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

*Isabel Gaivão, João Ferreira and Luisa María Sierra*

## **Abstract**

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*<sup>+</sup> 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*<sup>+</sup> SMART. We reviewed the state of the art of the *w*/*w*<sup>+</sup> 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.

**Keywords:** genotoxicity test, *w*/*w*<sup>+</sup> SMART, eye-spot test, *Drosophila melanogaster*, streptonigrin, genotoxic agent, oxidative stress, DNA damage, ROS inhibition, antigenotoxicity, antimutagens, dietary antioxidants

## **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].

with in vitro methods, *D. melanogaster* has the advantage of enabling a more solid

*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

**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*<sup>+</sup> SMART) [9]. The wing-spot test was firstly described by Graf and Würgler [10] and the *w*/*w*<sup>+</sup> SMART by Würgler and Vogel [11], with both showing high values of sensitivity, specificity and

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*<sup>+</sup> 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*<sup>+</sup> SMART, a standardized genotoxic agent, inducing a high

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*<sup>+</sup> 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*<sup>+</sup> SMART will be

genotoxicity without toxic effects, streptonigrin (further focused on the chapter) [17], is available and has proved its effectiveness. Nevertheless, since the wing-spot

extrapolation at the organism level [3].

*DOI: http://dx.doi.org/10.5772/intechopen.91630*

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)…*

quickly and inexpensively [2, 8].

accuracy.

focused.

**113**

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 *The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)… DOI: http://dx.doi.org/10.5772/intechopen.91630*

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 envi-

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

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.

optimal test conditions) for increasing their reproducibility and precision.

*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

**2.** *Drosophila melanogaster* **in toxicological research**

**112**

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

at least greatly reducing the produced toxic agents.

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

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.

This whole problem greatly affects DNA, causing DNA damage (genotoxicity),

ronmental substances [1].

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*<sup>+</sup> SMART) [9]. The wing-spot test was firstly described by Graf and Würgler [10] and the *w*/*w*<sup>+</sup> 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*<sup>+</sup> 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*<sup>+</sup> 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*<sup>+</sup> 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*<sup>+</sup> SMART will be focused.


**Reference SMART**

Fernández-Bedmar et al. [34]

Fernandez-Bedmar et al. [35]

Fernández-Bedmar et al. [36]

Guterres et al. [38]

Idaomar et al. [39]

Kylyc and Yesilada [40]

Laohavechvanich et al. [41]

Lozano-Baena et al. [42]

Marques et al. [43]

Martinez-Valdivieso et al.

Mateo-Fernandez et al. [45]

Merinas-Amo et al. [46]

Mezzoug et al. [47]

Niikawa et al. [48]

**115**

[44]

**type**

*DOI: http://dx.doi.org/10.5772/intechopen.91630*

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)…*

Graf et al. [37] Wing-spot Urethane (URE)

**Genotoxic agent Substance tested as**

Wing-spot Hydrogen peroxide Citrus juices

Wing-spot Hydrogen peroxide Tomato

Wing-spot Hydrogen peroxide Garlic

Methyl urea + sodium nitrite

Wing-spot Doxorubicin (DXR) *Momordica charantia*:

Wing-spot Urethane (URE) Essential oils from:

Wing-spot Mitomycin C (MMC) Dried mycelia from:

Wing-spot Urethane (URE) Bird pepper

Wing-spot Hydrogen peroxide *Brassica carinata*

Wing-spot Hydrogen peroxide Caramel color class IV +

Wing-spot Hydrogen peroxide Choline +

Wing-spot Urethane (URE) *Origanum compactum*

Wing-spot Mitomycin C (MMC) Salicylic acid

Eye-spot Streptonigrin (SN) *Ulva rigida*

Wing-spot Hydrogen peroxide Lutein

Ferreira et al. [3] Eye-spot Streptonigrin (SN) *Grateloupia turuturu*

**antigenotoxic**

Hesperidin Limonene

Lycopene

Diallyl disulphide Dipropyl disulphide

*Porphyra umbilicalis*

*Helichrysum italicum*, *Ledum groenlandicum*, *Ravensara aromatica*

*Trametes versicolor Pleurotus ostreatus*

Red chili spur pepper Green bell pepper Green sweet pepper

Sinigrin

*Fucus vesiculosus Gracilaria* species

β-Carotene Zeaxanthin Dehydroascorbic acid Yellow zucchini Light green zucchini

essential oil

acid

Salicyluric acid Gentisic acid Gentisuric acid 2,3-Dihydroxybenzoic

Instant coffee Ascorbic acid Catechin

aerial parts Fruit

Onion

**Response**

+ + +

+ +

+ + + +

+ +

+ + +

 +

+ + +

+ +

+ + + +

+ +

+ + +

+ + + + + +

+

 + + + +


**Reference SMART**

Aydemir et al. [25]

Demir and Marcos [27]

[28]

[29]

[30]

[32]

[33]

**114**

De Rezende et al.

De Rezende et al.

Drosopoulou et al.

El Hamss et al. [31]

Fernandes et al.

Fernandez-Bedmar and Alonso-Moraga **type**

Abraham [18] Wing-spot Cyclophosphamide (CPH)

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

Alaraby et al. [20] Wing-spot Potassium dichromate (PD)

Amkiss et al. [21] Eye-spot Methyl methanesulfonate

Cápiro et al. [26] Eye-spot Methyl methanesulfonate

(MMS)

Juglone (JG)

(DMBA)

Wing-spot Doxorubicin (DXR)

Benzo(a)pyrene (B(a)P)

Alaraby et al. [19] Wing-spot Potassium dichromate (PD) CeO2 NPs

(MMS)

Anter et al. [22] Wing-spot Hydrogen peroxide Virgin Olive oil

Anter et al. [23] Wing-spot Hydrogen peroxide Red table grapes + Anter et al. [24] Wing-spot Hydrogen peroxide Phenols: apigenin,

Ethylnitrosourea (ENU)

Dimethylbenz(a)anthracene

Wing-spot Potassium dichromate Boron nitride

Wing-spot Doxorubicin (DXR) Grape seed

Wing-spot Doxorubicin (DXR) Dibenzylbutyrolactolic

Wing-spot Mitomycin C (MMC) Chios mastic products:

Wing-spot Urethane (URE) Turmeric +

Wing-spot Hydrogen peroxide Green sweet pepper

Wing-spot Fotemustine Amifostine +

**Genotoxic agent Substance tested as**

Diethylnitrosamine (DEN) Mitomycin C (MMC) Procarbazine (PRO) Urethane (URE)

Ethyl methanesulfonate (EMS) Potassium dichromate (PD) Ethyl methanesulfonate (EMS) **antigenotoxic**

Cerium sulphate

Fennel plant fruit extracts

CuO NPs Copper oxide

Triolein Tyrosol Squalene

bisabolol, protocatechuic acid

nanotubes

verbenone α-terpineol linalool trans-pinocarveol

proanthocyanidins

lignan()-cubebin

Vitexin +

Red sweet pepper Green hot pepper Red hot pepper Capsaicin Capsanthin Lutein

*Cymbopogon citratus* +

Coffee +

**Response**

+ + +

+

+ +

+

+ + + +

+ + +

+ + +

+

+

+/

+ + + 

+

+ + + + + +


**3.** *w***/***w***<sup>+</sup> SMART (eye-spot test)**

**Reference SMART**

Tasset-Cuevas et al. [63]

Toyoshima et al.

Valadares et al.

*activity) is presented.*

[64]

[65]

**Table 1.**

**type**

*DOI: http://dx.doi.org/10.5772/intechopen.91630*

NDMA 4NQO

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)…*

(*w*/*w* with *w*<sup>+</sup>

**117**

*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

**Genotoxic agent Substance tested as**

Wing-spot Hydrogen peroxide Borage seed oil

Wing-spot Doxorubicin (DXR) Propolis (water

*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*

*Published studies focusing the antigenotoxic evaluation of several types of chemicals, nanoparticles and plants/*

Valente et al. [66] Eye-spot Streptonigrin (SN) Thalassotherapy

Wing-spot Sun and UV light Sunscreens:

**antigenotoxic**

SPF 20 SPF 40 SPF 60

extracts)

products

Gamma linolenic acid

**Response**

+ +

+ +

+ + +

+

+

The basis of the *w*/*w*<sup>+</sup> 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

crossed with white-eyed males (*w*/Y; eyes without pigmentation), or vice versa

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

/Y), a heterozygous offspring is developed for females (*w*<sup>+</sup>

Moreover, Vogel and Nivard [69, 70] designed a more refined, as well as timeconsuming, version of the *w*/*w*<sup>+</sup> 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

/*w*<sup>+</sup>

; red eyes) are

/*w*; red

simple to detect genetic alterations changing its pigmentation.

