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Action of Ponceau 4R (E-124) food dye on root meristematic cells of Allium cepa L./Acao do corante alimentar Ponceau 4R (E-124) sobre as celulas meristematicas de raizes de Allium cepa L.


Many additives are used in the food industry to enhance the sensory aspects and acceptance of food by the population. As an example, one can cite the synthetic food dyes, additives that do not add nutritional value, but directly influence consumer acceptance by restoring or intensifying the color of the food (SILVA; REED, 2011). However, these dyes are not recommended by experts in the health field as they contribute to the impoverishment of the diet and often cause adverse effects to the body (CHEESEMAN, 2012).

Azo dyes, a class of synthetic organic dyes that provide more vivid colors to foods, have in their composition a naphthalene ring bonded to a benzene ring by an azo bond (N = N), and the Ponceau 4R, also known in the food industry as New Coccine, Coccine Red and Food Red, is a representative of this class (PAN et al., 2011). This dye gives a red color, is marketed as a powder, has high stability in the presence of light, heat and acids in general, and has good water solubility (PARK et al., 2009). It is widely used in the coloring of candy, lozenges, sweets, gummy candies, gelatin candies, jellies, chewing gum, soy-based beverages, carbonated soft drinks and in almost all diet products (FAVERO et al., 2011).

The control of the use of food dyes is based on the Acceptable Daily Intake (ADI), which, in turn, is based on the research findings and the recommendations of the Codex Committee on Food Additives and Contaminants (CCFAC) (GANESAN et al., 2011). In Brazil, permission to use and the establishment of maximum tolerable levels of food additives is responsibility of the National Agency for Sanitary Surveillance (ANVISA) in partnership with the Ministry of Health (MOH), which performs this activity through the Standing Committee on Food Additives (CPAA) (BRASIL, 2005; FENG et al., 2012). According to Freitas (2012), the ADI established for the Ponceau 4R dye is 0.10 mg [kg.sup.-1].

Despite the required limits, the use of synthetic dyes in foods still raises a number of questions regarding their toxicity (FENG et al., 2012; YADAV et al., 2013). In the case of Ponceau 4R, according to Hamerski et al. (2013), this additive can cause hypersensitivity in the skin, severe anemia and hyperactivity in children, and glomerulonephritis in children and adults. However, these same authors reported the urgent need for studies to properly evaluate the actual toxicity of this dye at the cellular level. According to Prado and Godoy (2007), these evaluations are extremely important in terms of establishing the potential of chemicals to cause cytotoxicity, genotoxicity and mutagenicity, conditions that can greatly contribute to the development of cancer.

According to Polonio and Peres (2009), toxicity evaluations, in addition to warning about the permitted tolerance limits of dyes, have banned the use of some synthetic dyes worldwide, such as solid yellow (formerly used in jellies); orange GGN (formerly used in ice cream), solid red (formerly used in fillings and cookie coatings), alizarin blue (formerly used in emulsified oils and gelatins) and scarlet GN (formerly used in confectionery fillings), because these additives revealed cytotoxicity, genotoxicity and mutagenicity in various test systems. These same authors also noted that it is of great importance that the Ponceau 4R, as well as other food dyes, is evaluated in cells of various system tests, for example, animal and plant cells and cell cultures.

Bioassays with plants have been considered highly sensitive, rapid and simple for the monitoring of toxic effects of chemicals at the cellular level (USEPA) (IGANCI et al., 2006) and root meristem cells of Allium cepa (onion) have been shown as an efficient plant test organism for this type of evaluation (CARITA; MARINMORALES, 2009) for their kinetic properties of proliferation, large chromosomes few in number (2n = 16), which facilitates their analysis (HERRERO et al., 2012), for enabling verification of alterations in the cell division rate (mitotic index) and cell aberrations (TABREZ et al., 2011), and for demonstrating satisfactory similarity to the results obtained with other bioassays, such as in animals and in cell cultures (GERA'KIN et al., 2011).

