Antimutagenic and antirecombinagenic activities of noni fruit juice in somatic cells of Drosophila melanogaster

Noni, a Hawaiian name for the fruit of Morinda citrifolia L., is a traditional medicinal plant from Polynesia widely used for the treatment of many diseases including arthritis, diabetes, asthma, hypertension and cancer. Here, a commercial noni juice (TNJ) was evaluated for its protective activities against the lesions induced by mitomycin C (MMC) and doxorrubicin (DXR) using the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. Three-day-old larvae, trans-heterozygous for two genetic markers (mwh and fl r3), were co-treated with TNJ plus MMC or DXR. We have observed a reduction in genotoxic effects of MMC and DXR caused by the juice. TNJ provoked a marked decrease in all kinds of MMCand DXR-induced mutant spots, mainly due to its antirecombinagenic activity. The TNJ protective effects were concentration-dependent, indicating a dose-response correlation, that can be attributed to a powerful antioxidant and/or free radical scavenger ability of TNJ.


INTRODUCTION
Morinda citrifolia L. (Rubiaceae) -popularly known in Hawaii and Brazil as noni, is also called "indian mulberry", "ba ji tian", "nono", "nonu", "cheese fruit", and "nhau".It is one of the most traditional and popular medicinal plants in Polynesia, and its use has been recorded for over 2000 years (Earle 2001).Noni is native to Southeastern Asia (Indonesia) and Australia, this small evergreen tree or shrub is noted for its extremely wide range of environmental tolerances and now has a pantropical distribution, found even at Central and South America (from Mexico to Panama, Venezuela and Surinam) (Nelson 2006).All parts of the plant, especially the fruit, have been utilized as a food source or for its medicinal properties (Cardon 2003, Wang et al. 2002).As a medicinal plant, noni has been used to prevent and cure several diseases.Its therapeutic effects include antimicrobial, analgesic, hypotensive, anti-infl ammatory, anticancer and 585-594 LEONARDO P. FRANCHI et al. immunological system stimulation effects (Chan-Blanco et al. 2006, Wang et al. 2002, Yu et al. 2008).Currently, the use of Morinda citrifolia has become widespread, and its products are commercially available in health food stores, chain grocery stores specializing in natural foods, and on the internet.Both leaves and fruits are processed and sold as capsules, tea, and juice forms, although the fruit juice is the predominant formulation.
The anti-infl ammatory, analgesic, hypotensive and cardiovascular activities of Morinda citrifolia were reviewed in Chan-Blanco et al. (2006).Antimicrobial effects of ethanol and hexane extracts of noni have been described, including their antitubercular activity, with the extracts inhibiting the growth of Mycobacterium tuberculosis by 89-95% (Saludes et al. 2002).Immunomodulatory effects were demonstrated for commercially available juice (Tahitian Noni ® Juice -TNJ); polysaccharide-rich substance from noni juice (noni-ppt) and fruit juice concentrates, both in vivo and in vitro (Hirazumi and Furusawa 1999, Hirazumi et al. 1996, Palu et al. 2008), and recently Harada et al. (2009) detected neuroprotective effect of noni juice against the development of ischemic neuronal damage in mice.
Furthermore, noni-ppt showed antitumor activity in the Lewis lung peritoneal carcinomatosis model (Hirazumi and Furusawa 1999) and prophylactic and therapeutic potential against the immunomodulatorsensitive sarcoma 180 tumor system (Furusawa et al. 2003).The anticarcinogenic properties of the TNJ have been observed at the initiation stage of chemical carcinogenesis, by preventing the carcinogen-DNA adduct formation and/or by acting as an antioxidant (Wang and Su 2001).
Over 150 phytochemical compounds have already been identifi ed in the noni plant (for a review see Chan-Blanco et al. 2006), and the major micronutrients are phenolic compounds, organic acids and alkaloids.The fruit is described to have fl avonoids, lignans and coumarins (Potterat and Hamburger 2007).Although antraquinones occur nearly exclusively in the roots (Deng et al. 2007, Ohsawa andOhba 1993), a potent quinone reductase (QR) inducer, 2-methoxy-1,3,6trihydroxyanthraquinone, has been reported to be present in the fruit, which could account for the cancer chemopreventive activity exerted by noni (Pawlus et al. 2005).
Noni has been tested in various bioassays, in vitro and in vivo, to indicate the absence and/ or evaluate its genotoxic potential, including gene mutation (HPRT), unscheduled DNA synthesis (UDS), Comet assay, Ames test (Westendorf et al. 2007), micronucleus in mouse, chromosomal aberration in human lymphocytes (Edwards 2002(Edwards , 2003) ) and somatic mutation and homologous recombination in Drosophila melanogaster (Franchi et al. 2008).
The purpose of this study was to directly evaluate antimutagenic and/or antirecombinagenic effects of TNJ using the Somatic Mutation and Recombination Test (SMART).This assay is based on the loss of heterozigosity (LOH) induction that may occur through various mechanisms, such as point and chromosomal mutations, as well as mitotic recombination.This versatile short-term in vivo assay detects simultaneously mutational and mitotic recombination, being able to quantify the recombinagenic activity of a compound in a genotoxicity screening (Franchi et al. 2009, Téllez et al. 2007, Toledo et al. 2008).