*seaweeds/seeds/oils using somatic mutation and recombination tests (SMARTs).*

carcinogenesis [69]. Accordingly, when wild-type females (*w*<sup>+</sup>

will provide quantitative information on somatic recombination [9].


*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.*

#### **Table 1.**

**Reference SMART**

Niikawa et al. [49]

Patenkovic et al.

Patenkovic et al.

Romero-Jiménez et al. [58]

Sarıkaya et al. [59]

Sukprasansap et al. [61]

**116**

[53]

[54]

**type**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

Pádua et al. [52] Wing-spot Mitomycin C (MMC)

Prakash et al. [56] Wing-spot Methyl methanesulfonate

Savić et al. [60] Wing-spot Methyl methanesulfonate

Taira et al. [62] Wing-spot 2-AAF

**Genotoxic agent Substance tested as**

Wing-spot Mitomycin C (MMC) Salicylic acid

Oliveira et al. [50] Wing-spot Doxorubicin (DXR) Metformin + Orsolin et al. [51] Wing-spot Doxorubicin (DXR) Simvastatin +

Prakash et al. [55] Wing-spot Ethyl methanesulfonate (EMS) Caffeine +

Rizki et al. [57] Wing-spot Potassium dichromate (PD) Sodium selenite +

Wing-spot Hydrogen peroxide *Matricaria chamomilla*

Wing-spot Ethyl methanesulfonate (EMS) Boron +

Wing-spot Urethane (URE) Eggplants +

Wing-spot Methyl methanesulfonate (MMS)

Wing-spot Methyl methanesulfonate (MMS)

(MMS)

(MMS)

Aflatoxin B1 DMBA IQ MeIQx MNU NDMA 4NQO 2-AAF Aflatoxin B1 DMBA IQ MeIQx MNU NDMA 4NQO 2-AAF Aflatoxin B1 DMBA IQ MeIQx MNU

Ethyl methanesulfonate (EMS)

**antigenotoxic**

Salicyluric acid Gentisic acid Gentisuric acid 2,3-Dihydroxybenzoic

acid

*Terminalia actinophylla* extracts

Sage tea +

Gentian tea

*Dioscorea pentaphylla* +

*Tilia cordata Mentha piperita Mentha pulegium Uncaria tomentosa Valeriana officinalis*

Royal Sun Agaricus

*Agrocybe cylindracea Lentinula edodes Pleurotus ostreatus*

extract

**Response**

 + + + +

+ +

+ + + + + +

+ + + + + + + + - + + + + + + + + + +

*Published studies focusing the antigenotoxic evaluation of several types of chemicals, nanoparticles and plants/ seaweeds/seeds/oils using somatic mutation and recombination tests (SMARTs).*

## **3.** *w***/***w***<sup>+</sup> 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) [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*<sup>+</sup> 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*<sup>+</sup> /*w*<sup>+</sup> ; red eyes) are crossed with white-eyed males (*w*/Y; eyes without pigmentation), or vice versa (*w*/*w* with *w*<sup>+</sup> /Y), a heterozygous offspring is developed for females (*w*<sup>+</sup> /*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 [69, 70] designed a more refined, as well as timeconsuming, version of the *w*/*w*<sup>+</sup> 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

#### **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<sup>+</sup> 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*<sup>+</sup> 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 [74–76] (**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 pro-

*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*

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

[74–76]. 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

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 [78–80]. Since most of the possible antigenotoxic agents are components of natural products that could be included in the diet, the analysis of their properties

� and quinone regeneration. Hydroquinone can lead to the production

� + H2O2 ! OH + OH� + O2), leading to oxidative stress

duction of O2

**Figure 2.**

[76] (**Figure 3**).

**119**

Haber-Weiss reaction (O2

*when surrounded by pigmented/red ommatidia.*

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)…*

*DOI: http://dx.doi.org/10.5772/intechopen.91630*

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*<sup>+</sup> SMART.

## **3.1 Antigenotoxicity with** *w***/***w***<sup>+</sup> SMART**

*w*/*w*<sup>+</sup> 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, 71–73]. 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

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)… DOI: http://dx.doi.org/10.5772/intechopen.91630*

#### **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.*

ommatidia) without toxic effects (at 20 μM) in the *w*/*w*<sup>+</sup> 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 [74–76] (**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 [74–76]. 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**).

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 [78–80]. Since most of the possible antigenotoxic agents are components of natural products that could be included in the diet, the analysis of their properties

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

*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<sup>+</sup> 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*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

*/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*

*w*/*w*<sup>+</sup> 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, 71–73]. As such, alkylating agents, such as

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

methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and

performing *w*/*w*<sup>+</sup> SMART.

*chromosome with the wild-type allele.*

**Figure 1.**

*(w+*

**118**

**3.1 Antigenotoxicity with** *w***/***w***<sup>+</sup> SMART**

Focusing on the *w*/*w*<sup>+</sup> 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

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

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-

thalassotherapy products (containing seaweeds) [66].

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)…*

*DOI: http://dx.doi.org/10.5772/intechopen.91630*

scavenging actions [3, 66].

**121**

#### **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.*

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**).

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)… DOI: http://dx.doi.org/10.5772/intechopen.91630*

Focusing on the *w*/*w*<sup>+</sup> 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 antioxidantscavenging 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

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**).

*(SOD); (2) catalase (CAT); (3) glutathione peroxidases.*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

*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*

 *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*

**Figure 3.**

*O2*

**120**

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].

## **4. 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*<sup>+</sup> 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.

## **Acknowledgements**

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.

**Author details**

\*, João Ferreira1,2 and Luisa María Sierra<sup>3</sup>

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)…*

*DOI: http://dx.doi.org/10.5772/intechopen.91630*

Sciences (CITAB), UTAD, Vila Real, Portugal

\*Address all correspondence to: igaivao@utad.pt

provided the original work is properly cited.

1 Department of Genetics and Biotechnology and Animal and Veterinary Research Centre (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real,

2 Centre for the Research and Technology of Agro-Environmental and Biological

3 Department of Functional Biology, Genetics Area, Oncology University Institute (IUOPA) and Institute of Sanitary Research of Asturias (ISPA), University of

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Isabel Gaivão<sup>1</sup>

Oviedo, Spain

**123**

Portugal

## **Conflict of interest**

The authors declare no conflict of interest.

*The* w*/*w*<sup>+</sup> Somatic Mutation and Recombination Test (SMART)… DOI: http://dx.doi.org/10.5772/intechopen.91630*

## **Author details**

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

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*<sup>+</sup> 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)

approaches of SMARTs, see the protocol from Marcos and Sierra [9].

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

**4. Conclusions**

**Acknowledgements**

**Conflict of interest**

**122**

of Spain under the Project CTQ2016-80060-C2-1R.

The authors declare no conflict of interest.

Isabel Gaivão<sup>1</sup> \*, João Ferreira1,2 and Luisa María Sierra<sup>3</sup>

1 Department of Genetics and Biotechnology and Animal and Veterinary Research Centre (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal

2 Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), UTAD, Vila Real, Portugal

3 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

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[73] Gaivão I, Comendador MA. The *w*/*w*<sup>+</sup> 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

[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.

Genotoxicity of streptonigrin: A review. Mutation Research. 2001;**488**:25-37. DOI: 10.1016/S1383-5742(00)00062-4

(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

[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

[74] Donohoe TJ, Jones CR, Kornahrens AF, Barbosa LCA, Walport LJ, Tatton MR, et al. Total synthesis of the antitumor antibiotic ()-streptonigrin: First-and secondgeneration routes for de novo pyridine formation using ring-closing metathesis. American Chemical Society. 2013;**78**: 12338-12350. DOI: 10.1021/jo402388f

M112.344903

[76] Bolzán AD, Bianchi MS.

[77] Deepa PV, Akshaya AS, Solomon FDP. Anthracycline

(87)90010-0

[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):

[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,

[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

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[71] Vogel EW, Zijlstra JA. Mechanistic

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[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

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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

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[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

methanesulfonate caused genotoxicity in *Drosophila melanogaster*. International Journal of Medicinal Mushrooms. 2011;

mrgentox.2005.05.004

et al., extract on methyl

**13**(4):377-385. DOI: 10.1615/ intjmedmushr.v13.i4.80

**813**:39-45. DOI: 10.1016/j. mrfmmm.2018.12.004

mrgentox.2005.06.007

[61] Sukprasansap M, Sridonpai P, Phiboonchaiyanan PP. Eggplant fruits protect against DNA damage and mutations. Mutation Research. 2019;

[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.

