Mutagenicity, antioxidant potential, and ... - Wiley Online Library

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

Mutagenicity, Antioxidant Potential, and Antimutagenic Activity Against Hydrogen Peroxide of Cashew (Anacardium occidentale) Apple Juice and Cajuina Ana Ame ´lia Melo Cavalcante,1,3 Gabriel Rubensam,3 Jaqueline N. Picada,3,4 Evandro Gomes da Silva,2 Jose ´ Clau ´ dio Fonseca Moreira,2 and Joa ˜ o A.P. Henriques3* 1

Centro Federal de Educaça ˜ o Tecnolo´gica do Piauı´, CEFET-PI, Teresina, PI, Brasil 2 Centro de Estudos em Estress Oxidativo, Departamento de Bioquı´mica, Universidade Federal do Rio Grande do Sul, UFRGS, Porto Alegre, RS, Brasil 3 Departamento de Biofı´sica e Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brasil 4 Universidade Luterana do Brasil, Departamento de Farmaćia, Canoas, RS, Brasil Fresh and processed cashew (Anacardium occidentale) apple juice (CAJ) are among the most popular drinks in Brazil. Besides their nutritional benefits, these juices have antibacterial and antitumor potential. The chemical constituents of both the fresh juice and the processed juice (cajuina) were analyzed and characterized as complex mixtures containing high concentrations of vitamin C, various carotenoids, phenolic compounds, and metals. In the present study, these beverages exhibited direct and rat liver S9-mediated mutagenicity in the Salmonella/microsome assay with strains TA97a, TA98, and TA100, which detect frameshifts and base pair substitution. No mutagenicity was observed with strain TA102, which detects oxidative and alkylating mutagens and active forms of oxygen. Both CAJ and cajuina showed antioxidant activity as determined by a total radical-trapping potential assay. To test whether this antioxidant

potential might result in antimutagenesis, we used a variation of the Salmonella/microsome assay that included pre-, co-, and posttreatment of hydrogen peroxide-exposed Salmonella typhimurium strain TA102 with the juices. CAJ and cajuina protected strain TA102 against mutation by oxidative damage in co- and posttreatments. The antimutagenic effects during cotreatment with hydrogen peroxide may be due to scavenging free radicals and complexing extracellular mutagenic compounds. The protective effects in posttreatment may be due to stimulation of repair and/or reversion of DNA damage. The results indicate that CAJ and cajuina have mutagenic, radical-trapping, antimutagenic, and comutagenic activity and that these properties can be related to the chemical constituents of the juices. Environ. Mol. Mutagen. 41:360 –369, 2003. © 2003 Wiley-Liss, Inc.

Key words: cashew apple juice; cajuina; mutagenicity; antioxidant; antimutagenicity; Salmonella/microsome assay

INTRODUCTION In recent years the economic value of cashew (Anacardium occidentale) apple juice has increased. Fresh cashew apple juice (CAJ) and processed juice, called cajuina, are among the most popular natural products in Brazil, especially in the northeast region [Embrapa, 2001]. Chemical components in fruits and vegetables, such as micronutrients, phenols, and fiber, may protect against a number of degenerative diseases in humans, including cancer, cardiovascular diseases, cataracts, and brain dysfunction [Ames et al., 1993; Ames, 2001], while deficiencies in micronutrients are likely to cause DNA damage [Ames, 2001; Morrow et al., 2001]. For instance, polyphenols are present in both CAJ and cajuina [Agostini-Costa et al., 2000]. These compounds are a large and diverse class and many have antioxidant, antimutagenic, anticarcinogenic, © 2003 Wiley-Liss, Inc.

antiestrogenic, and antiinflammatory properties that might be beneficial in preventing diseases by improving genomic stability [Ferguson, 2001]. However, not all of the polyphenols, and not all the properties of polyphenols, are beneficial Grant sponsors: CEFET-PI (Centro Federal de Educaçaõ Tecnolo´gica do Piauı´, Brasil) and GENOTOX-Laborato´rio de Genotoxicidade, Centro de Biotecnologia, UFRGS. *Correspondence to: J.A.P. Henriques, GENOTOX–Laborato´rio de Genotoxicidade, Centro de Biotecnologia, UFRGS, Av. Bento Gonçalves, 9500, Pre´dio 43421; Campus do Vale; Caixa Postal 15005; CEP 91501-970, Porto Alegre, RS, Brasil. E-mail: [email protected] Received 2 October 2002; provisionally accepted 27 January 2003; and in final form 8 March 2003 DOI 10.1002/em.10158

