Open Access is an initiative that aims to make scientific research freely available to all. To date our community has made over 100 million downloads. It’s based on principles of collaboration, unobstructed discovery, and, most importantly, scientific progression. As PhD students, we found it difficult to access the research we needed, so we decided to create a new Open Access publisher that levels the playing field for scientists across the world. How? By making research easy to access, and puts the academic needs of the researchers before the business interests of publishers.
We are a community of more than 103,000 authors and editors from 3,291 institutions spanning 160 countries, including Nobel Prize winners and some of the world’s most-cited researchers. Publishing on IntechOpen allows authors to earn citations and find new collaborators, meaning more people see your work not only from your own field of study, but from other related fields too.
To purchase hard copies of this book, please contact the representative in India:
CBS Publishers & Distributors Pvt. Ltd.
www.cbspd.com
|
customercare@cbspd.com
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.
Department of Genetics and Biotechnology and Animal and Veterinary Research Centre (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Portugal
João Ferreira
Department of Genetics and Biotechnology and Animal and Veterinary Research Centre (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Portugal
Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), UTAD, Portugal
Luisa María Sierra
Department of Functional Biology, Genetics Area, Oncology University Institute (IUOPA) and Institute of Sanitary Research of Asturias (ISPA), University of Oviedo, Spain
*Address all correspondence to: igaivao@utad.pt
1. 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 approximately 4000 new substances being registered daily [1]. As a controlling measure, the EU Commission created, in 2004, a network (NORMAN network) of laboratories, research centres and organizations for monitoring the emerging environmental substances [1].
Environmental toxicology encompasses exposure to toxic substances whether through the air we breathe, the food we eat, the water we drink and the clothes we wear or through the skin, cosmetics, etc. There is also radiation exposure, which also has harmful effects, and is much more problematic today than some years ago. The planet is poisoned, affecting the air, the water, the soil and the food we produce, which causes serious problems to human health and ecosystems. It is hoped that worldwide awareness of this reality will be achieved, and the focus of humanity’s greatest concerns will be on the cleansing of the planet by eliminating or at least greatly reducing the produced toxic agents.
This whole problem greatly affects DNA, causing DNA damage (genotoxicity), affecting DNA repair mechanisms and causing mutations when damage is not properly repaired. In the short term, this genome instability leads to diseases such as cancer, degenerative diseases, fertility decrease and other problems. In the long term, we may see the emergence of new diseases due to new mutations in the germ line, which, if recessive, may take several generations until there is a chance of homozygosis, where rare diseases may arise. All combined may affect the life expectancy of several species, causing an environmental collapse. Preventive strategies are indispensable to reduce the heavy burden on national healthcare systems and families. The most effective is a healthy lifestyle including diet, as an antigenotoxic diet reduces DNA damage and all the associated diseases. Antigenotoxic activities include inactivation of genotoxic compounds, by several mechanisms and increasing repair capacity, decreasing the effectiveness of a genotoxic. While DNA damage is clearly implicated as the initiating event in most cancers, the link is not a simple one. Most damage is removed by repair enzymes before it can interfere with the process of DNA replication and introduce mutations. Given a carcinogenic exposure, the individual variation in the capacity for DNA repair is therefore likely to be an important factor in determining cancer risk.
Over the years, many investigations in DNA damage and DNA repair mechanisms were made, in vitro and in vivo, aiming to know our environment and thus identifying the harmful compounds to our genome, to our health, leading to preventive actions such as prohibiting the commercialization of certain drugs, construction materials, foods and drinks. Genotoxicological studies using cell cultures and animals are essential for increasing human’s wellbeing, since they display solid results in showing the genotoxicity of compounds and should be standardized (with optimal test conditions) for increasing their reproducibility and precision.
2. Drosophila melanogaster in toxicological research
Drosophila melanogaster is currently being used as one of the preferable organisms for toxicological research [2]. According to current knowledge, the use of D. melanogaster as a model organism respects the principles of animal welfare (3Rs), since ethical matters do not urge when using this organism [2, 3]. Considering the metabolic pathways responsible for dietary input (including nutrient uptake, digestion, absorption, storage and metabolism) [4], the xenobiotic metabolizing system, the antioxidant enzymes and the DNA repair systems of D. melanogaster, which are analogous to those of mammals, D. melanogaster emerges as an optimal replacer of higher animals in toxicological studies [2, 5]. Furthermore, contrasting 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].