[63] Tasset-Cuevas I, Fernández-Bedmar

Hanamura M, Okamoto K, Kobayashi H, Negishi T. Alternative methods to

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,

phytochemical assessment of *Terminalia actinophylla* ethanolic extract. Food and Chemical Toxicology. 2013;**62**:521-527.

[53] Patenkovic A, Stamenkovic-Radak

Antimutagenic effect of sage tea in the

[54] Patenkovic A, Stamenkovic-Radak

Andelkovic M. Synergistic effect of

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Dhananjaya BL. Protective effect of caffeine on ethyl methanesulfonateinduced wing primordial cells of *Drosophila melanogaster*. Toxicology International. 2014;**21**(1):96-100. DOI:

Dhananjaya BL. Antimutagenic effect of *Dioscorea pentaphylla* on genotoxic

methanesulfonate in the *Drosophila* wing spot test. Toxicology International. 2014;**21**(3):258-263. DOI: 10.4103/

[57] Rizki M, Amrani S, Creus A, Xamena N, Marcos R. Antigenotoxic properties of selenium: Studies in the

[58] Romero-Jiménez M, Campos-Sánchez J, Analla M, Muñoz-Serrano A,

wing spot test in *Drosophila*. Environmental and Molecular Mutagenesis. 2001;**37**:70-75. DOI: 10.1002/1098-2280(2001)37:1<

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M, Banjanac T, Andjelkovic M.

wing spot test of *Drosophila melanogaster*. Food and Chemical Toxicology. 2009;**47**:180-183. DOI:

10.1016/j.fct.2008.10.024

M, Nikolic D, Markovic T,

*Gentiana lutea* L. on methyl

[55] Prakash G, Hosetti BB,

10.4103/0971-6580.128814

[56] Prakash G, Hosetti BB,

effect induced by methyl

0971-6580.155341

[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*<sup>+</sup> 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 secondgeneration 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

**Chapter 8**

Agents

**Abstract**

important.

**131**

**1. Introduction**

Current Trends and Future

Perspectives of Antimutagenic

*Adel M. AbdelHakem and El-Shimaa M.N. Abdelhafez*

Mutation is the process leading to heritable changes in DNA caused mainly by internal and external factors. Recently, studies on mutagenic agents have been increased due to increasing in mutation-related disease. The antimutagenic effect is desired to prevent mutation on genes or to inactivate the mutagenic agent. It seems that the interest in antimutagenic substances displaying multiple mechanisms of action will be an important trend in the research and development of new

antimutagenic compounds in the near future. Therefore, this chapter displays various possible mechanisms of action for antimutagenic agent and introduces differ-

ent types of antimutagens, natural and synthetic, that are considered very

agriculture, industrial sources, and other contaminants [3].

**Keywords:** mutagenesis, antimutagenic, mechanism, natural, synthetic, DNA

Mutagenicity is the process of induction of permanent heritable changes in the DNA sequence of living systems [1]. It is caused mainly by the external factors, including chemical and physical agents, or can also occur spontaneously due to errors in DNA repair, replicationand recombination [2]. A number of mutagens have been recognized in our environment recently as many factors which modulate the toxic activities either in vitro or in vivo [3]. Agents contributing to mutagenesis in the environment could be from wide-spectrum applications of biocides in the

These mutagenic chemicals have severe drawbacks in humans such as cancer and various inherited diseases; therefore, it is important to detect such mutagenic agents precisely and rapidly and also look for solutions to combat them [2].

Natural occurring dietary antimutagens such as healthy protective foods such as

fruits and vegetables could strongly counteract the deleterious effect of these mutagens [4]. Additionally, the World Health Organization (WHO) revealed that one-third of all cancer death incidences are preventable depending on the diet type especially health protective phytochemicals that provide an effective solution to these concerns [4]. The current chapter will present the mutagenic events and a brief compilation of the existing scientific findings either from dietary sources or synthetic agents that have the potential activity to combat the disorders

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

## **Chapter 8**

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.

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

[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

antigenotoxic effects. Part 1: Fruits and polysaccharides. Nutrients. 2017;**9**(2):

some natural products with

102. DOI: 10.3390/nu9020102

10.1038/cddis.2016.105

**130**

[81] Panieri E, Santoro MM. ROS homeostasis and metabolism: A dangerous liason in cancer cells. Cell Death & Disease. 2016;**7**(6):e2253. DOI:

DOI: 10.3390/nu10121954

## Current Trends and Future Perspectives of Antimutagenic Agents

*Adel M. AbdelHakem and El-Shimaa M.N. Abdelhafez*

## **Abstract**

Mutation is the process leading to heritable changes in DNA caused mainly by internal and external factors. Recently, studies on mutagenic agents have been increased due to increasing in mutation-related disease. The antimutagenic effect is desired to prevent mutation on genes or to inactivate the mutagenic agent. It seems that the interest in antimutagenic substances displaying multiple mechanisms of action will be an important trend in the research and development of new antimutagenic compounds in the near future. Therefore, this chapter displays various possible mechanisms of action for antimutagenic agent and introduces different types of antimutagens, natural and synthetic, that are considered very important.

**Keywords:** mutagenesis, antimutagenic, mechanism, natural, synthetic, DNA

## **1. Introduction**

Mutagenicity is the process of induction of permanent heritable changes in the DNA sequence of living systems [1]. It is caused mainly by the external factors, including chemical and physical agents, or can also occur spontaneously due to errors in DNA repair, replicationand recombination [2]. A number of mutagens have been recognized in our environment recently as many factors which modulate the toxic activities either in vitro or in vivo [3]. Agents contributing to mutagenesis in the environment could be from wide-spectrum applications of biocides in the agriculture, industrial sources, and other contaminants [3].

These mutagenic chemicals have severe drawbacks in humans such as cancer and various inherited diseases; therefore, it is important to detect such mutagenic agents precisely and rapidly and also look for solutions to combat them [2].

Natural occurring dietary antimutagens such as healthy protective foods such as fruits and vegetables could strongly counteract the deleterious effect of these mutagens [4]. Additionally, the World Health Organization (WHO) revealed that one-third of all cancer death incidences are preventable depending on the diet type especially health protective phytochemicals that provide an effective solution to these concerns [4]. The current chapter will present the mutagenic events and a brief compilation of the existing scientific findings either from dietary sources or synthetic agents that have the potential activity to combat the disorders

caused by the mutagenic agents, putting in mind possible future perspectives and mechanism of antimutagenics [2].

## **2. Mechanisms of action**

Several classes of antimutagenic compounds may be distinguished based on their mechanism of action as the following:

#### **2.1 Antimutagens with antioxidant potency**

Reactive oxygen species (ROS) are generated by many mutagens; therefore, the removal of reactive molecules is considered an important strategy in the process of antimutagenesis. It is reported that compounds with antioxidant propertiescan remove ROS before these molecules react with DNA, resulting in a mutation [5].

**2.4 Multifunctionally acting antimutagens**

*Current Trends and Future Perspectives of Antimutagenic Agents*

*DOI: http://dx.doi.org/10.5772/intechopen.91689*

antimutagens is of great importance [9].

**2.5 Desmutagenesis**

*Bichalcophene derivatives.*

**Figure 3.**

**2.6 Bio-antimutagenesis**

**3. Antimutagenic agents**

*3.1.1 Isolated compounds*

*3.1.1.1 Cinnamaldehyde*

**133**

**3.1 Natural antimutagenic agents**

compounds in them or due to whole extract.

Various antimutagenic agents work through multiple mechanisms affording protection against several mutagens. Noteworthy, the ability of compounds to

antimutagenic effectiveness. Hence, searching for such multifunctionally acting

This way of preventing induced cellular mutagenesis depends on mutagens that

Damaged DNAusually requires fixation steps (e.g., DNAreplication and/or repair) before it can be expressed as stable and heritable mutant genes. Hence this mechanism relates to interference with some aspects of cellular DNA fixation pro-

Antimutagenic agents are able to combat the disorders caused by mutagens [10]. This group of agents includes both natural and synthetic compounds categories [1].

The antimutagenic effect of natural sources was investigated due to certain

It is the first naturally occurring organic antimutagen [11]; it has been involved

in screening and chemical studies of such biologically active substances [12]. Antimutagenic action is attributed to either by a selective killing effect of cells which have premutation lesion of DNA via inhibition of the errorprone SOS repair system, or by enhancement of the error-free DNA repair system (**Figure 4**) [13].

affect mutagens simultaneously in varied ways significantly enhances

are inactivated before they can attack the DNA in vitro [3].

cesses working on reducing genetic damage in DNA [3].

