Assessment of Genotoxicity of Potassium Nitrate and Sodium Benzoate in Drosophila melanogaster Using Smart and Comet Assays

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Modern food industry increasingly relies on food additives.The dramatic increase in the demand on preserved ready to eat food products making up to 75 percent of the diet of western society lead to neglectance of the harmful effects of the food additives on human health; among these are hypersensitivity, allergic reactions, genotoxicity, mutagenicity and more.This proposal investigates genotoxic effects of two commonly used food preservatives; sodium benzoate and potassium nitrate using the somatic mutation and recombination test (SMART) and comet assay.Two important end points of genotoxicity can be covered by in vivo assay systems; comet assay detects DNA damage, while the Drosophila transgenic animal model recognizes gene mutation and/or chromosomal aberrations.Both of the tested compounds showed significantly high levels of tumor induction and frequency compared to a negative control in SMART assay accompanied with a significant amount of DNA damage detected by the comet assay indicating their high potential of being genotoxic materials.
The carcinogenic risk of food additives cannot be neglected and may be attributed to various factors that may include; interaction of additives with some food ingredients, food processing may change the chemical formula of food additives to a formula like carcinogenic compound, a negative synergistic effects when combined with other additives, unsuitable storage conditions and unknown carcinogenic by-products occurring during the food processing (Gülsoy et al. 2015).
Potassium nitrate (E252) is a preservative commonly used in meat products, certain kinds of cheese and for reducing discoloration of vegetables and fruits such as dehydrated potatoes and dried apples (Binkerd and Kolari 1975).The carcinogenic potential of nitrates and nitrites used as preservatives and color-enhancing agents in meats was confirmed by (Nujić and Habuda-Stanić 2017).In vitro treatment of human peripheral blood cells with potassium nitrate caused a decrease in mitotic index MI as compared to control (Mpountoukas et al. 2008).Gömürgen (2005) study on the effect of potassium nitrate on root tips of Allium cepa showed the reduction in the MI, indicating mitotic inhibition and increased frequency of abnormal mitosis.The types of abnormalities observed were chromosome stickiness, c-metaphase, disturbed chromosomes of anaphase and telophase stages, anaphase and telophase bridges, anaphase lagging and forward chromosomes at anaphase and telophase and micronuclei formation at interphase cells.The same results were observed in root tips of Allium cepa treated with sodium nitrate (Pandy et al. 2014).
Sodium benzoate is one of the synthetic additives that is widely used in the food industry and is generally recognized as safe (GRAS) (Hanes 2017).It is widely used as a preservative against fungi, yeast and bacteria in food and soft drinks industry (Hong et al. 2009).Maximum recommended a concentration of sodium benzoate as a preservative according to WHO (1997) was in the range of 2000 mg/kg.Genotoxicity tests for benzyl alcohol, benzoic acid and sodium benzoate have mostly reported negative results but some assays were positive in cell lines and plants (Turkoglu 2007).Ishidate and Odashima (1977) reported chromosomal aberrations in Chinese hamster cell lines grown in culture with sodium benzoate.Onyemaobi et al. (2012) evaluated the effect of sodium benzoate and sodium metabisulphite with different concentrations on root length, chromosomal aberrations and MI of Allium cepa plant.The MI decreased with increasing concentration of both sodium benzoate and sodium metabisulphite.Clumping and fragmentation were the most common among the observed cytological aberrations.The percentage of chromosomal aberrations at mitosis increased as the concentration of the food preservatives increased.The irreversible cytotoxic effects induced by the two tested additives supports the call for banning these substances as food preservatives.
Currently, there are more than 100 genotoxicity assays published so far (Nohmi et al. 2012).The major end points of short-term genotoxicity assays include aneuploidy and chromosomal aberrations, DNA damage, and point mutations (Fairbairn et al. 1995, Nohmi et al. 2012).The use of alternative small organisms as models in toxicology has grown tremendously in the last decade.Drosophila has always been a premier model for both developmental biologists and geneticists, however, several recent toxicology studies have used this organism.
Currently, Drospohila is being used in studies of a number of priority environmental contaminants and toxicants (Rand et al. 2015).Using Drosophila as a genetic model, there are certain mutagenicity test systems to detect aneuploidy and chromosomal aberrations in germ line cells and in somatic cells, dominant lethal mutations, sex-linked and autosomal recessive lethal mutations, and translocations (Würgler andVogel 1986, Zimmering et al. 1990).One of the most widely accepted genotoxicity tests is the Somatic Mutations and Recombination Test (SMART) carried out in Drosophila melanogaster (Demir et al. 2013).This assay uses tumor suppressor gene warts which is a homolog to the mammalian tumor suppressor gene LATS (Nepomuceno 2015, Vasconcelos et al. 2017).Loss of the wts gene not only results in over proliferation but also in apical hypertrophy of epithelial cells, leading to abnormal deposition of extracellular matrix (cuticle) during adult development (Justice et al. 1995).The test for detection of epithelial tumor clones (warts) represents a rapid, very sensitive to different classes of agents and inexpensive assay to evaluate the carcinogenic activity of single compounds as well as of complex mixtures.Various protocols are available for the application of the test materials; single or combined as well as sequential treatments of larvae by feeding.Factors capable of inducing tumors in Drosophila instead of marker clones might directly adverse the risk of these factors for inducing cancer in humans (Sidorov et al. 2001).Genetic events that can lead to the tumor appearance in flies heterozygous for the wts gene and hence can be detected by SMART may include; gene mutations in the wts gene, multilocusdeletions (partial), chromosomal loss and somatic recombination collectively referred to as loss of heterozygosity (Eeken et al. 2002).
Comet assay is a microgel electrophoresis technique for DNA damage detection at the level of single cells.The most important advantage of this technique is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity (Sasaki et al. 2000).The DNA damage measured by DNA strand breakage in the form of single-or double-strand breaks performs as a reliable indicator of genotoxicity.A number of studies showed that comet assay is a rapid, sensitive and inexpensive test for detecting DNA damage which is widely used for detecting genotoxicity of chemical compounds under laboratory and field conditions in mice (Sasaki et al. 2000), zebra fish (Gülsoy et al. 2015), human germ cells (Pandir 2016), and Drosophila melanogaster (Eid et al. 2017).Comet assay has been described as one of the most promising methods for genotoxicity studies against environmental chemicals due to its rapidity and sensitivity.Moreover, the comet assay has been recently adapted to use in vivo in Drosophila to combine its advantages with those well-established of this fly (Mukhopadhyay et al. 2004, Shukla et al. 2011).Alkaline comet assay (pH>13), the most commonly used version, is able to detect all possible kinds of DNA damage (Tice et al. 2000).The assay is clearly useful as a tool for the evaluation of local genotoxicity, particularly organs or cell types, which can hardly be evaluated with other standard tests (Brendler-Schwaab et al. 2005).
The objective of this study was to evaluate the carcinogenic and genotoxic effects of two food additives; potassium nitrate, and sodium benzoate using SMART and comet assays in Drosophila melanogaster.

