was used for all sub-sequent experiments.
Detection of reverse and frameshift mutation in XV185-14c haploid strain
A suspension of 2 £ 108 cells/mL in stationary or exponen-tial growth phase was incubated in PBS for mutagenic eval-uation under non-growth conditions, or in liquid YPD for
mutagenic evaluation under growth conditions, with diVer-ent concentrations of biXorin at 30°C for 3 h. In order toverify biXorin antimutagenic activity, cells were pre-treatedwith biXorin at 30°C for 3 h. Cells were then washed andtreated with H2O2 (4 mM) at 30°C in the dark with agitationfor 1 h. After treatment, appropriate cell dilutions wereplated onto SC plates to determine cell survival, and cellsuspension aliquots were plated on the appropriate omis-sion media, lacking lysine (SC-lys), histidine (SC-his), orhomoserine (SC-hom). While his1-7 is a non-suppressiblemissense allele, and reversions result from mutation at thelocus itself, lys1-1 is a suppressible ochre nonsense mutantallele, which can be reverted either by locus-speciWc or byforward mutation in a suppressor gene. True reversions andforward (suppressor) mutations at the lys1-1 locus werediVerentiated according to Schuller and von Borstel (1974),where the reduced adenine content of the SC-lys mediumshows locus reversions as red colonies, and suppressormutations as white colonies. It is believed that hom3-10contains a frameshift mutation due to its response to aseries of diagnostic mutagens.
Detection of induced mitotic recombination in XS2316 diploid strain
Suspensions of exponential cells of diploid strain(2 £ 107 cells/mL) were incubated for 3 h at 30°C in PBS,which contained diVerent biXorin concentrations. When theprotective eVect was investigated, cells were incubated withbiXorin for 3 h, washed, and exposed to H2O2 (4 mM) at30°C in the dark with agitation for 1 h. After treatment,cells were diluted, plated on three diVerent media (SC,SC-leu and SC+cycloheximide 0.2%), and incubated at 30°Cfor 5–7 days. Colonies grown in SC medium indicated cellsurvival and colonies grown in SC-leu and SC+cyh werescored for intragenic mitotic recombination (gene conver-sion) and intergenic recombination (crossing-over), respec-tively. In order to measure the exact frequency of reciprocalcrossing-over, it was necessary to eliminate the possibilitythat some cycloheximide-resistant colonies were producedby reversion at the CYH2 locus, as well as by chromosomeVII monosomy. Therefore, cycloheximide-resistant colo-nies were replica-plated on a series of SC-lys, SC-met andSC-ade media plates, with subsequent screening of thesemarkers for cyh2.
V79 mammalian cell culture and treatments
V79 cells were grown as monolayers under standard condi-tions in DMEM supplemented with 10% heat-inactivatedFBS, 0.2 mg/mL L-glutamine, 100 IU/mL penicillin/100 �g/mL streptomycin; and kept in tissue-culture Xasksat 37°C in a humidiWed atmosphere containing 5% CO2.
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One day prior to treatment, cells were harvested by treat-ment with 0.15% trypsin-0.08% EDTA in PBS, and seededinto 5 mL DMEM complete medium in a 25-cm2 Xask.BiXorin was added to the FBS-free medium to achieve thediVerent designed concentrations, and the cells were incu-bated at 37°C for 3 h in a humidiWed atmosphere containing5% CO2. Oxidative challenge with 150 �M H2O2 was car-ried out for 1 h in the dark in FBS-free medium. All experi-ments with V79 cells were carried out at least three times.Treatments of cells with MMS or H2O2 were used as posi-tive control.
Cytotoxicity evaluation by colony-forming ability (clonogenic assay) in V79 cells
After biXorin treatment, cells (500 cells/per Xask) werewashed and incubated in complete medium at 37°C in ahumidiWed atmosphere containing 5% CO2 for 7 days. Col-onies were Wxed with 3% formaldehyde, stained with 1%crystal violet, counted, and the survival expressed as a per-centage relative to the negative control treatment.
