Mutagenicity, Antioxidant Potential, and AntimutagenicActivity Against Hydrogen Peroxide of Cashew

(Anacardium occidentale) Apple Juice and Cajuina

Ana Amelia Melo Cavalcante,1,3 Gabriel Rubensam,3

Jaqueline N. Picada,3,4 Evandro Gomes da Silva,2

Jose Claudio Fonseca Moreira,2 and Joao A.P. Henriques3*1Centro Federal de Educacao Tecnologica do Piauı, CEFET-PI, Teresina, PI, Brasil

2Centro de Estudos em Estress Oxidativo, Departamento de Bioquımica,Universidade Federal do Rio Grande do Sul, UFRGS, Porto Alegre, RS, Brasil

3Departamento de Biofısica e Centro de Biotecnologia, UFRGS,Porto Alegre, RS, Brasil

4Universidade Luterana do Brasil, Departamento de Farmacia, Canoas, RS, Brasil

Fresh and processed cashew (Anacardium occi-dentale) apple juice (CAJ) are among the mostpopular drinks in Brazil. Besides their nutritionalbenefits, these juices have antibacterial and antitu-mor potential. The chemical constituents of both thefresh juice and the processed juice (cajuina) wereanalyzed and characterized as complex mixturescontaining high concentrations of vitamin C, vari-ous carotenoids, phenolic compounds, and metals.In the present study, these beverages exhibiteddirect and rat liver S9-mediated mutagenicity in theSalmonella/microsome assay with strains TA97a,TA98, and TA100, which detect frameshifts andbase pair substitution. No mutagenicity was ob-served with strain TA102, which detects oxidativeand alkylating mutagens and active forms of oxy-gen. Both CAJ and cajuina showed antioxidantactivity as determined by a total radical-trappingpotential assay. To test whether this antioxidant

potential might result in antimutagenesis, we useda variation of the Salmonella/microsome assaythat included pre-, co-, and posttreatment of hydro-gen peroxide-exposed Salmonella typhimuriumstrain TA102 with the juices. CAJ and cajuinaprotected strain TA102 against mutation by oxida-tive damage in co- and posttreatments. The anti-mutagenic effects during cotreatment with hydro-gen peroxide may be due to scavenging freeradicals and complexing extracellular mutageniccompounds. The protective effects in posttreatmentmay be due to stimulation of repair and/or rever-sion of DNA damage. The results indicate that CAJand cajuina have mutagenic, radical-trapping, an-timutagenic, and comutagenic activity and thatthese properties can be related to the chemicalconstituents of the juices. Environ. Mol. Mutagen.41:360–369, 2003. © 2003 Wiley-Liss, Inc.

Key words: cashew apple juice; cajuina; mutagenicity; antioxidant; antimutagenicity; Salmo-nella/microsome assay

INTRODUCTION

In recent years the economic value of cashew (Anacar-dium occidentale) apple juice has increased. Fresh cashewapple juice (CAJ) and processed juice, called cajuina, areamong the most popular natural products in Brazil, espe-cially in the northeast region [Embrapa, 2001].

Chemical components in fruits and vegetables, such asmicronutrients, phenols, and fiber, may protect against anumber of degenerative diseases in humans, including can-cer, cardiovascular diseases, cataracts, and brain dysfunc-tion [Ames et al., 1993; Ames, 2001], while deficiencies inmicronutrients are likely to cause DNA damage [Ames,2001; Morrow et al., 2001]. For instance, polyphenols arepresent in both CAJ and cajuina [Agostini-Costa et al.,2000]. These compounds are a large and diverse class andmany have antioxidant, antimutagenic, anticarcinogenic,

antiestrogenic, and antiinflammatory properties that mightbe beneficial in preventing diseases by improving genomicstability [Ferguson, 2001]. However, not all of the polyphe-nols, and not all the properties of polyphenols, are beneficial

Grant sponsors: CEFET-PI (Centro Federal de Educacao Tecnologica doPiauı, Brasil) and GENOTOX-Laboratorio de Genotoxicidade, Centro deBiotecnologia, UFRGS.

*Correspondence to: J.A.P. Henriques, GENOTOX–Laboratorio de Geno-toxicidade, Centro de Biotecnologia, UFRGS, Av. Bento Goncalves, 9500,Predio 43421; Campus do Vale; Caixa Postal 15005; CEP 91501-970,Porto Alegre, RS, Brasil. E-mail: pegas@dna.cbiot.ufrgs.br

Received 2 October 2002; provisionally accepted 27 January 2003; and infinal form 8 March 2003

DOI 10.1002/em.10158

Environmental and Molecular Mutagenesis 41:360–369 (2003)

© 2003 Wiley-Liss, Inc.

and some of these compounds have mutagenic and prooxi-dant effects [Ferguson, 2001].