Mutagenicity and Antimutagenicity of CAJ

and some of these compounds have mutagenic and prooxidant effects [Ferguson, 2001]. Oxidative damage can originate from increased production of free radicals, caused by exogenous and/or endogenous sources [Halliwell and Gutteridge, 1998, 2000]. Oxidation is considered a highly significant DNA-damaging agent. During the metabolism of DNA, reactive chemical mutagens, carcinogens, or UV light can produce oxygen radicals 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 immunological dysfunction, premature aging, and cancer are associated with singlet molecular oxygen (1O2), hydroxyl radical (OH 䡠 ), superoxide anion (O2 䡠 -), and hydrogen peroxide (H2O2) production [Weisburger, 2001; Ferrer et al., 2002]. H2O2 is produced by endogenous metabolic and catabolic processes in cells and can induce mutations as a direct consequence of the radical generated upon its decomposition [Termini, 2000]. Studies on the free radical-scavenging properties of flavonoids have identified many of the major naturally occurring phenolic compounds as phytochemical antioxidants [Rice-Evans et al., 1997]. The flavonoid quercetin can reduce the DNA damage induced by H2O2 in isolated human lymphocytes by inhibiting DNA strand breakage [Duthie et al., 1997]. Antioxidant activity corresponds to the rate constant of a single antioxidant against a given free radical. The antioxidant capacity is measured as the moles of a free radical scavenged by a test solution, independent of the antioxidant activity of any one antioxidant present in the mixture [Ghiselli et al., 2000]. The aim of the present study was to investigate the mutagenic effects of CAJ and cajuina by the Salmonella/ microsome assay and to determine the possible antimutagenic activity of the juices against H2O2 using pre-, co-, and posttreatment of Salmonella typhimurium strain TA102, with and without metabolic activation. In addition, we evaluated the total antioxidant potential of juices by the total radical-trapping antioxidant potential (TRAP) assay and the principal chemical components of both fresh and processed juices. The identification of antimutagenic compounds and the elucidation of their mechanism of action deserve attention because of their possible significance in the protection of 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 juice sieved using sterile equipment. A sample was tested for the absence of microorganisms and the juice samples were frozen at –20°C. The production of cajuina from CAJ included centrifugation of fruits, clarification with

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

Chemicals Direct-acting mutagens were 4-nitroquinoline-1-oxide (4-NQO), methyl methanesulfonate (MMS), and H2O2; as indirect mutagens, we used benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1). All mutagens were dissolved in dimethylsulfoxide (DMSO). AAPH [2-2⬘–azobis (2 methyl propionamidine dihydrochloride)], isoluminol (6-amino-2, 3-dihydro-1,4-phthalazinedione), 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 Mortelmans and Zeiger [2000], were kindly supplied by Dr. B.N. Ames, University of California, Berkeley, CA, USA.

Microsomal Fraction The postmicrosomal S9 fraction, prepared from livers of Sprague-Dawley 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 to Maron and Ames [1983] and Mortelmans and Zeiger [2000].

Chemical Analysis of Juice The amount of quercetin in CAJ and cajuina was determined by HPLC as described by Careri et al. [2000], using a C18 narrow-bore column (Luna, 150 ⫻ 2.0 mm, 3 mm; Phenomenex, Torrance, CA, USA) and an isocratic 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 1050 delivered the mobile phase and measurements were taken at 370 nm. The total phenolic compounds in both juices were determined using the FolinDenis method described by Agostini-Costa et al. [2000]. The concentrations of these compounds were determined by comparison with a standard curve constructed with tannic acid (0 –10 mL and abs⫹ 0.06921 ⫻ conc. ⫹ 0.02213). Condensed tannins were quantified using the vanillin method described by Agostini-Costa et al. [1999]. Catequin was used to make a standard curve (Abs ⫽ 0.00614 ⫻ Conc ⫹ 0.00252). Anacardic acid was measured according to Agostini-Costa and Jales [2001]. The concentration in the products was quantified by a standard curve produced with anacardic acids extracted from cashews (Anacardium occidentale) (Abs⫹ 0.01205 ⫻ conc-0. 01974). Total carotenoids were assayed using a simplified method for carotenoid distribution in natural compounds [Cecchi and Rodrigues, 1977]. Vitamin C (ascorbic acid) was determined according to the protocol of Pearson and Cox [1976] for the chemical analysis of foods. All concentrations are expressed in mg/100g. Metals were determined by proton-induced X-ray emission (PIXE), a method applying X-ray protons [Kennedy et al., 1998, 1999]. The sample of CAJ and cajuina (from a single manufacturer) were vacuum-filtered onto regenerated cellulose acetate filters (Sartorious, 0.45 ␮m pore, 40 nm diameter) Three samples of each juice were prepared. A clean filter was used 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– 0.7 nmol of juice per cm2. The PIXE analysis was carried out at the 3MV Tandetron accelerator facility at IF-UFRGS. The membranes containing the juices, the blank membrane, and calibration membrane were placed in

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

Possible mechanism of modulation Intracellular reaction

Pretreatment Cotreatment

Extracellular reaction

Posttreatment

Effect on DNA repair

Procedures Juice ⫹ bacteria in fresh nutrient broth (4 hr), wash bacteria and add H2O2 ⫾ S9mix (20 min), wash bacteria and plate. 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 A—Bacteria ⫹ H2O2 ⫾ S9mix (20 min), wash bacteria, add the juice and plate B—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].

the trail receptacle, which accommodates up to 10 specimens. Each sample was located in the proton beam by means of the electrical-mechanical system. The characteristic X-rays induced by the proton beam were detected with an HPGe detector from EG&G (GLP series), with an energy resolution of 170 eV at 5 eV. A mylar filter of 280 ␮M was used in order to prevent low-energy X-rays from reaching the detector, considerably improving the dead time of the acquisition system.