2.1 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.
Published studies focusing the antigenotoxic evaluation of several types of chemicals, nanoparticles and plants/seaweeds/seeds/oils using somatic mutation and recombination tests (SMARTs).
The type of test, wing- or eye-spot, the used genotoxic agents, as well as the information about the antigenotoxic potential of the tested substances (response: + antigenotoxic activity; − no antigenotoxic activity or synergistic genotoxic activity) is presented.
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) [67]. Between each two ommatidia, six secondary pigment cells, three tertiary pigment cells and three mechanosensory bristle complexes are present [67]. 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 [68]. 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 [69]. 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].
Figure 1.
Scheme of the possible four types of genetic alterations that generate white ommatidia in a heterozygous D. 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 wild-type 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.
Figure 2.
Wild-type eyes of D. melanogaster (females) at the stereoscopic microscope (80× magnification). (A) An eye without mutant spots, (B) an eye with a dark spot affecting one to two ommatidium(a) (marked by a black arrow) and (C) an eye with a spot affecting innumerable ommatidia. White mutant spots appear as black when surrounded by pigmented/red ommatidia.
Moreover, Vogel and Nivard [69, 70] designed a more refined, as well as time-consuming, 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.
3.1 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, 71, 72, 73]. 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 [72]. 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 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 (Zn2+, Cu2+, Fe2+, Mn2+, Cd2+ and/or Au2+), binds to DNA establishing SN-metal-DNA complexes, known as DNA adducts [74, 75, 76] (Figure 3). Upon the binding, the quinone reduces, via one or two e− (NADH as a cofactor), producing a semiquinone or a hydroquinone, respectively. Semiquinone reacting with O2 leads to the production of O2− and quinone regeneration. Hydroquinone can lead to the production of H2O2, while quinone is regenerated (Figure 3). In consequence, OH can be produced by the Fenton reaction (H2O2 + Fe2+ → OH + OH− + Fe3+) and by the Haber-Weiss reaction (O2− + H2O2 → OH + OH− + O2), leading to oxidative stress [74, 75, 76]. The production of reactive oxygen species (ROS), and the prolonged SN linkage to DNA, can lead to the inhibition of DNA (and RNA) synthesis, induce unscheduled DNA synthesis, promote DNA strand breaks as well as inhibit topoisomerase II [77]. Chromosomal aberrations may occur upon mutagenic events, creating genomic instability that can culminate into carcinogenic events [76] (Figure 3).
Figure 3.
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 Fe2+) + DNA]. SN’s quinone groups are reduced (NADH as a cofactor) to semiquinone and hydroquinone that, in the presence of O2, lead to the formation of O2− and H2O2, 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.
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 [78, 79, 80]. Since most of the possible antigenotoxic agents are components of natural products that could be included in the diet, the analysis of their properties 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). Focusing on the w/w+ SMART performed for antigenotoxicity testing, there are a few studies evaluating the antigenotoxic potential of lemongrass extracts [26]; fennel plant fruit extracts [21]; red, green and brown seaweeds [3, 43]; and thalassotherapy products (containing seaweeds) [66].
Ferreira and Marques [3] and Marques and Ferreira [43] 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 H2O and/or chelating metal ions responsible for producing OH (Fenton reaction inhibition) [34, 81]. In line, using the same conditions, Valente and Borges [66] 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 antioxidant-scavenging actions [3, 66].
MMS (at 1 mM) was used as a genotoxic insult against a fennel plant fruit aqueous extract [21]. The positive control showed a great number of induced mutant ommatidia, proving the results from Vogel and Nivard [72], 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 [21]. In a similar way, Cápiro and Sánchez-Lamar [26] 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 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, 73]), 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].
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.