Measurement of lipid peroxidation in V79 cells
The extent of biXorin-induced lipid peroxidation was evalu-ated by the reaction of thiobarbituric acid (TBA) with mal-ondialdehyde (MDA), a product resulting from lipidperoxidation. The assays were carried out according toSalgo and Pryor (1996), with minor modiWcations. Aftertreatment, V79 cells (3 £ 106 cells) were lysed with Tris–HCl (15 mM for 1 h), 2 mL of 0.4 mg/mL TCA, and0.25 M HCl, followed by incubation with 6.7 mg/mL TBAat 100°C for 15 min. The mixture was centrifuged at 750£gfor 10 min and the supernatant was used for analyses. AsTBA reacts with products of lipid peroxidation other thanMDA, results were expressed in terms of thiobarbituricreactive species (TBARS), as determined by their absor-bance at 532 nm. Hydrolyzed tetramethoxypropane (TMP)was used as standard. The results were normalized for pro-tein content (Lowry et al. 1951).
Comet assay in V79 cells
Alkaline comet assay was performed as previouslydescribed (Collins 2004). After treatment, cells werewashed with ice-cold PBS, trypsinized, and resuspended incomplete medium. Then, 20 �L of cell suspension(3 £ 106 cells/mL) was dissolved in 0.75% low-meltingpoint agarose, and immediately spread onto a glass micro-scope slide pre-coated with a layer of 1% normal meltingpoint agarose. Agarose was allowed to set at 4°C for 5 min.Slides were then incubated in ice-cold lysis solution (2.5 MNaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100, and
10% DMSO, pH 10.0) at 4°C for at least 1 h in order toremove cell membranes, leaving DNA as “nucleoids”. Inthe modiWed Comet assay, slides were removed from thelysis solution, washed three times in enzyme buVer (40 mMHepes, 100 mM KCl, 0.5 Mm Na2EDTA, 0.2 mg/mL BSA,pH 8.0), and incubated with FPG (30 min 37°C) or EndoIII(30 min 37°C). Slides were placed in a horizontal electro-phoresis unit and incubated with fresh buVer solution(300 mM NaOH, 1 mM EDTA, pH 13.0) at 4°C for 20 minin order to allow DNA unwinding and the expression ofalkali-labile sites. Electrophoresis was conducted for20 min at 25 V (94 V/cm). All the above steps were per-formed under yellow light or in the dark in order to preventadditional DNA damage. Slides were then neutralized(0.4 M Tris, pH 7.5) and stained using the silver stainingprotocol described by Nadin et al. (2001). After the stainingstep, gels were left to dry at room temperature overnightand analyzed under a light microscope. One hundred cells(50 cells from each of two replicate slides for each treat-ment) were selected and analyzed for DNA migration.These cells were visually scored according to tail lengthinto Wve classes: (1) class 0: undamaged, without a tail; (2)class 1: with a tail shorter than the diameter of the headnucleus; (3) class 2: with a tail length 1–2£ the diameter ofthe head; (4) class 3: with a tail longer than 2£ the diameterof the head; and (5) class 4: comets with no heads. Thevalue of damage index was assigned to each sample. Dam-age index (DI) is an arbitrary score based on the number ofcells in the diVerent damage classes, which are visuallyscored by measuring DNA migration length and the amountof DNA in the tail. DI ranges from 0 (no tail: 100 cells £ 0)to 400 (with maximum migration: 100 cells £ 4) (Burlinsonet al. 2007). When selecting cells, edges and cells aroundair bubbles were disregarded.
