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Cytotoxicity of cheese and cheddar cheese food flavorings on Allim cepa L root meristems/Citotoxicidade de aromatizantes alimentares de queijo e queijo cheddar sob meristemas de raizes de Allim cepa L.

1. Introduction

Flavorings are food additives with aromatic and/or sapid properties capable of conferring or enhancing the aroma and flavor of foods without providing nutrition (Constant et al., 2007). They are classified as natural, synthetic identical to natural, synthetic artificial, reaction or conversion flavorings, and smoke flavorings (Honorato et al., 2013).

Diluents, antioxidants, defoamers, preservatives, emulsifiers, stabilizers, acidity regulators, flavor enhancers, anti-humectants, anti-caking agents, dyes, and extraction and processing solvents are present in the formulation of these additives approved for worldwide use by the European Food Safety Authority (EFSA), and nationally by the National Agency for Sanitary Vigilance (ANVISA, 2007).

However, due to the complex chemical formulation, flavorings, in general, are considered a controversial advancement of the food industry by many healthcare experts who claim that these compounds, along with food dyes, significantly contribute to the dietary impoverishment and the triggering or potentiation of pathologies (Cheeseman, 2012). Thus, researchers such as Shibamoto (2014), have reported that the use of flavorings, mainly synthetic, raises a number of questions regarding their toxicity on the systemic and cellular level, and declare that the carrying out of studies to properly evaluate the toxic potential of these flavors are necessary and urgent; confirming the appeal of ANVISA (2007), itself, for the improvement of the safe use of additives in the constitution of food to be constant and prioritary.

Bioassays with plants have been considered highly sensitive and simple for the monitoring of chemical compound cytotoxic effects (USEPA) (Iganci et al., 2006) and Allium cepa (onion) has been indicated as an effective test organism for evaluating toxicity at the cellular level (Carita and Marin-Morales, 2008) due to its kinetic properties of proliferation, for having large chromosomes and few in number (2n = 16), which facilitates their analysis in the detection of micronuclei and mitotic spindle abnormalities (Herrero et al., 2012) as well as the verification of changes in the cell division rate (mitotic index) (Tabrez et al., 2011). According to Carita and Marin-Morales (2008) the Allium system is an efficient bioassay for the initial cytotoxicity and mutagenicity screening of chemical compounds.

Furthermore, in most cases this test system demonstrates satisfactory similarity to the results obtained with other bioassays (Arung et al., 2011). As an example one can cite the work by Gomes et al. (2013) and Oliveira et al. (2013) who evaluated the toxic potential of dyes widely used in the food industry on A. cepa meristematic root cells and obtained results similar to those obtained in animal-system testing and in cell cultures.

In this context, this work aimed to evaluate the toxicity of Cheese and Cheddar Cheese savory synthetic food flavorings, identical to natural, individually and in combination, to A. cepa meristematic root cells at the cellular level. These food flavorings are widely used in the food industry in frozen meals, crackers, snacks and breads, and widely consumed by the population.

2. Material and Methods

2.1. Obtaining the flavorings and definition of study doses

Cheese and Cheddar Cheese flavor synthetic savory liquid food flavorings, identical to the natural, were obtained from a retailer, located in northeastern Brazil, specialized in the national and international commercialization of synthetic food additives. The flavorings, with an oily appearance, were packaged in 100 mL amber bottles and were within the expiration date. The label on these additives suggested the use of 1.0 mL of flavoring in 300g of dough. Thus, we selected large onion bulbs with an average of 300g and established a test doses of 1.0 and 2.0 mL for the two flavorings studied.

The doses established for these two food additives were also evaluated individually and in combination, as follows: 0.5 mL of the Cheese flavor associated with 0.5 mL of Cheddar cheese flavor; 1.0 mL of the Cheese flavor combined with 1.0 mL of the Cheddar cheese flavor. The associated evaluation manner was conducted because the Cheese and Cheddar flavorings are widely used in combination in the preparation of processed foods.

It is important to clarify that for the cytotoxicity and mutagenicity evaluation of the flavorings in question, individually and in combination, no dilution was performed to define the doses, i.e., the intention was to verify their toxicity to A. cepa root meristems directly in the solution present in the product vials. We chose to do it this way out of concern that the concentration of the compounds present in the flavorings could be changed since they have a complex chemical formulation.