This work was supported by the project UIDB/CVT/00772/2020, which was supported by the Portuguese Science and Technology Foundation (FCT), and by the Gobierno del Principado de Asturias (Oviedo, Spain) through Plan de Ciencia, Tecnología e Innovación (PCTI), co-financed by FEDER funds (Ref. FC-GRUPIN-IDI/2018/000242) and by the Ministerio de Economia y Competitividad (MINECO) of Spain under the Project CTQ2016-80060-C2-1R.
1.Dulio V, Bv B, Brorström-Lundén E, Harmsen J, Hollender J, Schlabach M, et al. Emerging pollutants in the EU: 10 years of NORMAN in support of environmental policies and regulations. Environmental Sciences Europe. 2018;30(5):1-13. DOI: 10.1186/s12302-018-0135-3
2.Mishra N, Srivastava R, Agrawal UR, Tewari RR. An insight into the genotoxicity assessment studies in dipterans. Mutation Research. 2017;773:220-229. DOI: 10.1016/j.mrrev.2016.10.001
3.Ferreira J, Marques A, Abreu MH, Pereira R, Rego A, Pacheco M, et al. Red seaweeds Porphyra umbilicalis and Grateloupia turuturu display antigenotoxic and longevity-promoting potential in Drosophila melanogaster. European Journal of Phycology. 2019;54(4):519-530. DOI: 10.1080/09670262.2019.1623926
4.Lemaitre B, Miguel-Aliaga I. The digestive tract of Drosophila melanogaster. Annual Review of Genetics. 2013;47:377-404. DOI: 10.1146/annurev-genet-111212-133343
5.Holmes AM, Creton S, Chapman K. Working in partnership to advance the 3Rs in toxicity testing. Toxicology. 2010;267(1-3):14-19. DOI: 10.1016/j.tox.2009.11.006
6.Neethu BK, Babu YR, Harini BP. Enriched nutrient diet shortens the developmental time—A transgenerational effect in Drosophila sulfurigaster sulfurigaster. Drosophila Information Service. 2013;96:98-102
7.Güler P, Ayhan N, Koşukcu C, Önder BŞ. The effects of larval diet restriction on developmental time, preadult survival, and wing length in Drosophila melanogaster. Turkish Journal of Zoology. 2015;39:395-403. DOI: 10.3906/zoo-1305-42
8.Stamenković-Radak M, Andjelković M. Studying genotoxic and antimutagenic effects of plant extracts in Drosophila test systems. Botanica Serbica. 2016;40(1):21-28. DOI: 10.5281/zenodo.48854
9.Marcos R, Sierra LM, Gaivão I. The SMART assays of Drosophila: Wings and eyes as target tissues. In: Sierra LM, Gaivão I, editors. Genotoxicity and DNA Repair: A Practical Approach. New York: Humana Press; 2014. pp. 283-295
10.Graf U, Würgler FE, Katz AJ, Frei H, Juon H, Hall CB, et al. Somatic mutation and recombination test in Drosophila melanogaster. Environmental Mutagenesis. 1984;6(2):153-188. DOI: 10.1002/em.2860060206
11.Würgler EE, Vogel EW. In vivo mutagenicity testing using somatic cells of Drosophila melanogaster. In: Serres FJd, editor. Chemical Mutagens, Principles and Methods for Their Detection. 10. New York: Plenum Press; 1986. p. 1-72
13.Kumar JP. Building an ommatidium one cell at a time. Developmental Dynamics. 2012;241(1):136-149. DOI: 10.1002/dvdy.23707
14.Tyler MS. Developmental Biology: A Guide for Experimental Study. 3.1 ed. Sunderland, Massachusetts, USA: Sinauer Assoc., Inc.; 2010
15.Vlastos D, Drosopoulou E, Efthimiou I, Gavriilidis M, Panagaki D, Mpatziou K, et al. Genotoxic and antigenotoxic assessment of chios mastic oil by the in vitro micronucleus test on human lymphocytes and the in vivo wing somatic test on Drosophila. PLoS One. 2015;10(6):e0130498. DOI: 10.1371/journal.pone.0130498
