Carcinogenic and Cytotoxic Effect of Some Food Additives on Drosophila melanogaster and Human Cell Lines

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mutagenic , genotoxic, human cell lines, Drosophila, food additives.Some food additives that are commonly used by humans were recently proved to be mutagenic.It is of significant importance to evaluate their genotoxic effects, since they are frequently consumed by humans in their daily meals.In this proposal, we investigated the effects of sodium sulphite, boric acid, and benzoic acid on human cell lines; liver cancer (HepG2), colon cancer (HCT-116), lung cancer (A-459), and normal lung (Wi38) and cells were evaluated using neutral red cytotoxicity assay and assessed using the somatic mutation and recombination test (SMART).These compounds at 100mM concentrations induced tumor induction and increased the frequency compared to a negative control in SMART assay.Also, they reduced the viability of the four examined cell lines cells using different concentrations (75, 150, 300 and 600µg/ml).Boric acid had the highest toxic effect while benzoic had the lowest on the examined cells.The toxicity effect of the tested food additives was higher on normal lung human cells than on lung cancer cells, therefore, these food additives may act as carcinogenic agents cells (chromosome aberrations in Chinese hamster fibroblast cell line (CHL) and Chinese hamster ovarian (CHO) cells, sister chromatid exchange in human lymphocytes) without metabolic activation (Oikawa et al., 1980).On the other hand, benzoic acid had a weak positive increase in chromosomal aberration test in CHO cells (Ishidate et al., 1984) and increased the somatic mutations in Drosophila SMART test (Sarikaya and Solak, 2003).Also, it increased the chromosomal aberration, sister chromatid exchange, and micronucleus frequency (200 and 500 µg/ml) in human peripheral lymphocytes without changing the pH medium in a dose dependent manner.While the mitotic index decreased as benzoic acid concentration increased (Yilmaz et al., 2009;Al-Tai et al., 2014).
Boric acid inhibits the proliferation of prostate cancer cell lines, DU-145, and LNCaP, in a dose-dependent manner.Also, it inhibited non-tumorigenic prostate cell lines, PWR-1E, and RWPE-1, and the cancer line PC-3, but required concentrations higher than observed human blood levels.Using DU-145 cells, boric acid stimulated cell death independent proliferative inhibition, with little effect on cell cycle stage distribution and mitochondrial function (Barranco and Eckhert, 2004).Boric acid addition decreased the genotoxicity and cytotoxicity caused by the anticancer drug (paclitaxel) used for treatments of breast, ovarian, and lung cancers (Turkez et al., 2010).Borax, a salt of boric acid, had inhibitory effect on HepG2 cell growth and induced apoptosis in a concentrationdependent manner (Wei et al., 2016).

The Somatic Mutations and Recombinations Test (SMART) in
Drosophila melanogaster is used successfully to detect carcinogenic compounds.This assay is preferable to researchers because it is rapid, inexpensive, and sensitive to different classes of agents.It uses tumor suppressor gene warts which is a homolog to the mammalian tumor suppressor gene LATS (Nepomuceno, 2015;Vasconcelos et al., 2017).Induction of tumors in Drosophila instead of marker clones may directly adverse the risk of these factors for inducing cancer in humans (Sidorov et al., 2001).In flies, heterozygous for the wts gene, the genetic events that can lead to the tumor include gene mutations in the wts gene, multilocus-deletions (partial), chromosomal loss, and somatic recombination collectively referred to as loss of heterozyygosity (Eeken et al., 2002).Fekrazad et al., (2017) mentioned that neutral red uptake assay is used for in vitro assessment of cytotoxicity of infectious agents, food additives, and pharmaceuticals.This assay has some advantages; it is rapid, economical, semiautomated, and can be used with a variety of cell types to provide quantitated data that can be used to rank test agents according to their potencies (Babich and Borenfreund, 1990).
The objective of this study was to evaluate the genotoxic effect of food additives such as, sodium sulphite, boric acid, and benzoic acid on somatic cells of Drosophila and human cell lines.

