The w / w + Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster for Detecting Antigenotoxic Activity

Genotoxicological studies are emerging as fundamental for knowing the hazards to our genome, to our health. Drosophila melanogaster is one of the preferable organisms for toxicological research considering its metabolic similarities (viz. on dietary input, xenobiotic metabolizing system, antioxidant enzymes and DNA repair systems) to mammals. Accordingly, somatic mutation and recombination tests (SMARTs) of D. melanogaster are fast and low-cost in vivo assays that have shown solid results evaluating genotoxicity. The w / w + SMART uses the white ( w ) gene as a recessive marker to monitor the presence of mutant ommatidia (eye units), indicating the occurrence of point mutations, deletions, mitotic recombination or/and nondisjunction. Additionally, several studies used SMARTs to assess antigenotoxicity, with some using the w / w + SMART. We reviewed the state of the art of the w / w + SMART used for antigenotoxicity analysis, focusing on published results, aiming to contribute to the conception of a reliable protocol in antigenotoxicity. As such, genotoxic agents with known action mechanisms, as streptonigrin (oxidative stress inducer), were used as a genotoxic insult for proving the antigenotoxic effects of natural substances (e.g. seaweeds), demonstrating the presence of antimutagens in their composition. These antigenotoxicity studies are crucial for promoting preventive measures against environmental genotoxics that affect humans daily.


Introduction
The environmental emergency is largely related to environmental toxicology. Each day, new molecules are synthesized, or natural molecules are intensively produced that enter in ecosystems and affect them at all levels. Nowadays there are circulating in living organisms thousands of substances that did not exist 100 years ago, with somewhat unpredictable consequences. As such, more than 159 million chemical substances are registered in the Chemical Abstracts Service (CAS), with with in vitro methods, D. melanogaster has the advantage of enabling a more solid extrapolation at the organism level [3].
D. melanogaster exposure to toxic agents leads to the alteration of simple life traits, which are perturbed negatively, such as development time, number of eclosed individuals, sex ratio, adult body size, fertility and others [6,7]. These life traits can be assessed as a way of measuring the toxicological effects of a given drug, food, drink and so on. However, as science progresses and hazards are targeted in a more specific way, genotoxicological studies with D. melanogaster were developed, aiming to identify environmental hazards inducing damages to genome, i.e. genotoxic agents. In this way, genotoxicological studies with D. melanogaster deal with the assessment of changes in genetic material through various assays, such as germ line mutation assays, somatic mutation assays, the chromosomal aberration assay, the micronucleus test, the comet assay and DNA sequence-based assays, among others. In particular, somatic mutation and recombination tests (SMARTs) have proven to be a good tool for detecting a broad range of genetic alterations quickly and inexpensively [2,8].

Somatic mutation and recombination tests of D. melanogaster
The somatic mutation and recombination tests of D. melanogaster have shown excellent results in assessing the genotoxicity of several and diversified compounds in somatic cells. Originally, in the 1980s, the SMART could be performed by four different assays, but only two of them made it through to the present day: the wing-spot test and the eye-spot test (or w/w + SMART) [9]. The wing-spot test was firstly described by Graf and Würgler [10] and the w/w + SMART by Würgler and Vogel [11], with both showing high values of sensitivity, specificity and accuracy.
Briefly, in the late embryogenesis, larval structures are set, and groups of diploid cells of undifferentiated epithelium, imaginal discs, are formed in the embryo [12]. Then, upon the ending of the larval stages, pupa emerges, and metamorphosis takes place upon systemic hormonal regulation, with the histolysis of the larval organs and differentiation of the imaginal discs into adult structures [13,14]. Accordingly, the exposure of imaginal discs to genotoxic agents may lead to genetic alterations (the product of DNA damage) capable of being transmitted to daughter cells upon mitosis. These genetic alterations can be phenotypically manifested in the adults in structures such as the wings and the eyes, which can be assessed according to the wing-spot test and the eye-spot test, respectively. The loss of heterozygosity (LOH) for specific genetic markers in heterozygous individuals allows the quantification of DNA damage/level of genotoxicity in the adult tissues by visual scoring [9,15].
Between the two types of SMART currently used, from the practical point of view, the w/w + SMART can be assayed with six different strains, as firstly shown by Vogel and Nivard [16], contrasting with only two strains available for the wing-spot one; in the w/w + SMART, a standardized genotoxic agent, inducing a high genotoxicity without toxic effects, streptonigrin (further focused on the chapter) [17], is available and has proved its effectiveness. Nevertheless, since the wing-spot test allows the visual scoring of wings over time, considering that wings are mounted/preserved on slides, opposite from what happens in the w/w + SMART, where eyes have to be analysed quickly since no preserving actions are available (time limited scoring), a greater number of studies have been performed using the wing-spot test (Table 1). Henceforward, as a measure of further exploring the potential of this test and increasing its number of studies, the w/w + SMART will be focused.