Somatic Mutation and Recombination Test (SMART) in D. melanogaster: Drosophila melanogaster Strains:
Two different Drosophila strains were used in this study; wild-type strain and a strain that carries wtsMT 4-1 , a lethal warts allele balanced on TM3, characterized by multiple inversions and marked by the dominant mutation stubble according to Eeken et al. (2002) and Fly Base (2006).The genetic structure of this strain, which was abbreviated wts/TM3, is; st p in ri wtsMT4-1/ TM3 Sb.Details about the various markers and the balancer chromosome was described by Lindsley and Zimm (1992).

Crosses and Treatments:
The wts/TM3 females were crossed to wild-type males resulting in two genotypes offspring, wts/+ and TM3, Sb wts +/+ .After 2 days, the parental flies were removed and 56-68 hours old larvae were washed with 20% glycerol, collected using a fine mesh sieve and transferred to four different vials representing the four test groups.For food additives, treated groups (potassium nitrate and sodium benzoate); the flies were transferred to a standard Drosophila medium to which a 100 mM of each food additive powder was added and properly dissolved at 50⁰C.The larvae were submitted to chronic treatment for approximately 24 hours, then they were transferred to standard Drosophila medium.The positive control group was transferred to a vial where 20 µg/ml of an appropriate mitomycin C (MMC) solution was mixed with a standard Drosophila medium, kept for 24 hours, then they were transferred to standard Drosophila medium.The negative control group was directly transferred to a standard Drosophila medium.Afterwards, larvae of all groups were left to feed on the medium until completion of their development when they leave the medium and pupate.All Drosophila stocks and crosses were maintained at 25⁰C.Only adult flies, without the chromosome balancer (TM3, Sb) with no truncated bristles were analysed.

