Antiproliferative and genotoxic effects of nature identical and artificial synthetic food additives of aroma and flavor/Efeito antiproliferativo e genotoxico de aditivos alimentares de aroma e sabor sinteticos, dos tipos identico ao natural e artificial.
Flavorings are food additives with aromatic properties capable of providing or enhancing the aroma and flavor of food without nutritional purposes. They are classified as natural, synthetic nature-identical and synthetic artificial (Koca et al., 2015). They have a complex chemical composition consisting of several classes of chemical compounds such as diluents, antioxidants, defoamers, preservatives, emulsifiers, stabilizers, acidity regulators, flavor enhancers, anti-caking agents, dyes, and extraction and processing solvents. Such a composition is approved for use worldwide by the European Food Safety Authority (EFSA) (Xu et al., 2015) and in Brazil by the National Sanitary Surveillance Agency (ANVISA) (Brasil, 2007).
Synthetic food additives of aroma and flavor, although essential for the food industry, are considered a controversial advancement in the area of food technology by many healthcare experts (Koca et al., 2015). These professionals reported that these ingredients contribute significantly to the dietary impoverishment and development of diseases such as allergies and alterations in the functioning of the digestive tract (Konishi et al., 2014, Oliveira et al., 2013). However, technical documents of food surveillance agencies do not specify which specific compounds and their concentrations are present in the composition of these additives and nor the Acceptable Daily Intake (ADI) of the flavorings for the population (Konishi et al., 2014). The information of the chemical constituents present in the formulation is also not presented on the labels of flavoring solutions marketed nor at the specialized sites selling these food additives.
However, ANVISA (Brasil, 2007) states that high doses of flavorings cause irritant and narcotic actions, and produce chronic toxicity to the digestive tract when taken long term, but the regulations of this agency does not mention what concentrations and what flavorings determined such a conclusion. It also highlights the continuing need for toxicological studies involving food additives in general, and emphasizes that the results obtained from these analyses are guiding the development or modification of action strategies of food safely agencies and activities of professionals responsible for monitoring food and nutrition of the population (Brasil, 2007; Konishi et al., 2014).
According to the International Programme on Chemical Safety (IPCS) and the United Nations Environment Programme (UNEP), the root meristematic zone of Allium cepa L. (onion) is an efficient test organism for the preliminary evaluation of toxicity at cellular level (Turkoglu, 2007; Tabrez et al., 2011; Lacerda et al., 2014). This organism has excellent kinetic properties of proliferation, large chromosomes in reduced number (2n = 16), which facilitates the detection of chromosomal aberrations and abnormalities in the mitotic spindle (Herrero et al., 2012; Cardoso et al., 2014). It also allows the verification of changes in cell division or mitotic index when exposed to chemical compounds with potential cytotoxic action (Carita and Marin-Morales, 2008; Neves et al., 2014). According to Herrero et al. (2012) and Tabrez et al. (2011), A. cepa system is very effective for initial assessment of cytotoxicity and genotoxicity of chemical compounds or for validation of these conditions after conducting research in other bioassays.
In this context, this study used A. cepa root meristem roots to evaluate the antiproliferative and genotoxic potential of synthetic flavorings Passion Fruit and Vanilla. These flavorings were chosen for analysis because they are widely used in the food industry in the preparation of processed sweet foods, and there is no scientific literature evaluating the toxicity of these flavorings.
2. Material and Methods
This work was conducted at the Laboratory of Animal and Plant Cytogenetics, Campus Senador Hei vi dio Nunes de Barros, Federal University of Piaui, between December 2014 and July 2015.
Flavor and aroma synthetic additives in the liquid form, nature identical of Passion Fruit and artificial of Vanilla, were obtained from an industry in the city of Recife, Pernambuco State, Brazil, specialized in manufacturing and domestic and international marketing of synthetic food additives. The manufacturer suggested, on the label of vials, the use of 1.0 mL of flavoring solution for 1.0 kg of mass. Onion bulbs selected for this study weighed on average 200 grams. Thus, proportionally to the recommended, it was initially set for analysis the volume of flavoring (dose) of 0.2 mL. Then, we defined two higher doses, 0.4 and 0.6 mL.