MATERIALS AND METHODS Somatic Mutation and Recombination Test (SMART) in D. melanogaster . Drosophila Crosses and Treatments:
For Detection of epithelial tumors in D. melanogaster, the wts/TM 3 females, the genetic structure of this strain is; st p in ri wtsMT4-1/ TM3 Sb,.Details about the various markers and the balancer chromosome can be found in Flybase (1999) and Lindsley and Zimm (1992).These wts/TM3 females crossed to wild type males.After 2 days, the parental flies were removed and 56-68 hours old larvae were transferred to a standard Drosophila medium containing 20 µg/ml of an appropriate Mitomycin C (MMC) solution for 24 hours, then they were transferred to standard Drosophila medium.For food additives (Sodium sulphite, Boric acid, and Benzoic acid) treatments; concentration of 100 mM powder was dissolved in standard Drosophila medium at 50⁰C.All Drosophila stocks and crosses were maintained at 25⁰C.

Scoring of Warts:
To score tumor of warts, later the males and females of the (wts + /+) genotype, which was wild type to be analyzed for tumor (wart) presence.Only tumors that were large enough to be unequivocally classified are recorded.The flies were observed using a Leica stereomicroscope used at a standard magnification of 25 X.Tumors were only included when large enough to be classified unambiguously.

Statistical Analysis:
The tumor frequency was calculated as the number of tumors/number of wts + /+ flies (Eeken et al., 2002) and Tumor induction = Number of tumors/ Number of tumor flies.The carcinogenic potential from compounds is identified by the Mann, Whitney, and Wilcoxon nonparametric U test, using α=0.05 level of significance, to evaluate the significant of difference between negative control and other treatments.

Neutral Red Cytotoxicity Assay:
Neutral red cytotoxicity assay based on the initial protocol described by Borenfreund and Puerner (1984) and modified by Fotakis and Timbrell (2006) was carried out.The cells from mother flasks were seeded in a 24-well microtitre plate (Corning) (1X106) cell/well.The plates were incubated at 37ºC in 5% CO2 for 24 hrs to achieve monolayer confluence.Culture medium containing different concentrations of each chemical compounds (75, 150, 300 and 600 µg/ml) were added in triplicate.Medium without chemical compounds served as untreated control.The dye-medium was removed and the plates were washed with formolcalcium (10 ml 40% formaldehyde, 10 ml 10% anhydrous calcium chloride, and 80 ml water).Five hundred µl of acetic acid-ethanol (one ml glacial acetic acid in 100 ml 50% ethanol) was added and the plates were kept for 15 min at room temperature to extract the dye.Plates were then shacked for a few seconds, so that complete dissolution was achieved.The absorbance of the extracted dye was measured by spectrophotometric reading (Spectra max 190-Molecular devices) using with 540 nm filter.The mean of three measurements for each concentration was determined (n=6).The viability % was calculated for having the concentration of the test chemical reflecting the half maximum concentration of the cell proliferation (IC50).

Calculations and Statistics:
Cytotoxicity assay was measured as optical density at 540 nm.Doseresponse curves were plotted, and 50% inhibitory concentrations of plants extracts (IC50) were calculated through Graph Pad Prism software program.Data are presented as mean ± SD.For statistical analysis of data, multiple comparisons were performed using oneway analysis of variance (ANOVA) followed by the LSD test for post hoc analysis.Statistical significance was accepted at a level of P < 0.05.Data were analyzed using SPSS.