Hypoxanthine/xanthine oxidase assay
The in vitro assay based on a previously described method(Owen et al. 1996) was used to determine the biXorin anti-oxidant potential. BrieXy, biXorin was dissolved in theassay buVer (1.0 mL hypoxanthine, Fe(III), EDTA, and sal-icylic acid) at a concentration of 5.0 mg/mL and appropri-ately diluted (in triplicate) in assay buVer to a Wnal volumeof 1.0 mL, resulting in a 0.025–2.0 mg/mL concentrationrange. A 5 �L aliquot of xanthine oxidase (18 mU) dis-solved in 3.2 mol/L (NH4)2SO4 was added to trigger thereaction. The sample tubes were incubated at 37°C for 3 h.A 30 �L aliquot of the reaction mixture was analyzed byHPLC using chromatographic conditions, as described byOwen et al. (1996). Chromatographic analysis was carriedout using a gradient based on methanol/water/acetic acidwith a �BondaPak C18 reverse phase column (Waters), anddetection at 325 nm. The HPLC equipment included a 2695
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separation module and UV detector 2487 (Waters). Theamount of dihydroxyphenol (2,5-dihydroxybenzoic acidand 2,3-dihydroxybenzoic acid) produced by the reaction ofsalicylic acid with the produced hydroxyl radicals (OH·)was determined based on the standard curves of the respec-tive dihydroxyphenols.
Statistical analysis
Mutagenicity data in bacteria were analyzed using Salmo-nel software. A compound was considered positive formutagenicity only when: (a) the number of revertants wasat least the double of the spontaneous yield (MI ¸ 2—mutagenic index (MI): number of induced colonies in thesample/number of spontaneous colonies in the negativecontrol); (b) a signiWcant response was determined by anal-ysis of variance (P · 0.05); and (c) a reproducible positivedose–response (P · 0.01) was present. An eVect was con-sidered cytotoxic when MI · 0.6.
Data from assays with S. cerevisiae and in V79 cellswere expressed as means and standard deviations and sta-tistically analyzed by one-way ANOVA with Tukey’s mul-tiple comparison test, using GraphPad Prism 4.00 Software.DiVerences were considered signiWcant when P < 0.05.
Results
Evaluation of mutagenesis in bacteria
BiXorin was Wrst tested for TA100 strain toxicity at concen-trations of 8–2,000 �g/plate in Salmonella/microsome
assay. The test-substance was considered toxic if the muta-genic index (colony counts on the test plate/average countson the negative control plates; MI) value was lower than0.60 in at least two of the tested concentrations. The resultsof the range-Wnder experiment were used to deWne the doserange to be applied in the mutagenicity test, which concen-tration range should be the highest allowed by the toxicityor the solubility of the test substance. The range Wndingresults indicate cytotoxicity in concentrations higher than333.3 �g/plate (data not shown), and therefore, the concen-trations of 66.6, 133.5, 200.1, 266.7, and 333.3 �g per platewere used in the mutagenicity assay. No biXorin mutage-nicity eVect was detected, even at the highest concentration,on TA97a (detects frameshift mutation in DNA target–C–C–C–C–C–C–; +1 cytosine), TA98 (detects frameshiftmutation in DNA target –C–G–C–G–C–G–C–G–), orTA100 (base-pair substitution mutation results from thesubstitution of a leucine [GAG] by a proline [GGG]), in theabsence or presence of metabolic activation. Also, biXorindid not show mutagenic eVect in TA102 cells that detectoxidative and alkylating mutagens by an ochre mutationTAA in the hisG gene, which can be reverted by all six pos-sible base-pair substitutions (Tables 1, 2).
Evaluation of mutagenesis, antimutagenesis, and recombinogenesis in yeast
BiXorin induced dose-dependent cytotoxic eVects both instationary and in exponential phase cultures of the haploidyeast S. cerevisiae (Fig. 2). Therefore, we used sub-cyto-toxic concentrations of this o-naphthoquinone (rangingfrom 31.25 to 500 �g/mL) to verify its mutagenic eVect on
Table 1 Induction of his+ revertants in TA98 and 97a S. typhimurium frameshift strains by biXorin with and without metabolic activation (S9 mix)
a 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
S. typhimurium TA98 TA97a
¡S9 +S9 ¡S9 +S9
Substance Dose (�g/plate)
Rev./platea MIb Rev./plate MI Rev./plate MI Rev./plate MI
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*** 24.70
BiXorin 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 1.32
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the XV-18514c strain of S. cerevisiae. BiXorin did notinduce locus non-speciWc, locus-speciWc, or frameshiftmutations in this strain in none of the concentrations andgrowth phases employed (Table 3). Stationary cells wereused to verify an intracellular protective eVect of biXorinagainst H2O2-induced oxidative stress. When compared tothe H2O2 treatment, biXorin pre-treatment enhanced cellsurvival and decreased mutation frequency at concentra-tions of 31.25, 62.5, and 125 �g/mL (Table 4). A protectiveeVect of 250 �g/mL biXorin on mutagenesis induction butnot on the H2O2-induced cytotoxicity was observed.