w/w + SMART (eye-spot test)
D. melanogaster presents two symmetrically positioned eyes in its head. Each eye consists of repeated hexagonal arrays of approximately 750-800 ommatidia (eye units formed upon differentiation of imaginal discs), homogenous in size and regularly spaced, with each ommatidium being constituted by 14 cells (8 photoreceptor cells, 4 cone cells and 2 primary pigment cells) [18]. Between each two ommatidia, six secondary pigment cells, three tertiary pigment cells and three mechanosensory bristle complexes are present [18]. The adult eye of D. melanogaster is particularly used in toxicological assays since subtle defects in ommatidia development are amplified, by mitosis, several hundred times in the eye [19]. Therefore, it is quite simple to detect genetic alterations changing its pigmentation.
The basis of the w/w + SMART is the white (w) gene located at the position 1.5 of the X chromosome. This gene is used as a recessive genetic marker to monitor the presence of mutant ommatidia/spots, indicating the occurrence of LOH by deletions, point mutations, mitotic/somatic recombination (the most frequent) or/and nondisjunction (chromosome losses) in somatic cells (Figures 1 and 2) [9,16]. These genetic events are known to display a significant role in the induction of carcinogenesis [20]. Accordingly, when wild-type females (w + /w + ; red eyes) are crossed with white-eyed males (w/Y; eyes without pigmentation), or vice versa (w/w with w + /Y), a heterozygous offspring is developed for females (w + /w; red eyes). However, if the offspring is exposed to genotoxic agents in its development phase, the presence of white/mutant phenotype spots in the red eyes may occur (Figures 1 and 2). In addition, when crossing wild-type females with white-eyed males, males' eyes can also be analysed, although somatic recombination should not be considered in this case [9]. The difference between females and males scoring will provide quantitative information on somatic recombination [9].
Moreover, Vogel and Nivard [20, 21] designed a more refined, as well as timeconsuming, version of the w/w + SMART, which allows the detection of chromosomal aberrations in late larval stages. However, and according to Marcos and Sierra [9], the ratio of results obtained/time consumption is low in comparison with the original version of the assay, making it less efficient in the laboratorial routine. Thus, the original version of the assay continues to be the main choice when performing w/w + SMART.

Antigenotoxicity with w/w + SMART
w/w + SMART was, in its original concept, used for the genotoxicological evaluation of several chemical agents, directed to unveiling the action mechanisms behind their genotoxic activities [17, 22-24]. As such, alkylating agents, such as methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethylnitrosourea (ENU), are between the chemicals that induce a great number of mutant ommatidia in D. melanogaster [23]. Even so, and considering the study from Gaivão and Sierra [17], a quinone-based antibiotic, streptonigrin (SN), showed its potential to induce a great level of genotoxicity (increased number of mutant melanogaster female for the white (w) gene. In the scheme, the heterozygous female cell has two X chromosomes with two chromatids each (duplicated DNA in interphase) and daughter cells have two X chromosomes but only one chromatid each (except for nondisjunction). The X chromosomes in red carry the w + allele (dominant) and those in white carry the w allele (recessive), however there are a few exceptions that will be described below.