For toxicological analysis, onion bulbs were allowed to root in flasks with distilled water at room temperature ([+ or -] 27 [degrees]C) to obtain 2.0 cm long roots. For the analysis of each treatment group (TG), we used five onion bulbs. The TG established for each flavor were: TG 0.2; TG 0.4 and TG 0.6 mL. Before placing the roots in contact with their respective doses, some roots were collected and fixed to serve as control of the bulb itself. Then, the remaining roots were placed into their respective flavoring solutions for 24 hours, a procedure called 24 hour exposure time (ET 24 h). After 24 hours, some roots were removed and fixed. After this, the remaining roots of each onion were returned to their respective solutions where they remained for additional 24 hours, which was called as 48-hour exposure time (ET 48 h).
Thereafter, roots were collected and fixed again. The fixation of roots occurred in Carnoy 3: 1 (ethanol: acetic acid) for 24 hours. Three roots per bulb were taken in each collection. On average 03 slides were mounted per bulb, following the protocol proposed by Guerra and Souza (2002), and analyzed under an optical microscope at 400 x magnification. For each onion bulb, we examined 1,000 cells, totaling 5,000 cells for control, ET 24 and ET 48 hours of each TG analyzed.
It was analyzed cells throughout the cell cycle and calculated the number of cells in interphase and in division for each control and exposure time and determined index of cell division or mitotic index (MI). It was also evaluated the toxic potential of doses by means of the number of micronucleated cells, colchicine metaphases, anaphase and telophase bridges. Data were analyzed by Chi-square test at 5% level.
3. Results and Discussion
Importantly, no dilution was performed to define the doses of flavorings, that is, it was tested the toxicity of these additives on A. cepa root meristem directly from the original solutions in vials of the products sold in retail trade. This because the additives have complex chemical formulation and so, the concentration and the action of compounds present in these ingredients could be changed if diluted. Also, ET 24h and 48h were set to evaluate the effect of these additives on more than one cell cycle. It is also important to mention that, according to the Technical Regulation on Flavorings/Aroma approved by ANVISA (Brasil, 1999) and still in force, the formulation of any synthetic food flavoring is standardized worldwide.
Table 1 presents the number of cells in interphase and at different stages of cell division, and the values of mitotic index obtained from meristematic cells of A cepa roots treated with water and food flavorings of Passion Fruit and Vanilla.
The results in Table 1 shows that, at a dose of 0.2 mL of the additive passion fruit, the cell division index observed for CO and ET 24 h are statistically similar. For the ET 48 h of this dose, there has was a sharp reduction in the mitotic index in relation to the respective CO and ET 24 h. For the doses 0.4 and 0.6 mL of this flavoring, the cell division index of ET 24 h and ET 48 h decreased significantly compared to the value observed for their respective controls. Also, for these two doses, the reduction in cell division was more pronounced in the ET 48 h. Thus, for the flavoring of passion fruit, under these analytical conditions, the dose of 0.2 mL at the ET 48 h, and the doses of 0.4 to 0.6 mL at the ET 24 and ET 48 h promoted a significant antiproliferative effect in the cell test system used.
The results obtained for cells exposed to the flavoring vanilla (Table 1) show that the three doses examined, 0.2; 0.4 and 0.6 mL, significantly reduced the cell division index when compared to the mitotic index observed for their respective controls, a condition that has been intensified in the ET 48 h. For all three doses, the cell division index registered for the ET 24 h was not statistically different from the values found in the ET 48 h. According to Gomes et al. (2013), inhibition of cell division caused by chemical compounds in normal cell lines without any mutation and/or cell changes causes malfunction of a tissue because it does not allow the replacement of cells, alter protein production and thus result in malfunction of the organ where it is located.
Table 2 lists the mitotic spindle aberrations and micronucleus found in root meristem cells of A. cepa treated with water and food flavorings of Passion Fruit and Vanilla.
The results in Table 2 show that all three doses, 0.2; 0.4 and 0.6 mL of passion fruit flavoring, at ET 24 h and 48 h, promoted alterations in mitotic spindle--colchicine metaphase and anaphase/telophase bridges--and formation of micronuclei in statistically significant number of cells of A cepa roots. It is important to emphasize that even at the dose of 0.2 mL, considered ideal for use according to the manufacturer, the passion fruit additive promoted the development of cellular alterations at significant numbers. Although the dose of 0.2 mL of this flavoring was not cytotoxic under the conditions of this study, it was genotoxic to cells of the test system used.