Detection of Carcinogenic Agents Using
Somatic Mutation and Recombination Test (SMART) in D. melanogaster.
The SMART assay in Drosophila melanogaster has been widely used with many different objectives.In the present study, we evaluated the carcinogenic potential of some food additives such as, sodium sulphite, boric acid, and benzoic acid.In the treatment procedure of that test, the larvae are treated for 24 h in vials containing medium with the test compound at a certain concentration.Then they were transferred to standard Drosophila medium.The larvae feed on this medium until completion of their development when they leave it and pupate.1) showed that investigation of five groups of crosses as following: a negative control group, MMC 20µg/ml, sodium sulfite 100mM, boric acid 100mM, and benzoic acid 100mM.The frequency of tumors in wts/+ negative control flies was 0.07 i.e., 7 flies with one warts in 100 flies scores nearly.Tumor induction in a negative control was low (1.1).On the other hand, MMC treatment recorded the highest frequency 1.33 associated with the highest tumor induction (2.18).These tumors were detected in every part of the examined flies.

Data in Table (
Concerning the effect of sodium sulphite on the frequency of warts tumor was statistically significant, showed increase (0.69), where the tumor induction was (1.55) as shown in Table (1).Boric acid treatment highly increased in tumor frequency (0.8) and was statistically highly significant, which showed (1.67) tumor induction.In case of benzoic treatment, the frequency of tumor and tumor induction were 0.73 and 1.2, respectively.Diagram represents the tumor induction of spontaneous and induced warts Epithelial tumors in +/wts flies after treatments with Mitomycin C (MMC), Sodium sulphite, Boric acid, and Benzoic acid as observed in (Fig. 1).

In vitro Assay for Cytotoxic Activity Human Cell Lines (Neutral Red Assay):
The effects of four different concentrations (600, 300, 150 and 75 µg/ml) of the three food additives; sodium sulphite, boric, and benzoic on the proliferation of colon cancer cells in comparison to a positive control (3µg/ml) were determined using the neutral red cytotoxic assay.
In general, the cell viability was decreased gradually as the concentration of the three tested food additives increased as illustrated in Table (2).The cytotoxicity and cell viability of sodium sulphite, boric, and benzoic with the concentrations (75, 150, 300 and 600 µg/ml) and a positive control 3µg/ml were evaluated in vitro against human liver cell lines (hepatoma cells HepG2).The viability of positive control was 62.85%, and the viability of HepG2 was reduced as the concentration increased of the three tested food additives, but the reduction was non-significant in sodium sulphite and benzoic.The significant reduction in the viability was observed in boric at 300 and 600 µg/ml.The Dose inducing 50% cell growth inhibition (IC50) against hepatoma cell line cells (HepG2) is presented in Table (2) and Dose-response curves for cell viability in Figure (2).
In human colon cell lines (HCT 116), a positive control attained 44.8% of viability.Boric was the most effective on the viability reduction among the three tested food additives where the all concentrations had significant effect while benzoic was the lowest without significant change.The IC50 values were 1219, 870.6, and 1986  The viability reduced significantly by sodium sulphite at 300 and 600µg/ml while benzoic, the viability slightly decreased without significant change.Using boric, the viability decreased significantly as the concentration increased except at the lowest concentration (75µg/ml).As mentioned above, boric acid gave the highest toxic effect among tested food additives.IC50 were 3637 in sodium sulphite, 1617 in boric, and 3289 in benzoic as observed in Table (2) and Dose-response curves for cell viability in Figure ( 4).
Data in Table (2) showed that cell survival rate of normal lung cell line (Wi38) was affected by sodium sulphite, boric, and benzoic with different concentrations and positive control (3µg/ml).The viability affected by positive control was 61.8%.Normal cell line (Wi38) viability did not induce significant change with 75 and 150µg/ml of the three tested food additives.Like A459, the viability in Wi38 was affected negatively with the concentration of the three tested food additives but the reduction in viability was higher than those in A-459.Dose-response curves for cell viability of human normal ling cell line treated with sodium sulphite, boric acid, and benzoic acid using a typical neutral red cytotoxicity, as shown in Figure (5).* Significant difference from the negative control at P<0.05 using one-way analysis of variance (ANOVA).

