Antigenotoxic activity and antioxidant properties of organic and aqueous extracts of pequi fruit ( Caryocar brasiliense Camb . ) pulp

The daily consumption of natural antioxidants protects against oxidative damage caused by reactive oxygen species (ROS), including DNA damage, and can reduce the risk of cancer, atherosclerosis and other degenerative diseases. The pulp of pequi (Caryocar brasiliense Camb.) fruit, a tree native to the Brazilian savannah, contains several compounds with antioxidant properties, including carotenoids, vitamin C, phenolic compounds such as flavonoids, saponins and tannins, and essential oils. In this work, we examined the ability of organic and aqueous extracts of pequi fruit pulp to protect against the genotoxicity induced by two antineoplastic drugs, cyclophosphamide (CP) and bleomycin (BLM). Micronucleus tests with mouse bone marrow cells and single-cell gel electrophoresis (comet assay) with peripheral blood leukocytes were used to examine the effects of CP and BLM, respectively. The antioxidant activity of the extracts was assessed by measuring lipid peroxidation with the TBARS method in mouse plasma. The fruit pulp extracts had no clastogenic or genotoxic effects in the cells studied, but both extracts protected against oxidative DNA damage caused by BLM or CP, indicating an ability to inhibit chemical mutagenesis in vivo. However, the protective effect against oxidative DNA damage depended on the dose of extract used. At the doses tested, the aqueous extract enhanced lipid peroxidation in mice of both sexes, especially in males. In contrast, the organic extract enhanced lipid peroxidation only in male mice, with no significant effect in females. These results suggest that, with adequate adjustment of the dose, an organic extract of pequi fruit pulp could be a useful dietary supplement with natural antioxidant activity, at least in females.

Natural antioxidants in the human diet can attenuate the effects of mutagens and genotoxic carcinogens.Some antioxidants, such as vitamins A, C and E, minimize the side effects of antineoplastic drugs and can improve cancer chemotherapy.An increase in the dietary content of antioxidants through the increased ingestion of fruits and vegetables rich in these compounds can decrease the oxidation of DNA by free radicals, thereby preventing cancer and other degenerative diseases.Oxidative damage to biomolecules caused by stress is one of the major risk factors for atherosclerosis, mainly through the oxidation of low density lipoprotein (LDL) in the blood.Thus, cancer, atherosclerosis and many other degenerative diseases share a common mechanism (Kong and Lillehei, 1998;Santos and Cruz, 2001;Dusinská et al., 2003).
Some dietary antioxidants can act as adjuvants in cancer therapy because of their ability to induce apoptosis in vitro.Antioxidants also show promise in cancer therapy because of their palliative action in reducing painful side effects associated with chemotherapy (Borek, 2004).Khouri et al. (2007) showed that an aqueous extract of pequi pulp can prevent mouse bone marrow cells against chromosomal aberrations induced by bleomycin.Experiments with cultured CHO-K1 cells also showed that this aqueous extract protected cells against the clastogenic effects of cyclophosphamide.
In this study, the anticlastogenic potential of an organic extract of pequi was evaluated in mice bone marrow cells in vivo by using the micronucleus assay.The antigenotoxic effects of aqueous (AEP) and organic (OEP) extracts of pequi pulp were also evaluated by single-cell gel electrophoresis (SCGE -comet assay) in mouse peripheral blood leukocytes.The antioxidant activity of AEP and OEP on lipid peroxidation in mouse blood was assessed by the thiobarbituric acid-reactive substances (TBARS) assay.

Plant material
Pequi fruit was obtained in natura from vendors in Brasília, DF (Brazil) and the surrounding region.The internal mesocarp was peeled or grated to obtain the pulp, which was then packed in a covered pot and frozen to -86 °C.

Extraction procedure
Pequi pulp was extracted by the soxhlet procedure under an argonium atmosphere using chloroform (organic extract of pequi pulp -OEP) or distilled water (aqueous extract of pequi pulp -AEP) as the solvent.After extraction, the extracts were submitted to evaporation under re-duced pressure to remove the solvents, dried at high vacuum and immediately frozen at -86 °C.

