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Toxicity at the cellular level of artificial synthetic flavorings/ Toxicidade em nivel celular de aromatizantes sinteticos artificiais.

Introduction

In food industry, food additives or microingredients have become essential to enhance sensory properties and extend the shelf life of processed foods. The most important additives include flavorings, substances with aromatic and taste properties able to confer and/or intensify the aroma and the taste of foodstuffs without nutritional purposes (Brasil, 2007; Koca, Erbay, & Kaymak-Ertelain, 2015). Classified as natural, synthetic nature-identical and synthetic artificial, aroma and taste microingredients have a complex formulation comprising a variety of chemical compounds, such as diluents, antioxidants, defoamers, preservatives, emulsifiers, stabilizers, acidity regulators, flavor enhancers, antiwetting agents, anti-caking agents, dyes, and extraction and processing solvents. Worldwide, flavoring substances are regulated and authorized for use by the Food and Agriculture Organization (FAO) (Xu et al., 2013), and in Brazil by the National Sanitary Surveillance Agency (ANVISA) by Resolution RDC 2 of January 15th, 2007 (Brasil, 2007).

Although conferring essential organoleptic properties to processed foods, flavoring additives are a controversial advancement in the area of food science and technology by many health professionals (Konishi, Hayashi, & Fukushima, 2011). Experts report that these ingredients contribute significantly to the impoverishment of the diet, cause severe disturbances in the functioning of the digestive tract and trigger allergic and narcotic reactions in the body, especially in children and elderly individuals (Konishi et al., 2011; Oliveira, Alves, Lima, Castro, & Peron, 2013). Also, to date, food surveillance agencies have not established the Acceptable Daily Intake (ADI) to ensure the safe use of these substances (Zeguin, Yuzbagioglu, Unal, Yilmaz, & Aksoy, 2011; More, Raza, & Vince, 2012). Because of such considerations, food safety experts emphasize the urgent need for studies on flavoring toxicity assessment, focusing on cytotoxicity, genotoxicity and mutagenicity (Brasil, 1999; Brasil, 2007; Konishi et al., 2011; Gomes, Oliveira, Carvalho, & Menezes, 2013; Xu et al., 2013). However, there is no research in the scientific literature assessing the toxicity at the cellular level of aroma and flavor additives.

Cytotoxic and/or genotoxic compounds have the potential to change vital cellular mechanisms, such as duplication and transcription, and promote mitotic spindle changes and chromosomal breaks. These changes can significantly impair cell division of the tissue or organ affected and initiate and/or potentiate cancerous processes (Valavanidis, Vlachogranni, Fiotakis, & Lionidas, 2013; Zilifdar, Alpes-Hayta, Yilmaz, Kaplan-Orzen, & Aylogn, 2014). According to Zaineddin et al. (2012), the development of the most common types of cancer results from the interaction between endogenous and environmental factors, especially the diet, especially when it consists of excess of processed foods. Bendino, Populine and Cerqueira (2012) and Louzada, et al. (2015) report that over 40% of various cancers are initiated or exacerbated due to inadequate diets, rich in food additives.

Root meristem cells of Allium cepa L. (onion) are effective bioassays for the initial screening of acute toxicity at the cellular level (Herrero et al., 2012; Lacerda, Malaquias, & Peron, 2014). This test 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 (Cardoso, Dantas, Sousa, & Peron, 2014). It also allows the verification of changes in cell division or mitotic index when exposed to chemical compounds with potential cytotoxic action. Furthermore, this test system has, in most cases, satisfactory similarity to the results obtained with other test systems (Tabrez et al., 2011). For instance, the studies carried out by Gomes et al. (2013) and Oliveira et al. (2013) evaluated, in root meristem cells of A. cepa, cytotoxic and geotoxic potential of artificial synthetic food dyes and obtained results similar to those observed in animal test systems and in cell cultures.

Based on this context, this study evaluated, in root meristem cells of A. cepa, individually and associated with each other, the cytotoxicity and genotoxicity of two artificial synthetic flavorings, tutti-frutti, found in candy, chewing gum, gum, ice cream, jelly and soft drinks, and cookie, found in cake dough and industrialized cookies. These additives are also commonly found in combination in cupcakes and processed cakes.