Oxidative damage can originate from increased produc-tion of free radicals, caused by exogenous and/or endoge-nous sources [Halliwell and Gutteridge, 1998, 2000]. Oxi-dation is considered a highly significant DNA-damagingagent. During the metabolism of DNA, reactive chemicalmutagens, carcinogens, or UV light can produce oxygenradicals and reactive oxygen or nitrogen species (ROS,RNS) [Weisburger, 2001]. Many types of chronic disease,including cardiovascular, neoplastic, neurodegenerative,Parkinson’s and Alzheimer’s diseases, as well as immuno-logical dysfunction, premature aging, and cancer are asso-ciated with singlet molecular oxygen (1O2), hydroxyl radi-cal (OH � ), superoxide anion (O2 � -), and hydrogen peroxide(H2O2) production [Weisburger, 2001; Ferrer et al., 2002].H2O2 is produced by endogenous metabolic and catabolicprocesses in cells and can induce mutations as a directconsequence of the radical generated upon its decomposi-tion [Termini, 2000].

Studies on the free radical-scavenging properties of fla-vonoids have identified many of the major naturally occur-ring phenolic compounds as phytochemical antioxidants[Rice-Evans et al., 1997]. The flavonoid quercetin can re-duce the DNA damage induced by H2O2 in isolated humanlymphocytes by inhibiting DNA strand breakage [Duthie etal., 1997]. Antioxidant activity corresponds to the rate con-stant of a single antioxidant against a given free radical. Theantioxidant capacity is measured as the moles of a freeradical scavenged by a test solution, independent of theantioxidant activity of any one antioxidant present in themixture [Ghiselli et al., 2000].

The aim of the present study was to investigate themutagenic effects of CAJ and cajuina by the Salmonella/microsome assay and to determine the possible antimuta-genic activity of the juices against H2O2 using pre-, co-, andposttreatment of Salmonella typhimurium strain TA102,with and without metabolic activation. In addition, we eval-uated the total antioxidant potential of juices by the totalradical-trapping antioxidant potential (TRAP) assay and theprincipal chemical components of both fresh and processedjuices. The identification of antimutagenic compounds andthe elucidation of their mechanism of action deserve atten-tion because of their possible significance in the protectionof human health.

MATERIALS AND METHODS

Preparation of Juice From Anacardium occidentale

To produce fresh CAJ, cashew fruits, obtained from the State of Piauı,Brazil, were washed and sterilized by soaking the fruit in 70% ethanol for�5 sec and flaming. The cashew apples were then macerated and the juicesieved using sterile equipment. A sample was tested for the absence ofmicroorganisms and the juice samples were frozen at –20°C. The produc-tion of cajuina from CAJ included centrifugation of fruits, clarification with

gelatin, filtration, and thermal treatment (1 hr at 100°C), according to themanufacturer’s protocol (Lili Doces, Teresina, PI, Brazil).

Chemicals

Direct-acting mutagens were 4-nitroquinoline-1-oxide (4-NQO), methylmethanesulfonate (MMS), and H2O2; as indirect mutagens, we used ben-zo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1). All mutagens were dissolvedin dimethylsulfoxide (DMSO). AAPH [2-2�–azobis (2 methyl propiona-midine dihydrochloride)], isoluminol (6-amino-2, 3-dihydro-1,4-phthala-zinedione), and quercetin were purchased from Sigma (St. Louis, MO,USA).

Bacterial Strains

Salmonella typhimurium strains TA97a (his 01242, bio chlD uvrb gal,rfa, pKM101); TA98 (his D3052, bio chlD uvrb gal, rfa, pKM101); TA100(his G46, bio chlD uvrb gal, rfa, pKM101), and TA102 (his G428, rfa,pKM101, pAQI), as described by Maron and Ames [1983] and Mortelmansand Zeiger [2000], were kindly supplied by Dr. B.N. Ames, University ofCalifornia, Berkeley, CA, USA.

Microsomal Fraction

The postmicrosomal S9 fraction, prepared from livers of Sprague-Daw-ley rats treated with the polychlorinated biphenyl mixture Aroclor 1254,was purchased from Molecular Toxicology (Maltox™, Annapolis, MD,USA). The S9 metabolic activation mixture was prepared according toMaron and Ames [1983] and Mortelmans and Zeiger [2000].

Chemical Analysis of Juice

The amount of quercetin in CAJ and cajuina was determined by HPLCas described by Careri et al. [2000], using a C18 narrow-bore column(Luna, 150 � 2.0 mm, 3 mm; Phenomenex, Torrance, CA, USA) and anisocratic solvent system (aqueous formic acid, pH 2.4 (A)-acetonitrile (B);80:20, v/v at a flow rate of 200 �L/min). A Hewlett Packard HP 1050delivered the mobile phase and measurements were taken at 370 nm. Thetotal phenolic compounds in both juices were determined using the Folin-Denis method described by Agostini-Costa et al. [2000]. The concentra-tions of these compounds were determined by comparison with a standardcurve constructed with tannic acid (0–10 mL and abs� 0.06921 � conc.� 0.02213). Condensed tannins were quantified using the vanillin methoddescribed by Agostini-Costa et al. [1999]. Catequin was used to make astandard curve (Abs � 0.00614 � Conc � 0.00252). Anacardic acid wasmeasured according to Agostini-Costa and Jales [2001]. The concentrationin the products was quantified by a standard curve produced with anacardicacids extracted from cashews (Anacardium occidentale) (Abs� 0.01205 �conc-0. 01974). Total carotenoids were assayed using a simplified methodfor carotenoid distribution in natural compounds [Cecchi and Rodrigues,1977]. Vitamin C (ascorbic acid) was determined according to the protocolof Pearson and Cox [1976] for the chemical analysis of foods. All con-centrations are expressed in mg/100g.