16.Vogel EW, Nivard MJM, Zijlstra JA. Variation of spontaneous and induced mitotic recombination in different Drosophila populations: A pilot study on the effects of polyaromatic hydrocarbons in six newly constructed tester strains. Mutation Research. 1991;250:291-298. DOI: 10.1016/0027-5107(91)90184-p
17.Gaivão I, Sierra LM, Comendador MA. The w/w+ SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds. Mutation Research. 1999;440:139-145. DOI: 10.1016/S1383-5718(99)00020-0
18.Abraham SK. Antigenotoxicity of coffee in the Drosophila assay for somatic mutation and recombination. Mutagenesis. 1994;9:383-386. DOI: 10.1093/mutage/9.4.383
19.Alaraby M, Hernández A, Annangi B, Demir E, Bach J, Rubio L, et al. Antioxidant and antigenotoxic properties of CeO2 NPs and cerium sulphate: Studies with Drosophila melanogaster as a promising in vivo model. Nanotoxicology. 2015;9(6):749-759. DOI: 10.3109/17435390.2014.976284
20.Alaraby M, Hernández A, Marcos R. Copper oxide nanoparticles and copper sulphate act as antigenotoxic agents in Drosophila melanogaster. Environmental and Molecular Mutagenesis. 2017;58(1):46-55. DOI: 10.1002/em.22068
21.Amkiss S, Dallouh A, Idaomar M, Amkiss B. Genotoxicity and anti-genotoxicity of fennel plant (Foeniculum vulgare Mill) fruit extracts using the somatic mutation and recombination test (SMART). African Journal of Food Science. 2013;7(8):193-197. DOI: 10.5897/AJFS2013.0999
22.Anter J, Campos-Sánchez J, Hamss RE, Rojas-Molina M, Muñoz-Serrano A, Analla M, et al. Modulation of genotoxicity by extra-virgin olive oil and some of its distinctive components assessed by use of the Drosophila wing-spot test. Mutation Research. 2010;703(2):137-142. DOI: 10.1016/j.mrgentox.2010.08.012
23.Anter J, de Abreu-Abreu N, Fernandez-Bedmar Z, Villatoro-Pulido M, Alonso-Moraga A, Munoz-Serrano A. Targets of red grapes: Oxidative damage of DNA and leukaemia cells. Natural Product Communications. 2011;6(1):59-64
24.Anter J, Romero-Jimenez M, Fernandez-Bedmar Z, Villatoro-Pulido M, Analla M, Alonso-Moraga A, et al. Antigenotoxicity, cytotoxicity, and apoptosis induction by apigenin, bisabolol, and protocatechuic acid. Journal of Medicinal Food. 2011;14(3):276-283. DOI: 10.1089/jmf.2010.0139
25.Aydemir N, Sevim N, Celikler S, Vatan O, Bilaloglu R. Antimutagenicity of amifostine against the anticancer drug fotemustine in the Drosophila somatic mutation and recombination (SMART) test. Mutation Research. 2009;679(1-2):1-5. DOI: 10.1016/j.mrgentox.2009.08.005
26.Cápiro N, Sánchez-Lamar A, Baluja L, Sierra LM, Comendador MA. Efecto de la Concentración de Cymbopogon Citratus (Dc) Stapf. Sobre la Genotoxicidad de Mutágenos Modelos, en el Ensayo Smart de Ojos W/W+ de Drosophila melanogaster. Revista CENIC Ciencias Biológicas. 2005:36
27.Demir E, Marcos R. Antigenotoxic potential of boron nitride nanotubes. Nanotoxicology. 2018;12(8):868-884. DOI: 10.1080/17435390.2018.1482379
28.De Rezende AA, Graf U, Guterres Zda R, Kerr WE, Spano MA. Protective effects of proanthocyanidins of grape (Vitis vinifera L.) seeds on DNA damage induced by doxorubicin in somatic cells of Drosophila melanogaster. Food and Chemical Toxicology. 2009;47(7):1466-1472. DOI: 10.1016/j.fct.2009.03.031
