The genotoxic effects of some food colorants on Zea mays L. var. saccharata Sturt

ABSTRACT

Carmoisine and sunset yellow are organic azo dyes and widely used in food products, drugs and cosmetics. The present study was conducted to evaluate the toxic effects of these colorants on Zea mays with the root and shoot inhibition test, chlorophyll content, α-amylase activity and RAPD PCR technique. In the root inhibition test, EC₅₀ values for carmoisine and sunset yellow were determined as 20 g l^–1 and 22.5 g l^–1, respectively. Chlorophyll a and chlorophyll b levels were decreased when compared to the control group for all of the carmoisine and sunset yellow concentrations. A slight increase was observed in enzyme activity in groups treated with 10 g l^–1 and 20 g l^–1 of carmoisine, while a decrease in enzyme activity was observed in the group treated with 40 g l^–1 of carmoisine. For sunset yellow, a decrease in enzyme activity was observed in all concentrations. In the RAPD test, 71% polymorphism was obtained for carmoisine treated groups. Polymorphism ratio was determined as 53% for the group that was treated with sunset yellow. We concluded that both carmoisine and sunset yellow change the biochemical parameters and have genotoxic effects on Z. mays.

KEYWORDS:

• Food colorants
• genotoxicity
• carmoisine
• RAPD PCR
• sunset yellow
• Zea mays

Introduction

Food safety is one of the most important issues of today. Food additives have great importance for long-term preservation of food quality. Therefore, the use of food additives has become inevitable. However, it is reported that food additives show toxicity in organisms (Pandey and Upadhyay 2007; Türkoğlu 2009). Food colorants are food additives used to color foods and drinks. They are used to strengthen the original color, to restore color lost during the manufacturing process or to color a product. Some of these colorants are prohibited, while some can be used in limited quantities as they have been shown to have toxic effects. Carmoisine and sunset yellow are food colorants which are allowed for use in Turkey. Acceptable daily intake for carmoisine and sunset yellow was determined as 4 mg kg^–1 bw and 2.5 mg kg^–1 bw, respectively (JECFA 1981).

Carmoisine and sunset yellow are used in many food products that are consumed daily such as ready-made soup, bread crumbs, confectionery, cake, ice cream, fruit yogurt, and ready-made jams. Carmoisine contains naphthionic acid and 1-naphtalene-sulfonic acid-3-amin-4-hydroxyl in its structure. Sunset yellow contains sulfanilic acid and 2-naphtalene-sulfonic acid-5 amin-6 hydroxyl. Carmoisine and sunset yellow been shown to cause a decrease in body weight in rats compared to a control group (Amin et al. 2010). Decrease in body weight is regarded as a reliable and sensitive symptom of toxicity (Ezeuko Vitalis et al. 2007). Amin et al. (2010) found that carmoisine increases the oxidative stress and leads to significant increase in serum proteins (ALT, AST, ALP, T protein, albumin, and globulin). Rats exposed to carmoisine for 30 days have been shown to have increased creatinine and urea because of the renal function damage. Carmoisine is a dye which belongs to the azo dye group and it is converted into aromatic amines by intestinal flora; these aromatic amines can react with nitrites or nitrates in foods or in the stomach to form reactive oxygen species (ROS). ROS, e.g. superoxide anion, hydroxyl radical and hydrogen peroxide, can be produced in nitrosamine metabolism and can increase oxidative stress (Bansal 2005).

Dierickx (1982) found that sulfanilic acid and its derivatives acetylated with nitrogen react with glutatyon S-transferase isoenzymes and inhibit these enzymes in rat liver. Osman et al. (2004) found that sunset yellow inhibits human erythrocytes and cholinesterase plasma. Sunset yellow may be responsible for causing an allergic reaction in people with an aspirin intolerance (Ibero et al. 1982) resulting in symptoms including diarrhea, urticaria, gastric upset, angioedema and vomiting (Ghoneim et al. 2011). In another study conducted by Gomes et al. (2013), it was reported that sunset yellow, Bordeaux red and tartrazine dyes cause a statistically significant number of mitotic aberrations and decreasing mitotic index in Allium cepa root cells compared with a control group. Macioszek and Kononowicz (2004) tested the effects of two food colorants, brilliant black and quinoline yellow, on Vicia faba with comet assay and micronuclei test and concluded that both food colorants decreased the mitotic index and demonstrated genotoxic effects compared with the control group.