The position of the alleles in the X chromosomes is represented in a purely illustrative, non-exact way. w* is a mutated wild-type expressing white phenotype. In the development phase of a heterozygous female for the w gene (w + /w), genetic alterations may be induced in the imaginal discs and, upon cell division, daughter cells with mutant/white phenotype ommatidia may appear. The genetic alterations that cause mutant phenotypes are: deletion in one X chromosome including the white locus (in the wild-type allele); point mutation in the wildtype allele by substitution, insertion, or deletion; mitotic recombination between chromatids of the homologous X chromosomes, that replaces the wild-type locus by a mutant locus; nondisjunction, that causes the loss of the chromosome with the wild-type allele.
ommatidia) without toxic effects (at 20 μM) in the w/w + SMART, making it a suitable genotoxic insult for this assay. SN, in the presence of certain metal cations (Zn 2+ , Cu 2+ , Fe 2+ , Mn 2+ , Cd 2+ and/or Au 2+ ), binds to DNA establishing SN-metal-DNA complexes, known as DNA adducts [25-27] (Figure 3). Upon the binding, the quinone reduces, via one or two e À (NADH as a cofactor), producing a semiquinone or a hydroquinone, respectively. Semiquinone reacting with O 2 leads to the production of O 2 À and quinone regeneration. Hydroquinone can lead to the production of H 2 O 2 , while quinone is regenerated (Figure 3) (Figure 3).
Among the processes related to genotoxicity, with an increased relevance in the last years, the analysis of antigenotoxicity is probably the most important one. The search for antigenotoxic agents that could prevent or counteract the harmful consequences of the exposure to DNA damaging agents has increased exponentially lately [29][30][31]. Since most of the possible antigenotoxic agents are components of natural products that could be included in the diet, the analysis of their properties The w/w + Somatic Mutation and Recombination Test (SMART)… DOI: http://dx.doi.org /10.5772/intechopen.91630 should be performed in in vivo experiments. As so, Drosophila fulfils all the requirements for this analysis, specifically when using SMARTs. In fact, there are numerous published studies using D. melanogaster in antigenotoxicity analyses, and most of them are using SMARTs, especially with the wing-spot test ( Table 1).