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 potent antioxidant activity, was used as a positive control to assess antioxidant potential. Statistics were performed using one-way ANOVA on the mean of three independent experiments. Dunnett’s multiple comparison test was carried out, accepting a probability of P ⱕ 0.05 as statistically significant.

Salmonella/Microsome Assay The mutagenicity of the juices was measured by the preincubation procedure [Maron and Ames, 1983], using S. typhimurium strains TA97a, TA98, TA100, and TA102 with and without S9 mix. The mixture, consisting of the juice samples to be tested, 500 ␮L of S9 mix (in tests with metabolism) 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 top agar (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 contents poured into a Petri dish containing minimal agar (1.5% agar, Vogel-Bonner E medium plus 2% glucose). All assays were carried out in triplicate. After incubation for 48 hr, colonies (his⫹ revertants) were counted and the results were expressed as mutagenic index (MI ⫽ number of his⫹ colonies induced in the sample/number of spontaneous his⫹ revertants in the negative control). Negative (appropriate solvent) and positive controls were included in each assay. Two ␮g MMS per plate for strains TA100 and TA102 and 0.5 ␮g 4-NQO per plate for TA98, TA97a, and TA102 were used as positive controls for assays conducted without S9. AFB1 (1 ␮g per plate) was used as positive control for TA102 and 1 ␮g per plate B[a]P for strains TA97a, TA98, and TA100 in assays conducted with S9. The juices were considered positive for mutagenicity when: 1) the number of revertants was at least double the spontaneous yield (MI ⱖ 2); 2) a significant response for analysis of variance (P ⱕ 0.05) was found; and 3) a reproducible positive dose-response (P ⱕ 0.01) was present, as evaluated by the Salmonel software [Myers et al., 1991].

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

Antimutagenicity Analysis Antimutagenicity of the CAJ and cajuina against H2O2 was assessed using the standard plate incorporation assay as described by Maron and Ames [1983] and Mortelmans and Zeiger [2000] with the methodological variations described by De Flora et al. [1992] (Table I). An overnight culture of TA102 was washed with 5 mL of 0.2 M phosphate-buffered saline (PBS, pH 7.4). In each experiment we included H2O2 as a positive control. The dose of H2O2 was 1 mM, while the doses of juices were selected in preliminary range-finding assays. We used the following controls: 1) for H2O2, H2O ⫹ H2O2 ⫹ bacteria ⫾ S9 mix; 2) for juice, H2O ⫹ juice ⫹ bacteria ⫾ S9 mix; 3) for S9 mix, juice ⫹ bacteria ⫹ H2O2, with omission of S9 fractions; and 4) for bacteria, H2O ⫹ bacteria ⫾ S9 mix. Incubation was at 37°C with continuous gentle shaking, followed by centrifugation 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 mutagenicity from the H2O2 control only in relation to that shown upon incubation with each juice. The percentage of inhibition for each dose of CAJ and cajuina against H2O2 was calculated according to Cabrera [2000] as follows: (I%) ⫽ [1ⴚ(B/A)] ⴛ 100, where A represents the number of revertants/ plate containing H2O2 and B represents the number of revertants/plate containing H2O2 and juices. The frequency of spontaneous revertants from the appropriate control was subtracted from all plates. The antimutagenic effect 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 in the number of revertant colonies on plates with juice and H2O2 in relation to the number of spontaneous revertants (negative control), the absence of a background lawn and/or complete absence of growth, and presence of pinpoint nonrevertants according to Mortelmans and Zeiger [2000]. Comutagenic effects were considered to have occurred when the number of revertants on the plates with juices and H2O2 were higher than those containing H2O2 only.


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

Total carotenoids Mean ⫾ SDb

Total phenols Mean ⫾ SD

Condensed tannins Mean ⫾ SD

Quercetin Mean ⫾ SD

Anacardic acid Mean ⫾ SD

CAJ Cajuina

0.32 ⫾ 0.0** 0.006 ⫾ 0.0**

11.9 ⫾ 0.3** 8.6 ⫾ 0.4**

61.1 ⫾ 0.5** 13.0 ⫾ 4.0**

0.232 ⫾ 0.30 0.279 ⫾ 0.35

17.9 ⫾ 0.4** 0.41 ⫾ 0.0**

Ascorbic acid Mean ⫾ SD 120.80 ⫾ 4.1** 1.56 ⫾ 0.4**

a

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

TABLE III. Elemental Concentrations Observed in Cashew Apple Juice (CAJ) and Cajuina Obtained by PIXE Analysisa Element (mg/mL) P S Cl K Ti Cr Mn Fe Co Ni Cu Zn Rb Sr Zr Cd Pb