CHEMICAL COMPOUNDS
In this study we used a commercial noni fruit juice produced by Morinda Inc ( The wing SMART is based on the identifi cation of wing hairs with mutant phenotypes that represent the occurrence of injuries at DNA level.Such alterations are primordially induced in cells of the imaginal discs, which mitotically divide to originate the adults' wings.Thereby, to obtain more detailed data about the antigenotoxic profi le of TNJ, we employed the standard version of the wing SMART in Drosophila melanogaster (Andrade et Drosophila melanogaster (Andrade et Drosophila melanogaster al. 2003, Graf et al. 1984).

FLY STOCKS
The following stocks of D. melanogaster, with genetic markers on the left arm of chromosome 3, were used: (i) mwh/mwh, carrying the wing cell marker 'multiple wing hairs' (mwh abbreviated) and (ii) fl r 3 /In(3LR)TM3, ri p p p p p sep bx 34e es Bd S es Bd S es Bd (fl r (fl r ( 3 abbreviated).
The wing cell marker 'fl are' (fl r The wing cell marker 'fl are' (fl r The wing cell marker 'fl are' ( 3) is a zygotic recessive lethal gene, which is maintained in the strain over the balancer chromosome TM3.

STANDARD (ST) CROSSING
The crossings were carried out en masse for 3 days, in glass vials containing standard culture medium, using 80 fl r using 80 fl r using 80 3 virgin females and 30 mwh males (Graf and van Schaik 1992).The following progeny was produced from this cross: marker-heterozygous fl ies (mwh +/+ fl r 3 ) with phenotypically wild-type wings; and balancer-heterozygous fl ies (mwh +/+ TM3, Bd S Bd S Bd ) with phenotypically serrate wings.S ) with phenotypically serrate wings.

TREATMENTS
Eggs derived from ST crossing were collected for 8 h on standard medium enriched with baker's yeast supplemented with sucrose.After 72 ± 4 h, third-instar larvae were collected by fl otation in running water and placed in bottles containing 0.9 g of Drosophila instant medium (Carolina Biological Supply Company, Burlington, NC, USA) rehydrated with 3 mL of the treatment solutions.
The co-treatment was carried out by mixing the mutagens, MMC (0.05 mM) or DXR (0.2 mM), with three concentrations of TNJ (25%, 50%, and 75% v/v).Larvae were fed on instant medium until pupation (about 48 h).After emergence, adult fl ies were collected from the treatment vials and stored in 70% ethanol.Their wings were mounted in Faure's solution on slides and wing hair mutations were analyzed under a 400× magnifi cation.

SCORING OF WINGS
The induction of LOH in the marker-heterozygous fl ies produce two mutant clones types: (i) single spots, either mwh or fl r 3 , resulting from point or chromosome mutations as well as mitotic recombination, and (ii) twin spots, consisting of both mwh and fl r 3 subclones, originating exclusively from mitotic recombination (Graf et al. 1984).In the balancer-heterozygous genotype, mwh spots refl ect predominantly somatic point mutation and chromosome mutation, since mitotic recombination involving the balancer chromosome and its structurally normal homologue is a lethal event (Vogel et al. 1999).
DATA COLLECTION AND STATISTICAL ANALYSIS The relative frequencies of each type of mutant clone per fl y in a treatment series were compared pair-wise (i.e., genotoxin versus genotoxin + TNJ) using the conditional binomial test according to Kastenbaum and Bowman (1970).The data was evaluated according to the multiple decision procedure proposed by Frei andWürgler (1988, 1995) resulting in four possible diagnostic: positive, negative, inconclusive or weak positive antigenotoxicity.