According to our knowledge, toxic effects of these two food colorants on Z. mays have not yet been examined. The present investigation was conducted to understand the root growth inhibiting effect of these two different food colorants on Z. mays seeds. The aim of this investigation was to determine the effects of different concentrations of the food colorants on DNA polymorphism, α-amylase activity and the amount of the photosynthetic pigments of Z. mays.

Material and methods

Determination of EC₅₀ and treatment of Z. mays seeds with food colorants

Carmoisine [disodium 4-hydroxy-2-[(E)-(4-sulfonato-1-naphthyl)diazenyl]naphthalene-1-sulfonate] and sunset yellow [disodium 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfonate] (Figure 1) are synthetic anionic azo dyes in powder form. Both are used to color foods or drinks, e.g. candy, gels, and pastes. Food colorants are also used in a variety of non-food applications including home craft projects, pharmaceuticals, medical devices and cosmetics (CFR Title 21 Part 70: Color Additive Regulations, FDA, retrieved 15 February 2012).

Figure 1. Molecular structure of (A) carmoisine and (B) sunset yellow.

Surface sterilized Z. mays seeds were allowed to produce roots in distilled water for 24 h. Twenty homogeneous seeds were transferred to the distilled water and six different concentrations of carmoisine and sunset yellow (15, 17.5, 20, 22.5, 25, and 30 g l^–1) for 72 h to determine EC₅₀ values. The root lengths in the non-treated and treated groups were measured and the relative reduction was calculated (T/C %) . After determination of EC₅₀, Z. mays seeds were placed in Petri dishes and treated with EC₅₀/2, EC₅₀ and 2×EC₅₀ concentrations of each food colorants. The control groups were treated only with distilled water. 0.5 cm and taller root tips were determined as germinated. All experiments were done in three replicate. Seed germinations were carried out in Petri dishes in laboratory conditions for 14 days.

Pigment analysis in leaves

Z. mays seeds were germinated in Petri dishes at 23 ± 2°C during seven days in laboratory conditions. Seedlings, which were measured each day, were collected on the seventh day for pigment analysis. Washed leaves were laid on filter paper to dry. For each concentration of solution 100 mg of leaves was weighed and ground with pestle and mortar by adding 80% acetone. This extraction was filtrated to Erlenmeyer flasks with Whatman filter paper. Filtered extraction was transferred to Falcon tubes and centrifuged at 3000 rpm for 10 min. Absorbance values of these chlorophyll extracts were calculated at 645 and 663 nm wavelengths with a Shimadzu Uvmini-1240 spectrophotometer (Duisburg, Germany). Chlorophyll concentrations were calculated from the spectrophotometric data using the formulas of Arnon (1949).

Determination of α-amylase activity

Dextrogenic method was used to measure α-amylase activity. This method is based on the reaction between starch and iodine. Purple color is observed when starch is treated with iodine, and this color gets lighter as starch breaks down. During activity measurements, 1% soluble starch was used as the substrate, and extracts obtained from treated and non-treated seeds were used as the enzyme sources. Activity measurements were performed by observing the lightening of the color of the enzyme–substrate mixture using a spectrophotometer. The α-amylase activity was calculated by the calculation of the amount of substrate loss. For this purpose, blank, control and sample tubes were prepared (Ekinci and Aktaç 1997). Preparation of the tubes is shown in Table 1. Sterilized Z. mays seeds were germinated for 48 h in laboratory conditions and radicles and coleoptiles removed. Then 0.4 gr Z. mays seed and 2 ml malate buffer were homogenized in a mortar. The obtained homogenate was incubated at room temperature for 15 min and centrifuged at 500 rpm for 8 min. Supernatant was used as the enzyme source.

Table 1. Preparation of tubes for α-amylase activity.

	Blank	Control	Sample
Malate buffer (ml)	9	7	7
Starch (0.1%) (ml)		2	2
Amylase (ml)	0.1	0.1	0.1
HCI (1N) (ml)		0.9	0.9*

*Added after incubation.

All of the tubes were allowed to incubate for 3 min at 30°C. Following the incubation 0.9 ml of 1 N HCl was added to the sample tubes and 1 ml of iodine was added to all of the tubes. Absorbance measurements were made against the blind tube of all samples at 620 nm wavelength on a Shimadzu UV mini-1240 spectrophotometer. The OD value of the control tube was subtracted from the OD value of the sample tubes, to find the difference OD that is the value of the starch disrupted by the enzyme. The amount of starch broken down was first converted to mg and then to unit/ml using the starch standard curve obtained using fresh iodine solution at a wavelength of 620 nm. One unit of α-amylase activity was defined as the amount of enzyme that degraded 0.1 mg of starch at 30°C per minute (Yarkın 2007).