Metals were determined by proton-induced X-ray emission (PIXE), amethod applying X-ray protons [Kennedy et al., 1998, 1999]. The sampleof CAJ and cajuina (from a single manufacturer) were vacuum-filtered ontoregenerated cellulose acetate filters (Sartorious, 0.45 �m pore, 40 nmdiameter) Three samples of each juice were prepared. A clean filter wasused for background counts. The mass of the samples varied from 0.005–0.35 mg (dry weight), giving rise to a filter density in the range of 0.1–0.7nmol of juice per cm2. The PIXE analysis was carried out at the 3MVTandetron accelerator facility at IF-UFRGS. The membranes containingthe juices, the blank membrane, and calibration membrane were placed in

Mutagenicity and Antimutagenicity of CAJ 361

the trail receptacle, which accommodates up to 10 specimens. Each samplewas located in the proton beam by means of the electrical-mechanicalsystem. The characteristic X-rays induced by the proton beam were de-tected with an HPGe detector from EG&G (GLP series), with an energyresolution of 170 eV at 5 eV. A mylar filter of 280 �M was used in orderto prevent low-energy X-rays from reaching the detector, considerablyimproving the dead time of the acquisition system.

Salmonella/Microsome Assay

The mutagenicity of the juices was measured by the preincubationprocedure [Maron and Ames, 1983], using S. typhimurium strains TA97a,TA98, TA100, and TA102 with and without S9 mix. The mixture, con-sisting of the juice samples to be tested, 500 �L of S9 mix (in tests withmetabolism) and 100 �L of the bacterial suspension (1–2 � 109 cells/mL),was incubated for 20 min at 37°C without shaking. Two mL of molten topagar (0.55% agar, 0.55% NaCl, 50 �M L-histidine, 50 �M biotin, pH 7.4,45°C) was then added to the test tubes, the tubes mixed, and the contentspoured into a Petri dish containing minimal agar (1.5% agar, Vogel-BonnerE medium plus 2% glucose). All assays were carried out in triplicate. Afterincubation for 48 hr, colonies (his� revertants) were counted and theresults were expressed as mutagenic index (MI � number of his� coloniesinduced in the sample/number of spontaneous his� revertants in the neg-ative control). Negative (appropriate solvent) and positive controls wereincluded in each assay. Two �g MMS per plate for strains TA100 andTA102 and 0.5 �g 4-NQO per plate for TA98, TA97a, and TA102 wereused as positive controls for assays conducted without S9. AFB1 (1 �g perplate) was used as positive control for TA102 and 1 �g per plate B[a]P forstrains TA97a, TA98, and TA100 in assays conducted with S9. The juiceswere considered positive for mutagenicity when: 1) the number of rever-tants was at least double the spontaneous yield (MI � 2); 2) a significantresponse for analysis of variance (P � 0.05) was found; and 3) a repro-ducible positive dose-response (P � 0.01) was present, as evaluated by theSalmonel software [Myers et al., 1991].

Total Radical-Trapping Antioxidant Potential(TRAP) Assay

A modified TRAP assay was used for determining the capacity of CAJand cajuina to trap a flow of water-soluble peroxyl radicals produced at aconstant rate by the thermal decomposition of AAPH [Ghisellia et al.,2000]. Briefly, the reaction mixture (4 mL), containing the free radicalsource (10 mM AAPH), 10 �L of the samples to be tested and luminol (10�M) as an external probe for monitoring radical production, was incubatedin glycine buffer (0.1 M, pH 8.6) at room temperature. Chemiluminescence

was measured in out-of-coincidence mode of a liquid scintillation counter(Wallac 1409, Beckman, Palo Alto, CA, USA). Trolox (0.75 �M, Aldrich,Milwaukee, WI, USA), a water-soluble analog of vitamin E with potentantioxidant activity, was used as a positive control to assess antioxidantpotential. Statistics were performed using one-way ANOVA on the meanof three independent experiments. Dunnett’s multiple comparison test wascarried out, accepting a probability of P � 0.05 as statistically significant.