Simplistic scheme of the genotoxic activity of streptonigrin (SN) on an animal cell. Cell exposure to SN leads to the formation of DNA adducts [SN + metal cation (such as Fe 2+ ) + DNA]. SN's quinone groups are reduced (NADH as a cofactor) to semiquinone and hydroquinone that, in the presence of O 2 , lead to the formation of O 2 À and H 2 O 2 , respectively, both with quinone regeneration (vicious cycle). Thus, by chemical reactions (such as the Fenton and Haber-Weiss ones), OH is produced, the most severe reactive oxidative species (ROS). In this case, the antioxidants (endogenous enzymatic and non-enzymatic, and dietary inputs) are not capable of avoiding excessive ROS formation and progression, as well as communicating to repair mechanisms for repairing the induced genetic damages that may lead to chromosomal aberrations. (1) Superoxide dismutase (SOD); (2) catalase (CAT); (3) glutathione peroxidases.
Focusing on the w/w + SMART performed for antigenotoxicity testing, there are a few studies evaluating the antigenotoxic potential of lemongrass extracts [32]; fennel plant fruit extracts [33]; red, green and brown seaweeds [3,34]; and thalassotherapy products (containing seaweeds) [35]. Ferreira and Marques [3] and Marques and Ferreira [34] studied the exposure of D. melanogaster [Oregon-K (OK) strain] to a chronic treatment (from egg to adult eclosion) with media (Formula 4-24 ® Instant Drosophila Medium) supplemented with red, green or brown seaweeds and SN (at 20 μM). Reductions in the number of mutant ommatidia were shown in individuals cotreated with seaweed and SN in relation to the positive control. Thus, protective properties of seaweeds were exerted against the genotoxic insult of SN, demonstrating antigenotoxic potential. Even more, some species displayed antigenotoxic effects against the spontaneous genotoxicity (without SN insult) of D. melanogaster. The authors also refer the possible phytochemicals acting as antimutagens that include vitamins, phenolic compounds, pigments and polysaccharides. These phytochemicals, which may promote their action in a synergetic way, may inhibit ROS triggered by SN activity, acting as dietary antioxidants [3] (Figure 3). Their mechanisms of action may include ROS scavenging, donation of electrons and/or protons to endogenous enzymatic and/or non-enzymatic antioxidants for converting ROS to H 2 O and/or chelating metal ions responsible for producing OH (Fenton reaction inhibition) [36,37]. In line, using the same conditions, Valente and Borges [35] showed the antigenotoxicity of thalassotherapy products (with seaweeds) against SN. Once more, the potential of seaweeds as dietary antioxidants/antimutagens, as well as the potential of SN as an optimal inducer of chromosomal aberrations quantifiable by the SMART, was demonstrated. Longevity-promoting properties were also displayed upon seaweed supplementation which, according to free radical and mitochondrial theories of ageing, may be a collateral effect of the dietary antioxidants that modulate the enzymatic antioxidants and exert direct antioxidantscavenging actions [3,35].
MMS (at 1 mM) was used as a genotoxic insult against a fennel plant fruit aqueous extract [33]. The positive control showed a great number of induced mutant ommatidia, proving the results from Vogel and Nivard [23], and the fennel extract showed antigenotoxic activity against MMS. According to the authors, and considering the mutagenic activity of MMS, an alkylating agent, consisting of direct interactions with DNA bases that induce mutagenic events, fennel may possess antimutagens that interact directly with the methyl radical groups of MMS and inactivate them in such a manner that they cannot bind to DNA as effectively to induce their mutagenic activity. The antimutagenic properties displayed by fennel may be related to components of its essential oil [33]. In a similar way, Cápiro and Sánchez-Lamar [32] demonstrated the antigenotoxic potential of lemongrass decoction extracts against different genotoxics, MMS, ENU, juglone (JG) and dimethylbenz(a)anthracene (DMBA), that exhibit different mechanisms of action. According to the authors, the lemongrass extract modulated the genotoxic action of the alkylating agents MMS and ENU by interacting with them directly or/and with their mutagenic derivatives. Regarding JG, a naphthoquinone that induces ROS production in an analogous way to SN, damages were reduced upon exposure to the decoction extract by probably inhibiting ROS production, by sequestrating/ inhibiting ROS activity or/and activating intracellular defence mechanisms. For DMBA, as it needs metabolic activation by microsomal enzymes, the extract may have interfered with the microsomal enzymatic system for avoiding DMBA activation. Overall, lemongrass extract acted as an antimutagen in the protection of DNA.
In fact, SMART can be assayed using different test conditions, including the D. melanogaster strain (OK strain has potential for genotoxicity testing; presents high 11 The w/w + Somatic Mutation and Recombination Test (SMART)… DOI: http://dx.doi.org /10.5772/intechopen.91630 susceptibility to ROS, mainly due to a low activity of antioxidant enzymes, being more sensitive to increase its antioxidant status upon intake of dietary antioxidants [3,24]), treatment method (chronic or acute and pre-, co-and post-treatments), genotoxic agent (should always be chosen among those with a known mechanism of action; an example is SN) and sample size. For more details on the methodological approaches of SMARTs, see the protocol from Marcos and Sierra [9].

Conclusions
In vitro and especially in vivo genotoxicity testing of substances such as foods, drinks, drugs and herbicides is fundamental for increasing humans' knowledge on the hazards that we may be exposed to. In this way, upon the identification of a substance/compound as genotoxic, priorities should be focused on avoiding this genotoxic or, at least, when the exposure is unavoidable, preventing our metabolism from damages to DNA that can culminate in mutagenic events and, in a later stage, on carcinogenesis. Upon in vitro testing, in vivo genotoxicological assays, such as w/w + SMART in D. melanogaster, are great tools for evaluating the antigenotoxic potential of a given substance/compound, considering optimal test conditions. The ultimate objective of these tests is to promote the dietary intake of antimutagens, since they are essential for reinforcing our metabolic defences towards genotoxic events, especially the ones that may be produced by strong exogenous agents. Foods, teas, nutraceuticals and others who are richly composed of dietary antimutagens should be of daily intake, considering that there is an increasing threat of new chemical substances with genotoxic potential every day.