The recombinogenic eVects of biXorin were investigatedin XS2316 diploid yeast strain (Table 5, upper panel).BiXorin did not induce signiWcant recombinogenic events
of crossing-over and gene conversion at the concentrationrange employed. As observed in mutagenicity assay, thepre-treatment with biXorin at concentrations of 33.25–250 �g/mL also prevented the recombinogenic eventsinduced by H2O2 (Table 5, lower panel).
BiXorin cytotoxicity and protective eVects against hydrogen peroxide in V79 cells
BiXorin did not show signiWcant cytotoxic eVects on V79cells in the clonogenic survival assay at concentrationslower than 10 �g/mL (Fig. 3). The pre-treatment at non-toxic concentrations of 5 and 10 �g/mL biXorin preventsH2O2 toxicity (Fig. 3).
The degree of lipid peroxidation in V79 cells can beobserved in Fig. 4. BiXorin itself does not induce anincrease in TBARS levels at concentration range of1–10 �g/mL. In addition, the pre-treatment with biXorinreduces the extent of H2O2-induced lipid peroxidation, sug-gesting that this molecule can present antioxidant activity(Fig. 4).
BiXorin genotoxicity and antigenotoxic potential against hydrogen peroxide in V79 cells
The in vitro alkaline (pH > 13) comet assay detects DNA-strand breaks and alkali-labile sites and can be performedwith a variety of cell types, including V79 cells (Collins2004). Our results showed that biXorin does not generateDNA-strand breaks at doses lower than 10 �g/mL in V79cells. At cytotoxic concentrations of 20 and 30 �g/mL
Table 2 Induction of his+ revertants in TA100 and TA102 S. typhimurium base-substitution strains by biXorin with and without metabolicactivation (S9 mix)
a 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)
S. typhimurium TA100 TA102
¡S9 +S9 ¡S9 +S9
Substance Dose (�g/plate)
Rev./platea MIb Rev./plate MI Rev./plate MI Rev./plate MI
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*** 4.20
BiXorin 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 0.93
Fig. 2 BiXorin-induced cytotoxicity in XV185-14c haploid straintreated during stationary-growth phase (open square) and exponential-growth phase (open circle) in PBS after 3 h of treatment
0 125 250 375 500
1
10
100
1000 1500 2000
Exponential phase
Stationary phase
Biflorin (μg/mL)
% s
urvi
val
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(Fig. 3), it induces signiWcant DNA damage (Fig. 5a).BiXorin pre-treatment at concentrations below 10 �g/mLsigniWcantly reduced the H2O2-induced DNA damage(Fig. 5b). The pre-treatment at concentration of 30 �g/mLbiXorin (that induces DNA damage by itself, Fig. 5a)slightly enhances the DNA migration after H2O2 treatment(Fig. 5b).
While the alkaline version of the comet assay detectsDNA single- and double-strand breaks and alkali-labilesites, the modiWed comet assay is more speciWc than thisstandard method. In the modiWed version, there is an incu-bation step with lesion-speciWc enzymes, which recognize
certain damaged bases and create breaks. In the presentstudy, we used FPG, that is speciWc for oxidized purines,including 8-oxo-7,8-dihydroguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopy-rimidine, and other ring-opened purines, as well as EndoIII,that recognizes oxidized pyrimidines, including thymineglycol and uracil glycol. The levels of EndoIII and FPGsensitive sites were calculated as the score obtained withenzymes minus the score without enzymes (only withenzyme buVer) after treatment with biXorin or H2O2. Theresults indicate that biXorin does not induce signiWcantoxidative damage at concentration 2.5–30 �g/mL (Fig. 6a).