RESULTS
Prior to the antigenotoxicity assessment, TNJ was submitted to a dose range test, demonstrating that concentrations from 25 to 100% v/v do not exert toxic effects (Franchi et al. 2008).The concentrations used to assess TNJ antigenotoxic effects ranged from 25 to 75% v/v and were coadministered with 0.05 mM of MMC or 0.2 mM of DXR.These concentrations induce genotoxic effects without affecting fl y survival, as also observed in previously published results (Rezende et al. 2011, Santos 1999).
The antigenotoxic effects of TNJ, measured by the wing SMART, after chronic co-exposure to MMC and DXR are summarized in Table I.TNJ showed a statistically signifi cant (p showed a statistically signifi cant (p showed a statistically signifi cant ( <0.05) inhibitory effect against MMC and DXR in the frequencies of total spots for both genotypes (mwh/fl r /fl r / 3 and mwh/ TM3,Bd S Bd S Bd ), although only a weak positive effect S ), although only a weak positive effect S was observed in fl ies treated with the lowest dose of TNJ + MMC.
For the mwh/fl r /fl r / 3 fl ies, the total spot frequency was reduced by 41, 68 and 85% in MMC cotreatments, and 66, 79 and 90% in DXR co-treatment, for concentrations of 25, 50 and 75% of TNJ, respectively.TNJ also had a signifi cant (p<0.05)positive antimutagenic action against MMC and DXR in the mwh/TM3,Bd S Bd S Bd fl ies, S fl ies, S considering the total spot category.Mutant clones induced by recombination are not recovered in the balancer-heterozygous fl ies, indicating that the spots detected in this genotype are all of mutational origin (point and/or chromosomal).
The inhibition observed was of 63, 71 and 97% for MMC-treated fl ies; and of 83, 92 and 96% for DXR-treated fl ies, for concentrations of 25, 50 and 75% of TNJ, respectively.The effect on mutation and recombination induced by both genotoxins, and its modulation by TNJ is shown in Figure 1.MMC (0.05 mM) induced a total of 24.17 spots per fl y through a combination of mutation and recombination, while 25, 50 and 75% concentrations of TNJ decreased this frequency to 14.60, 8.25 and 4.40, respectively.MMC alone induced 5.31 spots per fl y due exclusively to mutation, and 18.86 due to mitotic recombination.TNJ administered in the culture media reduced the MMC mutational frequency to 2.27, 1.86 and 0.60, while the MMCrecombinational frequency was decreased to 12.33, 6.39 and 3.80 respectively, demonstrating a doseresponse effect for both MMC-genotoxic events.DXR (0.2 mM) induced a total frequency of 10.37 spots, but when TNJ (25, 50 and 75%) were co-administered this frequency dropped to 3.80, 2.43 and 1.35 (Table I).DXR induced 0.85 spots per fl y due exclusively to mutation and all TNJ concentrations reduced DXR mutation to a similar value of ~0.30 spots (Figure 1).We observed a clear dose response behavior to DXR recombinational frequency that changed from 9.49 to 3.41, 2.10 and 1.05, in TNJ concentrations of 25, 50 and 75%, respectively.In terms of TNJ antigenotoxic potential these frequencies represent a drop of 56-66% and 63-87% in the frequency of mutation and recombination induced by DXR.

Total spots m=2
Total

TABLE I Effect of the co-treatment (TNJ + MMC and TNJ + DXR) in somatic cells of
Drosophila melanogaster using the wing SMART standard cross.Drosophila melanogaster using the wing SMART standard cross.