DNA extraction and RAPD PCR

Germinated roots were ground in liquid nitrogen and total genomic DNA was extracted according to Dneasy Plant Mini Kit (Qiagen, Hilden, Germany). DNA concentrations was determined by using a BMG Labtech Spectrostar Nano spectrometer (Allmendgrün, Ortenberg, Germany) and the quality was verified by electrophoresis on 1% agarose gel. Ten primers (OPA1, OPA2, OPA3, OPA4, OPA5, OPA6, OPA7, OPA8, OPA9, and OPA10, Operon Technologies (Alameda, California, USA)) were used for RAPD analysis, and PCR conditions were optimized according to Williams et al. (1990). The amplifications were carried out in triplicate. The molecular weights of the amplification products were calculated using 1 kb DNA ladder standards. RAPD banding patterns were visualized using a UV transilluminator and the size of each amplification product was automatically estimated using the Vision Works LS Version 6.8 (Cambridge, UK). Amplicons (bands) were scored as 1 (presence) or 0 (absence). Genetic similarity coefficients (phenetic numerical analysis) among the untreated control and treated germinated root tips of Z. mays seeds were estimated from Nei’s unbiased measure (Nei 1978) in POPGENE version 1.31 (https://sites.ualberta.ca/~fyeh/popgene.html).

Statistical analysis

The statistical analysis of data was performed using SPSS for Windows version 22.0 statistical software (SPSS Inc., Chicago, IL, USA). Statistically significant differences between the groups were compared using one-way analysis of variance (ANOVA) and Duncan’s test. The data were displayed as means ± standard deviation (SD), and p-values less than 0.05 were considered statistically significant.

Results and discussion

Determination of EC₅₀ values

EC₅₀ values were determined 20 g l^–1 for carmoisine (Figure 2), and 22.5 g l^–1 for sunset yellow (Figure 3). All experimental procedures were carried out using 10 g l^–1 (EC₅₀/2), 20 g l^–1 (EC₅₀) and 40 g l^–1 (2×EC₅₀) concentrations for carmoisine, 11.25 g l^–1 (EC₅₀/2), 22.5 g l^–1 (EC₅₀) and 45 g l^–1 (2×EC₅₀) concentrations for sunset yellow.

Figure 2. Growth curve and EC₅₀-value for carmoisine.

Figure 3. Growth curve and EC₅₀-value for sunset yellow.

Three concentrations (EC₅₀/2, EC₅₀ and 2×EC₅₀) of both carmoisine (Figure 4) and sunset yellow (Figure 5) were applied to Z. mays seeds and seedling lengths were measured at the end of the seventh day. When the length of seedlings was compared with the control group, the lowest seedling length value was shown with the highest concentration of sunset yellow. Sunset yellow had more inhibitory effects on Z. mays seedlings than carmoisine.

Figure 4. Seedling length percentages after seven days carmoisine treatment.

Figure 5. Seedling length percentages after seven days sunset yellow treatment.

Carmoisine and sunset yellow are widely used food colorants. Studies have been carried out to determine possible toxic effects of food colorants on various organisms with various tests and it has been determined that these colorants and other azo dyes show cytotoxic and genotoxic effects. However, studies on plant systems have been limited to a few plant species.

As a result of our study, we found that root lengths were decreased in Z. mays seeds treated with carmoisine and sunset yellow when compared to the control group. It was observed that root lengths decreased with increasing concentration. In previous studies which evaluated the toxic effects of carmoisine, sunset yellow and other azo dyes on plants, it was determined that these colorants cause DNA damage, apoptotic cell death and mitotic abnormalities by reducing root length and cell division (Gomes et al. 2013; Dwivedi and Kumar 2015; Vazhangat and Thoppil 2016). The maximum inhibition of root lengths was observed at the highest concentrations. These results are in accordance with Vazhangat and Thoppil (2016) who found a significant decrease in A. cepa root length.

The effects of different carmoisine and sunset yellow concentrations on the chlorophyll contents of Z. mays leaves

The effect of different carmoisine concentrations on the chlorophyll content of Z. mays leaves can be seen in Tables 2 and 3. It can be observed that highest concentration of carmoisine caused the greatest effect on chlorophyll content of Z. mays.

Table 2. The effect of different concentrations of carmoisine on the chlorophyll content of Z. mays leaves.