CAJ

Cajuina

0.61 ⫾ 0.05 0.82 ⫾ 0.02 0.09 ⫾ 0.01 3.05 ⫾ 0.03 (0.007 ⫾ 0.002)b (0.006 ⫾ 0.001)b 0.011 ⫾ 0.001 0.21 ⫾ 0.02 ⬍LOD (0.003 ⫾ 0.001)b 0.075 ⫾ 0.004 1.02 ⫾ 0.02 ⬍LOD ⬍LOD ⬍LOD 0.36 ⫾ 0.03 ⬍LOD

⬍LOD 0.006 ⫾ 0.001 (0.0012 ⫾ 0.0006)b 2.57 ⫾ 0.05 (0.0006 ⫾ 0.0001)b ⬍LOD 0.0023 ⫾ 0.0002 0.0014 ⫾ 0.0002 (0.0004 ⫾ 0.0002)b ⬍LOD 0.0174 ⫾ 0.0006 0.0009 ⫾ 0.0002 (0.0008 ⫾ 0.0004)b (0.0013 ⫾ 0.0005)b (0.0007 ⫾ 0.0006)b ⬍LOD (0.0005 ⫾ 0.0002)b

a

The uncertainties quoted above stem from the fitting procedure of the spectra analyzed with Gupix code [Campbell et al., 2000], which takes into account the statistical uncertainty of each photopeak b May be present near the limit of detection (LOD). Means of at least three independent experiments.

RESULTS Chemical Analysis Table II summarizes the results of chemical analysis of the CAJ and cajuina. The concentrations of the analyzed chemical compounds in cajuina were lower than in the fresh juice, especially for ascorbic and anacardic acids, which were about 77- and 43-fold lower in cajuina, respectively. There were no significant differences (P ⬎ 0.1) between the quantity of quercetin in CAJ and cajuina. The results of the PIXE analysis for metals content are shown in Table III. The concentrations of S, Cl, Ti, Mn, Fe, Cu, and Zn in cajuina were reduced. The metals Rb, Sr, Zr, Co, and Pb were detected only in the CAJ and the metals P, Ni, Cr, and Cd only in cajuina. The differences in metal content between CAJ and cajuina may have resulted from the processing of these two products. Cajuina is clarified with gelatin and heat-treated. Metals could be removed by this treat-

ment. Both juices were produced by the same manufacturer from 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 metabolic activation CAJ induced mutagenicity in TA97a (detects frameshift mutation in -C-C-C-C-C-C-; ⫹ 1 cytosine) and cajuina in TA97a and TA100 (basepair substitution mutation results from the substitution of a leucine (GAG) by a proline (GGG)). In the presence of S9 mix, CAJ induced mutations in the TA97a, TA98 (detects frameshifts in DNA target -C-G-C-G-C-G-C-G-) and TA100 strains, while cajuina was only mutagenic in TA98, showing a pronounced mutagenic response (MI ⬎ 15). Neither juice showed any mutagenic activity in TA102, which detects oxidative, alkylating mutagens, and ROS [Levin et al., 1982]. CAJ showed significant toxicity at doses ⬎100 ␮L/plate, both in the presence and absence of metabolic activation (data not shown). Evaluation of Antioxidant Potential of CAJ and Cajuina by the TRAP Assay The pure juices showed excellent antioxidant potential based on their capacity to scavenge free peroxyl radicals produced by AAPH (Fig. 1A,B). To evaluate the relative antioxidant potential of the juices, we diluted each in distilled water. A 5-fold dilution showed no change in relation to the pure juice. At 10-fold dilution the antioxidant potential decreased both in CAJ and in cajuina after 70 min. At a 20-fold dilution, a significant decrease in the antioxidant potential was observed after 40 min and at 50-fold dilution we observed a loss in the antioxidant properties of both juices after 10 min (Fig. 1C,D). Antimutagenic Evaluation of CAJ and Cajuina in a Modified Salmonella/Microsome Assay The antimutagenic effects of CAJ and cajuina against H2O2 in TA102 are shown in Table V. In order to determine the possible presence of promutagens and/or antimutagenic metabolites in the juices, we also used S9 metabolic acti-

Number of his⫹ revertants/plate: mean values of at least three experiments ⫾ SD. Mutagenic Index: no. of his⫹ induced in the sample/no. of spontaneous his⫹ in the negative control. c NC: Negative control: sterile distilled water; positive control (S9) 4NQO (0.5 ␮g/plate) for TA97a (647 ⫾ 25) and TA98 (246 ⫾ 135); MMS (2 ␮g/plate) for TA100 (517 ⫾ 95) and TA102 (634 ⫾ 105); (⫹S9) B[a]P (1 ␮g/plate) for TA97a (349 ⫾ 50), TA98 (189 ⫾ 23) and TA100 (295 ⫾ 1) and AFB1(1 ␮g/plate) for TA102 (765 ⫾ 21). d Negative; ⫹ Positive (ANOVA). *P ⱕ 0.05; dose–response curve, **P ⱕ 0.01; MI ⱖ 2). b

Response

a

1.1 1.4 1.4 – 215 ⫾ 24 248 ⫾ 21 303 ⫾ 28 307 ⫾ 67 145 ⫾ 23 128 ⫾ 12 142 ⫾ 21 212 ⫾ 32 1.0 1.2 2.0 ⫹ 117 ⫾ 16 125 ⫾ 13 144 ⫾ 17 237 ⫾ 14** 1.2 33 16 ⫹