It is also important to report, as EFSA states, that the formulation of any natural or synthetic food flavoring is standardized worldwide, with no changes in chemical formulations for different product brands. However, this regulatory agency, as well as the manufacturers of food additives, do not distinguish the concentrations of each compound present in the formulation of these additives.

2.2. Obtaining root meristem cells of A. cepa for cytogenetic analysis

Bulbs of A. cepa (Common onion, Baia variety) were placed to root in flasks of distilled water at room temperature ([+ or -] 25[degrees]C) and aerated to obtain roots of about 2.0 cm. For analysis of each dose (treatment) an experimental group was established with five onion bulbs. Before placing the roots in contact with their respective doses, some roots were collected and fixed to serve as control (CO) of the bulb itself. The remaining roots were then placed in their respective solutions for 24 hours, a procedure called 24 hour exposure time (24h ET).

After this time some roots were removed and fixed. On completing this procedure, the roots remaining from each bulb were returned to their respective solutions where they remained for 24 hours, which is called the 48 hour exposure time (48h ET). After this period, roots were again collected and fixed. Exposure times of 24 and 48 were chosen in order to evaluate the effects of these doses on more than one cell cycle.

In the flask of each bulb under study 1.0 or 2.0 mL of flavoring (s) to be tested was added, taking care to ensure that all roots were were in adequate contact with the solution under study. The root fixations occurred in Carnoy 3:1 (ethanol:acetic acid) at room temperature for 24 hours. On average, three roots per bulb were removed for each root collection.

2.3. Preparation and reading of the slides and statistical analysis

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

Cells in interphase, prophase, metaphase, anaphase and telophase were observed. The number of cells in interphase and under division, in each control and exposure time was calculated and the mitotic index determined. We also evaluated the action of doses by the number of micronucleated cells in colchicine metaphases, anaphase-telophase bridges, gene amplifications, cells with adhesions, nuclear buds and multipolar anaphases. The data analysis was performed by Chi-square ([chi square]) using the Prism software version 5.0 (GraphPad Software).

3. Results and Discussion

Table 1 presents the number of cells in interphase and under different stages of cell division and MI values obtained from meristematic root tissue cells of A. cepa treated with water (CO) and Cheese flavor at doses of 1.0 and 2.0 mL (TR), at ET of 24 and 48 h. The significant [chi square] values are presented in the results description.

From the results in Table 1, it was seen that the Cheese flavor, at the two doses, reduced the cell division rate of A. cepa root meristem cells in a statistically significant manner when compared to the MIs obtained from their respective controls, thus presenting as cytotoxic. When considering the MIs, among themselves, of the ETs of the same dose it was found that values were statistically similar. Furthermore, the two doses of this flavoring studied did not promote mitotic spindle abnormalities nor micronuclei in the test the system used. No published papers on the toxicity of this flavoring on the cellular and systemic level have been found.

Table 2, presents the number of undifferentiated cells in interphase and during different phases of cell division and the mitotic index values obtained from the root meristem cells of A. cepa treated with water and Cheddar Cheese food flavoring at doses of 1.0 and 2.0 mL at ETs of 24 and 48 hours. Significant [chi square] values were also presented.

The results described in Table 2 showed that the two Cheddar flavor doses evaluated had significant antiproliferative effect at both ETs, as they drastically reduced the A. cepa root tip cell MI when compared with MI data obtained for the respective controls, thus presenting as cytotoxic. It is also possible to observe that at the 1.0 mL dose cell division rates for the 24h ET were statistically higher than MI obtained at 48 h ET. As for the 2 mL dose, the MIs, when compared with those obtained for the 24 and 48 ETs, were statistically equal.

No changes were observed in the mitotic spindle nor micronuclei in any of the doses evaluated for the flavoring in question. As with the Cheese flavoring, no published papers on the toxicity of Cheddar Cheese flavoring on the cellular and systemic level were found.

In Table 3, the number of interphase cells and at different stages of cell division and MI values obtained from meristematic tissue cells of the roots of Allium cepa treated with water is presented, and with the Cheese and Cheddar Cheese flavors at doses of 0.5 mL of Cheese flavor associated with 0.5 mL of Cheddar Cheese flavor; and 1.0 mL Cheese flavor associated with 1.0 mL of Cheddar Cheese flavor. Significant [chi square] values were also presented in the description of results.