Concentration (g l^–1)	Chlorophyll a (mg g^–1) ± SD	Chlorophyll b (mg g^–1) ± SD	Total chlorophyll (mg g^–1) ± SD
Control	7.10 ± 1.26	0.95 ± 0.22	8.04 ± 1.15
10	5.30 ± 1.25	0.47 ± 0.25	5.77 ± 1.01
20	4.39 ± 0.48	0.38 ± 0.15	4.76 ± 0.56
40	3.04 ± 0.23	0.25 ± 0.10	3.29 ± 0.31

Table 3. The effect of different concentrations of sunset yellow on the chlorophyll content of Z. mays leaves.

Concentration (g l^–1)	Chlorophyll a (mg g^–1) ± SD	Chlorophyll b (mg g^–1) ± SD	Total chlorophyll (mg g^–1) ± SD
Control	7.10 ± 1.26	0.95 ± 0.22	8.04 ± 1.15
11.25	5.48 ± 0.0	0.57 ± 0.0	6.05 ± 0.0
22.5	3.50 ± 0.14	0.39 ± 0.08	3.90 ± 0.10
45	3.24 ± 0.12	0.30 ± 0.06	3.55 ± 0.15

Chlorophyll a, chlorophyll b and total chlorophyll levels were found to decrease in the carmoisine and sunset yellow treated groups when compared to the control group. The azo dyes, including carmoisine and sunset yellow, are industrially the most commonly used chemical dyes (Fitzgerald and Bishop 1995), are not biodegradable and is considered permanent (Mohan et al. 2005).

As described by (Karanlık 2001), there is an increase in the levels of free radicals in plants when they are exposed to stress, and these free radicals damage cells and restrict the activity of photosynthesis. The free oxygen radicals cause reductions in chlorophyll levels, as they also destroy cell membrane components (Fridovich 1986; Davies 1987) such as protein membrane lipids and nucleic acids and chlorophyll. Previous studies have also reported that chlorophyll levels are reduced in plants when exposed to various stresses (Gadallah 1996; Homayoun et al. 2011; Soltangheisi et al. 2013; Abd El-Ghany et al. 2015).

In an investigation of the toxic effects of an azo dye RP₂B on Anabeana, it was determined that the concentration of chlorophyll a decreased with increasing concentration of RP₂B (Hu and Wu 2001). Application of an anionic dye, Metomega Chrome Orange GL (MCO), to Nostoc muscorum at a concentration of 20 mg l^–1 resulted in a decrease in the level of chlorophyll a. Some anionic pigments can inhibit the growth of cyanobacteria because they can interact with physiologically important metallic ions such as Mg⁺², Ca⁺², Zn⁺² and Ni⁺² (Shukla and Gupta 1994). Carmoisine and sunset yellow are also anionic dyes and they are thought to interact with Mg in the chlorophyll structure and to cause a decrease in the amount of chlorophyll.

The effects of different carmoisine and sunset yellow concentrations on α-amylase activity of Z. mays seeds

All the concentrations of sunset yellow caused a decrease in α-amylase activity (Table 4), whereas only one concentration of carmoisine caused a reduction in α-amylase activity (Table 5) when compared with the control. But there was no significant difference between the concentrations.

Table 4. The effect of carmoisine concentrations on a-amylase activity of Z. mays seeds.

Concentration (g l^–1)	Amylase activity (U ml^–1)	Starch (mg ml^–1) ± SD
Control	64.34	0.012 ± 0.007
10	65.00	0.011 ± 0.006
20	65.75	0.011 ± 0.002
40	43.00	0.042 ± 0.005

Table 5. The effect of carmoisine concentrations on a-amylase activity of Z. mays seeds.

Concentration (g l^–1)	Amylase activity (U ml^–1)	Starch (mg ml^–1) ± SD
Control	72.5	0.001 ± 0.001
11.25	70.80	0.003 ± 0.001
22.5	64.34	0.012 ± 0.004
45	56.91	0.022 ± 0.003