Antimutagenicity Analysis

Antimutagenicity of the CAJ and cajuina against H2O2 was assessedusing the standard plate incorporation assay as described by Maron andAmes [1983] and Mortelmans and Zeiger [2000] with the methodologicalvariations described by De Flora et al. [1992] (Table I). An overnightculture of TA102 was washed with 5 mL of 0.2 M phosphate-bufferedsaline (PBS, pH 7.4). In each experiment we included H2O2 as a positivecontrol. The dose of H2O2 was 1 mM, while the doses of juices wereselected in preliminary range-finding assays. We used the following con-trols: 1) for H2O2, H2O � H2O2 � bacteria � S9 mix; 2) for juice, H2O �juice � bacteria � S9 mix; 3) for S9 mix, juice � bacteria � H2O2, withomission of S9 fractions; and 4) for bacteria, H2O � bacteria � S9 mix.Incubation was at 37°C with continuous gentle shaking, followed bycentrifugation at 3,000 rpm for 20 min (RT6000, Sorvall Instruments,Dupont, Rockville, MD, USA). All tests were performed in triplicate.Antimutagenic activity was calculated as the difference in mutagenicityfrom the H2O2 control only in relation to that shown upon incubation witheach juice. The percentage of inhibition for each dose of CAJ and cajuinaagainst H2O2 was calculated according to Cabrera [2000] as follows:(I%) � [1�(B/A)] � 100, where A represents the number of revertants/plate containing H2O2 and B represents the number of revertants/platecontaining H2O2 and juices. The frequency of spontaneous revertants fromthe appropriate control was subtracted from all plates. The antimutageniceffect of CAJ and cajuina was characterized at nontoxic doses by the ID50,the dose causing a 50% reduction of mutagenicity in the test system.Toxicity in the Salmonella/microsome assay was observed as a decrease inthe number of revertant colonies on plates with juice and H2O2 in relationto the number of spontaneous revertants (negative control), the absence ofa background lawn and/or complete absence of growth, and presence ofpinpoint nonrevertants according to Mortelmans and Zeiger [2000]. Co-mutagenic effects were considered to have occurred when the number ofrevertants on the plates with juices and H2O2 were higher than thosecontaining H2O2 only.

TABLE I. Salmonella/Microsome Assay Antimutagenesis Treatment Protocols Used in This Study

Treatmentmode

Possible mechanism ofmodulation Procedures

PretreatmentIntracellular reaction Juice � bacteria in fresh nutrient broth (4 hr), wash bacteria and add H2O2 � S9mix

(20 min), wash bacteria and plate.Cotreatment Extracellular reaction A—Bacteria � juice and H2O2 � S9mix (20 min), wash bacteria and plate.

B—Juice � H2O2 � S9mix (20 min), add to the bacteria and plate.C—H2O2 � S9mix (20 min), add the juice (20 min), add bacteria and plate

Posttreatment Effect on DNA repair A—Bacteria � H2O2 � S9mix (20 min), wash bacteria, add the juice and plateB—Bacteria � H2O2 � S9mix (20 min), wash and incubate with juice in fresh broth

(30 min), wash bacteria and plate.C—Bacteria � H2O2 � S9mix (20 min), wash and further incubate in fresh broth

(30 min), add juice and plate.

Adapted from De Flora et al. [1992].

362 Melo Cavalcante et al.

RESULTS

Chemical Analysis

Table II summarizes the results of chemical analysis ofthe CAJ and cajuina. The concentrations of the analyzedchemical compounds in cajuina were lower than in the freshjuice, especially for ascorbic and anacardic acids, whichwere about 77- and 43-fold lower in cajuina, respectively.There were no significant differences (P � 0.1) between thequantity of quercetin in CAJ and cajuina. The results of thePIXE analysis for metals content are shown in Table III.The concentrations of S, Cl, Ti, Mn, Fe, Cu, and Zn incajuina were reduced. The metals Rb, Sr, Zr, Co, and Pbwere detected only in the CAJ and the metals P, Ni, Cr, andCd only in cajuina. The differences in metal content be-tween CAJ and cajuina may have resulted from the process-ing of these two products. Cajuina is clarified with gelatinand heat-treated. Metals could be removed by this treat-

ment. Both juices were produced by the same manufacturerfrom the same raw materials.

Mutagenicity of CAJ and Cajuina in the Salmonella/Microsome Assay

As can be seen in Table IV, in the absence of metabolicactivation CAJ induced mutagenicity in TA97a (detectsframeshift mutation in -C-C-C-C-C-C-; � 1 cytosine) andcajuina in TA97a and TA100 (basepair substitution muta-tion results from the substitution of a leucine (GAG) by aproline (GGG)). In the presence of S9 mix, CAJ inducedmutations in the TA97a, TA98 (detects frameshifts in DNAtarget -C-G-C-G-C-G-C-G-) and TA100 strains, while ca-juina was only mutagenic in TA98, showing a pronouncedmutagenic response (MI � 15). Neither juice showed anymutagenic activity in TA102, which detects oxidative, al-kylating mutagens, and ROS [Levin et al., 1982]. CAJshowed significant toxicity at doses �100 �L/plate, both inthe presence and absence of metabolic activation (data notshown).

Evaluation of Antioxidant Potential of CAJ andCajuina by the TRAP Assay

The pure juices showed excellent antioxidant potentialbased on their capacity to scavenge free peroxyl radicalsproduced by AAPH (Fig. 1A,B). To evaluate the relativeantioxidant potential of the juices, we diluted each in dis-tilled water. A 5-fold dilution showed no change in relationto the pure juice. At 10-fold dilution the antioxidant poten-tial decreased both in CAJ and in cajuina after 70 min. At a20-fold dilution, a significant decrease in the antioxidantpotential was observed after 40 min and at 50-fold dilutionwe observed a loss in the antioxidant properties of bothjuices after 10 min (Fig. 1C,D).