From the results shown in Table 3, it can be seen that in cells treated with 0.5 mL of Cheese flavor associated with 0.5 mL of Cheddar Cheese flavor, as well as cells under the treatment with 1.0 mL Cheese flavor associated with 1.0 mL of Cheddar Cheese flavor at ETs of 24 and 48, there was a statistically significant reduction in the cell division rate in relation to MIs obtained for cells treated only with water.

One can also observe that MIs obtained for the 48h ET to the two doses of combined flavors studied were significantly lower than those obtained for their respective 24h ETs. As such, the combined doses of the two flavorings were highly cytotoxic to the test system in question. Similar to the results obtained for the flavorings tested individually, the doses in combination did not induce cell aberrations.

As shown in Tables 1, 2, and 3, the studied flavorings resulted in toxicity at the lowest manufacturer suggested dose, causing significant changes in the A. cepa meristematic root cell cell division rate. As for their evaluation in combination, it was found that in all treatments, the antiproliferative effect is intensified with increasing ET.

Food flavoring, on a systemic level, can be quite toxic when used for prolonged periods, promoting hyperactivity in children with and without attention deficit (Stevens et al., 2014), a significant decrease in hemoglobin concentration in the blood, drastic changes in liver function, a significant decrease in the weight of mice (Shibamoto, 2014), allergies, cutaneous hypersensitivity and poor digestion in humans (Anderson et al., 2013).

However, few studies to date have been conducted to evaluate the toxicity of food flavorings at the cellular level, unlike some dyes used in the food industry, which have been widely studied in different bioassays and have well defined Acceptable Daily Intake (ADI) values. Thus, for food flavorings Honorato et al. (2013) and Curwin et al. (2014) state that these food additives are the least studied regarding cytotoxic potential.

In Brazil, the Agencia Nacional de Vigilancia Sanitaria (ANVISA, 2007) states that high doses of flavorings can cause irritant and narcotic action within the body. They can also produce long-term chronic toxicity to the digestive tract, when used indiscriminately. However, this regulatory body, as well as EFSA, fails to inform what the acceptable daily intake limits for these additives are and also does not report the doses which are considered high, nor which flavorings cause such damage to the body.

Salinas (2002), states that the use of flavorings in low doses does not cause danger to human health. However, when doses are high, this author states that flavorings can cause irritant and narcotic actions and chronic cellular toxicity over the long term, when used in doses higher than those recommended. However, as with ANVISA (2007), the doses considered ideal for use, as well as those considered toxic, are not specified, nor are the flavorings with such action defined.

Thus, according to Honorato et al. (2013), it appears that although the use of flavoring is permitted by the Ministry of Health and ANVISA, studies are necessary and urgent to determine, with propriety, the toxic potential of these food additives, and thus guide the regulators as well as the food industry to define the IDA for safe use of these chemical compounds.

4. Conclusion

From the results obtained in this work there is an urgent need for more studies on the toxicity of these additives at the cellular level in other test systems, especially those using animals, since the A. cepa bioassay was only the first cytotoxicity screening for Cheese and Cheddar Cheese flavorings.

http://dx.doi.org/10.1590/1519-6984.20514

References

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A. G. Moura (a), G. M. Santana (a), P M. P. Ferreira (b), J. M. C. Sousa (a) and A. P Perona, (c) *

(a) Nucleo de Pesquisa Aplicada a Saude e ao Meio-ambiente-NUPBSAM, Laboratorio de Citogenetica Vegetal e Animal-LCVA, Universidade Federal do Piaui-UFPI, Campus Senador Helvidio Nunes de Barros-CSHNB, Rua Cicero Duarte, 940, Bairro Junco, CEP 64607-670, Picos, PI, Brazil

(b) Laboratorio de Cancerologia Experimental-LabCancer, Departamento de Biofisica e Fisiologia, Centro de Ciencias da Saude-CCS, Universidade Federal do Piaui-UFPI, Campus Ministro Petronio Portella-CMPP, Bairro Ininga, CEP 64049-550, Teresina, PI, Brazil

(c) Programa de Pos-graduacao em Genetica e Melhoramento-PPGM, Centro de Ciencias Agrarias-CCA, Universidade Federal do Piaui-UFPI, Campus Ministro Petronio Portella-CMPP, Bairro Ininga, lado impar, CEP 64049-550, Teresina, PI, Brazil

* e-mail: anapaulaperon@ufpi.edu.br

Received: September 30, 2014--Accepted: March 5, 2015--Distributed: May 31, 2016
Table 1. Number of cells observed for each phase of the cell cycle in
meristematic tissue of Allium cepa roots treated with 1.0 or 2.0 mL of
Cheese flavor food flavoring at 24 and 48 hour ETs, and Mitotic
Indices obtained for each food flavoring dose evaluated.