Peron et al. (2008), Fachinetto and Tedesco (2009) and Geras'kin et al. (2011) reported that even though the plant metabolism is different from that of animals, the results obtained by this system test are good toxicity analysis parameters at the cellular level, and have long been used to alert the population about the consumption of some foods and some synthetic and natural medicines.

Therefore, due to the wide use of Ponceau 4R dye in the food industry, the need for additional studies evaluating its toxic effects at the cellular level, and considering the A. cepa system as a suitable bioassay for cytotoxicity evaluation of chemicals, this study aimed to evaluate the toxicity of this food dye on the root meristem cells of A. cepa.

Material and methods

This work was developed at the Plant and Animal Cytogenetics Laboratory, Senator Helvidio Nunes de Barros Campus, Federal University of Piaui, Municipality of Picos, Piaui State, from May to September 2013.

Obtaining the Ponceau 4R dye and definition of concentrations

The pure Ponceau 4R dye (E-124) was purchased from a distributor specialized in national and international marketing of food synthetic additives located in Northeastern Brazil. Each vial contained 50 g of the dye product, and as described on the label, it was recommended to dilute 0.25 g of Ponceau in 1l of water. Comparing with other manufacturers, this dilution is that recommended by most of the Brazilian industries manufacturing this dye.

Thus, for this study, the first concentration analyzed was 0.25 g [L.sup.1]. The second and third concentrations, 0.50 and 0.75 g [L.sup.1], were established doubling and tripling the amount of the first Ponceau 4R concentration.

Obtaining root meristematic cells of A. cepa for cytogenetic analysis

Onions were allowed to root in flasks containing distilled water at room temperature ([+ or -] 25[degrees]C) and aerated until obtaining roots of about 2.5 cm in length. For analysis of each concentration, an experimental group with five onion bulbs was established.

Before placing the roots in contact with their respective solutions, some roots were collected and fixed to serve as a control (CO) of the bulb itself. The remaining roots were then placed in their respective concentrations for 24 hours, a procedure denominated 24 hour-exposure time (24h ET).

After, some roots were removed and fixed. This procedure being completed, the remaining roots from each bulb were returned to their respective solutions where they remained for 24h, which is denominated 48 hour-exposure time (48h ET). Next, roots were again collected and fixed. Exposure times of 24 and 48h were chosen in order to evaluate the effect of the three concentrations along more than one cell cycle.

In the flasks for each bulb analyzed, 30 mL of its concentration were added, taking care to make sure that all roots were in proper contact with the test solution. The fixation of the roots was in Carnoy 3:1 (ethanol: acetic acid) at room temperature for 24h. For each root collection, three roots per bulb on average were removed.

Preparation and reading of the slides, and data analysis

The slides, 3 per bulb on average, were prepared following the protocol proposed by Guerra and Souza (2002). Each slide was stained with two drops of 2% acetic orcein and examined under an optical microscope at 40X. For each bulb 1,000 cells were analyzed, totaling 5,000 cells for each control and exposure time.

Cells were observed in interphase, prophase, metaphase, anaphase and telophase. We counted the number of cells in interphase and under division for each control and exposure time and the Mitotic Index (MI) was calculated. We also evaluated the potential of cell concentrations to cause cellular aberrations, such as micronuclei, colchicine metaphases, telophase and anaphase bridges, gene amplifications, cell adhesions, nuclear buds and multipolar anaphases. The statistical analysis was performed by the Chi-square ([chi square]) test, with a probability level < 0.05, by means of the BioEstat 3.0 statistical software (AYRES et al., 2007).

Results and discussion

Given the variety of staining dyes, the list of dyes permitted for foods in each country contrasts substantially. By current legislation, Resolution 388 from August 9, 1999, ANVISA, in Brazil only eleven synthetic dyes in food and beverages is permitted. They are: Erythrosin included in the class of xanthene dyes; Blue indigotine included in the indigotin class of dyes, Patent Blue V, Fast Green and Brilliant Blue in class of triphenylmethane dyes, Bordeaux Red, Ponceau 4R, Red 40, Azorubine, Tartrazine Yellow and Sunset Yellow, included in the class of azo dyes (POLONIO; PERES, 2009).