This
study evaluated the mutagenicity and carcinogenicity of three food additives commonly used in food industry by testing SMART of D. melanogaster and cytotoxicity of these food additives on some human cell lines cells.Loss of heterozygosity (LOH) in somatic cells is a major step involved in the formation of tumors can be determined by SMART assay in Drosophila.Not only does the mechanism involve mutation, but also chromosome loss and somatic recombination.The genetic events leading to LOH can be induced in cells of the imaginal disks of Drosophila larva and the resulting changes scored as clones of mutant cells in the adult.It is known to us the cell cycle in imaginal disk cells is very similar to that in somatic mammalian cells, and many of the regulation genes are participated between human and Drosophila.So, we cannot ignore the negative effect of these compounds with that concentration on the human health.From the results, it was obvious that the cytotoxicity of sodium sulphite, boric, and benzoic at 100mM concentration on D. melanogaster.In this trend, Sarikaya and Solak (2003) evaluated the genotoxicity of benzoic acid with 50, 75 and 100mM in the wing SMART of D. melanogaster.They found a positive correlation between total mutation and the number of mutated wings.Also, Demir et. al,. (2008) evaluated the genotoxicity of four benzyl derivatives; benzaldehyde, benzyl acetate, benzyl alcohol, and benzoic acid at different concentrations (0.1, 0.5, 1, 10, 25 and 50mM) used as flavor ingredients in the wing SMART of D. melanogaster they ordered these compounds according to their genotoxic effect as benzaldehyde, benzyl acetate, benzyl alcohol, and benzoic.Njagi and Gopalan (1982) reported that sodium sulphite and sodium benzoate inhibit DNA synthesis and induce the anaphase bridges, chromosome condensation in Vicia faba root meristems.
Regarding human cell lines cells, for benzoic, the concentration 75-600 µg/ml have no dangerous effect on all human cell lines checked except at 300 and 600 µg/ml on Wi38.Also, sod.sulphite is not toxic on Hep-G2 till 600µg/ml concentration.Moreover, the concentrations 75 and 150µg/ml of sod.sulphite are not toxic on the four examined cells except HCT 116, the 150µg/ml is toxic.The toxicity of sod.sulphite starts from higher than 150 µg/ml.The toxicity degree among the four checked cell lines can be ranked in decreasing order as colon cancer (HCT166), Wi38, A459, and Hep-G2.The genotoxicity of sod.benzoate on human lymphocytes was studied by (Patel and Ramani, 2017) using chromosomal aberration and sister chromatid exchange assay.They concluded that sod.benzoate can induce chromosomal aberration and sister chromatid exchange at 0.5, 1 and 1.5 mg/ml concentrations.Also, it can decrease the cell cycle proliferation index.Benzoic at 500 µg/ml decreased the mitotic index and increased the frequency of chromosomal aberration in human lymphocytes (Murli, 2003;Al-Tai, 2014).The impact of sod.metabisulphite and boric on somatic cells of Vicia faba L. was studied by (Pandey and Upadhyay, 2007).They found a significant decrease in mitotic index and an increase in the abnormality percentage with increasing concentrations.Sod.metabisulphite stimulated a significant decrease in mitotic index in human lymphocytes (Meng and Zang, 1992;Rencuzogullari et al., 2001).
Boric and its derivatives like borax are used as preservatives in foods and medicines but these compounds became of harmful effect on human health at high consumption (See et al., 2010).The possible lethal doses in babies are in range of 3-6g, whereas 15-20g in adults (Litovitz et al., 1988).In our present studies, the concentration 75 µg/ml of boric is not dangerous on Hep-G2, A459 and Wi38.The HCT 116 scored the highest toxicity where IC50 was 870 as affected by boric.This indicates that boric hasn't only have an effect on cancer human cells but also on normal cells.In addition to that, boric can cause tumor in normal lung cell