As shown in Table 2, only the dose of 0.6 mL, at both ET evaluated, caused mitotic spindle aberrations and micronuclei in the cells of the test system. Still in this table, for the genotoxic doses of the two flavorings, at the ET 48 h, the number of cellular alterations is significantly lower compared to the number observed for their respective ET 24 h. This result confirms the results in Table 1, where the number of dividing cells for the doses of 0.2; 0.4 and 0.6 mL of Passion Fruit and 0.6 mL of Vanilla was significantly lower compared to their respective ET 24 h.
Turkoglu (2007) argues that cells with mitotic spindle alterations and/or chromosomal breaks are likely to generate daughter cells with different chromosomal number, which impairs the functioning of the tissue where they are located. Such changes if not repaired or discarded can alter the cell cycle control mechanism, thus leading to the uncontrolled inhibition or proliferation of cells.
Marques et al. (2015) investigated toxicity at cellular level of flavorings of chocolate, strawberry and condensed milk using root meristem cells of A cepa, and reported that these three substances, in the same way as the aroma and flavor additives studied herein, were cytotoxic to root meristems, as they caused significant reduction of the mitotic index of the cells of the tissues analyzed. Nevertheless, none of these flavorings evaluated by these researchers promoted cell aberrations of the test system used.
In the scientific literature, there are studies evaluating toxicity of some constituents of the classes of chemical compounds that may be present in the formulation of food flavorings in general. Among the preservatives are potassium benzoate, sodium benzoate, potassium nitrate (Brasil, 1999), which, according to Mpountoukas et al. (2010) and Zequin et al. (2011), were clastogenic, mutagenic, and cytotoxic to normal human peripheral blood cells; boric acid, citric acid, potassium citrate and sodium citrate (Brasil, 1999) which, in agreement with Ttikoglu (2007), were cytotoxic to root meristems cells of A cepa.
For the class of diluents, Demir et al. (2010) found that benzoic alcohol at higher concentrations promoted significant damage to the mitotic spindle in human peripheral blood cells. In turn, a gene mutation assay in rat lymphoma showed that the diluent diacetyl (2,3-butadione) caused significant damage to loci of the chromosome 11 of these cells, causing loss of expression of the genes for thymidine kinase enzyme (Whittaker et al., 2008). It also led to the replacement of thymine with guanine in euchromatin, and the disruption of hydrogen and disulfide bonds in the tertiary structure of enzymes involved in cell division in mice cells (More et al., 2012).
Meanwhile, to date, the data obtained by these researchers cannot be directly attributed to the results obtained herein for flavorings Passion Fruit and Vanilla, once we have no detailed information on what chemical compounds representing preservatives and diluents in the chemical composition of these food additives.
The doses of 0.2 mL (ET 48h), 04 and 0.6 mL (ET 24 and 48 h) of the passion fruit flavoring, and the doses of 0.2, 0.4, and 0.6 mL (ET 24 and 48 h) of the additive vanilla were cytotoxic to root meristem cells of A cepa.
The three doses of passion fruit flavoring (ET 24 and 48 h) and the dose of 0.6 mL of vanilla were genotoxic to the cell test system used.
The results allow us to infer that although the use of flavorings in food is permitted by food surveillance agencies, it is of great importance a more detailed analysis of the toxic potential of these additives, with emphasis on Vanilla and Passion Fruit, since these flavoring solutions are present in many processed foods and are freely traded in retail markets and websites specialized in the sale of food additives.
BRASIL. Agencia Nacional de Vigilancia Sanitaria--ANVISA, 2007 [viewed 08 February 2014]. Resolucao da diretoria colegiada RDC no. 05, de 15 de Janeiro de 2007. Brasilia: ANVISA. Available from: http://www.anvisa.gov.br/legis/resol/2007/rdc/02_170107rdc.pdf.