Antimutagenic Evaluation of CAJ and Cajuina in aModified Salmonella/Microsome Assay

The antimutagenic effects of CAJ and cajuina againstH2O2 in TA102 are shown in Table V. In order to determinethe possible presence of promutagens and/or antimutagenicmetabolites in the juices, we also used S9 metabolic acti-

TABLE II. Chemical Components of Cashew Apple Juice (CAJ) and Cajuina

JuicesaTotal carotenoids

Mean � SDbTotal phenolsMean � SD

Condensed tanninsMean � SD

QuercetinMean � SD

Anacardic acidMean � SD

Ascorbic acidMean � SD

CAJ 0.32 � 0.0** 11.9 � 0.3** 61.1 � 0.5** 0.232 � 0.30 17.9 � 0.4** 120.80 � 4.1**Cajuina 0.006 � 0.0** 8.6 � 0.4** 13.0 � 4.0** 0.279 � 0.35 0.41 � 0.0** 1.56 � 0.4**

aConcentrations expressed in mg/100g.bMean value of at least three independent experiments � SD.Statistical significance, one-way ANOVA followed by Dunnett’s Multiple Comparison Test.**P 0.01.

TABLE III. Elemental Concentrations Observed in CashewApple Juice (CAJ) and Cajuina Obtained by PIXE Analysisa

Element(mg/mL) CAJ Cajuina

P 0.61 � 0.05 LODS 0.82 � 0.02 0.006 � 0.001Cl 0.09 � 0.01 (0.0012 � 0.0006)b

K 3.05 � 0.03 2.57 � 0.05Ti (0.007 � 0.002)b (0.0006 � 0.0001)b

Cr (0.006 � 0.001)b LODMn 0.011 � 0.001 0.0023 � 0.0002Fe 0.21 � 0.02 0.0014 � 0.0002Co LOD (0.0004 � 0.0002)b

Ni (0.003 � 0.001)b LODCu 0.075 � 0.004 0.0174 � 0.0006Zn 1.02 � 0.02 0.0009 � 0.0002Rb LOD (0.0008 � 0.0004)b

Sr LOD (0.0013 � 0.0005)b

Zr LOD (0.0007 � 0.0006)b

Cd 0.36 � 0.03 LODPb LOD (0.0005 � 0.0002)b

aThe uncertainties quoted above stem from the fitting procedure of thespectra analyzed with Gupix code [Campbell et al., 2000], which takes intoaccount the statistical uncertainty of each photopeakbMay be present near the limit of detection (LOD). Means of at least threeindependent experiments.

Mutagenicity and Antimutagenicity of CAJ 363

vation. With the pretreatment procedure (Table VI), CAJpotentiated the mutagenicity of H2O2, both in the presenceand absence of S9 mix, suggesting a stimulation of muta-genicity or comutagenic effect. However, cajuina did notshow any statistically significant promutagenicity, but wassignificantly toxic at 2 mL/plate. In cotreatment A, weobserved a decrease in the number of the his� revertantsbelow that of the spontaneous controls, suggesting toxiceffects of this treatment for both juices. In contrast, CAJ incotreatment B showed high antimutagenic potential, with 50�L/plate plus S9 mix inhibiting 100% of the mutagenicityinduced by H2O2, and 10 and 25 �L/plate inhibiting almost60% without S9 mix. Cajuina also had high antimutagenicpotential with the same treatment protocol, showing�100% inhibition with and without S9 mix. In the presenceof metabolic activation with cotreatment C, the juicesshowed a pronounced antimutagenic effect, with about 58%and 97% inhibition. In posttreatments A, B, and C, CAJappeared to be toxic in the absence of S9 mix. However, inthe presence of S9 mix, we observed antimutagenic effectsin posttreatment B at 50 �L/plate (91%) and in posttreat-ment C at almost all the doses, with inhibition reachingalmost 100%. Cajuina showed similar toxicity in the post-treatments and also inhibited the mutagenicity induced byH2O2, mainly at the higher dose, both with and without S9mix.

DISCUSSION

In this study, both CAJ and cajuina were mutagenic in theTA98 and TA97a strains of Salmonella. The effects weremore pronounced in the presence of metabolic activation.This observation indicates that the juices appear to induceframeshifts and not base substitutions and that metabolicactivation enhances their mutagenicity. Therefore, thesejuices contain substances that act as indirect genotoxic andmutagenic agents in prokaryotic organisms. Phenolic com-pounds, including quercetin, ascorbic acid, and some met-als, present in these juices (Tables II, III) could contribute tothese mutagenic activities.

S9 mix has also been shown to increase the genotoxicactivity in TA98 of aqueous extracts of Achyrocline sat-ureoides and the positive responses were related to thepresence of quercetin and caffeic acid [Vargas et al., 1990].Quercetin occurs mainly in a promutagenic form in plantsand the mutagenic activity is induced by microsomal hy-drolysis or by glycosidases [Vargas et al., 1990]. Variousstudies have demonstrated that quercetin is a strong frame-shift mutagen in the Salmonella/microsome assay, mainlyafter metabolic activation, and that it has lower activity instrains detecting basepair substitution mutations [MacGre-gor and Wilson, 1988; Czeczot et al., 1990; Gaspar et al.,1993]. Quercetin and other phenolic compounds present inhydrolysates of citrus fruit juices have been shown to beresponsible for their mutagenic activity in S. typhimuriumTA

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TA98, TA97, TA100, and TA1530 [Patrineli et al., 1996a;b; Franke et al., 2002]. In the present study, the quercetinconcentrations in doses exhibiting mutagenicity were 0.116�g/plate (50 �L/plate) for CAJ and 0.232 �g/plate (100�L/plate), 1.16 �g/plate (500 �L/plate), and 4.64 �g/plate(2,000 �L/plate) for cajuina. Quercetin at these concentra-tions could contribute to the observed mutagenicity of thejuices, since they are comparable with concentrations re-ported as mutagenic in previous studies [Schimmer et al.,1988; MacGregor and Wilson, 1988]. Similar quercetinconcentrations were also found in red wine (approximately0.603 �g/plate), where quercetin was demonstrated to be amajor mutagen in TA98 with S9 [Gaspar et al., 1993].