It was reported that the antigenotoxic effects of Lipoic acid (LA) (**Figure 1**) against mitomycin-C induced chromosomal aberrations, sister chromatid exchanges, and micronucleus formation was observed in human peripheral lymphocytes. Moreover, LA exhibits both anticlastogenic and antimutagenic activity [6].

#### **2.2 Interaction with mutagen**

A potential protective mechanism against mutagenesis is related to the direct chemical interaction between a mutagen and an antimutagenic compound before it induces DNA damage leading to the inhibition of their damaging activity. Sulfhydryl compounds, such as cysteine, can inactivate 3-chloro-4-(dichloromethyl)-5 hydroxy-2(5H)-furanone (MX) (**Figure 2**) [7].

#### **2.3 Antimutagen as blocking agents**

The mechanism of action for this type of antimutagenics is to prevent mutagenic compounds from reaching target sites such as nucleophilic bichalcophenes (**Figure 3**). They might be able to bind to DNA and, therefore, protect genetic materials from electrophilic mutagenic agents [8].

**Figure 1.** *Lipolic acid.*

**Figure 2.** *(a) Mutagen (MX) and (b) antimutagen (cysteine).*

*Current Trends and Future Perspectives of Antimutagenic Agents DOI: http://dx.doi.org/10.5772/intechopen.91689*

**Figure 3.** *Bichalcophene derivatives.*

caused by the mutagenic agents, putting in mind possible future perspectives

Several classes of antimutagenic compounds may be distinguished based on their

Reactive oxygen species (ROS) are generated by many mutagens; therefore, the removal of reactive molecules is considered an important strategy in the process of antimutagenesis. It is reported that compounds with antioxidant propertiescan remove ROS before these molecules react with DNA, resulting in a mutation [5]. It was reported that the antigenotoxic effects of Lipoic acid (LA) (**Figure 1**) against mitomycin-C induced chromosomal aberrations, sister chromatid exchanges, and micronucleus formation was observed in human peripheral lymphocytes. Moreover, LA exhibits both anticlastogenic and antimutagenic activity [6].

A potential protective mechanism against mutagenesis is related to the direct chemical interaction between a mutagen and an antimutagenic compound before it induces DNA damage leading to the inhibition of their damaging activity. Sulfhydryl compounds, such as cysteine, can inactivate 3-chloro-4-(dichloromethyl)-5-

The mechanism of action for this type of antimutagenics is to prevent mutagenic

compounds from reaching target sites such as nucleophilic bichalcophenes (**Figure 3**). They might be able to bind to DNA and, therefore, protect genetic

and mechanism of antimutagenics [2].

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

mechanism of action as the following:

**2.1 Antimutagens with antioxidant potency**

hydroxy-2(5H)-furanone (MX) (**Figure 2**) [7].

materials from electrophilic mutagenic agents [8].

**2.3 Antimutagen as blocking agents**

*(a) Mutagen (MX) and (b) antimutagen (cysteine).*

**Figure 1.** *Lipolic acid.*

**Figure 2.**

**132**

**2. Mechanisms of action**

**2.2 Interaction with mutagen**

## **2.4 Multifunctionally acting antimutagens**

Various antimutagenic agents work through multiple mechanisms affording protection against several mutagens. Noteworthy, the ability of compounds to affect mutagens simultaneously in varied ways significantly enhances antimutagenic effectiveness. Hence, searching for such multifunctionally acting antimutagens is of great importance [9].

#### **2.5 Desmutagenesis**

This way of preventing induced cellular mutagenesis depends on mutagens that are inactivated before they can attack the DNA in vitro [3].

#### **2.6 Bio-antimutagenesis**

Damaged DNAusually requires fixation steps (e.g., DNAreplication and/or repair) before it can be expressed as stable and heritable mutant genes. Hence this mechanism relates to interference with some aspects of cellular DNA fixation processes working on reducing genetic damage in DNA [3].

## **3. Antimutagenic agents**

Antimutagenic agents are able to combat the disorders caused by mutagens [10]. This group of agents includes both natural and synthetic compounds categories [1].

#### **3.1 Natural antimutagenic agents**

The antimutagenic effect of natural sources was investigated due to certain compounds in them or due to whole extract.

#### *3.1.1 Isolated compounds*

#### *3.1.1.1 Cinnamaldehyde*

It is the first naturally occurring organic antimutagen [11]; it has been involved in screening and chemical studies of such biologically active substances [12]. Antimutagenic action is attributed to either by a selective killing effect of cells which have premutation lesion of DNA via inhibition of the errorprone SOS repair system, or by enhancement of the error-free DNA repair system (**Figure 4**) [13].

## *3.1.1.2 Punicalagin (PC) and ellagic acid (EA)*

Punicalagin is an ellagitannin found in the fruit peel of *Punica granatum*. PC and EA (**Figure 5a, b**) had antioxidant and antigenotoxic properties which dosedependently and markedly antagonized the effect of tested mutagens such as NaN3, benzo[a]pyrene, 2-aminoflourine, and methyl methanesulfonate (EMS), with 90% mutagenicity inhibition [14].

the inhibition capability by blocking 9-aminoacridine binding to DNA [15]. In addition, the inhibition effects against ethyl methanesulfonate may be related to the protection against DNA double-strand breaks or EMS alkylating action

*Annona crassiflora Mart*. (AcM) is a Brazilian plant, *araticum*, which is widely used as a therapeutic medicine to treat several diseases such as rheumatism, diarrhea, and syphilis. Ethanolic extract were evaluated for antimutagenic and cytotoxic effects. The results indicated an antimutagenic activity of the AcM due to the

Pinocembrin and cardamonin (**Figure 8**) are found in Sozuku (Chinese drug from dried seed of *Alpinia katsumadae* HAYATA). These compounds showed potent antimutagenic activity against 2-amino3,4-dimethylimidazo-[4,5-f] quinolone (MeIQ) mutagenesis in Ames test using the *S. typhimurium* TA100 and TA98

It isa type of iridoid glycoside. HS (**Figure 9**) is considered as the main active component extracted from *Harpagophytumprocumbens* (HP) which is used as antiinflammatory and analgesic particularly against painful osteoarthritis. The extract wastested to evaluate the antimutagenic activity of HS and HP against mutagenic

activity of 1-nitropyrene (1-NPy) that is one of the most abundant nitropolycyclic aromatic hydrocarbons particularly in diesel exhausts. The results showed that HS significantly reduced the mutagenicity of 1-NPy in pretreatment and particularly in co-treatment. Moreover, HP extract significantly reduced the

presence of acetogenins (**Figure 7**) and other flavonoids [17].

*Current Trends and Future Perspectives of Antimutagenic Agents*

*DOI: http://dx.doi.org/10.5772/intechopen.91689*

(**Figure 6**) [16].

*3.1.1.4 Acetogenins*

strains [18].

*3.1.1.6 Harpagoside (HS)*

genotoxicity [19].

**Figure 7.** *Acetogenins.*

**Figure 8.**

**135**

*Pinocembrin and cardamonin.*

*3.1.1.5 Pinocembrin and cardamonin*

## *3.1.1.3 Luteolin derivatives*

Luteolin derivatives (luteolin 7-O-rutinoside, luteolin 7-O-glucoside, and luteolin 7-O-glucuronide) (**Figure 6**) are isolated from *Mentha longifolia* (L.) to evaluate the antimutagenic activities by using Ames *Salmonella* test (TA 1535 and TA1537 strains). The antimutagenic activity on TA1537 was 87.63, 84.03, and 67.77%, respectively. The antimutagenic activity of these compoundscan be due to

**Figure 4.** *Cinnamaldehyde.*

**Figure 6.** *Luteolin derivatives.*

## *Current Trends and Future Perspectives of Antimutagenic Agents DOI: http://dx.doi.org/10.5772/intechopen.91689*

the inhibition capability by blocking 9-aminoacridine binding to DNA [15]. In addition, the inhibition effects against ethyl methanesulfonate may be related to the protection against DNA double-strand breaks or EMS alkylating action (**Figure 6**) [16].

## *3.1.1.4 Acetogenins*

*3.1.1.2 Punicalagin (PC) and ellagic acid (EA)*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

mutagenicity inhibition [14].