29.De Rezende AA, Silva ML, Tavares DC, Cunha WR, Rezende KC, Bastos JK, et al. The effect of the dibenzylbutyrolactolic lignan (−)-cubebin on doxorubicin mutagenicity and recombinogenicity in wing somatic cells of Drosophila melanogaster. Food and Chemical Toxicology. 2011;49(6):1235-1241. DOI: 10.1016/j.fct.2011.03.001
30.Drosopoulou E, Vlastos D, Efthimiou I, Kyrizaki P, Tsamadou S, Anagnostopoulou M, et al. In vitro and in vivo evaluation of the genotoxic and antigenotoxic potential of the major Chios mastic water constituents. Scientific Reports. 2018;8:12200. DOI: 10.1038/s41598-018-29810-y
31.El Hamss R, Analla M, Campos-Sanchez J, Alonso-Moraga A, Munoz-Serrano A, Idaomar M. A dose dependent anti-genotoxic effect of turmeric. Mutation Research. 1999;446(1):135-139. DOI: 10.1016/s1383-5718(99)00140-0
32.Fernandes LM, Da Rosa Guterres Z, Almeida IV, Vicentini VEP. Genotoxicity and antigenotoxicity assessments of the flavonoid vitexin by the Drosophila melanogaster somatic mutation and recombination test. Journal of Medicinal Food. 2017;20(6):601-609. DOI: 10.1089/jmf.2016.0149
33.Fernandez-Bedmar Z, Alonso-Moraga A. In vivo and in vitro evaluation for nutraceutical purposes of capsaicin, capsanthin, lutein and four pepper varieties. Food and Chemical Toxicology. 2016;98(Pt B):89-99. DOI: 10.1016/j.fct.2016.10.011
34.Fernández-Bedmar Z, Anter J, Cruz-Ares SL, Muñoz-Serrano A, Alonso-Moraga Á, Pérez-Guisado J. Role of citrus juices and distinctive components in the modulation of degenerative processes: Genotoxicity, antigenotoxicity, cytotoxicity, and longevity in Drosophila. Journal of Toxicology and Environmental Health. 2011;74(15-16):1052-1066. DOI: 10.1080/15287394.2011.582306
35.Fernandez-Bedmar Z, Anter J, Alonso Moraga A. Anti/genotoxic, longevity inductive, cytotoxic, and clastogenic-related bioactivities of tomato and lycopene. Environmental and Molecular Mutagenesis. 2018;59(5):427-437. DOI: 10.1002/em.22185
36.Fernández-Bedmar Z, Demyda-Peyrás S, Merinas-Amo T, Del Río-Celestino M. Nutraceutic potential of two Allium species and their distinctive organosulfur compounds: A multi-assay evaluation. Foods. 2019;8(6):222. DOI: 10.3390/foods8060222
37.Graf U, Abraham SK, Guzman-Rincon J, Würgler FE. Antigenotoxicity studies in Drosophila melanogaster. Mutation Research. 1998;402:203-209. DOI: 10.1016/S0027-5107(97)00298-4
38.Guterres ZR, Zanetti TA, Sennes-Lopes TF, Da Silva AF. Genotoxic and antigenotoxic potential of Momordica charantia Linn (Cucurbitaceae) in the wing spot test of Drosophila melanogaster. Journal of Medicinal Food. 2015;18(10):1136-1142. DOI: 10.1089/jmf.2014.0099
39.Idaomar M, El Hamss R, Bakkali F, Mezzoug N, Zhiri A, Baudoux D, et al. Genotoxicity and antigenotoxicity of some essential oils evaluated by wing spot test of Drosophila melanogaster. Mutation Research. 2002;513(1):61-68. DOI: 10.1016/S1383-5718(01)00287-X
40.Kylyc A, Yesilada E. Preliminary results on antigenotoxic effects of dried mycelia of two medicinal mushrooms in Drosophila melanogaster somatic mutation and recombination test. International Journal of Medicinal Mushrooms. 2013;15(4):415-421. DOI: 10.1615/intjmedmushr.v15.i4.90
41.Laohavechvanich P, Kangsadalampai K, Tirawanchai N, Ketterman AJ. Effect of different Thai traditional processing of various hot chili peppers on urethane-induced somatic mutation and recombination in Drosophila melanogaster: Assessment of the role of glutathione transferase activity. Food and Chemical Toxicology. 2006;44(8):1348-1354. DOI: 10.1016/j.fct.2006.02.013