Animals
Swiss white mice of both sexes (60 days old, 30 ± 2 g) obtained from the Central Animal Facility of the University of Brasília were housed in plastic cages (6 or 8/cage) at room temperature (20 ± 2 °C) on a 12 h light/dark cycle with lights on at 6 a.m. and free access to food and water.The experimental protocols described here were approved by the institutional Ethics Committee for Animal Research (Institute of Biological Science, University of Brasília).

Anticlastogenic and antigenotoxic assays Micronucleus (MN) test
For the MN test, only the OEP was used since the AEP has already been studied by Khouri et al. (2007).Mice of both sexes were randomly allocated to groups M1 to M12 (n = 8 mice/group; Table 1).OEP (0.5 mL kg -1 or 1 mL kg -1 of body weight, respectively, corresponding to 15 μL and 30 μL of extract per mouse) was administered orally (per os, p.o.) by gavage daily for 10 days either alone or in combination with a single intraperitoneal (i.p.) injection of bleomycin diluted in 0.9% NaCl or aqueous cyclophosphamide (see Table 1 for concentrations).Control mice received filtered water in place of extract and no clastogen was administered.When required, the mice were killed by cervical dislocation and slides of bone marrow cells were prepared by a standard method (Schmid, 1975).Two thousand erythrocytes were counted per mouse (1000 normochromatic erythrocytes, NCE, and 1000 polychromatic erythrocytes, PCE) and the frequency of micronuclei (MN) in PCE and NCE, and the percentage of polychromatic erythrocytes (%PCE) were calculated.

Single-cell gel electrophoresis (SCGE) assay (comet assay)
The SCGE assay was done essentially as for the MN test, using mice of both sexes randomly allocated to groups C1 to C9 (n = 8 mice/group; Table 2).The comet assay was done after the MN test, using OEP (0.5 mL hg -1 ) and AEP (1 mL kg -1 ) administered p.o. daily for 10 days, either alone or in combination with a single injection of bleomycin (25 mg kg -1 , i.p.) or two injections of cyclophosphamide (at 30 mg kg -1 , i.p.48 h and 24 h before the mice were killed) (Table 2).
The comet assay (alkali method) proposed by Singh et al. (1988) and improved by Tice (1995) was used.Microscope slides were dipped briefly into 1.5% hot (60 °C) normal melting agarose prepared in phosphate-buffered saline (PBS).The slides were dried overnight at room temperature and then stored at 4 °C until used.Subsequently, freshly collected, heparinized peripheral blood (20 μL) was suspended in 120 μL of 0.5% low melting point agarose in PBS (Gibco BRL) at 37 °C and pipetted onto a microscope slide pre-coated with a layer of normal melting point agarose.This mixture was allowed to set at 4 °C for 10 min and the slides then immersed in a freshly prepared cold (4 °C) lysis solution (2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Tris and 1% lauroyl sarcosine, and the pH adjusted to pH 10.0-10.5 with NaOH; 1% Triton X-100 and 10% dimethyl sulfoxide were added immediately before use) at 4 °C for 1 h.After lysis, the slides were placed in an electrophoretic tank containing 300 mM NaOH and 1 mM EDTA, pH > 13.0 for 30 min, and electrophoresis was then done at 25 V, 350 mA for 40 min.Soon after neutralization (3 x 5 min in 0.4 M Tris, pH 7.5 at 4 °C), the slides were stained with ethidium-bromide (20 μg/mL), fixed in 100% ethanol for 5 min and analyzed with a Zeiss Axioskop 2 fluorescence microscope (filter 510-560 nm, barrier filter 590 nm) with a final magnification of 400x.All of the slides were prepared and analyzed in duplicate.
SCGE analysis: One hundred comets on each slide were scored visually and assigned to classes 0-4, as proposed by Collins (1995).Nucleoids with bright heads and no apparent tails were assigned to category 0, whereas comets with very small heads and long, diffuse tails were assigned to category 4. Comets with intermediate features between classes 0 and 4 were assigned to classes 1, 2 and 3.The number of comets in each category was counted and the average DNA damage (DD, expressed in arbitrary units, a.u.) was calculated according to Jalonszynski et al. (1997), as follows: where n 1 -n 4 = the number of comets in categories 1-4, and Σ = the sum of all comets, including category 0.