20 ⫾ 5 24 ⫾ 4 668 ⫾ 23** 316 ⫾ 41** 1.1 1.2 2.0 – 16 ⫾ 4 19 ⫾ 8 20 ⫾ 6 34 ⫾ 20 0.9 1.4 1.2 –

221 ⫾ 33 215 ⫾ 39 304 ⫾ 5 281 ⫾ 52 85 ⫾ 11 110 ⫾ 12 188 ⫾ 18** 152 ⫾ 5** 100 500 2000 Responsed NCc Cajuina

10 25 50 100 NC CAJ

c

1.2 2.2 1.7 ⫹

151 ⫾ 58 236 ⫾ 32 797 ⫾ 11** 713 ⫾ 35** 304 ⫾ 20* 53 ⫾ 15 74 ⫾ 6 112 ⫾ 17* 122 ⫾ 23* 170 ⫾ 71

1.4 2.1 2.3 3.2 ⫹

MIb

0.8 0.9 1.4 –

192 ⫾ 21 189 ⫾ 63 166 ⫾ 19 200 ⫾ 30 256 ⫾ 34

0.9 0.7 0.7 2.5 ⫹ 138 ⫾ 6 133 ⫾ 0 103 ⫾ 4 133 ⫾ 32 343 ⫾ 17** 1.4 1.0 1.0 1.0 – 203 ⫾ 1 290 ⫾ 34 218 ⫾ 25 209 ⫾ 39 206 ⫾ 80

1.2 1.4 11 18 ⫹ 13 ⫾ 1 16 ⫾ 4 19 ⫾ 6 142 ⫾ 32** 243 ⫾ 41** 0.9 1.2 0.8 0.8 – 1.5 5.2 4.7 2.0 ⫹

10 ⫾ 4 10 ⫾ 2 12 ⫾ 7 8.3 ⫾ 2 8.6 ⫾ 28

MI ⫺S9

Rev/plate MI

⫹S9

Rev/plate

⫺S9

Rev/platea Conc. (␮l/plate)

S. typhimurium

Juices

213 ⫾ 17 296 ⫾ 15 320 ⫾ 0 334 ⫾ 91

254 ⫾ 46 202 ⫾ 3 385 ⫾ 20 275 ⫾ 41 307 ⫾ 28 0.9 0.8 1.0 1.3 –

Rev/plate

⫹S9 ⫺S9 ⫹S9 ⫺S9

Rev/plate MI

⫹S9

Rev/plate

MI

Rev/plate

MI

Rev/plate

MI

TA102 TA100 TA98 TA97a

TABLE IV. Mutagenicity of Cashew Apple Juice (CAJ) and Cajuina in the Salmonella/Microsome Assay

1.3 1.5 1.5 –

0.7 1.5 1.1 1.7 –

Melo Cavalcante et al. MI

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vation. With the pretreatment procedure (Table VI), CAJ potentiated the mutagenicity of H2O2, both in the presence and absence of S9 mix, suggesting a stimulation of mutagenicity or comutagenic effect. However, cajuina did not show any statistically significant promutagenicity, but was significantly toxic at 2 mL/plate. In cotreatment A, we observed a decrease in the number of the his⫹ revertants below that of the spontaneous controls, suggesting toxic effects of this treatment for both juices. In contrast, CAJ in cotreatment B showed high antimutagenic potential, with 50 ␮L/plate plus S9 mix inhibiting 100% of the mutagenicity induced by H2O2, and 10 and 25 ␮L/plate inhibiting almost 60% without S9 mix. Cajuina also had high antimutagenic potential with the same treatment protocol, showing ⬃100% inhibition with and without S9 mix. In the presence of metabolic activation with cotreatment C, the juices showed a pronounced antimutagenic effect, with about 58% and 97% inhibition. In posttreatments A, B, and C, CAJ appeared to be toxic in the absence of S9 mix. However, in the presence of S9 mix, we observed antimutagenic effects in posttreatment B at 50 ␮L/plate (91%) and in posttreatment C at almost all the doses, with inhibition reaching almost 100%. Cajuina showed similar toxicity in the posttreatments and also inhibited the mutagenicity induced by H2O2, mainly at the higher dose, both with and without S9 mix. DISCUSSION In this study, both CAJ and cajuina were mutagenic in the TA98 and TA97a strains of Salmonella. The effects were more pronounced in the presence of metabolic activation. This observation indicates that the juices appear to induce frameshifts and not base substitutions and that metabolic activation enhances their mutagenicity. Therefore, these juices contain substances that act as indirect genotoxic and mutagenic agents in prokaryotic organisms. Phenolic compounds, including quercetin, ascorbic acid, and some metals, present in these juices (Tables II, III) could contribute to these mutagenic activities. S9 mix has also been shown to increase the genotoxic activity in TA98 of aqueous extracts of Achyrocline satureoides and the positive responses were related to the presence of quercetin and caffeic acid [Vargas et al., 1990]. Quercetin occurs mainly in a promutagenic form in plants and the mutagenic activity is induced by microsomal hydrolysis or by glycosidases [Vargas et al., 1990]. Various studies have demonstrated that quercetin is a strong frameshift mutagen in the Salmonella/microsome assay, mainly after metabolic activation, and that it has lower activity in strains detecting basepair substitution mutations [MacGregor and Wilson, 1988; Czeczot et al., 1990; Gaspar et al., 1993]. Quercetin and other phenolic compounds present in hydrolysates of citrus fruit juices have been shown to be responsible for their mutagenic activity in S. typhimurium