42.Lozano-Baena M-D, Tasset I, Obregón-Cano S, Haro-Bailon A, Muñoz-Serrano A, Alonso-Moraga Á. Antigenotoxicity and tumor growing inhibition by leafy Brassica carinata and sinigrin. Molecules. 2015;20(9):15748-15765. DOI: 10.3390/molecules200915748
43.Marques A, Ferreira J, Abreu MH, Pereira R, Rego A, Serôdio J, et al. Searching for antigenotoxic properties of marine macroalgae dietary supplementation against endogenous and exogenous challenges. Journal of Toxicology and Environmental Health. Part A. 2018;81(18):939-956. DOI: 10.1080/15287394.2018.1507856
44.Martinez-Valdivieso D, Font R, Fernandez-Bedmar Z, Merinas-Amo T, Gomez P, Alonso-Moraga A, et al. Role of zucchini and its distinctive components in the modulation of degenerative processes: Genotoxicity, anti-genotoxicity, cytotoxicity and apoptotic effects. Nutrients. 2017;9(7). DOI: 10.3390/nu9070755
45.Mateo-Fernandez M, Alves-Martinez P, Del Rio-Celestino M, Font R, Merinas-Amo T, Alonso-Moraga A. Food safety and nutraceutical potential of caramel colour class IV using in vivo and in vitro assays. Foods. 2019;8(9). DOI: 10.3390/foods8090392
46.Merinas-Amo T, Tasset-Cuevas I, Diaz-Carretero AM, Alonso-Moraga A, Calahorro F. Role of choline in the modulation of degenerative processes: In vivo and in vitro studies. Journal of Medicinal Food. 2017;20(3):223-234. DOI: 10.1089/jmf.2016.0075
47.Mezzoug N, Elhadri A, Dallouh A, Amkiss S, Skali NS, Abrini J, et al. Investigation of the mutagenic and antimutagenic effects of Origanum compactum essential oil and some of its constituents. Mutation Research. 2007;629(2):100-110. DOI: 10.1016/j.mrgentox.2007.01.011
48.Niikawa M, Nakamura T, Nagase H. Effect of cotreatment of aspirin metabolites on mitomycin C-induced genotoxicity using the somatic mutation and recombination test in Drosophila melanogaster. Drug and Chemical Toxicology. 2006;29(4):379-396. DOI: 10.1080/01480540600820528
49.Niikawa M, Shin S, Nagase H. Suppressive effect of post- or pre-treatment of aspirin metabolite on mitomycin C-induced genotoxicity using the somatic mutation and recombination test in Drosophila melanogaster. Biomedicine & Pharmacotherapy. 2007;61(2-3):113-119. DOI: 10.1016/j.biopha.2006.07.094
50.Oliveira VC, Constante SAR, Orsolin PC, Nepomuceno JC, De Rezende AAA, Spano MA. Modulatory effects of metformin on mutagenicity and epithelial tumor incidence in doxorubicin-treated Drosophila melanogaster. Food and Chemical Toxicology. 2017;106(Pt A):283-291. DOI: 10.1016/j.fct.2017.05.052
51.Orsolin PC, Silva-Oliveira RG, Nepomuceno JC. Modulating effect of simvastatin on the DNA damage induced by doxorubicin in somatic cells of Drosophila melanogaster. Food and Chemical Toxicology. 2016;90:10-17. DOI: 10.1016/j.fct.2016.01.022
52.Pádua PFMR, Dihl RR, Lehmann M, Abreu BRR, Richter MF, Andrade HHR. Genotoxic, antigenotoxic and phytochemical assessment of Terminalia actinophylla ethanolic extract. Food and Chemical Toxicology. 2013;62:521-527. DOI: 10.1016/j.fct.2013.09.021
53.Patenkovic A, Stamenkovic-Radak M, Banjanac T, Andjelkovic M. Antimutagenic effect of sage tea in the wing spot test of Drosophila melanogaster. Food and Chemical Toxicology. 2009;47:180-183. DOI: 10.1016/j.fct.2008.10.024