In agreement with Rutkunas et al. (2010), studies on the adverse health effects at the cellular level caused by synthetic dyes, especially the azo class, are quite insufficient and contradictory, and almost all these evaluations are on Tartrazine, Sunset Yellow and Red Bordeaux dyes, which warrant further studies on the effect of Ponceau 4R at the cellular level.

In this present work (Table 1), the concentrations of 0.25 and 0.50 g [L.sup.1] of Ponceau 4R at 48h ET, caused a statistically significant reduction in MI when compared with the MI obtained from their respective CO and 24h ET. However, the cell division rates obtained for the CO and 24h ET for the two concentrations did not differ significantly to each other (p < 0.05).

At a concentration of 0.75 g [L.sup.1] (Table 01), we observed that the MI obtained in the 24 and 48h ETs were statistically different compared with the MI of their respective controls, with a significant reduction in cell division in these two exposure times. The MI obtained for their ETs were not significant among themselves. From these results, it is worth noting that the inhibition of cell division had already occurred at the lowest concentration, 0.25 g [L.sup.1], the concentration suggested by the manufacturer on the label of the dye used for this work. It is also important to emphasize that the cell division rate decreased dramatically with increasing ET, for the three concentrations investigated.

Regarding cellular aberrations (Table 2), all the three concentrations studied, at both ET evaluated, showed a statistically significant number of cellular aberrations compared with their respective controls. Within each concentration, the number of aberrations at each ET was not significant to each other. The aberrations observed were micronuclei, gene amplifications, anaphase bridges and telophase bridges.

Under the conditions studied, therefore, the Ponceau 4R was cytotoxic to root meristematic cells of A. cepa. As reported in the literature, Shimada et al. (2010) applied the Comet Assay to evaluate the effect of Ponceau 4R, at 1 and 10 mg [kg.sup.-1] in chronic intraperitoneal treatment, on the bone marrow cells of mice and Wistar rats, and found that the higher concentration caused significant damage to DNA molecules of these organisms, evidencing its genotoxicity. Based on a few studies conducted in the 60s and 80s, Xie et al. (2012) reported that the Ponceau 4R dye, at high concentrations and long exposure, is carcinogenic, teratogenic and mutagenic. Nevertheless, despite the findings of these authors, these were the only reports found on the action of this dye at the cellular level.

Freitas (2012) observed that, in the 90s, the Ponceau 4R dye was a topic of discussion in the food industry worldwide, due to the lack of studies proving its safety to the human body. This controversy has led some states of Japan and the United States to restrict the use of this dye in foods. However, in Brazil, the use of this additive has never been restricted, even with the lack of studies on its toxicity. According to Xie et al. (2012), this situation is worrying, as this author states that almost all foods consumed by Brazilians currently consist of azo dyes, with emphasis on Tartrazine, Ponceau 4R and Red 40 dyes.

Corroborating Freitas (2012), Polonio and Peres (2009) pointed out that although the limits established by national regulatory bodies for the use of food dyes, they do not actually control how much of these additives the food industries add to food and how much the manufacturing industries of these dyes suggest to be added to food. This was verified by Prado and Godoy (2007), who assessed the levels of artificial dyes in food through liquid chromatography and found that almost all artificial food dyes used by the food industry in Brazil, including the Ponceau 4R dye, show levels above those allowed by Brazilian law. They also reported that most processed imported foods marketed in Brazil do not comply with Brazilian law as to the permitted food dye limits.

Thus, the Ponceau 4R dye, as well as all food coloring, must be kept under observation and be periodically reassessed on cells of various test organisms, such as mammals, plants and in vitro cell cultures at various concentrations and exposure times. It is expected that cytotoxicity results obtained with this dye in this study arouse the interest of other researchers to evaluate its toxicity. These studies are important because they can greatly assist the regulators and additive manufacturing companies in establishing optimal and safe concentrations for use by the population.