Flavoring/Dose    ET   Undifferentiated     P     M     A    T
                       Cells/ Interphase

Cheese 1 mL       CO         3.888         856   115   93   48
                 24h         4.425         498    21   30   26
                 48h         4.470         477    23   18   12

Cheese 2 mL       CO         4.208         595    90   66   41
                 24h         4.713         173    69   26   19
                 48h         4.667         177    72   59   25

Flavoring/Dose           Cells    MI (%)
                          ET      under
                       division

Cheese 1 mL       CO      256      5.0 (a)
                 24h       77      1.5 (b)
                 48h       53      1.1 (b)

Cheese 2 mL       CO      792      15.8 (a)
                 24h      287      5.7 (b)
                 48h      333      6.7 (b)

ET--Exposure Time; CO--Control; MI--Mitotic Index; Means followed by
the same letter within the same treatment do not differ significantly
at 5% by the [chi square] test.

Table 2. Number of cells observed for each phase of the cell cycle in
meristematic tissue of Allium cepa roots treated with 1.0 or 2.0 mL
Cheddar Cheese flavor food flavoring at 24 and 48 hour ETs, and
Mitotic Indices obtained for each dose of food flavoring evaluated.

     Flavoring         ET   Undifferentiated     P      M     A
                            Cells/ Interphase

Cheddar Cheese 1 mL    CO         3575          1309   187   95
                      24h         4158          632    113   54
                      48h         4669          123    101   52

Cheddar Cheese 2 mL    CO         3303          1567   73    30
                      24h         4557          301    57    41
                      48h         4576          352    40    15

     Flavoring         ET    T   Cells under   MI (%)
                                   division

Cheddar Cheese 1 mL    CO   34       1625      32.5 (a)
                      24h   43       842       16.8 (b)
                      48h   55       331       6.6 (c)

Cheddar Cheese 2 mL    CO   27       1697      33.9 (a)
                      24h   44       443       8.7 (b)
                      48h   17       424       8.5 (b)

ET--Exposure Time; CO--Control; MI--Mitotic Index; Means followed by
the same letter within the same treatment do not differ significantly
at 5% by the [chi square] test.

Table 3. Number of cells observed for each phase of the cell cycle in
meristematic tissue of Allium cepa roots treated with 0.5 mL of Cheese
flavor food flavoring associated with 0.5 mL of Cheddar Cheese food
flavoring; and 1.0 mL Cheese food flavoring associated with 1.0 mL of
Cheddar Cheese food flavoring, at ETs of 24 and 48 hours.

   Flavorings/doses      ET   Undifferentiated     P     M    A
                              Cells/ Interphase

Cheese/Cheddar Cheese    CO         3914           851   101   86
0.5 mL + 0.5 mL         24h         4786          162    22   14
                        48h         4944           23    19   07

1.0 mL + 1.0 mL          CO         3272          642    42   20
                        24h         4476          469    24   16
                        48h         4731          225    24   10

   Flavorings/doses      ET   T    Cells under   MI (%)
                                     division

Cheese/Cheddar Cheese    CO   48       1086       21.7 (a)
0.5 mL + 0.5 mL         24h   16       214        4.3 (b)
                        48h   07        56        1.1 (c)

1.0 mL + 1.0 mL          CO   24       1728       34.5 (a)
                        24h   15       524        10.5 (b)
                        48h   10       269        5.4 (c)

ET--Exposure Time; CO--Control; MI--Mitotic Index; Means followed by
the same letter within the same treatment do not differ significantly
at 5% by the [chi square] test.
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Title Annotation:Original Article
Author:Moura, A.G.; Santana, G.M.; Ferreira, P.M.P.; Sousa, J.M.C.; Perona, A.P.
Publication:Brazilian Journal of Biology
Date:Apr 1, 2016
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