54.Patenkovic A, Stamenkovic-Radak M, Nikolic D, Markovic T, Andelkovic M. Synergistic effect of Gentiana lutea L. on methyl methanesulfonate genotoxicity in the Drosophila wing spot test. Journal of Ethnopharmacology. 2013;146(2):632-636. DOI: 10.1016/j.jep.2013.01.027
55.Prakash G, Hosetti BB, Dhananjaya BL. Protective effect of caffeine on ethyl methanesulfonate-induced wing primordial cells of Drosophila melanogaster. Toxicology International. 2014;21(1):96-100. DOI: 10.4103/0971-6580.128814
56.Prakash G, Hosetti BB, Dhananjaya BL. Antimutagenic effect of Dioscorea pentaphylla on genotoxic effect induced by methyl methanesulfonate in the Drosophila wing spot test. Toxicology International. 2014;21(3):258-263. DOI: 10.4103/0971-6580.155341
57.Rizki M, Amrani S, Creus A, Xamena N, Marcos R. Antigenotoxic properties of selenium: Studies in the wing spot test in Drosophila. Environmental and Molecular Mutagenesis. 2001;37:70-75. DOI: 10.1002/1098-2280(2001)37:1<70::AID-EM1007>3.0
58.Romero-Jiménez M, Campos-Sánchez J, Analla M, Muñoz-Serrano A, Alonso-Moraga A. Genotoxicity and anti-genotoxicity of some traditional medicinal herbs. Mutation Research. 2005;585(1-2):147-155. DOI: 10.1016/j.mrgentox.2005.05.004
59.Sarıkaya R, Erciyas K, Kara MI, Sezer U, Erciyas AF, Ay S. Evaluation of genotoxic and antigenotoxic effects of boron by the somatic mutation and recombination test (SMART) on Drosophila. Drug and Chemical Toxicology. 2016;39:400-406. DOI: 10.3109/01480545.2015.1130719
60.Savić T, Patenković A, Soković M, Glamoclija J, Andjelković M, van Griensven LJ. The effect of royal sun agaricus, Agaricus brasiliensis S. Wasser et al., extract on methyl methanesulfonate caused genotoxicity in Drosophila melanogaster. International Journal of Medicinal Mushrooms. 2011;13(4):377-385. DOI: 10.1615/intjmedmushr.v13.i4.80
61.Sukprasansap M, Sridonpai P, Phiboonchaiyanan PP. Eggplant fruits protect against DNA damage and mutations. Mutation Research. 2019;813:39-45. DOI: 10.1016/j.mrfmmm.2018.12.004
62.Taira K, Miyashita Y, Okamoto K, Arimoto S, Takahashi E, Negishi T. Novel antimutagenic factors derived from the edible mushroom Agrocybe cylindracea. Mutation Research. 2005;586(2):115-123. DOI: 10.1016/j.mrgentox.2005.06.007
63.Tasset-Cuevas I, Fernández-Bedmar Z, Lozano-Baena MD, Campos-Sánchez J, de Haro-Bailón A, Muñoz-Serrano A, et al. Protective effect of borage seed oil and gamma linolenic acid on DNA: In vivo and in vitro studies. PLoS One. 2013;8(2):e56986. DOI: 10.1371/journal.pone.0056986
64.Toyoshima M, Hosoda K, Hanamura M, Okamoto K, Kobayashi H, Negishi T. Alternative methods to evaluate the protective ability of sunscreen against photo-genotoxicity. Journal of Photochemistry and Photobiology. B. 2004;73(1-2):59-66. DOI: 10.1016/j.jphotobiol.2003.09.005
65.Valadares BL, Graf U, Spanó MA. Inhibitory effects of water extract of propolis on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster. Food and Chemical Toxicology. 2008;46(3):1103-1110. DOI: 10.1016/j.fct.2007.11.005
66.Valente N, Borges J, Baptista A, Gaivão I. Benefits of thalassotherapy in the Portuguese coast: A study in Drosophila melanogaster. In: Annual Portuguese Drosophila Meeting; Tomar, Portugal. 2014
67.Lyer J, Wang Q, Le T, Imai Y, Srivastava A, Troisí BL, et al. Quantitative assessment of eye phenotypes for functional genetic studies using Drosophila melanogaster. G3: Genes|Genomes|Genetics. 2016;6:1427-1437. DOI: 10.1534/g3.116.027060