BRASIL. Ministerio da Saude--MS. Agencia Nacional de Vigilancia Sanitaria--ANVISA, 1999 [viewed 13 April 2014]. Regulamento tecnico sobre aditivos aromatizantes/aroma. Resolucao no. 104 de 14 de maio de 1999. Brasilia: MS. Available from: http://www.engetecno.com.br/port/legislacao/norqual_aditivos_aromatizantes.htm
CARDOSO, G.H.S., DANTAS, E.B.S., SOUSA, F.R. and PERON, A.P., 2014. Cytotoxicity of aqueous extracts of Rosmarinus officinalis L. (Labiatae) in plant test system. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 74, no. 4, pp. 886-889. http://dx.doi.org/10.1590/1519-6984.07313. PMid:25627599.
CARITA, R. and MARIN-MORALES, M.A., 2008. Induction of chromosome aberrations in the Allium cepa test system caused by the exposure of seeds to industrial effluents contaminated with azo dyes. Chemosphere, vol. 72, no. 5, pp. 722-725. http://dx.doi.org/10.1016/j.chemosphere.2008.03.056. PMid:18495201.
DEMIR, E., KOCAOGLU, S. and KAYA, R., 2010. Assessment genotoxic effects of benzyl derivatives by comet assay. Food and Chemical Toxicology, vol. 48, no. 5, pp. 1239-1242. http://dx.doi.org/10.1016/j.fct.2010.02.016. PMid:20170703.
GOMES, K.M.S., OLIVEIRA, M.V.G.A., CARVALHO, F.R.S., MENEZES, C.C. and PERON, A.P., 2013. Citotoxicity of food dyes sunset yellow (E-110), bordeax red (E-123), and tatrazine yellow (E-102) on Allium cepa L. root meristematic cells. Ciencia e Tecnologia de Alimentos, vol. 33, no. 1, pp. 218-223. http://dx.doi.org/10.1590/S0101-20612013005000012910.
GUERRA, M. and SOUZA, M.J., 2002. Como observar os cromossomos: um guia de tecnicas em citogenetica vegetal, animal e humana. Ribeirao Preto: FUNPEC.
HERRERO, O., MARTIN, J.P., FREIRE, P.F., LOPEZ, L.C., PEROPADRE, A. and HAZEN, M.J., 2012. Toxicological evaluation of three contaminant of emerging concern by use of Allium cepa test. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, vol. 743, no. 1-2, pp. 24-34. http://dx.doi.org/10.1016/j.mrgentox.2011.12.0282122. PMid:22249112.
KOCA, N., ERBAY, Z. and KAYMAK-ERTEKIN, F., 2015. Effects of spray-drying conditions on the chemical, physical, and sensory properties of cheese powder. Journal of Dairy Science, vol. 98, no. 5, pp. 2934-2943. http://dx.doi.org/10.3168/jds.2014-9111. PMid:25771045.
KONISHI, Y., HAYASHI, S.M. and FUKUSHIMA, S., 2014. Regulatory forum opinion piece*: supporting the need for international harmonization of safety assessments for food flavoring substance. Toxicologic Pathology, vol. 42, no. 6, pp. 949-953. http://dx.doi.org/10.1177/0192623313495603. PMid:23881932.
LACERDA, L.P., MALAQUIAS, G. and PERON, A.P., 2014. Antiproliferative action of aqueous extracts of Hymenaea stigonocarpa Mart. (Fabaceae) on the cell cycle of Allium cepa L. Anais da Academia Brasileira de Ciencias, vol. 86, no. 3, pp. 1147-1150. http://dx.doi.org/10.1590/0001-3765201420130163. PMid:25029361.
MARQUES, G.S., SILVA, S.I.O., SOUSA, J.M.C., FERREIRA, P.M.P. and PERON, A.P., 2015. Cytotoxicity and mutagenic potential of liquid synthetic food flavoring evaluated individually and in association. Food Science and Technology, vol. 35, no. 1, pp. 183-188.
MORE, S.S., RAZA, A. and VINCE, R., 2012. The butter flavorant, diacetyl, forms a covalent adduct with 2-deoxyguanosine, uncoils DNA, and leads to cell death. Journal of Agricultural and Food Chemistry, vol. 60, no. 12, pp. 3311-3317. http://dx.doi.org/10.1021/jf300180e. PMid:22385266.