CAJ also contains a high concentration of ascorbic acid(Table II) that could contribute to the positive response inTA97a with S9 metabolism. A correlation between themutagenic responses for TA97a in the presence of S9 mixand the amount of ascorbic acid has been shown for orangejuice [Franke et al., 2002]. It is known that high concentra-tions of ascorbic acid are mutagenic in the Salmonella/microsome assay [Norkus et al., 1993] and that, in thepresence of transition metal ions, ascorbic acid may damageDNA by formation of ROS through the Fenton reaction

[Halliwell and Gutteridge, 2000]. In spite of the presence ofFe and Cu (Table III) in both CAJ and cajuina, we did notdetect mutagenic activity in TA102 (Table IV), suggestingthat the metals are bound by other chemical components,thus impairing their participation in reactions generatingROS. Indeed, transition metals such as Fe and CU canparticipate in the generation of ROS, but these metals maybind with phenolic antioxidants such as quercetin and tannicacid in the juices, reducing their effects. Two mechanismsare commonly proposed to explain the antioxidant role ofphenolic compounds: metal chelation and/or free radicalscavenging, which can decrease the oxygen toxicity to cells[Khokhar et al., 2003]. However, some of the metals presentin cajuina, such as Cr, Cd, and Ni (Table III), have beenreported to be carcinogenic and/or mutagenic in animalstudies and in short-term tests [Rojas et al., 1999]. It hasbeen suggested that Ni can enhance 8-hydroxydeox-yguanosine formation in the presence of H2O2 and ascorbicacid, which could promote base substitution mutations (G:C3T:A transversion) [Rojas et al., 1999; Brozmanova etal., 2001].

Neither juice was mutagenic in TA102 (Table IV), pos-sibly because they do not form ROS. In contrast, owing to

Fig. 1. Evaluation of antioxidant activity of CAJ and cajuina by theTRAP assay expressed as moles of free radical scavenged by the juices.2-2�-Azo-bis (2-amidinopropane) (AAPH, 10 mM) was used as the freeradical and TROLOX (0.75 �M) as a positive antioxidant control. In A and

B, undiluted CAJ and cajuina. In C and D, CAJ and cajuina diluted 5-, 10-,20-, and 50-fold. Statistical significance, one-way ANOVA followed byDunnett’s Multiple Comparison Test. *P 0.05 and **P 0.01 comparedto AAPH. Data are means � SD of three separate determinations.

Mutagenicity and Antimutagenicity of CAJ 365

the presence of the various antioxidant compounds such asascorbic acid, carotenoids, and polyphenols (Table II), thejuices proved to be very efficient scavengers of peroxylradicals (Fig. 1). Despite the higher concentration of con-densed tannins and ascorbic acid in CAJ (Table II), we didnot observe any difference in the antioxidant potential of thejuices, possibly due to the higher concentration of metals(e.g., Fe and Cu; Table III) in CAJ. The lower concentrationof these metals in cajuina suggests that during its processingsome metals may have been complexed with the gelatinused for clarification. We observed that even 20- and 50-fold dilutions of the juices showed antioxidant activityequivalent to the 0.75 �M Trolox solution used as a positiveantioxidant control. The results demonstrate that the 50-folddilution of CAJ had higher antioxidant properties than ca-juina, as shown in Figure 1C,D. Protection against oxidativedamage is a commonly described property of polyphenolsthat is ascribed to binding minerals and scavenging ROS[Thompson and Williams, 1976; Ferguson, 2001]. Also, the

presence of tannic acid, which forms complexes with fer-rous ions, could inhibit the Fenton reaction [Lopes et al.,1999] and thus contribute to the observed antioxidant po-tential. In addition, ascorbic acid has considerable antioxi-dant activity in vitro, in part because of its ease of oxidationand because the semidehydroascorbate radical derived fromit is of low reactivity [Halliwell, 2001].

The high antioxidant potential of the juices generallycorrelated with their activity against the mutagenicity ofH2O2 in Salmonella strain TA102, but only when exposureto the juices occurred during and after the H2O2 treatment(Table V). In pretreatment experiments, exposure to CAJcaused an increase in H2O2 mutagenicity or a comutageniceffect (Table VI). Washing of bacteria with phosphatebuffer (pH 7.4) before plating may cause loss of nutrientsand may alter the pH of the bacterial suspension and thiscould cause inactivation of antimutagenic compounds, i.e.,the antioxidant components present in the juices (Table II).Also, it is known that phenolic compounds, especially at pH

TABLE V. Effects of Cotreatment and Posttreatment with Cashew Apple Juice (CAJ) and Cajuina on the Mutageneicity ofH2O2 in TA102