Scoring of Warts:
After metamorphosis, the adult flies were transferred to flasks containing 70% ethanol.Flies were analysed for tumor presence using a Leica stereomicroscope used at a standard magnification of 25 X and entomological tweezers.Only tumors that were large enough to be unequivocally classified are recorded (Eeken et al. 2002).

Statistical Analysis:
For the evaluation of the observed genotoxic effects, in the wing, eye and whole body spots, the frequencies of spots per individual of a treated series were compared to its concurrent negative control series using χ 2 -test.A multiple-decision procedure was used to decide whether a result is positive, weakly positive, inconclusive, or negative (Frei and Würgler 1996).

DNA Fragmentation by Comet Assay
(Single Cell Gel Electrophoresis, SCGE): 2 nd instar larvae of the isogenic strain w 1118 of Drosophila melano-gaster were treated for 24 hours with the same concentration of tested compounds as before in the SMART assay.Both treated and untreated (control) adults were assessed using the alkaline comet assay to measure the extent of DNA strand breaks in all types of cells (Singh et al. 1988).Adult flies were frozen in liquid nitrogen, around 100 flies were gently homogenized into powder, and then an alkaline comet assay was performed as described by Tice et al. (2000). 1 g of crushed samples were transferred to 1 ml icecold PBS.This suspension was stirred for 5 min and filtered.Cell suspension (100 µ1) was mixed with 600 µ1 of low-melting agarose (0.8% in PBS). 100 µ1 of this mixture was spread on pre-coated slides.The coated slides were immersed in lysis buffer (0.045 M TBE, pH 8.4, containing 2.5% SDS) for 15 min.The slides were placed in the electrophoresis chamber containing 0.045 M TBE buffer.The run conditions were 2 V/cm for 2 min and 100 mA.Slides were then stained with ethidium bromide 20µg/m1 at 4ºC.The observation was carried out while the samples still humid, the DNA fragment migration patterns of 100 cells for each dose level were evaluated with a fluorescence microscope using 40X objective (With excitation filter 420-490nm [issue 510nm]).A Komet™ analysis system 5.0 developed by Kinetic Imaging, LTD (Liverpool, UK) linked to a CCD camera was used to measure the length of DNA migration (tail length, in μm) (TL) and the percentage of migrated DNA (DNA %).Finally, the program calculated tail moment.Fifty to one hundred randomly selected cells were analyzed per sample (at least 25 cells per slide and 3 slides per treatment were evaluated).Three different parameters were used as indicators of DNA damage: tail moment (TM) (arbitrary units), tail DNA (%), and tail length (mm).