MPOUNTOUKAS P., PANTAZAKI A., KOSTARELI E., CHRISTODOULOU P., KARELI D., POLILIOU S. and LIALIARIS, T., 2010. Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine. Food Chemical and Toxicology, vol. 48, no. 10, pp. 2934-2944. http://dx.doi.org/10.1016/fct.2010.07.030111219.
NEVES E.S., FERREIRA P.M.P., LIMA LH G.M. and PERON A.P., 2014. Action of Aqueous Extracts of Phyllanthus niruri L. (Euphorbiaceae) leaves on Meristematic Root Cells of Allium cepa L. Anais da Academia Brasileira de Ciencias, vol. 86, no. 1, pp. 1131-1135. http://dx.doi.org/10.1590/0001-373765201420130170383914.
OLIVEIRA, M.V.A., ALVES, D.D.L., LIMA, L.H.G.M., CASTRO, J.M.C. and PERON, A.P., 2013. Cytotoxicity of erytrosine (E-127), brilliant blue (E-133) and red 40 (E-129) food dyes plant test system. Acta Scientiarum Biological Science, vol. 35, no. 4, pp. 557-562. http://dx.doi.org/10.4025/actascibiolsci.v35i4.1841945.
TABREZ, S., SAHKIL, S., UROOJ, M., DAMANHORI, G.A., ABUZENADAH, A.M. and AHMAD, D., 2011. Genotoxicity testing and biomarker studies on surface water: an over view of the techniques and their efficacies. Journal Environmental Science Health. Part C, vol. 29, no. 3, pp. 250-275. http://dx.doi.org/10.1080/10590501.2011.601849464716.
TURKOGLU, S., 2007. Genotoxicity of five food preservatives tested on root tips of Allium cepa L. Mutation Research, vol. 626, no. 1-2, pp. 4-14. http://dx.doi.org/10.1016/j.mrgentox.2006.07.006. PMid:17005441.
WHITTAKER P., CLARKE J.J., SAN R.H., BEGLEY T.H. and DUNKEL V.C., 2008. Evaluation of the butter flavoring chemical diacetyl and a fluorochemical paper additive for mutagenicity and toxicity using the mammalian cell gene mutation assay in L5178Y mouse lymphoma cells. Food Chemical and Toxicology, vol. 46, no. 8, pp. 2928-2933. http://dx.doi.org/10.1016/j.fct.2008.06.001262723.
XU, Z., GU, C., WANG, K., JU, J., WANG, H., RUAN, K. and FENG, Y., 2015. Arctigenic acid, the key substance responsible for t he hypoglycemic activity of Fructus Arctii. Phytomedicine, vol. 22, no. 1, pp. 128-137. http://dx.doi.org/10.1016/j.phymed.2014.11.006. PMid:25636881.
ZEQUIN, N., YUZBASIOGLU, D., UNAL, F., YILMAZ, S. and AKSOY, H., 2011. The evaluation of the genotoxicity of two food preservatives: sodium benzoate and potassium benzoate. Food Chemical and Toxicology, vol. 49, pp. 763-769. http://dx.doi.org/101016/j.fct.2010.11.040151620.