*3.1.1.3 Luteolin derivatives*

**Figure 4.** *Cinnamaldehyde.*

**Figure 5.**

**Figure 6.** *Luteolin derivatives.*

**134**

*(a) Punicalagin (b) ellagic acid.*

Punicalagin is an ellagitannin found in the fruit peel of *Punica granatum*. PC and

EA (**Figure 5a, b**) had antioxidant and antigenotoxic properties which dosedependently and markedly antagonized the effect of tested mutagens such as NaN3, benzo[a]pyrene, 2-aminoflourine, and methyl methanesulfonate (EMS), with 90%

Luteolin derivatives (luteolin 7-O-rutinoside, luteolin 7-O-glucoside, and luteolin 7-O-glucuronide) (**Figure 6**) are isolated from *Mentha longifolia* (L.) to evaluate the antimutagenic activities by using Ames *Salmonella* test (TA 1535 and TA1537 strains). The antimutagenic activity on TA1537 was 87.63, 84.03, and 67.77%, respectively. The antimutagenic activity of these compoundscan be due to

*Annona crassiflora Mart*. (AcM) is a Brazilian plant, *araticum*, which is widely used as a therapeutic medicine to treat several diseases such as rheumatism, diarrhea, and syphilis. Ethanolic extract were evaluated for antimutagenic and cytotoxic effects. The results indicated an antimutagenic activity of the AcM due to the presence of acetogenins (**Figure 7**) and other flavonoids [17].

## *3.1.1.5 Pinocembrin and cardamonin*

Pinocembrin and cardamonin (**Figure 8**) are found in Sozuku (Chinese drug from dried seed of *Alpinia katsumadae* HAYATA). These compounds showed potent antimutagenic activity against 2-amino3,4-dimethylimidazo-[4,5-f] quinolone (MeIQ) mutagenesis in Ames test using the *S. typhimurium* TA100 and TA98 strains [18].

## *3.1.1.6 Harpagoside (HS)*

It isa type of iridoid glycoside. HS (**Figure 9**) is considered as the main active component extracted from *Harpagophytumprocumbens* (HP) which is used as antiinflammatory and analgesic particularly against painful osteoarthritis. The extract wastested to evaluate the antimutagenic activity of HS and HP against mutagenic activity of 1-nitropyrene (1-NPy) that is one of the most abundant nitropolycyclic aromatic hydrocarbons particularly in diesel exhausts. The results showed that HS significantly reduced the mutagenicity of 1-NPy in pretreatment and particularly in co-treatment. Moreover, HP extract significantly reduced the genotoxicity [19].

**Figure 7.** *Acetogenins.*

**Figure 8.** *Pinocembrin and cardamonin.*

*3.1.2 Plant extract*

(EMS) [22].

under study [23].

*3.1.2.1* Date palm *fruit aqueous extract*

*DOI: http://dx.doi.org/10.5772/intechopen.91689*

*3.1.2.2* Maytenus ilicifolia *and* Peltastes peltatus *extract*

*Current Trends and Future Perspectives of Antimutagenic Agents*

*3.1.2.3* Citrus limonum *fruit residues (CLFR)*

can act as antioxidant and anitmutagenic [24].

*3.1.2.4* Mimosa tenuiflora *(MT) extract*

treatment of diseases [25].

damage [26].

**137**

*3.1.2.5* Albeofructus *(ADA) extract*

*3.1.2.6* Anemopsis californica *(AC)*

premutagens and precarcinogens [27].

It was found that *date palm* extract displays strong antimutagenic activity against ultraviolet (UV) radiation, and mitomycin C-induced mutagenesis, when it was analyzed using *E. coli* RNA polymerase β-based rifampicin resistance assay, but did not show any significant antimutagenesis against ethyl methane sulfonate

These two plants are both rich in compounds of the tanninand flavonoid groups

Aqueous and acidified methanol extracts of CLFR were evaluated for their total phenolic contents and antioxidant and antimutagenic activities. Antimutagenic potential of the extracts was done by Ames test. The results supported that the extracts from CLFR were mutagenically safe due to its high phenolic content which

The genotoxic effect of MT was investigated by using both micronucleus test and Ames test in *Salmonella typhimurium* TA97, TA98, TA100, and TA102, respectively. The results showed that the extract did not induce mutations in any strain. Further studies of toxicity were performed to investigate the use of this plant in the

It is an extract of *Acanthopanax divaricatus* which possesses antimutagenic activity against direct-acting mutagenic agents through the rapid elimination of mutagenic compounds from the cells before the induction of genetic material

Although *A. californica* (AC) possesses therapeutic uses, so it could be useful for reducing genotoxic risk generating from ROS-agents exposure and provide protec-

tion against poly-cyclic aromatic hydrocarbons which are well known as

and frequently employed in folk medicine. Antimutagenicity was determined against known mutagenic substances such as 4-oxide-1-nitroquinoline, NaN3, aflatoxin B1, 2-aminofluorene and 2-aminoanthracene, and 2-nitrofluorene using the *Salmonella*/microsome assay. There was a significant decrease in mutagenicity for the tested extract by 75%.The mechanism of antimutagenicity of this extract is still

**Figure 11.**

*Glycyrrhiza aspera root extract.*

## *3.1.1.7 Lycopene*

Natural oleoresin is rich in lycopene (**Figure 10**), which was obtained from two types of tomato (Zedona and Gironda). The antimutagenic activity of oleoresin was tested against aflatoxin B1 (AFB1), and both varieties had awfully high antimutagenic potential against AFB1 (60–66%) [20].

#### *3.1.1.8 Compounds extracted from* Glycyrrhiza aspera *root*

The powdered extract of *G. aspera* root was assayed for antimutagenic activity against N-methyl-N-nitrosourea (MNU) in S. typhimurium TA1535. Five components that were extracted by using ethanol which had antimutagenic activity against MNU were identified as glyurallin A, glyasperin B, licoricidin, 1-methoxyphaseollin, and licoisoflavone B (**Figure 11**). These components were demonstrated to possess an antigenotoxic effect against carcinogenic MNU. So this extract can be used to prevent DNA damage by N-nitrosamines for cancer chemoprevention [21].

*Current Trends and Future Perspectives of Antimutagenic Agents DOI: http://dx.doi.org/10.5772/intechopen.91689*

## *3.1.2 Plant extract*

## *3.1.2.1* Date palm *fruit aqueous extract*

It was found that *date palm* extract displays strong antimutagenic activity against ultraviolet (UV) radiation, and mitomycin C-induced mutagenesis, when it was analyzed using *E. coli* RNA polymerase β-based rifampicin resistance assay, but did not show any significant antimutagenesis against ethyl methane sulfonate (EMS) [22].

## *3.1.2.2* Maytenus ilicifolia *and* Peltastes peltatus *extract*

These two plants are both rich in compounds of the tanninand flavonoid groups and frequently employed in folk medicine. Antimutagenicity was determined against known mutagenic substances such as 4-oxide-1-nitroquinoline, NaN3, aflatoxin B1, 2-aminofluorene and 2-aminoanthracene, and 2-nitrofluorene using the *Salmonella*/microsome assay. There was a significant decrease in mutagenicity for the tested extract by 75%.The mechanism of antimutagenicity of this extract is still under study [23].

## *3.1.2.3* Citrus limonum *fruit residues (CLFR)*

Aqueous and acidified methanol extracts of CLFR were evaluated for their total phenolic contents and antioxidant and antimutagenic activities. Antimutagenic potential of the extracts was done by Ames test. The results supported that the extracts from CLFR were mutagenically safe due to its high phenolic content which can act as antioxidant and anitmutagenic [24].

## *3.1.2.4* Mimosa tenuiflora *(MT) extract*

The genotoxic effect of MT was investigated by using both micronucleus test and Ames test in *Salmonella typhimurium* TA97, TA98, TA100, and TA102, respectively. The results showed that the extract did not induce mutations in any strain. Further studies of toxicity were performed to investigate the use of this plant in the treatment of diseases [25].

## *3.1.2.5* Albeofructus *(ADA) extract*

It is an extract of *Acanthopanax divaricatus* which possesses antimutagenic activity against direct-acting mutagenic agents through the rapid elimination of mutagenic compounds from the cells before the induction of genetic material damage [26].

## *3.1.2.6* Anemopsis californica *(AC)*

Although *A. californica* (AC) possesses therapeutic uses, so it could be useful for reducing genotoxic risk generating from ROS-agents exposure and provide protection against poly-cyclic aromatic hydrocarbons which are well known as premutagens and precarcinogens [27].