Considering the conditions of this study, the concentrations 0.25 and 0.50 g [L.sup.-1] at 48h ET, and the concentration 0.75 g [L.sup.-1] at 24 and 48h ET, reduced the cell division rate in a statistically significant number of root meristematic cells of A. cepa. All concentrations analyzed caused a significant number of cellular aberrations in the cells of this test system. Thereby, under the conditions studied, the tested concentrations of the Ponceau 4R dye were cytotoxic.

Doi: 10.4025/actascibiolsci.v37i1.23119


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Received on February 24, 2014.

Accepted on August 8, 2014.

License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gleuvania Santana Marques, Josefa Janaina do Anjos Sousa and Ana Paula Peron *

Campus Senador Helvidio Nunes de Barros, Universidade Federal do Piaui, Rua Cicero Eduardo, s/n, 64600-000, Picos, Piaui, Brazil. * Author for correspondence: E-mail:
Table 1. Number of cells in interphase and at different stages of cell
division in the cell cycle of A. cepa root tips treated with water and
Ponceau 4R dye at concentrations of 0.25, 0.50 and 0.75 g [L.sup.-1],
at exposure times of 24 and 48h. 5,000 cells were analyzed for each CO
and ET.

Ponceau 4R                     Cells
(g [L.sup.-1])     ET      in Interphase      P        M

                   CO          3163          1284     178
0.25               24h         3388          1227     119
                   48h         4421          465       19
                   CO          3841          834      151
0.50               24h         3826          969      123
                   48h         4511          451       22
                   CO          3688          822      284
0.75               24h         4291          400      133
                   48h         4608          215       69

Ponceau 4R                             Cells
(g [L.sup.-1])      A        T      in division      MI (%)

                   174      201         1837        36.7 (a)
0.25               105      161         1612        32.2 (a)
                    16       79         579         11.6 (b)
                    70      104         1159        23.2 (a)
0.50                64       08         1164        23.3 (a)
                    12       04         489         9.8 (b)
                    88      118         1312        26.2 (a)
0.75                85       91         709         14.2 (b)
                    53       55         392         7.8 (b)

CO--Control; ET--Exposure time; h--hour; P--prophase;
M--metaphase; A--Anaphase; T--Telophase; MI--Mitotic Index.
Means followed by the same letter, within the same
concentration, are not significantly
different at 5% by the [chi square] test.

Source: Pessoal.

Table 2. Total number of cells analyzed, micronucleated cells, gene
amplifications and telophase and anaphase bridges, and total number
of aberrant cells found in each control and at concentrations of 0.25,
0.50 and 0.75g [L.sup.-1] of Ponceau 4R dye, at exposure times of 24
and 48h. 5,000 cells were analyzed for each CO and ET.

Concentration                                       Gene
(g [L.sup.-1])      ET      Micronucleated     amplifications

                    CO            00                 00
0.25               24h            19                 31
                   48h            22                 21
                    CO            00                 00
0.50               24h            34                 22
                   48h            28                 17
                    CO            00                 00
0.75               24h            20                 20
                   48 h           45                 20

Concentration        Metaphase and           Total
(g [L.sup.-1])     telophase bridges     aberrant cells

                          00                  00a
0.25                      23                  73b
                          29                  72b
                          00                  00a
0.50                      29                  85b
                          27                  72b
                          00                  00a
0.75                      45                  85b
                          34                  99b

CO--Control; ET--Exposure time. Means followed by the same letter
are not significantly different at 5% by the [chi square] test.

Source: Pessoal.
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Title Annotation:texto en ingles
Author:Marques, Gleuvania Santana; Sousa, Josefa Janaina do Anjos; Peron, Ana Paula
Publication:Acta Scientiarum. Biological Sciences (UEM)
Date:Jan 1, 2015
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