R. D. M. Nunes (a), I. M. S. Sales (a), S. I. O. Silva (a), J. M. C. Sousa (b) and A. P. Peron (b,c) *
(a) Curso de Ciencias Biologicas, Departamento de Ciencias Biologicas, Universidade Federal do Piaui--UFPI, Campus Senador Helvidio Nunes de Barros, Avenida Cicero Duarte, 950, Bairro Junco, CEP 64600-000, Picos, PI, Brazil
(b) Departamento de Ciencias Biologicas, Universidade Federal do Piaui--UFPI, Campus Senador Helvidio Nunes de Barros, Avenida Cicero Duarte, 950, Bairro Junco, CEP 64600-000, Picos, PI, Brazil
(c) Programa de Pos-graduacao em Genetica e Melhoramento--PPGM, Centro de Ciencias Agrarias, Campus Ministro Petronio Portella, Avenida Universitaria, Bairro Ininga, lado impar, CEP 64049-550, Teresina, PI, Brazil
* e-mail: firstname.lastname@example.org
Received: August 10, 2015--Accepted: October 5, 2015--Distributed: February 28, 2017
Table 1. Number of observed cells for each stage of the cell cycle in root meristem tissue of Allium cepa treated with water and synthetic flavorings Passion fruit and Vanilla at doses of 0.2, 0.4 and 0.6 mL at the exposure times of 24 and 48 hours. PASSION FRUIT FLAVORING TG/Dose ET Ten P M A T TCD MI (%) CO 4,660 211 75 39 15 340 6.8 (a) 0.2 mL 24h 4,696 147 85 58 14 304 6.4 (a) 48h 4,937 18 25 17 03 63 1.2 (b) CO 4,564 139 123 103 71 436 8.7 (b) 0.4 mL 24h 4,846 69 55 29 01 154 3.1 (b) 48h 4,955 29 14 01 01 45 0.9 (b) CO 4,228 416 170 122 64 772 15.4 (a) 0.6 mL 24h 4,894 58 31 10 07 106 2.1 (b) 48h 4,952 25 11 10 02 48 0.9 (b) VANILLA FLAVORING TG/Dose ET Ten P M A T TCD MI (%) CO 4,103 484 235 145 33 897 17.9 (a) 0.2 mL 24h 4,736 188 44 18 14 264 5.3 (b) 48h 4,892 63 20 11 14 108 2.1 (b) CO 4,579 276 62 64 19 421 8.4 (a) 0.4 mL 24h 4,891 62 24 07 16 109 2.2 (b) 48h 4,948 21 19 04 08 52 1.0 (b) CO 4,672 222 144 130 32 498 10.0 (a) 0.6 mL 24h 4,846 61 36 30 27 154 3.1 (b) 48h 4,948 11 22 06 13 52 1.0 (b) TG: Treatment group; TCII: Total number of cells in interfase and of undifferentiated cells; P: Prophase; M: Metaphase; A: Anaphase; T: Telophase; ET: Exposure Time; CO: Control; MI: Mitotic Index; TCD: Total number of dividing cells. Within the same treatment; MI values followed by different letters are significantly different at 5% by [chi square] test. Table 2. Mitotic spindle alterations and micronuclei observed in root meristem cells of Allium cepa treated with water and synthetic flavorings of Passion Fruit at doses of 0.2, 0.4 and 0.6 mL, and Vanilla at the dose 0.6 mL at the exposure times of 24 and 48 hours. PASSION FRUIT FLAVORING TG/Dose ET Colchicine Anaphase Telophase Micronuclei (mL) metaphase bridge bridge CO 01 00 00 00 0.2 24h 34 18 05 43 48h 07 00 00 11 CO 00 01 00 00 0.4 24h 27 12 13 58 48h 05 00 00 09 CO 00 01 00 00 0.6 24h 24 13 13 28 48h 01 00 00 10 VANILLA FLAVORING Dose ET Colchicine Anaphase Telophase Micronuclei (mL) metaphase bridge bridge CO 00 00 00 02 0.6 24h 09 09 07 22 48h 00 00 01 18 PASSION FRUIT FLAVORING TG/Dose ET Binucleate TAC (mL) cell CO 00 01 (a) 0.2 24h 00 100 (b) 48h 00 18 (c) CO 00 01 (a) 0.4 24h 00 110 (b) 48h 00 14 (c) CO 00 01 (a) 0.6 24h 02 78 (b) 48h 00 11 (c) VANILLA FLAVORING Dose ET Binucleate TAC (mL) cell CO 00 02 (a) 0.6 24h 00 47 (b) 48h 99 19 (c) TG: Group Treatment; ET: Exposure Time; CO: Control; TAC: Total Cell Alterations. Values followed by different letters are significantly different at 5% by [chi square] test.
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Original Article|
|Author:||Nunes, R.D.M.; Sales, I.M.S.; Silva, S.I.O.; Sousa, J.M.C.; Peron, A.P.|
|Publication:||Brazilian Journal of Biology|
|Date:||Jan 1, 2017|
|Previous Article:||Anatomy and micromorphometric analysis of leaf Catasetum x apolloi benelli & grade with addition of potassium silicate under different light...|
|Next Article:||Toxicity of clove essential oil and its ester eugenyl acetate against Artemia Salina/Toxicidade do oleo essencial de cravo e seu ester acetato de...|