*3.1.1.7 Lycopene*

*Glycyrrhiza aspera root extract.*

**Figure 11.**

**136**

**Figure 10.** *Lycopene.*

**Figure 9.** *Harpagoside (HS)*

Natural oleoresin is rich in lycopene (**Figure 10**), which was obtained from two types of tomato (Zedona and Gironda). The antimutagenic activity of oleoresin was

The powdered extract of *G. aspera* root was assayed for antimutagenic activity against N-methyl-N-nitrosourea (MNU) in S. typhimurium TA1535. Five components that were extracted by using ethanol which had antimutagenic activity against MNU were identified as glyurallin A, glyasperin B, licoricidin, 1-methoxyphaseollin, and licoisoflavone B (**Figure 11**). These components were demonstrated to possess an antigenotoxic effect against carcinogenic MNU. So this extract can be used to prevent DNA damage by N-nitrosamines for cancer chemoprevention [21].

tested against aflatoxin B1 (AFB1), and both varieties had awfully high

antimutagenic potential against AFB1 (60–66%) [20].

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

*3.1.1.8 Compounds extracted from* Glycyrrhiza aspera *root*

#### *3.1.2.7* Citrus sinensis *and* Citrus latifolia

The essential oils of *Citrus sinensis* and *Citrus latifolia* showed antimycotic besides antimutagenic and antioxidant activity. Their main components are R-(+)-limonene, α-myrcene, β-thujene, and γ-terpinene [28].

Brazilian fruit, possesses the antimutagenic potential of copaiba powder (dose of

*Ganoderma lucidum* was extracted with hot water (GLW) and then partially purified with crude glycoside extract (GLG) and crude polysaccharide extract (GLP). The extract was tested to evaluate the antioxidant and antimutagenic activity. The results showed that the extract has antimutagenic activity due to *β*-glucan

It was demonstrated that ethyl acetate extract of macro fungus showed the in vitro antimutagenic activity of *Phellinus rimosus*. The activity of the extract against direct-acting mutagens may result from the direct inactivation of mutagens. It is probable that, due to stimulation of the transmembrane export system in bacteria, mutagenic compounds are removed from the cells before they influence the DNA structure [37]. Additionally, in the case of doxorubicin (DXN), the extract

Synthetic antimutagens is another important trend in the area of antimuta-

Bile acids have either a co- or an antimutagenic activity toward various directand indirect-acting mutagens [38]. It was reported that steroidal hormones could inhibit the genotoxicity of both direct- and indirect-acting mutagens [39]. For example, both ethinyl oestradiol and mestranol (**Figure 12**), which are synthetic derivatives of 3-estradiol largely used in contraceptive pills, are strong

It could act as a nucleophile to scavenge the electrophilic mutagens. It was implied that gallic acid (**Figure 13**) can bind or insert into the outer membrane

content and antioxidant action due to the presence of high polyphenolic

of *P. rimosus* may affect the intercalation of mutagens to genetic material.

mutagenic inhibitors acting at nanomolar concentrations [39].

100 mg/kg) showing great reduction of micronuclei [35].

*Current Trends and Future Perspectives of Antimutagenic Agents*

*DOI: http://dx.doi.org/10.5772/intechopen.91689*

*3.1.2.13 Other sources*

content [36].

*3.1.2.13.2 Macro fungus*

genicity research.

*3.2.2 Gallic acid*

**Figure 12.**

**139**

*Steroidal hormonal molecules*

**3.2 Synthetic antimutagenic agents**

*3.2.1 Steroidal hormonal molecules*

*3.1.2.13.1 Ganoderma lucidum*

## *3.1.2.8* Heterotheca inuloides *(HI) extract*

The methanolic extract of HI reduced the mutagenicity of benzo[a]pyrene, norfloxacin, and 2-aminoanthracene. The antigenotoxic properties could be due to the antioxidant properties of component into extract such as catenanes, sterols, polyacetylenes, triterpenes, sesquiterpenes, flavonoids, and flavonoid glycosides [29].

#### *3.1.2.9 Extracts* of Acacia salicina

Literatures revealed that this extract displayed potent antioxidant and antimutagenic activities [30]. Also chloroform extract showed antimutagenic effect against both direct- and indirect-acting mutagens, as the extract may act as a blocking agent that is capable of influencing the activities of enzymes engaged in the metabolism of mutagens and carcinogens. Moreover, the tested extract displayed the ability to react directly with the mutagens electrophilic metabolite sand was capable of protecting against oxidative DNA damage [30].

## *3.1.2.10 Wheat bran*

It was reported that wheat bran provides antimutagenic effects that related to the presence of the antioxidant phytic acid. It was demonstrated that phytic acid may intercept carcinogenic azoxymethane, inhibiting it even before it can damage DNA. Moreover, antioxidants included in wheat bran are able tomodulate DNA repair enzymes [31].

#### *3.1.2.11 Vegetables*

Activity was displayed by beets, chives, horseradish, onions, rhubarb, and spinach. All cruciferous vegetables showed strong to moderate antimutagenic activities, except Chinese cabbage, which displayed weak activity. Moderate antimutagenicity was found in green beans and tomatoes, whereas weak activities in egg plant, garden cress, many types of lettuces, leeks, mangold, cucumber, pumpkin, radish, and summer squash. However, some vegetables such as *Asparagus*, carrots, fennel leaves, parsley, green pepper, and radishes were not found to display any antimutagenicity [32].

Antimutagenic activity of many vegetable juiceswere earlier studied againstmutagenicity induced by2-amino-3-methyl[4,5-f]-quinoline (IQ), 2-amino-3,4dimethylimidazo[4,5-f] quinoline (MeIQ) or 2-amino3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) in S.typhimurium TA98 and TA100 [33].

#### *3.1.2.12 Fruits*

Current research all over the world has focused on health protectiveproperties of fruits including antimutagenic potential of different fruittypes and their cultivars. Concerning apple fruit, its antioxidant and radioprotective properties were found to be better correlated with its antimutagenic effect [34]. Recently, copaiba, an exotic

Brazilian fruit, possesses the antimutagenic potential of copaiba powder (dose of 100 mg/kg) showing great reduction of micronuclei [35].

## *3.1.2.13 Other sources*

*3.1.2.7* Citrus sinensis *and* Citrus latifolia

*3.1.2.8* Heterotheca inuloides *(HI) extract*

*3.1.2.9 Extracts* of Acacia salicina

glycosides [29].

*3.1.2.10 Wheat bran*

repair enzymes [31].

antimutagenicity [32].

*3.1.2.12 Fruits*

**138**

*3.1.2.11 Vegetables*

The essential oils of *Citrus sinensis* and *Citrus latifolia* showed antimycotic besides antimutagenic and antioxidant activity. Their main components are

The methanolic extract of HI reduced the mutagenicity of benzo[a]pyrene, norfloxacin, and 2-aminoanthracene. The antigenotoxic properties could be due to the antioxidant properties of component into extract such as catenanes, sterols,

polyacetylenes, triterpenes, sesquiterpenes, flavonoids, and flavonoid

sand was capable of protecting against oxidative DNA damage [30].

Literatures revealed that this extract displayed potent antioxidant and antimutagenic activities [30]. Also chloroform extract showed antimutagenic effect against both direct- and indirect-acting mutagens, as the extract may act as a blocking agent that is capable of influencing the activities of enzymes engaged in the metabolism of mutagens and carcinogens. Moreover, the tested extract displayed the ability to react directly with the mutagens electrophilic metabolite

It was reported that wheat bran provides antimutagenic effects that related to the presence of the antioxidant phytic acid. It was demonstrated that phytic acid may intercept carcinogenic azoxymethane, inhibiting it even before it can damage DNA. Moreover, antioxidants included in wheat bran are able tomodulate DNA

Activity was displayed by beets, chives, horseradish, onions, rhubarb, and spinach. All cruciferous vegetables showed strong to moderate antimutagenic activities, except Chinese cabbage, which displayed weak activity. Moderate antimutagenicity was found in green beans and tomatoes, whereas weak activities in egg plant, garden cress, many types of lettuces, leeks, mangold, cucumber, pumpkin, radish, and summer squash. However, some vegetables such as *Asparagus*, carrots, fennel

Antimutagenic activity of many vegetable juiceswere earlier studied againstmu-

Current research all over the world has focused on health protectiveproperties of fruits including antimutagenic potential of different fruittypes and their cultivars. Concerning apple fruit, its antioxidant and radioprotective properties were found to be better correlated with its antimutagenic effect [34]. Recently, copaiba, an exotic

leaves, parsley, green pepper, and radishes were not found to display any

tagenicity induced by2-amino-3-methyl[4,5-f]-quinoline (IQ), 2-amino-3,4dimethylimidazo[4,5-f] quinoline (MeIQ) or 2-amino3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) in S.typhimurium TA98 and TA100 [33].

R-(+)-limonene, α-myrcene, β-thujene, and γ-terpinene [28].