Table 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
a 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)
Concentration (�g/mL)
Survival (%)
HIS1 revertants/107 survivorsa
LYS1 revertants/107 survivorsb
HOM3 revertants/107 survivorsa
Treatment of stationary-phase cells under non-growth conditions
DMSOd 100 12.1 § 2.0c 1.3 § 0.7c 2.2 § 0.7c
4NQOe 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 § 1.0
Treatment of exponential-phase cells under non-growth conditions
DMSOd 100 18.7 § 0.9 8.1 § 1.3 6.7 § 0.3
4NQOe 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 § 2.0
Treatment under growth conditions
DMSOd 100 10.3 § 0.9 3.0 § 0.5 4.8 § 0.9
4NQOe 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 § 0.0
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These results suggest that the elevated DNA-strand breaksobserved after treatment at 20 and 30 �g/mL in the alkalinecomet assay (Fig. 5a) do not have oxidative origin. Further-
more, the DNA migration was also evaluated after incuba-tion with these enzymes in cells pre-treated with biXorinand then exposed to H2O2. The extent of oxidative damage
Table 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
a 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)
Concentration Survival (%)
HIS1 revertants/107 survivors
LYS1 revertants/107 survivorsb
HOM3 revertants/107 survivorsa
DMSOd 100 10.3 § 0.9e 3.0 § 0.5e 4.8 § 0.9e
H2O2e 4 mM 41.0 41.4 § 1.3*** 37.7 § 1.8*** 36.0 § 0.9***
BiXorin pre-treatment plus H2O2 4 mM
31.25 �g/mL 66.4 7.4 § 1.8*** 3.2 § 0.4*** 4.5 § 1.4***
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 § 3.7
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
a 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 test
Concentration Survival (%)
Crossing over/105 survivors
Gene conversion/105 survivors
DMSOb 100 9.1 § 0.8a 1.5 § 0.2
4NQOc 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
H2O2d 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
125 �g/mL 71.9 33.1 § 9.3AAA 9.9 § 1.3AAA
250 �g/mL 63.0 55.0 § 6.5AAA 12.0 § 3.3AAA
500 �g/mL 49.3 82.0 § 8.4 45.1 § 4.1
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Arch Toxicol
recognized by the lesion-speciWc enzymes signiWcantlydecreased after pre-treatment at low biXorin concentrations(2.5, 5.0, and 10.0 �g/mL), indicating that the antigenotoxiceVects of this compound can be related to its antioxidantproperties (Fig. 6b).
BiXorin free-radical scavenging ability
BiXorin antioxidant capacity was determined by monitoringthe production of hydroxylated benzoic acid (DHBA)resulting from the attack of ROS on salicylic acid in the
hypoxanthine/xanthine oxidase assay. The decrease in totaloxidation products as a function of the amount of biXorinadded to the assay is shown in Fig. 7, indicating that biXo-rin presents signiWcant antioxidant capacity in a dose-dependent manner.
Discussion
Quinones are secondary metabolites found in several livingcells and are widely used as anticancer, antibacterial, or
Fig. 3 Clonogenic survival of V79 cells after treatment with biXorinfor 3 h or pre-treated with biXorin for 3 h and challenged with H2O2(150 �M) for 1 h at 37°C in the dark, at the indicated concentrations.Data are presented as mean § SD of three independent experiments.*SigniWcantly diVerent when compared to the control (dimethyl sulf-
oxide, used as solvent) at p < 0.05; ***p < 0.001; #p < 0.05, whenbiXorin pretreated cells were compared to cells exposed to challengemutagen—H2O2 (150 �M). Statistical analyses were carried out usingone-way ANOVA and Tukey’s multiple comparison test
DMSO
1.0 5.