958
DNA protective effects of pequi fruit pulp extracts  was prepared by dissolving TBA (Merck; final concentration 29 mmol/L) in acetic acid (8.75 mol/L; Merck).The stock standard solution of MDA was prepared by dissolving 480 μL of 1,1,3,3-tetraethoxypropane (Sigma) in 100 mL of ethanol (Merck).Immediately before use, the solution was diluted in Milli-Q water to yield a working solution of 10 μmol/L.The other reagents for the assay were purchased from Sigma (n-butanol) or Merck (EDTA, glutathione -GSH, and hydrochloric acid).Working aqueous solutions of EDTA (67.3 mmol/L) and GSH (32.5 mmol/L) were prepared freshly immediately before use (to avoid GSH oxidation).
Blood was collected by cardiac puncture using na insulin syringe containing EDTA as anticoagulant.After centrifugation (1500 x g, 10 min, 4 °C), the plasma was carefully removed and EDTA and GSH were added to final concentrations of 1.34 mmol/L and 0.65 mmol/L, respectively.The samples were then quickly frozen in liquid nitrogen and stored at -80 °C until used.
For the TBARS test, 50 μL of plasma or an equal volume of MDA working standard was added to 10 mL glass tubes containing 1 mL of Milli-Q water, followed by 1 mL of solution containing TBA (29 mmol/L) in acetic acid (pH of the reaction mixture, 2.4-2.6),mixing, and heating in a water bath for 1 h at 95°-100 °C.The samples were then cooled and 25 μL of 5 mol/L HC1 was added (final pH 1.6-1.7),followed by extraction with 3.0 mL of n-butanol and vortex mixing for 30 s.The butanol phase was separated by centrifugation (1500 x g, 10 min) and its fluorescence was measured with a Jasco FP-777 spectrofluoro-meter (excitation: 525 nm, emission: 547 nm).A standard curve was prepared with MBA (0-0.15 μmol/L).

Statistical analysis
The results were expressed as the mean ± SD.Statistical comparisons were done by using the Mann-Whitney U-test, with p < 0.05 indicating significance.

Anticlastogenic and antigenotoxic assays Micronucleus (MN) test
Neither of the doses of OEP (1 mL kg -1 -group M3 or 0.5 mL kg -1 -group M8) had any clastogenic effect on mouse bone marrow cells when compared with the control group (M1) (Table 1).However, the cellular proliferation index in mouse bone marrow cells, expressed as the percentage of PCE, increased in groups M3 and M8 (p = 0.0117 and p = 0.0460, respectively).There was no significant difference in the frequencies of MN-PCE (p = 0.5780), MN-NCE (p = 0.7004) and the cellular proliferation index (p = 0.9163) between groups M3 and M8.Cyclophosphamide (30 mg kg -1 , group M2) increased the frequency of MN-PCE, indicating this drugs clastogenic effect.Concomitant treatment with OEP (0.5 mL kg -1 , group M9) did not significantly affect the percentage of PCE compared to M2.In contrast, concomitant treatment with OEP (1 mL kg -1 , group M4) increased the frequency of MN-PCE to a greater extent than seen with CP or OEP alone (p = 0.0309).

Antioxidant activity (TBARS assay)
There was no significant difference in the TBARS levels of female (group T1) and male (group T2) mice (Table 3).There were also no significant differences between T1 and T3, T3 and T5 or T4 and T6.However, mice treated with AEP showed significant lipid peroxidation (p = 0.0248 for T1 vs. T5, and p = 0.0001 for T2 vs. T6), indicating that AEP (1 mL kg -1 ) enhanced oxidative stress in both sexes, especially in males.A similar result was observed between control males and males treated with OEP (0.5 mL kg -1 ) (T2 vs. T4, p = 0.00002).Thus, at the doses used, neither of the extracts protected males against lipid peroxidation.