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

## *3.1.2.13.1 Ganoderma lucidum*

*Ganoderma lucidum* was extracted with hot water (GLW) and then partially purified with crude glycoside extract (GLG) and crude polysaccharide extract (GLP). The extract was tested to evaluate the antioxidant and antimutagenic activity. The results showed that the extract has antimutagenic activity due to *β*-glucan content and antioxidant action due to the presence of high polyphenolic content [36].

## *3.1.2.13.2 Macro fungus*

It was demonstrated that ethyl acetate extract of macro fungus showed the in vitro antimutagenic activity of *Phellinus rimosus*. The activity of the extract against direct-acting mutagens may result from the direct inactivation of mutagens. It is probable that, due to stimulation of the transmembrane export system in bacteria, mutagenic compounds are removed from the cells before they influence the DNA structure [37]. Additionally, in the case of doxorubicin (DXN), the extract of *P. rimosus* may affect the intercalation of mutagens to genetic material.

## **3.2 Synthetic antimutagenic agents**

Synthetic antimutagens is another important trend in the area of antimutagenicity research.

## *3.2.1 Steroidal hormonal molecules*

Bile acids have either a co- or an antimutagenic activity toward various directand indirect-acting mutagens [38]. It was reported that steroidal hormones could inhibit the genotoxicity of both direct- and indirect-acting mutagens [39]. For example, both ethinyl oestradiol and mestranol (**Figure 12**), which are synthetic derivatives of 3-estradiol largely used in contraceptive pills, are strong mutagenic inhibitors acting at nanomolar concentrations [39].

## *3.2.2 Gallic acid*

It could act as a nucleophile to scavenge the electrophilic mutagens. It was implied that gallic acid (**Figure 13**) can bind or insert into the outer membrane

**Figure 12.** *Steroidal hormonal molecules*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

**Figure 13.** *Gallic acid.*

a product of MNNG that is related to its mutagenic effect. Both compounds also abolished mutagenesis induced by 9-AA that binds to DNA noncovalently by

This category of antimutagenics acts against mutagens via either intracellularor extracellular mechanisms [44]. The extracellular mechanism showed interference with the cytochrome P450-mediated metabolismof these mutagens and the interaction with active mutagenicmetabolites [8]. Moreover, the antimutagenic potency of these compounds may be relatedto DNA protection from mutagens presenting

Hydroxyphenyliminoligands and their metal complexes [Cu(II), Co(II), Ni(II) and Mn(II) complexes] of usnic acid (**Figure 16**) which is isolated from *Usnea longissima*, were synthesized by Schiff base method with *O-, P-,* and *M-*

aminophenol compounds to determine their antimutagenic activity against different bacteria species. The results showed that the Co and Mn complexes of the

New polymeric microspheres containing azomethine were designed and synthesized to evaluate their antimutagenic activity against NaN3, among of them; a new polymeric microspheres containing azomethine (**Figure 17**) which contains

Chitosan derivatives containing quaternary ammonium groups and di (tertbutyl) phenol (TBPh) (**Figure 18**) in the polymer side chain improved the

Hydrazone derivatives were synthesized to study their antioxidant and antimutagenic activity against 4-NPD and NaN3 in *S. typhimurium* TA98 and TA100, respectively, among of them; the hydrazone derivative (**Figure 19**)

intercalation [43].

**Figure 15.**

**Figure 16.** *Usnic acid.*

**141**

*β-Aminoketones and mutagens.*

*Current Trends and Future Perspectives of Antimutagenic Agents*

*DOI: http://dx.doi.org/10.5772/intechopen.91689*

*3.2.5 Phenolic agents*

electrophilicproperties [8].

ligands possess potent antimutagenic activity [45].

R = CH3 had potent antimutagenic effect against NaN3 [46].

antimutagenic efficiency of the polymer from 48 to 93% [47].

transporters leading to the blockage of a mutagen that was transferred intothe cytosol [40]. One of the mutagenic substances that gallic acid affects is NaN3. It is widely used in agriculture, industry, and medicine, but it is a highly toxicsubstance. If sodium azide is found in the intracellular milieu, azide ions bind Fe3þ in hemoglobin and inhibit the respiratory chain of metabolism [41].

## *3.2.3 Tannic acid*

The anticlastogenic effect of tannic acid (**Figure 14**) was studied *in vivo* in the mouse micronucleus test. Moreover, the antimutagenic effect of tannic acid was investigated *in vivo* in the mouse spot test using male PW and female C57BL/10 mice. The results showed that tannic acid can act as an anticlastogen and antimutagen in vivo [42].

#### *3.2.4 Synthesized β- aminoketones*

Theantigenotoxic potential of two newly synthesized β-aminoketones such as2- {(4-bromophenyl)[(4-methylphenyl) amino] methyl} cyclohexanone and 2-{(4 chlorophenyl)[(4-methylphenyl) amino] methyl} cyclohexanone compounds was tested against the mutagenN-methyl-N-nitro-N-nitrosoguanidine (MNNG), acting by DNAmethylation (**Figure 15**) [9]. The antimutagenic potential of these compounds may be related to the inhibition of the production of O6-methylguanine,

*Current Trends and Future Perspectives of Antimutagenic Agents DOI: http://dx.doi.org/10.5772/intechopen.91689*

#### **Figure 15.**

*β-Aminoketones and mutagens.*

#### **Figure 16.** *Usnic acid.*

transporters leading to the blockage of a mutagen that was transferred intothe cytosol [40]. One of the mutagenic substances that gallic acid affects is NaN3. It is widely used in agriculture, industry, and medicine, but it is a highly toxicsubstance. If sodium azide is found in the intracellular milieu, azide ions bind Fe3þ in hemo-

The anticlastogenic effect of tannic acid (**Figure 14**) was studied *in vivo* in the mouse micronucleus test. Moreover, the antimutagenic effect of tannic acid was investigated *in vivo* in the mouse spot test using male PW and female C57BL/10 mice. The results showed that tannic acid can act as an anticlastogen and

Theantigenotoxic potential of two newly synthesized β-aminoketones such as2- {(4-bromophenyl)[(4-methylphenyl) amino] methyl} cyclohexanone and 2-{(4 chlorophenyl)[(4-methylphenyl) amino] methyl} cyclohexanone compounds was tested against the mutagenN-methyl-N-nitro-N-nitrosoguanidine (MNNG), acting by DNAmethylation (**Figure 15**) [9]. The antimutagenic potential of these compounds may be related to the inhibition of the production of O6-methylguanine,

globin and inhibit the respiratory chain of metabolism [41].

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

*3.2.3 Tannic acid*

**140**

**Figure 14.** *Tannic acid.*

**Figure 13.** *Gallic acid.*

antimutagen in vivo [42].

*3.2.4 Synthesized β- aminoketones*

a product of MNNG that is related to its mutagenic effect. Both compounds also abolished mutagenesis induced by 9-AA that binds to DNA noncovalently by intercalation [43].

## *3.2.5 Phenolic agents*

This category of antimutagenics acts against mutagens via either intracellularor extracellular mechanisms [44]. The extracellular mechanism showed interference with the cytochrome P450-mediated metabolismof these mutagens and the interaction with active mutagenicmetabolites [8]. Moreover, the antimutagenic potency of these compounds may be relatedto DNA protection from mutagens presenting electrophilicproperties [8].

Hydroxyphenyliminoligands and their metal complexes [Cu(II), Co(II), Ni(II) and Mn(II) complexes] of usnic acid (**Figure 16**) which is isolated from *Usnea longissima*, were synthesized by Schiff base method with *O-, P-,* and *M*aminophenol compounds to determine their antimutagenic activity against different bacteria species. The results showed that the Co and Mn complexes of the ligands possess potent antimutagenic activity [45].

New polymeric microspheres containing azomethine were designed and synthesized to evaluate their antimutagenic activity against NaN3, among of them; a new polymeric microspheres containing azomethine (**Figure 17**) which contains R = CH3 had potent antimutagenic effect against NaN3 [46].

Chitosan derivatives containing quaternary ammonium groups and di (tertbutyl) phenol (TBPh) (**Figure 18**) in the polymer side chain improved the antimutagenic efficiency of the polymer from 48 to 93% [47].

Hydrazone derivatives were synthesized to study their antioxidant and antimutagenic activity against 4-NPD and NaN3 in *S. typhimurium* TA98 and TA100, respectively, among of them; the hydrazone derivative (**Figure 19**)

**Figure 17.**

*New polymeric microspheres containing azomethine.*

**Figure 18.** *Chitosan derivatives containing quaternary ammonium groups.*

**Figure 19.** *Hydrazone derivatives*

had high antimutagenic activity. The strongest antimutagenic activity was observed at 5 mg/plate concentration against *S. typhimurium* TA100 strain [48].