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Fig. 1. Evaluation of antioxidant activity of CAJ and cajuina by the TRAP assay expressed as moles of free radical scavenged by the juices. 2-2⬘-Azo-bis (2-amidinopropane) (AAPH, 10 mM) was used as the free radical 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 by Dunnett’s Multiple Comparison Test. *P ⬍ 0.05 and **P ⬍ 0.01 compared to AAPH. Data are means ⫾ SD of three separate determinations.

TA98, TA97, TA100, and TA1530 [Patrineli et al., 1996a; b; Franke et al., 2002]. In the present study, the quercetin concentrations 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 concentrations could contribute to the observed mutagenicity of the juices, since they are comparable with concentrations reported as mutagenic in previous studies [Schimmer et al., 1988; MacGregor and Wilson, 1988]. Similar quercetin concentrations were also found in red wine (approximately 0.603 ␮g/plate), where quercetin was demonstrated to be a major 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 in TA97a with S9 metabolism. A correlation between the mutagenic responses for TA97a in the presence of S9 mix and the amount of ascorbic acid has been shown for orange juice [Franke et al., 2002]. It is known that high concentrations of ascorbic acid are mutagenic in the Salmonella/ microsome assay [Norkus et al., 1993] and that, in the presence of transition metal ions, ascorbic acid may damage DNA by formation of ROS through the Fenton reaction

[Halliwell and Gutteridge, 2000]. In spite of the presence of Fe and Cu (Table III) in both CAJ and cajuina, we did not detect mutagenic activity in TA102 (Table IV), suggesting that the metals are bound by other chemical components, thus impairing their participation in reactions generating ROS. Indeed, transition metals such as Fe and CU can participate in the generation of ROS, but these metals may bind with phenolic antioxidants such as quercetin and tannic acid in the juices, reducing their effects. Two mechanisms are commonly proposed to explain the antioxidant role of phenolic compounds: metal chelation and/or free radical scavenging, which can decrease the oxygen toxicity to cells [Khokhar et al., 2003]. However, some of the metals present in cajuina, such as Cr, Cd, and Ni (Table III), have been reported to be carcinogenic and/or mutagenic in animal studies and in short-term tests [Rojas et al., 1999]. It has been suggested that Ni can enhance 8-hydroxydeoxyguanosine formation in the presence of H2O2 and ascorbic acid, which could promote base substitution mutations (G: C3 T:A transversion) [Rojas et al., 1999; Brozmanova´ et al., 2001]. Neither juice was mutagenic in TA102 (Table IV), possibly because they do not form ROS. In contrast, owing to

366


TABLE V. Effects of Cotreatment and Posttreatment with Cashew Apple Juice (CAJ) and Cajuina on the Mutageneicity of H2O2 in TA102 Number of his⫹ revertant colonies/plate (Mean ⫾ SD)a CAJ Procedure Cotreatment A

Cotreatment B

Cotreatment C

Posttreatment A

Posttreatment B

Posttreatment C

Positive controlf Spont. revertants S9 mix control

Cajuina

Doseb

⫺S9mix

I%d

⫹S9mix

I%

Dosec

⫺S9mix

I%

⫹S9mix

I%

10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 10 25 50 100

148 ⫾ 51c** 101 ⫾ 6e** 94 ⫾ 10e** 397 ⫾ 56** 393 ⫾ 58** 681 ⫾ 81 NT NT NT 63 ⫾ 18e** 57 ⫾ 6e** 152 ⫾ 1e** 206 ⫾ 18e** 222 ⫾ 72e** 214 ⫾ 36e** 96 ⫾ 14e** 222 ⫾ 6e** 229 ⫾ 46e** 634 ⫾ 40 257 ⫾ 23 NT NT NT

— — — 62 63 15 — — — — — — — — — — — — — — — — —

214 ⫾ 89c** 188 ⫾ 57e** 148 ⫾ 74e** 422 ⫾ 2** 296 ⫾ 20** 285 ⫾ 33** 296 ⫾ 91** 424 ⫾ 22** 432 ⫾ 20** 160 ⫾ 62e** 164 ⫾ 8e** 136 ⫾ 35e** 234 ⫾ 30e** 250 ⫾ 38e** 316 ⫾ 36** 296 ⫾ 12** 331 ⫾ 51** 467 ⫾ 19** 636 ⫾ 28 284 ⫾ 24 284 ⫾ 63 263 ⫾ 63 298 ⫾ 36