68.Gonzalez C. Drosophila melanogaster: A model and a tool to investigate malignancy and identify new therapeutics. Nature Reviews. Cancer. 2013;13:172-183. DOI: 10.1038/nrc3461
69.Vogel EW, Nivard MJ. Parallel monitoring of mitotic recombination, clastogenicity and teratogenic effects in eye tissue of Drosophila. Mutation Research. 2000;455(1-2):141-153. DOI: 10.1016/s0027-5107(00)00067-1
70.Vogel EW, Nivard MJ. A novel method for the parallel monitoring of mitotic recombination and clastogenicity in somatic cells in vivo. Mutation Research. 1999;431(1):141-153. DOI: 10.1016/s0027-5107(99)00198-0
71.Vogel EW, Zijlstra JA. Mechanistic and methodological aspects of chemically induced somatic mutation and recombination in Drosophila melanogaster. Mutation Research. 1987;182:243-264. DOI: 10.1016/0165-1161(87)90010-0
72.Vogel EW, Nivard MJM. Performance of 181 chemicals in a Drosophila assay predominantly monitoring interchromosomal mitotic recombination. Mutagenesis. 1993;8(1):57-81. DOI: 10.1093/mutage/8.1.57
73.Gaivão I, Comendador MA. The w/w+ somatic mutation and recombination test (SMART) of Drosophila melanogaster for detecting reactive oxygen species: Characterization of 6 strains. Mutation Research. 1996;360:145-151. DOI: 10.1016/0165-1161(96)00003-9
74.Donohoe TJ, Jones CR, Kornahrens AF, Barbosa LCA, Walport LJ, Tatton MR, et al. Total synthesis of the antitumor antibiotic (±)-streptonigrin: First-and second-generation routes for de novo pyridine formation using ring-closing metathesis. American Chemical Society. 2013;78:12338-12350. DOI: 10.1021/jo402388f
75.Troxell B, Xu H, Yang XF. Borrelia burgdorferi, a pathogen that lacks iron, encodes manganese-dependent superoxide dismutase essential for resistance to streptonigrin. The Journal of Biological Chemistry. 2012;287(23):19284-19293. DOI: 10.1074/jbc.M112.344903
76.Bolzán AD, Bianchi MS. Genotoxicity of streptonigrin: A review. Mutation Research. 2001;488:25-37. DOI: 10.1016/S1383-5742(00)00062-4
77.Deepa PV, Akshaya AS, Solomon FDP. Anthracycline (epirubicin) induced mutation studies in Drosophila melanogaster. Drososphila Information Service. 2011;94:53-61
78.López-Romero D, Izquierdo-Vega JA, Morales-González JA, Madrigal-Bujaidar E, Chamorro-Cevallos G, Sánchez-Gutiérrez M, et al. Evidence of some natural products with antigenotoxic effects. Part 2: Plants, vegetables, and natural resin. Nutrients. 2018;10(12):1954. DOI: 10.3390/nu10121954
79.Słoczyńska K, Powroźnik B, Pękala E, Waszkielewicz AM. Antimutagenic compounds and their possible mechanisms of action. Journal of Applied Genetics. 2014;55:273-285. DOI: 10.1007/s13353-014-0198-9
80.Izquierdo-Vega JA, Morales-González JA, Sánchez-Gutiérrez M, Betanzos-Cabrera G, Sosa-Delgado SM, Sumaya-Martínez MT, et al. Evidence of some natural products with antigenotoxic effects. Part 1: Fruits and polysaccharides. Nutrients. 2017;9(2):102. DOI: 10.3390/nu9020102
81.Panieri E, Santoro MM. ROS homeostasis and metabolism: A dangerous liason in cancer cells. Cell Death & Disease. 2016;7(6):e2253. DOI: 10.1038/cddis.2016.105
Written By
Isabel Gaivão, João Ferreira and Luisa María Sierra
Submitted: 11 October 2019Reviewed: 05 February 2020Published: 12 March 2020
Open access peer-reviewed chapter