Discussion
Micronuclei in interphase cells result from chromosomal breaks or chromosomal lagging (Schmid, 1975;Natarajan and Obe, 1982;FDA, 2000), and DNA damage to eukaryotic organisms or individual cells is frequently assessed with the comet assay (Henderson et al., 1998;Garcia et al., 2004).In the present study, the MN test and comet assay showed that OEP (0.5 mL kg -1 or 1 mL kg -1 ) had no clastogenic or genotoxic effect on mouse bone marrow cells or peripheral blood lymphocytes, a finding that corroborated the MN test results reported by Khouri et al. (2007).
Bleomycin sulphate (BLM), a radiomimetic glycopeptide routinely used in cancer chemotherapy, produces MN in human, rat and mouse cells, mainly through oxidative damage to DNA (Erexson et al., 1995).Bleomycinmediated DNA degradation requires the presence of a redox-active metal ion such as Fe 2+ or Cu 2+, as well as molecular oxygen, to produce reactive oxygen species (ROS) that are toxic and mutagenic in a variety of biological models in vitro and in vivo (Hoffmann et al., 1993;Anderson et al., 1995;Erexson et al., 1995;Hecht, 2000).

960
DNA protective effects of pequi fruit pulp extracts Table 3 -Lipid peroxidation (TBARS levels) in plasma of Swiss white mice treated orally an organic (OEP, 0.5 mL kg -1 ) or aqueous (AEP, 1 mL kg -1 ) extract of pequi (Caryocar brasiliense) fruit pulp.As shown here, the prior administration of OEP (1 mL kg -1 ) clearly protected mouse bone marrow cells against lysis induced by BLM (50 mg kg -1 ); a lower dose of OEP (0.5 mL kg -1 ) also protected against the natural clastogenic effects, although the decrease in MN-NCE was not significant.Hence, both of the OEP doses used offered some protection against the clastogenicity of BLM.This finding was corroborated by the comet assay.AEP (1 mL kg -1 ) offered greater protection against BLM (25 mg kg -1 )-induced DNA damage than OEP (0.5 mL kg -1 ); the reduction in DNA damage by the latter was not significant.