— — — 61 97 100 97 60 58 — — — — — 91 97 87 48 — — — — —

100 500 2000 100 500 2000 100 500 2000 100 500 2000 100 500 2000 100 500 2000 100 100 100 500 2000

144 ⫾ 13c** 84 ⫾ 27e** 70 ⫾ 16e** 388 ⫾ 24** 336 ⫾ 31** 321 ⫾ 28** NT NT NT 206 ⫾ 51e** 281 ⫾ 16e** 386 ⫾ 32** 120 ⫾ 17e** 146 ⫾ 9e** 156 ⫾ 24e 184 ⫾ 14e** 202 ⫾ 73e** 364 ⫾ 64** 672 ⫾ 40 290 ⫾ 27 NT NT NT

— — — 74 88 92 — — — — — 75 — — — — — 81 — — — — —

12 ⫾ 00c** 58 ⫾ 08e** 38 ⫾ 04e** 437 ⫾ 30** 569 ⫾ 16** 524 ⫾ 80** 473 ⫾ 30** 489 ⫾ 23** 502 ⫾ 08** 278 ⫾ 34e** 390 ⫾ 50e** 674 ⫾ 64** 210 ⫾ 34e** 261 ⫾ 57e** 180 ⫾ 28e** 286 ⫾ 58e** 422 ⫾ 95** 658 ⫾ 47** 923 ⫾ 51 408 ⫾ 34 580 ⫾ 76 574 ⫾ 62 476 ⫾ 10

— — — 94 69 77 87 84 82 — — 48 — — — — 97 51 — — — — —

10 25 50

a

Mean of three plates. Dose of CAJ in ␮L/plate. c Dose of cajuina in ␮L/plate. d Percentage of inhibition (1–100%). After 48 hr of incubation the number of revertants was counted and percentage of inhibition was calculated according to Cabrera [2000]. I% ⴝ [1ⴚ(B/A)] ⴛ 100, where A represents plates containing H2O2 and B represents the plate containing H2O2 and juice. I% ⱖ 50% was considered to show antimutagenicity. e The decrease in the number of his⫹ revertant colonies was less than the number of spontaneous his⫹ revertant colonies (negative control) suggesting toxicity. f Plates containing only H2O2. NT: not tested. Statistical significance, one-way ANOVA followed by Dunnett’s Multiple Comparison Test. **P ⱕ 0.01. b

the presence of the various antioxidant compounds such as ascorbic acid, carotenoids, and polyphenols (Table II), the juices proved to be very efficient scavengers of peroxyl radicals (Fig. 1). Despite the higher concentration of condensed tannins and ascorbic acid in CAJ (Table II), we did not observe any difference in the antioxidant potential of the juices, possibly due to the higher concentration of metals (e.g., Fe and Cu; Table III) in CAJ. The lower concentration of these metals in cajuina suggests that during its processing some metals may have been complexed with the gelatin used for clarification. We observed that even 20- and 50fold dilutions of the juices showed antioxidant activity equivalent to the 0.75 ␮M Trolox solution used as a positive antioxidant control. The results demonstrate that the 50-fold dilution of CAJ had higher antioxidant properties than cajuina, as shown in Figure 1C,D. Protection against oxidative damage is a commonly described property of polyphenols that is ascribed to binding minerals and scavenging ROS [Thompson and Williams, 1976; Ferguson, 2001]. Also, the

presence of tannic acid, which forms complexes with ferrous ions, could inhibit the Fenton reaction [Lopes et al., 1999] and thus contribute to the observed antioxidant potential. In addition, ascorbic acid has considerable antioxidant activity in vitro, in part because of its ease of oxidation and because the semidehydroascorbate radical derived from it is of low reactivity [Halliwell, 2001]. The high antioxidant potential of the juices generally correlated with their activity against the mutagenicity of H2O2 in Salmonella strain TA102, but only when exposure to the juices occurred during and after the H2O2 treatment (Table V). In pretreatment experiments, exposure to CAJ caused an increase in H2O2 mutagenicity or a comutagenic effect (Table VI). Washing of bacteria with phosphate buffer (pH 7.4) before plating may cause loss of nutrients and may alter the pH of the bacterial suspension and this could 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


367

TABLE VI. Effects of Pretreatments With Cashew Apple Juice (CAJ) and Cajuina on the Mutagenicity of H2O2 in the TA102 Number of his⫹ revertant colonies/plate (Mean ⫾ SD)a CAJ Procedure Pretreatment

Positive controld Negative control S9mix control

Cajuina

Doseb

⫺S9mix

⫹S9mix

Dosec

⫺S9mix

⫹S9mix

10 25 50

1096 ⫾ 118** 1474 ⫾ 98** 1228 ⫾ 118** 634 ⫾ 40 257 ⫾ 23 NT NT NT

2239 ⫾ 198** 1842 ⫾ 141** 2296 ⫾ 85** 636 ⫾ 28 284 ⫾ 24 284 ⫾ 63 263 ⫾ 63 298 ⫾ 36