Material and methods

Obtaining food flavorings, setting and analysis of the doses

Artificial synthetic aroma and flavor additives, tutti-frutti and cookies, were obtained from a manufacturing industry of food microingredients in the city of Recife, state of Pernambuco, Brazil, specialized in national and international marketing of synthetic food additives.

The label of both additives suggested the use of 3 mL of flavoring for 1.0 kg of mass. Onion bulbs selected for this study weighed on average 200 g. Thus, proportionally to that recommended, it was initially set for analysis the dose of 0.6 mL. Then, we defined two doses, 0.3 and 0.9 mL. We also assessed the combination of the two flavorings, as follows: for each tutti-frutti flavoring dose set it was added the same dose of the cookie flavoring.

Obtaining root meristem cells of A. cepa and cytogenetic analysis

Onion bulbs were allowed to root in bottles with aerated distilled water at room temperature ([+ or -] 27[degrees]C) until obtaining 2.0 cm long roots. For analysis of each dose and combined doses (combination treatment), we set up an experimental group with five onion bulbs. Before placing the roots in contact with their respective treatments, some roots were collected and fixed to serve as control of the bulb itself. Then, the remaining roots were returned to their respective solutions for 24 hour, a procedure called 24 hour exposure time (ET 24 hour).

After 24 hours, some roots were taken and fixed. Next, the remaining roots of each onion roots were returned to their respective doses or treatments, where they remained for more 24 hours, which was called as 48 hour exposure time (ET 48 hour). Thereafter, roots again were collected and fixed. Roots were fixed in Carnoy 3: 1 (ethanol: acetic acid) for 24 hour. Three roots per bulb were taken in each collection.

Preparation and reading of slides and statistical analysis

Slides, on average, 03 per bulb, were prepared following the protocol proposed by Guerra and Souza (2002), and analyzed by light microscopy at 40x magnification. For each onion bulb, we examined 1,000 cells, totaling 5,000 cells for the control, ET 24 hour and ET 48 hour in each treatment group. Cells were observed in interphase, prophase, metaphase, anaphase and telophase. We calculated the number of interphase and dividing cells in each control and exposure time and thus determined the index of cell division or mitotic index (MI). It was also evaluated the action of doses by means of the number of micronucleated cells, colchicine metaphases, anaphase and telophase bridges. The data were submitted to statistical Chi-square at 5%.

Results and discussion

First, it is important to mention that, according to the Technical Regulation on flavorings/aroma and flavor approved by ANVISA in 1999 and still in force, the formulation of any synthetic food flavoring is globally standardized and inspection of the composition is the responsibility of food safety agencies (Brasil, 1999, 2007). In addition, for this research, no dilution was performed to obtain the doses of flavorings. This because the flavorings, in general, have complex chemical formulation and so, the concentration and the action of compounds present in these ingredients could be changed if diluted. Furthermore, after an extensive search in sites specialized in domestic and international marketing of aroma and flavor additives, the ideal dose for consumption, recommended by different manufacturers, was almost unanimously the same as that used in this study, 3 mL tutti frutti or cookie flavoring for 1 kg of mass. The exposure times of 24 hour and 48 hour were established in order to evaluate the effect of these additives on root meristems on more than one cell cycle.

Table 1 lists the number of cells in interphase and at different stages of cell division, and the values of mitotic index obtained from root meristem cells of A. cepa treated with water and food flavorings cookie and tutti frutti. These additives were evaluated alone and in combination in two exposure times. The description of the results also presents the significant values of [chi square].

Table 1 shows that mitotic indices of cells treated with the three doses of cookie flavoring were significantly lower than cell division indices obtained for the respective controls. The cell division observed for doses 0.3 and 0.6 mL in ET 48 hour, this aditive were statistically lower than those observed mitotic indices for respective ET 24 hour. Otherwise, the mitotic index registered for ET 48 hour with the dose of 0.9 mL of the cookie additive was significantly lower in relation to its specific cell division index in ET 24 hour. Thus, under the conditions analyzed, doses of the cookie flavoring proved to be cytotoxic as they significantly inhibited cell division in root meristems of the test system.