RESULTS AND DISCUSSION Detection of Mutagenic Agents Using Somatic Mutation and Recombination Test (SMART) in D. melanogaster:
The F1 generation of the crossed flies was divided into four treatment groups: a negative control group transferred to basic Drosophila medium, positive control group transferred to a medium containing 20µg/ml MMC) and two treatment groups each was transferred to a medium containing 100mM of one of the tested compounds; potassium nitrate and sodium benzoate.
The frequency of tumors which is calculated as the ratio of the number of scored tumors to a total number of the scanned flies was 0.07 in negative control flies.Tumor induction in the negative control samples, calculated as the ratio of the number of tumors scored to the number of flies bearing the tumors, was also low (1.03).In contrast, MMC treatment scored the highest frequency (0.81) accompanied with the highest tumor induction (1.7).These tumors were found in every part of the examined flies.
As observed from Table (1) the frequency was increased significantly by either potassium nitrate or sodium benzoate as compared with the negative control.Potassium nitrate treatment showed higher tumor frequency (0.65) than sodium benzoate (0.52) while the reverse was observed in tumor induction 1.36 and 1.53, respectively, as represented by Fig. (1).(2) only small number of cells are needed to carry out the assay (<10,000); (3) the assay can be performed on virtually any eukaryotic cell type; (4) and it is faster and more sensitive than the alkaline elution method for detecting DNA damage DNA (Singh et al. 1988).
In the current study, DNA damage in a homogenous strain w 1118 of adult Drosophila emerged from 2 rd larval instar exposed to the potassium nitrate and sodium benzoate was assessed by comet assay.Data presented in Table (2) showed DNA damage parameters; tailed%, untailed %, tails length, tail DNA% and tail moment.According to the obtained data, both of the two tested salts caused significant DNA damage.Moreover, an increase in tail length was observed in Potassium nitrate and sodium benzoate treated groups as compared to control group (Fig. 2) as an indication of DNA degradation and strand breaks.Migration length is considered to be directly related to fragment size and proportional to the level of single-stranded breaks and alkalilabile sites (Tice et al. 2000).
Potassium nitrate exhibited a significantly higher deleterious effect on DNA of D. melanogaster (about 6 folds tails length as compared with control) than sodium benzoate (about 3.4 folds tails length as compared with control).Tail DNA percentage was 1.48% in control, 6.23% in potassium nitrate treatment and 3.36% in sodium benzoate treatment as recorded in Table (2   This study evaluated the potential genotoxicity and carcinogenicity of two food additives commonly used in the food industry using SMART and comet assays on Drosophila melanogaster system.The obtained results clearly imply the genotoxic potential of both of the tested materials. High frequency of tumor formation in Drosophila SMART assay strongly indicates Loss of heterozygosity (LOH) in somatic cells.Further studies will be carried out to investigate the mechanism of LOH that might involve mutation, chromosome loss or somatic recombination.The use of Drosophila model for understanding the human condition under stress of toxicants has been widely accepted on the basis of the presence of numerous highly conserved genes and pathways controlling the development of stress response across these two divergent species (Mackay and Anholt 2006, Misra et al. 2011, Sykiotis and Bohmann 2010).The evolutionary conservation of tumor suppressor genes among Drosophila and mammals has prompted studies of tumor induction in Drosophila, such studies have contributed to the understanding of cancer in human (Potter et al. 2000, Eeken et al. 2002).This striking resemblance implies that the potential risk of these tested compounds on human health cannot be ignored.
The current results are consistent with the findings of Sarikaya and Cakir (2005) who evaluated the genotoxicity of four food preservatives; sodium nitrite, sodium nitrate, potassium nitrite and potassium nitrate in SMART wing spot test.Exposure to 50, 75 and 100 mM of each tested substance increased the frequency of small single, large single and total spots.The genotoxic effect increased as concentration used increased.The four tested chemicals were ranked according to their genotoxic and toxic effects as sodium nitrite, potassium nitrite, sodium nitrate and potassium nitrate.