DISCUSSION
In a previous report we have demonstrated that TNJ (25, 50, 75 and 100% v/v) has no genetic toxicity effects in somatic cells of Drosophila melanogaster (Franchi et al. 2008), indicating the absence of TNJ mutational and recombinational actions.Previous studies have also shown that TNJ did not induce gene mutations at the HPRT locus (in 0.003 to 3 μL/mL dose range), in presence and absence of S9 mix.In the same study, no mutagenic activity of ethyl-acetate extract from noni juice (100-fold concentrated) was observed in Chinese hamster V79-cell line (Westendorf et al. 2007).There was no increase in micronuclei nor any evidence of systemic toxicity in mice ascribed to dehydrated TNJ (10 g/kg body weight) when administered via oral gavage (Edwards 2002).Also, no signifi cant increases were noted in the frequency of chromosome aberrations in cultured cells from human lymphocytes (625, 1250, 2500, and 5000 μg/mL) in presence or absence of S9 mix (Edwards 2003, Ratanavalachai et al. 2008, West et al. 2006).
Considering these fi ndings, we decided to evaluate the antimutagenic and/or antirecombinagenic action of TNJ.In our study, the cotreatment with different concentrations of TNJ plus DXR or MMC induced a statistically signifi cant dose-response reduction in the frequencies of spots in fl ies.LEONARDO P. FRANCHI et al.
DXR and MMC are two well known antineoplasic agents used in the treatment of solid tumors (Begleiter 2000, Minotti et al. 2004).DXR inhibits the activity of the enzyme topoisomerase II, inducing DNA strand breaks (Islaih et al. 2005, Resende et al. 2006), while MMC acts primarily by promoting DNA crosslinkage (Efi mov and Fedyunin 2010, Riley and Workman 1992, Tomasz et al. 1987).Through these mechanisms, MMC and DXR are able to induce mutations and chromosomal aberrations in both tumor and nontumor cells.Moreover, cellular enzymes are capable of converting DXR and MMC into free radical metabolites (Benchekroun et al. 1993, Dusre et al. 1989, Menegola et al. 2001), which in turn induce damage to several molecules, such as DNA.
Many studies have suggested the coadministration of antineoplasic agents and free radical scavengers, such as antioxidants, to reduce the genotoxicity of such treatments in non-tumor   2007, Antunes and Takahashi 1998, Costa and Nepomuceno 2006, Fragiorge et al. 2007, Gentile et al. 1998, Tavares et al. 2006).So, the noni juice may be promising in this scenario.
In this study, TNJ produced a marked decrease in all kinds of MMC-and DXR-induced mutant spots.However, the largest effect observed in the co-administration of TNJ with MMC and DXR was antirecombinagenic, resulting in 82 and 87% reductions, respectively.
The mechanisms by which TNJ exerts its antigenotoxic activity are not clear at the present.However, antioxidant and/or free radical scavenger activities could be suggested.The radical scavenging activity has been measured in vitro using the terazolium nitroblue (TNB) assay, by assessing the juice's potential capacity to protect cells or lipids from oxidative alteration promoted by a superoxide anion radical (SAR).The SAR scavenging activity of TNJ was 2.8 times higher than that of vitamin C, and 1.4 times higher than that of pycnogenol and grape seed powder (Wang and Su 2001).TNJ's protective effects were proportional to the concentrations applied, indicating a dose-response correlation.
Alternatively, the antigenotoxic activity detected for TNJ could be attributed to the presence of QR inducers.These compounds, such as 2-methoxy-1,3,6-trihydroxyanthraquinone (Pawlus et al. 2005), scopoletin and quercetin (Nitteranon et al. 2011), have been described in noni fruit.QR is a phase II metabolizing enzyme that is induced in conjunction with other protective phase II enzymes.This induction of phase II enzymes, such as QR, is considered as a cancer chemopreventive -since potential oxidative and electrophilic molecules can be more readily metabolized and excreted before its interaction with cellular macromolecules, such as DNA.QR is also responsible for maintaining the reduced states of antioxidants such as α-tocopherol LEONARDO P. FRANCHI et al. and coenzyme Q10.QR inducers are sometimes referred to as "indirect antioxidants", and this activity is considered protective at the initiation stage of carcinogenesis (Dinkova-Kostova andTalalay 2000, Kensler 1997).
This study has successfully used the SMART to demonstrate the protective effects of TNJ on the genotoxicity of DXR and MMC.We conclude that TNJ provides greater protection against these drugs, and that antirecombinagenic activity was the predominant effect.A dose-response relationship was also observed and might be attributed mainly to their powerful scavenger ability.Nevertheless, further experiments should be carried out to gain a better understanding of the mechanism of action of noni phytocompounds and to ensure their safe clinical use.

TABLE I (CONTINUATION) Spots per fl y (nº of spots) statistical diagnosis a
C = 48,800, i. e., approach number of cells examined for individual.f Numbers between keys are the corrected frequencies of induction in relation incidence estimate f Numbers between keys are the corrected frequencies of induction in relation incidence estimate a Statistical diagnosis according to Frei and Würgler (1988): +, positive; -, negative; i, inconclusive.m, factor of multiplication for evaluation of results signifi cantly negatives.Levels of signifi cance α= 0,05.b Including rare single spots fl r 3 .c Considering clones mwh for single spots mwh and for twin spots.d Calculated accordance with Frei et al. (1992).e