Regarding the tutti-frutti flavoring (Table 1), mitotic indices observed for the three doses in both exposure times investigated were statistically similar to the mitotic index obtained for their controls. In the same way, when comparing the cell division indices of ET 48 hour with ET 24 hour for doses of this flavoring, we found no statistically difference. Therefore, the tutti-frutti additive was not cytotoxic to cells of the test organism considered.

Still in Table 01, data for the treatments from the association between doses of cookie and tutti-frutti flavorings showed that in the treatment 0.3 mL + 0.3 mL, cell division indices for the control and ET 24 hour presented no significant differences. Nevertheless, the mitotic index obtained for this association in ET 48 hour was significantly lower than mitotic indices obtained for the respective control and ET 24 hour. For the other two associations, 0.6 mL + 0.6 mL and 0.9 mL + 0.9 mL, the observed mitotic indices for ET 24 and 48 hour were significantly different from the values of their respective controls. Likewise, when comparing the mitotic indices obtained for ET 48 hour of these two treatments with their specific ET 24 hour, there was a statistically significant reduction. Thus, treatments regarding the associations between the two flavoring additives significantly promoted antiproliferative effect of the analyzed meristems, proving to be cytotoxic.

Table 2 presents the number and types of cellular abnormalities found in meristematic cells of A. cepa roots treated with water and food flavorings cookie and tutti-frutti, alone and in combination in ET 24 hour and 48 hour at doses of 0.3 or 0.6 or 0.9 mL. The description of the results also presents the significant values of [chi square].

The results in Table 2 indicate that the doses of cookie and tutti-frutti additives, for both forms of assessment, alone and in combination, resulted in a significant number of micronuclei and mitotic spindle changes, such as colchicine metaphase, anaphase and telophase bridges in meristematic cells of A. cepa roots. Therefore, in the present study, doses of cookie and tutti-frutti flavorings, as well as treatments combining such substances were genotoxic. Still in Table 2, for the three combination treatments at ET 48 hour, the number of cell changes was significantly lower compared to the number verified for their ET 24 hour. This result confirms the results described in Table 1, in which the number of dividing cells for the mentioned treatments was significantly lower in relation to their specific ET 24 hour.

Regulations of food surveillance agencies EFSA and ANVISA do not explain specifically what compounds and concentrations are flavoring additives. This information is also not available on the labels of flavoring solutions marketed or on websites specialized in the sale of these substances. Despite being limited the number of toxicity studies, at the cellular level, of food flavorings found in the scientific literature, there are some cytotoxicity evaluation studies of some chemical constituents of diluents and preservatives found in the composition of these microingredients. Such compounds are allowed and mentioned in technical documentation of food safety agencies.

Among them, stands out benzyl alcohol, diluent responsible for maintaining uniformity and facilitating incorporation and dispersion of flavor concentrated in food products. An analysis of the action of this diluent at the cellular level conducted by Demir, Kocaoglu and Kaya (2010) found that this alcohol, at high concentrations, led to significant damage to the mitotic spindle and therefore cell division in human peripheral blood cells. Other diluent commonly used in the formulation of flavorings is the diacetyl (2,3-butadione). Whittaker, Clarke, San, Begley and Dunkel (2008) reported, in gene mutation assay in rat lymphoma, that this compound caused significant damage to loci on chromosome 11 of these cells, causing loss of expression of genes for thymidine kinase in these animals. Additionally, More et al. (2012) verified that the diluent diacetyl had the potential to replace thymine with guanine in euchromatin regions and cause the disruption of hydrogen and disulfide bonds in the tertiary structure of enzymes involved in the cell division process.

Preservatives in food flavorings include potassium benzoate, sodium benzoate and potassium nitrate (Brasil, 1999), which, according to Mpountoukas, Pantazaki, Kostareli, Christodoulou, and KarelI (2010) and Zeguin et al. (2011), were clastogenic, mutagenic, and cytotoxic to normal human peripheral blood cells. Also present are boric acid, citric acid, potassium citrate and sodium citrate (Brasil, 1999), which led to a significant reduction in the cell division index of root meristem cells of A. cepa, proving to be cytotoxic to this test system (Turkoglu, 2007).