α-Amylase is an enzyme induced by gibberellic acid, which plays a role in the conversion of starch to sugar by catalyzing the hydrolysis of α-1,4 glycosidic bonds during seed germination, and is commonly used in the food industry (Gupta et al. 2003). In the literature, there has been no study investigating the effect of azo dyes, including carmoisine and sunset yellow, on α-amylase activity on Z. mays. Plants exposed to a variety of stresses have generally been found to have decreased α-amylase activity and root lengths (Khan et al. 1989; Ashraf et al. 2002). α-Amylases have a key role in the mobilization of starch during germination and seedling establishment (Smith et al. 2005). During seed germination, α-amylase in the aleurone layer hydrolyzes the endosperm starch into metabolizable sugars, which provide the energy for the growth of roots (Beck and Ziegler 1989). The starch particles are initially digested to soluble starch by α-amylase. For the complete degradation of soluble starches, α-amylase, debranching enzyme (R-enzyme), β-amylase, and α-glucosidase enzymes work together. The glucose produced in the endosperm is taken up and converted to sucrose in the scutellum, then the sucrose is used in the growing tissues, such as the young root and shoot tissues (Itsui et al. 2010)

The decrease in root length of the seeds in the treated groups was found to be in parallel with the decrease in the amount of α-amylase enzyme. In conclusion, our data showed that reduced α-amylase activity plays a crucial role in physiological and biochemical processes. It has been found that higher food colorant concentrations decreased the α-amylase activity, and so seed germination and root growth.

Assessment of genotoxicity by RAPD PCR

RAPD analysis was performed with genomic DNA which was extracted from the Z. mays roots from each replicate treated with carmoisine, sunset yellow and the control. The list of polymorphic and monomorphic RAPD primers for carmoisine and sunset yellow, and the polymorphism percentages of primers for carmoisine and sunset yellow, were determined.

Ten primers of 60–70% GC content were utilized for screening Z. mays genome for changes and all the primers showed polymorphism for each concentration of carmoisine-treated Z. mays gDNAs. Only three primers showed monomorphism for 45 g l^–1 concentration of sunset yellow. The total number of bands was 25 (control group) and 107 (treated groups) ranging from 367 to 1977 base pairs for carmoisine concentrations. The total number of bands was 26 (control group) and 88 (treated groups) ranged from 314 to 1884 base pairs for sunset yellow concentrations.

In this study, RAPD analysis was performed on genomic DNA extracted from roots of three carmoisine treated and three sunset yellow treated and control plants. RAPD profiles generated by these primers revealed differences between control and treated plants, with visible changes in the number and size of amplified DNA fragments(Figure 6). New PCR products may result from mutations in some oligonucleotide priming sites, changes due to large deletions and homolog recombination. Loss of normal PCR products may be caused by genotoxin-induced DNA damage (e.g. DNA-protein cross-links, modified bases, single and double strand breaks, and oxidized bases), point mutations and complex chromosomal rearrangements (Atienzar et al. 1999, 2000; Cenkci et al. 2010).

Figure 6. PCR products of control and treated groups: (A) carmoisine; (B, C and D) sunset yellow).

Vazhangat and Thoppil 2016 reported that tartrazine and orange red (carmoisine + sunset yellow) cause DNA damage, micronucleus formation, chromosomal abnormalities and inhibition root elongation and cell division in A. cepa. Similar results have been found in studies in which the sunset yellow was applied to A. cepa (Gomes et al. 2013) and Trigonella foenum-graceum (Kumar and Srivastava 2011). In studies investigating the effects of sunset yellow in root cells of Brassica campestris L., mitotic abnormalities such as micronucleus formation, chromosomal adhesion, and chromosomal bridge formation were detected (Dwivedi and Kumar 2015). Additionally, Mpountoukas et al. (2010) reported that some food colorants from azo dyes are capable of binding dsDNA and resulting its degradation.

It is reported that azo dye food colorants can generate reactive oxygen species (ROS) (Bansal 2005; Demirkol and Zhang 2012) and overproduction of these ROS induces cell oxidative injury, such as DNA damage, oxidation of proteins and lipid peroxidation (Matés 2000). It may be suggested that the genotoxic effects of carmoisine and sunset yellow probably depends on the ROS-induced oxidative DNA damage at higher concentrations.

Conclusion

Azo dyes may cause adverse effects on humans and the environment, because both dyes and their metabolites can show toxic effects, mainly resulting in DNA damage. Azo dyes have a wide range of usage by different industries, and these dyes are discharged into the environment and so into ground waters. The azo dyes are an important class of environmental mutagens, and hence it is needed to increase the use of natural dyes and development of nongenotoxic dyes in order to avoid human and environmental exposure to these chemicals and decrease the adverse effects they can have on humans and other organisms.

Acknowledgments

This study is part of the MSc thesis titled “Bazı Gıda Renklendiricilerinin Zea Mays L. var. Saccharata Sturt. Üzerindeki Genotoksik Etkileri” and funded by Kocaeli University Research Council.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Kocaeli University [KOU BAP ARGE];