Number of his� revertant colonies/plate (Mean � SD)a

CAJ Cajuina

Procedure Doseb S9mix I%d �S9mix I% Dosec S9mix I% �S9mix I%

Cotreatment A 10 148 � 51c** — 214 � 89c** — 100 144 � 13c** — 12 � 00c** —25 101 � 6e** — 188 � 57e** — 500 84 � 27e** — 58 � 08e** —50 94 � 10e** — 148 � 74e** — 2000 70 � 16e** — 38 � 04e** —

Cotreatment B 10 397 � 56** 62 422 � 2** 61 100 388 � 24** 74 437 � 30** 9425 393 � 58** 63 296 � 20** 97 500 336 � 31** 88 569 � 16** 6950 681 � 81 15 285 � 33** 100 2000 321 � 28** 92 524 � 80** 77

Cotreatment C 10 NT — 296 � 91** 97 100 NT — 473 � 30** 8725 NT — 424 � 22** 60 500 NT — 489 � 23** 8450 NT — 432 � 20** 58 2000 NT — 502 � 08** 82

Posttreatment A 10 63 � 18e** — 160 � 62e** — 100 206 � 51e** — 278 � 34e** —25 57 � 6e** — 164 � 8e** — 500 281 � 16e** — 390 � 50e** —50 152 � 1e** — 136 � 35e** — 2000 386 � 32** 75 674 � 64** 48

Posttreatment B 10 206 � 18e** — 234 � 30e** — 100 120 � 17e** — 210 � 34e** —25 222 � 72e** — 250 � 38e** — 500 146 � 9e** — 261 � 57e** —50 214 � 36e** — 316 � 36** 91 2000 156 � 24e — 180 � 28e** —

Posttreatment C 10 96 � 14e** — 296 � 12** 97 100 184 � 14e** — 286 � 58e** —25 222 � 6e** — 331 � 51** 87 500 202 � 73e** — 422 � 95** 9750 229 � 46e** — 467 � 19** 48 2000 364 � 64** 81 658 � 47** 51

Positive controlf 100 634 � 40 — 636 � 28 — 100 672 � 40 — 923 � 51 —Spont. revertants 257 � 23 — 284 � 24 — 100 290 � 27 — 408 � 34 —S9 mix control 10 NT — 284 � 63 — 100 NT — 580 � 76 —

25 NT — 263 � 63 — 500 NT — 574 � 62 —50 NT — 298 � 36 — 2000 NT — 476 � 10 —

aMean of three plates.bDose of CAJ in �L/plate.cDose of cajuina in �L/plate.dPercentage of inhibition (1–100%). After 48 hr of incubation the number of revertants was counted and percentage of inhibition was calculated accordingto Cabrera [2000]. I% � [1�(B/A)] � 100, where A represents plates containing H2O2 and B represents the plate containing H2O2 and juice. I% � 50%was considered to show antimutagenicity.eThe decrease in the number of his� revertant colonies was less than the number of spontaneous his� revertant colonies (negative control) suggestingtoxicity.fPlates containing only H2O2. NT: not tested.Statistical significance, one-way ANOVA followed by Dunnett’s Multiple Comparison Test. **P � 0.01.

366 Melo Cavalcante et al.

values � 7, deprotonate, and in this form react with O2,giving rise to superoxide anions and subsequently to hydro-gen peroxide. In addition, the autooxidation of phenolsoccurs preferentially at pH values � 7 [Rueff et al., 1988].Furthermore, many of the chemicals described as antimuta-gens may also act as comutagens, e.g., vanillin and tannicacid [Ferguson, 2001]. These factors may contribute to theenhanced mutagenicity seen in our experiments. In contrast,Ferrer et al. [2002], using the Salmonella assay and theexperimental pretreatment approach employed in our as-says, showed that an extract of the medicinal plant Phyl-lanthus orbicularis protects bacterial cells from oxidativedamage and mutation by H2O2, irrespective of the antioxi-dant activity.

In cotreatment A, the juices decreased the number ofrevertants both with and without S9 (Table V). For thepurpose of evaluating the results of this study, mutagenicresponses below those seen in the negative control plateswere assumed to be caused by toxicity, although such re-sponses could conceivably be due to antimutagenicity thataffected both H2O2-induced and spontaneous mutations. Itis known that under certain experimental conditions, aninteraction between different mutation inhibitors can induceadverse effects, including toxicity [De Flora et al., 1992].Similarly, many antioxidants can, depending on the redoxpotential, either accept or donate electrons, which mayrender them either protective or toxic [De Flora, 1998; DeFlora et al., 2001]. In addition, the presence of anacardicacids in the juices (Table II) could have produced toxicity.Anacardic acid from CAJ acts as an antimicrobial and as acytotoxic agent against BT-20 breast carcinoma and HeLaepithelioid cervix carcinoma cells [Kubo et al., 1993a,b].Whether or not the reductions in revertant frequency seen inour experiments were truly due to toxicity should be estab-lished by additional experimentation.