Nitrite or nitrate in food may react with endogenous amines, forming mutagenic and carcinogenic compounds (Ertuğrul 1998).Nitrates can be converted into nitrites, which can in turn react with secondary amines or amides to produce mutagenic and carcinogenic compounds i.e. nitrosoamines (Loon et al. 1998, Ertuğrul 1998).The genotoxicity of Nnitrosamines like N-nitrosodimethyl amine, N-nitrosodiethyl amine, Nnitrosodi-n-butyl amine, Nnitrosopiperidine and N-nitrosopyrrolidine in Drosophila somatic cells was detected by (Negishi et al. 1991).The genotoxic effects arising from the in vivo exposure to nitrosation precursor in D. melanogaster could be retarded by co-treatment with catechin (nitrosation inhibitor) (Rincon et al. 1998).Gömürgen (2005) found potassium nitrate to be mitotoxic, and that at higher doses it was able to induce chromosomal aberrations.Benli and Turkoglu (2017) found that sodium benzoate among ten other tested preservatives had a significant effect on decreasing survival and longevity in D. melanogaster using 5, 10, 15 and 20 ppm concentrations.Sarikaya and Solak (2003) declared that 50, 75 and 100 mM concentrations of benzoic acid induced mutations and decreased the life period in D. melanogaster.Deepa et al. (2012) reported that benzaldehyde which is used to give aroma to foods have mutagenic and genotoxic effects on D. melanogaster via SMART assay.Saatci et al. (2016) evaluated the effect of sodium benzoate on lymphocytes of pregnant rats and their fetuses.They declared that Sodium benzoate usage increased micronuclei formation.Sodium benzoate was also found to inhibit DNA synthesis and induce the anaphase bridges, chromosomal condensation in root meristems of Vicia faba (Njagi and Gopalan 1982).Enzymes activity in the mitochondria and cytosol of rat liver hepatocytes were suppressed at doses ≥ 500 µg/ml of Sodium benzoate and DNA synthesis was suppressed at 100 µg/ml of sodium benzoate (Oyanagi et al. 1987).
The single cell gel electrophoresis test, or comet assay, was originally developed by Östling and Johanson (1984) as a microelectrophoretic technique to visualize DNA damage in single cells.Subsequently it was improved by Singh et al. (1988), and since then it was so extensively used that some working-groups were created to standardize its application to mammal and human cells studies (Burlinson et al. 2007, Karlsson 2010, Azqueta and Collins 2013, Ersson et al. 2013, Godschalk et al. 2013, Collins et al. 2014).Its usefulness and easy performance lead to its rapid application to several fields including genotoxicity analyses (Tice et al. 2000, Hartmann et al. 2003, Collins 2004).Surprisingly, its application to Drosophila melanogaster was rather late, despite the fact that this organism is one of the most valuable higher eukaryotic model organism, for all kind of processes and situations related to human health (Reiter et al. 2001, Koh et al. 2006, Wolf et al. 2006, Khurana et al. 2006, Rand 2010), including the in vivo DNA damage response processes (Søndergaard 1993, Sekelsky et al. 2000, Vecchio 2014).In the first published work, the comet assay was performed with brain ganglia cells from third instar larvae (Bilbao et al. 2002).
As with other organisms, several cell types, apart from the brain cells, have been used to carry out this assay in Drosophila in vivo, such as midgut cells (Mukhopadhyay et al. 2004, Siddique et al. 2005, Sharma et al. 2011), hemocytes (Carmona et al. 2011), and imaginal disk cells (Verma et al. 2012).Most of these authors used the comet assay for its original purpose, the in vivo analyses of genotoxicity and DNA repair.Suzuki and inukai (2006) reported that nitrite and nitrate may play a role in enhancing the genotoxic effects and DNA damage of UV light in humans.Saatci et al. (2016) found that sodium benzoate usage may cause DNA damage in liver cells of pregnant rats and their fetuses.They declared that and increase micronuclei formation.Ishidate et al. (1984) observed inhibition of DNA synthesis in rat liver cells following application of 100 mg/mL of Sodium benzoate.Zengin et al. (2011) investigated the in vitro effects of Sodium benzoate and potassium benzoate on cultured human peripheral lymphocytes.They found a significant DNA damage in Sodium benzoate treated groups, providing further evidence for a cytotoxic, mutagenic and clastogenic activity of sodium benzoate.Sasaki et al. (2000) determined the genotoxicity of 39 chemicals used as food additives, most of them induced DNA damage in gastrointestinal organs.

CONCLUSIONS
It can be safely concluded from the present and previous work that both of the tested compounds has a noticeable genotoxic and cytotoxic potential that cannot be ignored while using such chemicals in the food industry.
Further thorough investigations are recommended before continuing using these substances in food and cosmetics as additives.

Table 1 :
Frequencies of induced tumor in trans-heterozygous (wts/+) offspring after larvae feeding treatments with concentrations of Potassium Nitrate (P.N) and Sodium benzoate (S.B) compared to both the MMC as the positive control and the negative control.*and ** significant, highly significant difference from the negative control at P<0.05.Frequency (No. of Tumor/fly) = Number of tumors/Total number of tested flies.Tumor induction = Number of tumors/ Number of tumor flies.

Table 2 :
Detection of DNA damage by the comet assay, assessed as the tail moment (TM) in whole body cells of white eye adult Drosophila treated with the Potassium nitrate (P.N) and sodium benzoate (S.B).and c Different superscript letters in the same column of tail length showed significant difference at P< 0.05.

Fig. 2 :
Fig. 2: Comet images representing DNA damage in adult Drosophila whole body cells.(A and B) represents DNA strand breaks of control, while (C) and (D) represents those after the exposure to Potassium nitrate and sodium benzoate, respectively.