*3.2.8 Organoselenium*

**Figure 20.** *Xanthone.*

*Current Trends and Future Perspectives of Antimutagenic Agents*

*DOI: http://dx.doi.org/10.5772/intechopen.91689*

**Figure 21.**

**Figure 22.** *Indolizine.*

*Novel polymeric-Schiff bases.*

radicals production [53].

*3.2.9 Bichalcophenes*

*3.2.10 Others*

**143**

Scientists demonstrated that this series of compounds are protected against genotoxicity and oxidative stress induced by an indirect-acting mutagen CP [52]. This is attributed to effect of CP on DNA through its alkylating properties and free

The novel bichalcophenes significantly decreased the mutagenicity induced by two mutagens, namely, NaN3 and BP [54]. It was found that the antimutagenic potential of the compounds could be attributed to their antioxidant activity [55].

New zerumbone-bicarbonyl analogues were synthesized to determine their antimutagenic activity against *Salmonella* tester strains. Zerumbal (**Figure 23**) had

significant higher antimutagenic activity than zerumbone [56].

#### *3.2.6 Xanthones*

The potential antimutagenic of xanthonesis attributed to different mechanisms, such as the rapid elimination of mutagens from bacteria; the interaction between antimutagens and the reactive intermediates of mutagens; and the influence on microsomal enzymes against direct mutagen 4-nitroquinoline-N-oxide (NQNO) (**Figure 20**) [49].

#### *3.2.7 Indols*

Novel polymeric-Schiff bases including indol (L1, L2, L3) (**Figure 21**) exhibited the antigenotoxic properties against sodium azide in human lymphocyte cells by micronuclei (MN) and sister chromatid exchange tests [50].

A series of indolizine derivatives have been synthesized to determine their antimutagenic activity, the indolizine derivative (**Figure 22**) had the highest activity [51]. *Current Trends and Future Perspectives of Antimutagenic Agents DOI: http://dx.doi.org/10.5772/intechopen.91689*

**Figure 20.** *Xanthone.*

**Figure 21.** *Novel polymeric-Schiff bases.*

#### **Figure 22.** *Indolizine.*

had high antimutagenic activity. The strongest antimutagenic activity was observed

The potential antimutagenic of xanthonesis attributed to different mechanisms, such as the rapid elimination of mutagens from bacteria; the interaction between antimutagens and the reactive intermediates of mutagens; and the influence on microsomal enzymes against direct mutagen 4-nitroquinoline-N-oxide (NQNO)

Novel polymeric-Schiff bases including indol (L1, L2, L3) (**Figure 21**) exhibited the antigenotoxic properties against sodium azide in human lymphocyte cells by

A series of indolizine derivatives have been synthesized to determine their antimutagenic activity, the indolizine derivative (**Figure 22**) had the highest activity [51].

at 5 mg/plate concentration against *S. typhimurium* TA100 strain [48].

micronuclei (MN) and sister chromatid exchange tests [50].

*3.2.6 Xanthones*

**Figure 17.**

**Figure 18.**

**Figure 19.** *Hydrazone derivatives*

*New polymeric microspheres containing azomethine.*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

*Chitosan derivatives containing quaternary ammonium groups.*

(**Figure 20**) [49].

*3.2.7 Indols*

**142**

## *3.2.8 Organoselenium*

Scientists demonstrated that this series of compounds are protected against genotoxicity and oxidative stress induced by an indirect-acting mutagen CP [52]. This is attributed to effect of CP on DNA through its alkylating properties and free radicals production [53].

## *3.2.9 Bichalcophenes*

The novel bichalcophenes significantly decreased the mutagenicity induced by two mutagens, namely, NaN3 and BP [54]. It was found that the antimutagenic potential of the compounds could be attributed to their antioxidant activity [55].

## *3.2.10 Others*

New zerumbone-bicarbonyl analogues were synthesized to determine their antimutagenic activity against *Salmonella* tester strains. Zerumbal (**Figure 23**) had significant higher antimutagenic activity than zerumbone [56].

Two newly synthesized oxadiazoles: 1,3-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M1) and 1,4-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M2) (**Figure 25**) were synthesized and studied in *Salmonella typhimurium* strains TA97, TA100, TA102 and TA1537 in the presence and absence of S9mix. The antimutagenicity of M1 and M2 against H2O2, NaN3, and 4-nitro-o-phenylene diamine (NPD) using the tester strains, was also investigated. The two compounds were found to be nonmutagenic [58].

#### **Figure 23.**

*1,4-Dihydropyridines (1,4-DHP) (Figure 24) possessed antioxidant and antimutagenic activities. The compounds modified the activity of DNA repair enzymes, to protect the DNA in living cells against peroxynitrite-induced damage [57].*

#### **Figure 24.**

*1,4-Dihydropyridines (1,4-DHP) derivatives.*

**Figure 25.** *Oxadiazole derivatives.*

Dihydrothienoquinoline derivatives were designed and synthesized to evaluate

antimutagenicity. The results for compounds (**Figure 26**) were found to be statisti-

A series of novel azacrown ether Schiff bases have been synthesized, and they were investigated for their antimutagenic activities using the spot test and Ames test using strains TA1535, TA100, and TA97a of *Salmonella typhimurium*. The results showed that compounds 1 and 2 (**Figure 27**) were antimutagenic [59].

their antimutagenicity using Ames test. Several compounds showed good

*Current Trends and Future Perspectives of Antimutagenic Agents*

*DOI: http://dx.doi.org/10.5772/intechopen.91689*

cally significant (P = 0) [58].

*Azacrown ether Schiff bases.*

**Figure 27.**

**Conflict of interest**

**Author details**

Minia, Egypt

**145**

The authors declare no conflict of interest.

Adel M. AbdelHakem\* and El-Shimaa M.N. Abdelhafez

provided the original work is properly cited.

Faculty of Pharmacy, Department of Medicinal Chemistry, Minia University,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: pharmacist\_adel2010@yahoo.com

**Figure 26.** *Dihydrothienoquinoline derivatives.*

*Current Trends and Future Perspectives of Antimutagenic Agents DOI: http://dx.doi.org/10.5772/intechopen.91689*

**Figure 27.** *Azacrown ether Schiff bases.*

Two newly synthesized oxadiazoles: 1,3-bis(5-benzylthio-1,3,4-oxadiazol-2-yl)

benzene (M1) and 1,4-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M2) (**Figure 25**) were synthesized and studied in *Salmonella typhimurium* strains TA97, TA100, TA102 and TA1537 in the presence and absence of S9mix. The antimutagenicity of M1 and M2 against H2O2, NaN3, and 4-nitro-o-phenylene diamine (NPD) using the tester strains, was also investigated. The two compounds were

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

*1,4-Dihydropyridines (1,4-DHP) (Figure 24) possessed antioxidant and antimutagenic activities. The compounds modified the activity of DNA repair enzymes, to protect the DNA in living cells against*

found to be nonmutagenic [58].

*peroxynitrite-induced damage [57].*

*1,4-Dihydropyridines (1,4-DHP) derivatives.*

**Figure 23.**

**Figure 24.**

**Figure 25.**

**Figure 26.**

**144**

*Dihydrothienoquinoline derivatives.*

*Oxadiazole derivatives.*

Dihydrothienoquinoline derivatives were designed and synthesized to evaluate their antimutagenicity using Ames test. Several compounds showed good antimutagenicity. The results for compounds (**Figure 26**) were found to be statistically significant (P = 0) [58].

A series of novel azacrown ether Schiff bases have been synthesized, and they were investigated for their antimutagenic activities using the spot test and Ames test using strains TA1535, TA100, and TA97a of *Salmonella typhimurium*. The results showed that compounds 1 and 2 (**Figure 27**) were antimutagenic [59].

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Adel M. AbdelHakem\* and El-Shimaa M.N. Abdelhafez Faculty of Pharmacy, Department of Medicinal Chemistry, Minia University, Minia, Egypt

\*Address all correspondence to: pharmacist\_adel2010@yahoo.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

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mutagenicity assay. Drug and Chemical

e4609

**150**

*Edited by Sonia Soloneski and Marcelo L. Larramendy*

This edited book, "*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*", aims to present the latest developments from different fields, highlighting the detrimental influence that mutagenic and genotoxic agents inflict on DNA and how antimutagenic and anticarcinogenic modulators are able to reduce the negative impact of such toxic agents on living species.

Published in London, UK © 2021 IntechOpen © yucelyilmaz / iStock

Genotoxicity and Mutagenicity - Mechanisms and Test Methods

Genotoxicity and

Mutagenicity

Mechanisms and Test Methods

*Edited by Sonia Soloneski* 

*and Marcelo L. Larramendy*