Cotreatments B and C, which involved reaction of the

juices with H2O2, followed by addition of bacteria andplating, led to inhibition, suggesting that the antimutagenicactivity might be due to phenolic compounds forming com-plexes or to dilution and/or deactivation of H2O2 by chem-ical reactions (Table VII). Possible mechanisms involved insuch modulation of mutagenicity of H2O2 could include freeradical-scavenging (Fig. 1) or extracellular enzymes react-ing with H2O2 or with components of an exogenous meta-bolic system [De Flora, 1998; De Flora et al., 2001]. Thesimilar effects with both juices in the absence and presenceof metabolic activation imply that the inhibitory effectswere not caused by the S9 mix, but by antioxidant compo-nents of the juices, i.e., ascorbic acid and phenolic com-pounds (Table II). The inhibition of H2O2-induced mu-tagenesis by posttreatments B and C for CAJ and byposttreatments A and C for cajuina in the presence of S9(Table V) suggests a possible interaction of the phenoliccompounds in the juices with S9 enzymes. These resultsfavor DNA repair and/or the reversion of DNA damage [DeFlora, 1998; De Flora et al., 2001] as the mechanism in-volved in the inhibitory action of the juices (Table VII).Indeed, a number of phenolic compounds, including vanil-lin, anthocyanins [Agostine-Costa et al., 1999, 2000], tannicacid, and quercetin (Table II), act as antimutagens by mod-ifying DNA replication and/or DNA repair [Ohta, 1993].The antimutagenicity was greater at lower doses of CAJ andcajuina in posttreatment and cotreatment C. This lack of adose-dependent protection against H2O2-induced mutationsuggests competition between the pro- and antimutagenicactivities of some components, such as total phenols, con-densed tannins, and quercetin under these test conditions.

In summary, while CAJ and cajuina were bacterial mu-tagens, they also displayed strong antimutagenic potentialagainst H2O2 in co- and posttreatment protocols. This anti-mutagenic activity could be due to phenolic compoundssuch as quercetin, tannin, and anthocyanins, as well as to the

TABLE VI. Effects of Pretreatments With Cashew Apple Juice (CAJ) and Cajuina on the Mutagenicity ofH2O2 in the TA102

Number of his� revertant colonies/plate (Mean � SD)a

CAJ Cajuina

Procedure Doseb S9mix �S9mix Dosec S9mix �S9mix

Pretreatment 10 1096 � 118** 2239 � 198** 100 724 � 70 1050 � 5625 1474 � 98** 1842 � 141** 500 824 � 00 970 � 9450 1228 � 118** 2296 � 85** 2000 266 � 18** 97 � 28**

Positive controld 634 � 40 636 � 28 672 � 40 923 � 51Negative control 257 � 23 284 � 24 290 � 27 408 � 34S9mix control 10 NT 284 � 63 100 NT 580 � 76

25 NT 263 � 63 500 NT 574 � 6250 NT 298 � 36 2000 NT 476 � 10

aMean of three plates.bDose of CAJ in �L/plate.cDose of cajuina in �L/plate. NT: not tested.dAssays conducted with only H2O2. After 48 hr of incubation the number of revertants was counted. Statistical significance, one-way ANOVA followedby Dunnett’s Multiple Comparison Test. **P � 0.01.

Mutagenicity and Antimutagenicity of CAJ 367

presence of carotenoids and ascorbic acid. Moreover, de-pending on experimental conditions such as route or time ofexposure relative to H2O2 treatment, the juices appear to betoxic and/or comutagenic. Thus, CAJ and cajuina may notonly be nutrients, but may also be a source of chemicalcompounds with antioxidant, mutagenic, antimutagenic,and comutagenic properties. The present study shows thatunder certain conditions CAJ and cajuina may have animportant role in protecting DNA from damage induced byROS generated by intra- and extracellular mechanisms.Clearly, these results warrant further studies to characterizethese proprieties in vivo.

ACKNOWLEDGMENTS

We thank Embrapa (Empresa Brasileira de Pesquisa Agr-opecuaria–Embrapa Agropecuaria Tropical, Fortaleza, CE,Brasil) and PUC-RS (Pontifıcia Universidade Catolica doRio Grande do Sul) for chemical analysis of the juices, andDr. Johnny Ferraz Dias and Dr. Maria Lucia Dias, Institutode Fısica da Universidade Federal do Rio Grande do Sul,Brasil, for determination of metals by proton-induced X-rayemission (PIXE). The authors thank Dr. Martin Brendel andDr. Cristina Gaylarde for review and constructive sugges-tions in improving the manuscript.

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TABLE VII. Possible Antimutagenic Mechanisms of CAJ and Cajuina as Suggested by the Results

Approach Possible mechanisms of antimutagenesisPossible active

compounds

Pretreatment No activity —CotreatmentA No activity —B Interaction with H2O2-forming complexes Total phenols

Dilution and/or deactivation of H2O2 and free radical scavengersInhibition of cytocrome P450 and formation of complex.Reduction of active metabolic processes through downregulation of relevant phase IInhibition of oxidative damage

C Action on metabolites of H2O2 Ascorbic acidPrevention of oxidative metabolism via cytocrome P450 systems CarotenoidsInhibition of the production of electrophilic metabolites

PosttreatmentA, B, and C Interactions with S9 enzymes Total phenols

Modification of DNA replication and/or DNA repair TanninsPromotion of DNA excision repair activity Quercetin

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Accepted by—W.D. Sedwick

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