100 500 2000

724 ⫾ 70 824 ⫾ 00 266 ⫾ 18** 672 ⫾ 40 290 ⫾ 27 NT NT NT

1050 ⫾ 56 970 ⫾ 94 97 ⫾ 28** 923 ⫾ 51 408 ⫾ 34 580 ⫾ 76 574 ⫾ 62 476 ⫾ 10

10 25 50

100 500 2000

a

Mean of three plates. Dose of CAJ in ␮L/plate. c Dose of cajuina in ␮L/plate. NT: not tested. d Assays conducted with only H2O2. After 48 hr of incubation the number of revertants was counted. Statistical significance, one-way ANOVA followed by Dunnett’s Multiple Comparison Test. **P ⱕ 0.01. b

values ⬎ 7, deprotonate, and in this form react with O2, giving rise to superoxide anions and subsequently to hydrogen peroxide. In addition, the autooxidation of phenols occurs preferentially at pH values ⬎ 7 [Rueff et al., 1988]. Furthermore, many of the chemicals described as antimutagens may also act as comutagens, e.g., vanillin and tannic acid [Ferguson, 2001]. These factors may contribute to the enhanced mutagenicity seen in our experiments. In contrast, Ferrer et al. [2002], using the Salmonella assay and the experimental pretreatment approach employed in our assays, showed that an extract of the medicinal plant Phyllanthus orbicularis protects bacterial cells from oxidative damage and mutation by H2O2, irrespective of the antioxidant activity. In cotreatment A, the juices decreased the number of revertants both with and without S9 (Table V). For the purpose of evaluating the results of this study, mutagenic responses below those seen in the negative control plates were assumed to be caused by toxicity, although such responses could conceivably be due to antimutagenicity that affected both H2O2-induced and spontaneous mutations. It is known that under certain experimental conditions, an interaction between different mutation inhibitors can induce adverse effects, including toxicity [De Flora et al., 1992]. Similarly, many antioxidants can, depending on the redox potential, either accept or donate electrons, which may render them either protective or toxic [De Flora, 1998; De Flora et al., 2001]. In addition, the presence of anacardic acids in the juices (Table II) could have produced toxicity. Anacardic acid from CAJ acts as an antimicrobial and as a cytotoxic agent against BT-20 breast carcinoma and HeLa epithelioid cervix carcinoma cells [Kubo et al., 1993a,b]. Whether or not the reductions in revertant frequency seen in our experiments were truly due to toxicity should be established by additional experimentation. Cotreatments B and C, which involved reaction of the

juices with H2O2, followed by addition of bacteria and plating, led to inhibition, suggesting that the antimutagenic activity might be due to phenolic compounds forming complexes or to dilution and/or deactivation of H2O2 by chemical reactions (Table VII). Possible mechanisms involved in such modulation of mutagenicity of H2O2 could include free radical-scavenging (Fig. 1) or extracellular enzymes reacting with H2O2 or with components of an exogenous metabolic system [De Flora, 1998; De Flora et al., 2001]. The similar effects with both juices in the absence and presence of metabolic activation imply that the inhibitory effects were not caused by the S9 mix, but by antioxidant components of the juices, i.e., ascorbic acid and phenolic compounds (Table II). The inhibition of H2O2-induced mutagenesis by posttreatments B and C for CAJ and by posttreatments A and C for cajuina in the presence of S9 (Table V) suggests a possible interaction of the phenolic compounds in the juices with S9 enzymes. These results favor DNA repair and/or the reversion of DNA damage [De Flora, 1998; De Flora et al., 2001] as the mechanism involved in the inhibitory action of the juices (Table VII). Indeed, a number of phenolic compounds, including vanillin, anthocyanins [Agostine-Costa et al., 1999, 2000], tannic acid, and quercetin (Table II), act as antimutagens by modifying DNA replication and/or DNA repair [Ohta, 1993]. The antimutagenicity was greater at lower doses of CAJ and cajuina in posttreatment and cotreatment C. This lack of a dose-dependent protection against H2O2-induced mutation suggests competition between the pro- and antimutagenic activities of some components, such as total phenols, condensed tannins, and quercetin under these test conditions. In summary, while CAJ and cajuina were bacterial mutagens, they also displayed strong antimutagenic potential against H2O2 in co- and posttreatment protocols. This antimutagenic activity could be due to phenolic compounds such as quercetin, tannin, and anthocyanins, as well as to the

368


TABLE VII. Possible Antimutagenic Mechanisms of CAJ and Cajuina as Suggested by the Results Approach Pretreatment Cotreatment A B

C

Posttreatment A, B, and C

Possible mechanisms of antimutagenesis No activity

Possible active compounds —

No activity Interaction with H2O2-forming complexes Dilution and/or deactivation of H2O2 and free radical scavengers Inhibition of cytocrome P450 and formation of complex. Reduction of active metabolic processes through downregulation of relevant phase I Inhibition of oxidative damage Action on metabolites of H2O2 Prevention of oxidative metabolism via cytocrome P450 systems Inhibition of the production of electrophilic metabolites

— Total phenols

Interactions with S9 enzymes Modification of DNA replication and/or DNA repair Promotion of DNA excision repair activity

Total phenols Tannins Quercetin

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Accepted by— W.D. Sedwick