010
.0 20 30 Mμ 1
50
2O2H
1.0
5.0
10.0
0
25
50
75
100
Biflorin ( μg/mL) Biflorin ( μg/mL) pre-treatment plus H2O2exposure
*#
#
***
Su
rviv
al (
%)
Fig. 4 Determination of thiobarbituric acid reactive substances(TBARS) in V79 cells pretreated with biXorin at the indicated concen-trations for 3 h and subsequently exposed to H2O2 (150 �M). Data arepresented as mean § SD of three independent experiments. *SigniW-cantly diVerent when compared to the control (dimethyl sulfoxide,
used as solvent) at p < 0.05; # p < 0.05, when biXorin pretreated cellswere compared to cells exposed to H2O2 (150 �M). Statistical analyseswere carried out using one-way ANOVA and Tukey’s multiple com-parison test
DMSO Mμ
150
2O2H
1.0 5.
010
.0 1.0
5.0
10.0
0
10
20
30
40
50
60
Biflorin ( μg/mL) Biflorin ( μg/mL) pre-treatment plus H2O2exposure
*
#
#
#
MD
A e
qu
ival
ents
(nm
ol M
DA
/mg
pro
tein
)
123
Arch Toxicol
antimalarial drugs. In fact, naphthoquinones, mainly repre-sented by �-lapachone, are a promising group of com-pounds with antitumor properties (Asche 2005). Inaddition, quinone moieties are present in many drugs, suchas anthracyclines daunorubicin, doxorubicin, mitomycin,mitoxantrones, and saintopin, which are clinically used intherapy of solid cancers (Verma 2006). In the present study,we investigated the cytotoxic, genotoxic, mutagenic, andantimutagenic eVects of biXorin using diVerent approacheson prokaryotic and eukaryotic cells systems.
BiXorin is a 1,4-o-napthoquinone isolated from C. biX-ora that presents in vivo and in vitro antitumor eVects;however, its exact mechanism of action is still unknown(Vasconcellos et al. 2007). We showed here that biXorin
has a dose-dependent cytotoxic eVect on diVerent eukary-otic cells (Figs. 2, 3) but it is not mutagenic to bacteria andyeast (Tables 1, 2, 3, 4 and 5). Employing TBARS determi-nation and modiWed comet assay with ENDOIII and FPGrepair proteins, we found that biXorin did not induce oxida-tive damage in V79 cells (Figs. 4, 6a). Moreover, biXorinreduced DNA damage and mutation triggered by H2O2 inyeast (Tables 4, 5) and V79 cells (Figs. 5b, 6b), suggestingan antioxidant eVect. BiXorin protective capacity againstH2O2-induced oxidative stress was also conWrmed byreductions in lipid peroxidation in V79 cells (Fig. 4).Hydrogen peroxide is an important ROS compound that, incombination with reduced trace metals such as iron or cop-per, is transformed via Fenton reaction into the highly reac-tive hydroxyl radicals (OH·), which causes damage to
Fig. 5 a Evaluation of genotoxicity of biXorin treatment for 3 h usingcomet assay in V79 cells. b EVect of the pre-treatment with biXorin for3 h on hydrogen peroxide (150 �M) induced DNA damage in V79cells using comet assay. Bars represent the mean § SD of three inde-pendent experiments. ***DiVerences are signiWcant when compared tothe control in a (dimethyl sulfoxide, used as solvent) or in b to H2O2 atp < 0.001; **p < 0.01 with Tukey’s multiple comparison test at one-way ANOVA–Tukey’s multiple comparison test
DMSO Mμ
MM
S 40 2.
55.
010
.0 2030
.00
50
100
150
200
250
300
350
Biflorin (μg/mL)
*********
AD
amag
e In
dex
Mμ15
0
2 O2H
2.5 5.