Group
Since the cytotoxicity of BLM involves the production of ROS (Hoffmann et al., 1993;Anderson et al., 1995;Erexson et al., 1995;Hecht, 2000), our results suggest that the protection against BLM-induced oxidative damage to DNA was mediated by antioxidant components in the C. brasiliense extracts.Indeed, previous studies have indicated that compounds with antioxidant activities may modulate BLM-induced genotoxicity (Povirk and Austin, 1991;Povirk, 1996;Khouri et al., 2007).The results of the comet assay corroborated those of the MN test and showed that the ability of the two extracts to protect against BLM was limited by the dose of extract used.The greater protection seen with AEP probably reflected its higher content of antioxidants compared to that of OEP at the doses used.
Chemotherapy with CP can cause secondary tumors in humans by activating hepatic mixed function oxidases.Phosphoramide mustard, the major antineoplastic metabolite of CP, is an alkylating agent that induces a variety of changes in DNA (IARC, 1981;Krishna et al., 1987) through its ability to form labile covalent DNA adducts and cross-linkages (Anderson et al., 1995).
Compounds with antioxidant properties, such as vitamin C (present in AEP) and β-carotene (present in OEP) can have antioxidant and oxidant effects that are dosedependent (Antunes and Takahashi, 1999;Paolini et al., 2003).This two-sided or Janus effect reflects the dual nature of certain chemical substances such as antioxidants and drugs used to treat cancer.As shown here, OEP had a Janus effect: at 0.5 mL kg -1 OEP effectively protected cells against the genotoxicity of CP (comet assay) without significantly protecting against chromosomal aberrations (MN test) whereas at 1 mL kg -1 OEP increased the frequency of MN in the presence of CP (30 mg kg -1 ).Similar results were reported by Khouri et al. (2007) who studied the combination of AEP (1 mL kg -1 ) plus CP (33 mg kg -1 or 66 mg kg -1 ).
With the treatment schedule used here, it was not possible to determine whether the protection offered by the extracts was related to a reduction in the efficacy of BLM or CP (by affecting the pharmacokinetics of these compounds), or to a direct effect on the cells themselves.However, previous studies have shown that natural antioxidants can prevent the oxidation of biomolecules, including DNA, without decreasing the effectiveness of chemotherapeutic compounds.According to Liu et al. (2003) and Berger (2005), an antioxidant dietary supplement can reduce the level of oxidative damage to DNA and protect normal cells against the adverse side-effects of some chemotherapeutic agents.Nutritional therapy with antioxidants concomitant with chemotherapy reduces the frequency and severity of adverse effects associated with many drugs in cancer patients, thereby allowing the treatment to be continued (the toxicity of anti-cancer drugs is a frequent limitation to their extended use) (Borek, 2004).As shown here, OEP and AEP protected against oxidative DNA damage caused by BLM or CP, which agrees with the ability of antioxidants to inhibit chemical mutagenesis in vivo and with studies showing that plant extracts contain antioxidants that can protect DNA against damage by ROS (Cano et al., 2003;Lee et al., 2003;Owen et al., 2003;Khouri et al., 2007).However, our results indicate that this protection is related to the dose of extract used.
Organisms are constantly exposed to the action of ROS and the presence of antioxidants in the diet can protect against oxidative processes (Ferreira and Matsubara, 1997;Degáspari and Waszczynskyj, 2004).In contrast, the use of nutritional supplements with antioxidant properties is controversial.Antunes and Takahashi (1999) showed that vitamin C had antimutagenic activity only at certain concentrations; at high concentrations this compound did not protect against mutations but was cytotoxic to lymphocytes.Paolini et al. (2003) reported that β-carotene prevented oxidative lesions in DNA but stimulated cytochrome P450 enzymes, leading to the bioactivation of pro-carcinogenic agents such as those found in cigarette smoke.In agreement with these studies, the OEP of pequi fruit may contain high concentrations of many antioxidant compounds capable of exerting a pro-oxidative effect and increasing DNA lesions when combined with CP or BLM.
The TBARS assay is a simple, reliable and reproducible fluorometric method for assessing lipid peroxidation in serum, and is based on the reaction between malondialdehyde (MDA), a byproduct of lipid peroxidation, and thiobarbituric acid.Oxidative attack of cellular components by ROS is a common phenomenon in the pathogenesis of several human diseases, including cardiovascular diseases (e.g., atherosclerosis), diabetes, various liver disorders, and inflammatory rheumatic diseases.For this reason, there is considerable interest in the quantification of MDA in body fluids or cells (Wasowicz et al., 1993;Jentzsch et al., 1996).The TBARS results obtained here indicate that high doses of plant extracts rich in antioxidant compounds can produce unexpected effects.Since the 1980s, there has been a considerable increase in the search for natural antioxidants that can be included in the diet as a substitute for synthetic antioxidants (Degáspari and Waszczynskyj, 2004).The TBARS results obtained here in female mice suggest that the antioxidant properties of OEP make this product a potentially useful dietary supplement, in addition to its other nutritional properties.
The anticlastogenic and antigenotoxic activities of OEP seen in this study were attributable to its content of antioxidants.However, the comet assay and MN test results obtained here and by Khouri et al. (2007) indicate that, at the doses used, AEP was more effective than OEP in protecting DNA against damage by clastogenic agents.However, the ability of OEP and AEP to protect against BLMor CP-induced oxidative damage to DNA depended on the dose of extract used.
a p = 0.0117 compared to M1. b p = 0.0003 compared to M5. c p = 0.0460 compared to M1. d p = 0.01003 compared to M1. e p = 0.0357 compared to M11. f p = 0.0357 compared to M1. g p = 0.0006 compared to M1. h p = 0.0309 compared to M2. i p = 0.0006 compared to M1. j p = 0.0003 compared to group M5.k p = 0.0009 compared to M1. l p = 0.0172 compared to M1. m p = 0.0006 compared to M1. n p = 0.0009 compared to M1. o p = 0.0332 compared to M7.

Table 1 -
Micronuclear evaluation of bone marrow cells from Swiss white mice treated orally with an organic extract of pequi (Caryocar brasiliense) fruit pulp (OEP; 0.5 mL kg -1 MN-PCE and MN-NCE -micronucleus test results for PCE and NCE, respectively.The results are expressed as the mean±SD.*Extensive cell lysis was seen in bone marrow, with few cells and no reticulocytes.