line (Wi38) rather than antitumor in lung cancer (A459) cells.In this field, Hep-G2 cells were affected by borax lead to inhibit proliferation and promotion of apoptosis, using MTT and annexin V/P1 staining, respectively, (Weil et al., 2016).Another study, Centurk et al., (2016) evaluated the effect of boric on an acute leukemia cell line (HL-60) and healthy human lymphocytes using MMT, Neutral Red, transmission microscope, and flow cytometry methods.They noticed that boric at 500µM concentration caused double nucleus and micronucleus formation in both HL-60 cells and lymphocytes in addition to appearance of an expansion in mitochondrial dimension and deformation in cristas.Kumar and Srivastava (2011) observed that boric at (0.25, 0.5, 0.75 and 1%) used for 3 hours produced mitoinhibitory effect and increase in chromosomal aberrations in root tips of Trigonella foenum-graecum.The most observed aberrations were stickiness at metaphase and anaphase, scattering at metaphase and bridges at anaphase.
The mechanism of boric and borax genotoxicity was assessed on zebrafish Denio rerio for 24, 48, 72 and 96-hours acute exposure level; 1, 4, 16, 64mg/l in semi-static bioassay experiment.Peripheral erythrocytes drawn from caudal vein were used and Comet assay was applied to assess genotoxicity.The genotoxicity for boric was found as concentration dependent and borax as concentration and time dependent manner.The highest damage in DNA was at 96h for borax and 24h for boric concentrations in peripheral blood of D. rerio.For boric, the maximum increase in % tail DNA at 24h and reduction at 48, 72 and 96h were seen.However, the reduction in 96h values, it was still higher than negative control level at all doses.This limited decrease indicated that cytoprotective and tolerant mechanisms or repair of damaged DNA in the cell (Gülsoy et al., 2015).On the other hand, Murmu et al., (2002) reported that the two new boron compounds, guanidine biboric acid and dihydroxy boron hydrochloride monohydrate adduct have antitumor effect.Also, Gallordo-Williams et al., (2003) observed that mice receiving 1.7 or 90mg/kg daily dosage of boric produced a decrease in tumor size by 38% and 25%, respectively.
It could be concluded that boric acid has the highest toxic effect among the three studied compounds on Drosophila SMART, Hep-G2, HCT116, A459, and Wi38 human cell lines.These compounds induce higher toxic effect on normal lung (Wi38) cells than in lung cancer (A459) cells; this indicates that they have cytotoxic activity.However, sodium sulphite, benzoic, and boric are genotoxic in the other short-term genotoxicity tests.For this reason, it is necessary to be careful when using these substances in food and cosmetics as additives.
Cytotoxic effects of food additives and pharmaceuticals on cells in culture as determined with the

*
and ** significant, highly significant difference from the negative control at P<0.05 using Mann, Whitney and Wilcoxon nonparametric U test.Frequency (No. of Tumor/fly) = Number of tumors/Total number of tested flies.Tumor induction = Number of tumors/ Number of tumor flies.

Fig
Fig. (1): Diagram represents the tumor induction of spontaneous and induced warts Epithelial tumors in +/wts flies after treatments with Mitomycin C (MMC), Sodium sulphite, Boric acid, and Benzoic acid.

Fig. ( 2
Fig. (2): Dose-response curves for cell viability of human liver cancer (Hep G2) cell lines treated with sodium sulphite, boric acid, and benzoic acid using a typical neutral red cytotoxicity.

Table ( 1
): Frequencies of induced tumor in trans-heterozygous (wts/+) after larvae feeding treatments with three concentrations of Sodium sulphite, Boric acid, and Benzoic acid compared with the MMC as a positive control and negative control.

Table ( 2
): The cell viability percentage and IC50 of human cell lines tested by Sodium sulphite, Boric acid, and Benzoic acid compared with Positive control using neutral red cytotoxicity assay.