010
.0 2030
.00
50
100
150
200
250
300
350
Biflorin( μg/mL) pre treatmentplus H2O2150 μM
*******
B
Dam
age
Ind
ex
Fig. 6 DNA damage measured by comet assay in V79 cells exposedto biXorin at 37°C for 3 h with subsequent treatment with buVer, Endo-III or FPG. The levels of EndoIII and FPG sensitive sites were calcu-lated as the score obtained with enzymes minus the score withoutenzymes (buVered) after treatment with biXorin or H2O2. Means andstandard deviation values were determined from an average of 100cells per replicate, with three replicates per concentration. **SigniW-cantly diVerent when compared to the positive control (H2O2) atp < 0.01 and ***p < 0.001, as determined by one-way Anova andTukey’s multiple comparison test
FPGEndoIII0
20
40
60
80
100 DMSObiflorin 2.5 μg/mLbiflorin 5.0 μg/mLbiflorin 10 μg/mLbiflorin 20 μg/mLbiflorin 30 μg/mL
A
Oxi
dat
ive
dam
age
sco
re
0
20
40
60
80
100
H2O2 150 μMbiflorin 2.5 μg/mL plus H2O2
biflorin 5.0 μg/mL plus H2O2
biflorin 10.0 μg/mL plus H2O2
biflorin 20.0 μg/mL plus H2O2
biflorin 30.0 μg/mL plus H2O2
***
***
*********
**
B
Oxi
dat
ive
dam
age
sco
re
FPGEndoIII
123
Arch Toxicol
virtually all macromolecules (Halliwell and Gutteridge2000). Semiquinones, which often act reducing O2 to O2
·¡
(Fenton intermediary), in aqueous solution can favor O2·¡
dismutation reaction (Halliwell and Gutteridge 2000). Thein vitro hypoxanthine-xanthine oxidase assay in this studyshowed that biXorin can interfere with the Fenton reaction,acting as O2
·¡ and/or OH· scavenger, suggesting that theprotection against H2O2–induced damage provided bybiXorin 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 cellsoccurred only in the lower concentrations tested whereas athigh concentration biXorin was cytotoxic and inducedDNA-strand breaks as evidenced by the comet assay. TheDNA damage induction by biXorin could justify itsanti-proliferative potential (Vasconcellos et al. 2007).Therefore, it is possible that the toxicity observed in con-centrations higher than 250 �g/mL in haploid yeast strainand 10 �g/mL in V79 cells is a result of the DNA damageinduced by this 1,4-naphthoquinone. The biXorin-inducedDNA lesions in V79 mammalian cells, at concentrationsabove 10 �g/mL (Fig. 5a), do not include oxidized basesthat are substrate of the ENDOIII and FPG enzymes(Fig. 6a). Also the absence of point mutation induction inbacteria and yeast reinforced our results obtained in V79cells, indicating that biXorin does not induce oxidativedamage. Most of the antitumour quinones belong to thegroups of DNA intercalating and/or alkylating agents andalso can be eVective inhibitors of DNA topoisomerase(Asche 2005). Previous study has reported that the�-lapachone does not intercalate into DNA (Pardee et al.
2002). As biXorin does not induce frameshift mutation inbacteria and yeast in our study, we supposed that it is notintercalating agent as well. Another mechanism leading toDNA-strand breaks induction is the interaction with DNAtopoisomerase. It has been shown that �-lapachone andderivatives can stimulate DNA double-strand breaks forma-tion in the presence of topoisomerase II� (Frydman et al.1997). Similar mechanism of action can explain the strandbreaks induction in V79 cells observed in comet assay aftertreatment at higher biXorin concentrations (Fig. 5a). Thetopoisomerase II poisoning could be responsible for thecytotoxic eVect of biXorin, contributing for the anticanceractivity.
In summary, our Wndings indicate that the cellular eVectsof biXorin are concentration dependent. At a range of lowdoses, the hydroxyl radical-scavenging property of biXorincan contribute to the signiWcant antioxidant and protectiveeVects against the cytotoxicity, genotoxicity, mutagenicity,and intracellular lipid peroxidation induced by H2O2. Onthe other hand, at higher concentrations biXorin is cyto-toxic, possibly due to its ability to induce DNA-strandbreaks.
Acknowledgments We are grateful to Dr. Marc François Richter forassistance with HPLC analysis. This research was supported by theBrazilian Agencies FINEP, CNPq, BNB/FUNDECI, PRONEX,GENOTOX-Royal Institute—Genotoxicity Laboratory, UniversidadeFederal do Rio Grande do Sul and CAPES.
ConXict of interest statement The authors declare that they have noconXicts of interest.
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