For food flavorings, the only class of compounds with usage restrictions by regulatory bodies to some of its constituents is the extraction solvent class, where the agaric acid, aloin, beta-azarone, berberine, coumarin, hydrocyanic acid, hypericin, pulegone, quassine, safrole and isosafrole, santonin and tuyona alpha and beta have maximum tolerable limits discriminated in documents (Brasil, 1999; Brasil, 2007). In the meanwhile, for the manufacture of any food flavoring, it is necessary to join more than 20 classes of chemical compounds (Konishi et al., 2011; Xu et al., 2013).

Therefore, from the results obtained, along with the evaluation of toxicity at the cellular level already performed, while the use of aroma and flavor additives is permitted by EFSA and ANVISA, there is an urgent need for more detailed studies in the medium and long term, to determine properly the cytotoxicity of these substances and/or classes of chemical compounds that constitute them. It is worth mentioning that from cyto- and toxicological evaluation, in the short and medium term, developed in the 80s, food flavorings sparteine, allyl hexanoate and quinine have been banned for use in processed foods by ANVISA in the early 90s.

Conclusion

The cookies flavoring and combination treatments were cytotoxic and genotoxic, and the tutti-frutti flavoring, although non-cytotoxic demonstrated a genotoxic potential.

Our findings show the great need for more effective participation of food surveillance agencies as for the possible cytological and toxicological risks of flavoring additives to consumers, with emphasis on the flavorings cookie and tutti-frutti.

Similar studies can effectively assist health surveillance agencies to rethink and/or reorganize the content present in the normative documents of the agencies responsible for the regulation of these food additives.

Acknowledgements

To the Programa Institucional de Bolsas de Iniciacao Cientifica (PIBIC). To Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq). To Campus Senador Helvidio Nunes de Barros (CSHNB), Universidade Federal do Piaui (UFPI).

References

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More, S. S., Raza, A., & 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, 60(12), 3311-3317.

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Xu, Z., Gu, K., Wang, J., Ju, H., Wang, K., Ruan, K. (2013). Arctigenic acid, the key substance responsible for the hypoglycemic activity of Fructus Arctii. Phytomedicine, 22(1), 128-137.

Whittaker, P., Clarke, J. J., San, R. H., Begley, T.H., & 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, 46(8), 2928-2933.

Zaineddin, A. K., Buck, K., Vrieling, A., Heinz, L., Flesch-Janys, D., Linseisen, J., & Chang-Claude J. (2012). The association between dietary lignans, phytoestrogen-rich foods, and fiber intake and postmenopausal breast cancer risk: a German case-control study. Nutrition Cancer, 64(5), 652-665.

Zeguin, N., Yuzbagioglu, D., Unal, F., Yilmaz, S., & Aksoy, H. (2011). The evaluation of the genotoxicity of two food preservatives: sodium benzoate and potassium benzoate. Food Chemical and Toxicology, 49(4), 763-769.

Zilifdar, F., Alpes-Hayta, S., Yilmaz, C., Kaplan-Orzen, E., & Aydogan, N. (2014). Genotoxic potential and eukaryotic DNA topoisomerase inhibitory effects of some benzoxazine derivative. Medical Chemistry Research, 23(5), 480-486.

Received on January 26, 2016.

Accepted on May 10, 2016.

Doi: 10.4025/actascibiolsci.v38i3.30751

Ila Monize Sousa Sales (1), Jussara Damascena de Oliveira (1), Fabelina Karollyne Silva dos Santos (1), Lidiane de Lima Feitoza (2,3), Joao Marcelo de Castro e Sousa (1) and Ana Paula Peron (1,3) *

(1) Departamento de Ciencias Biologicas, Campus Senador Helvidio Nunes de Barros, Universidade Federal do Piaui, Avenida Cicero Duarte, 940, 64607-670, Picos, Piaui, Brazil. (2) Departamento de Ciencias Biologicas, Campus Ministro Petronio Portella, Universidade Federal do Piaui, Teresina, Piaui, Brazil. (3) Centro de Ciencias Agrarias, Campus Ministro Petronio Portella, Universidade Federal do Piaui, Teresina, Piaui, Brazil.

* Author for correspondence. E-mail: anapaulaperon@ufpi.edu.br
Table 1. Number of cells observed for each stage of the cell cycle
root meristem cells of Allium cepa treated with water and artificial
synthetic flavorings cookie and tutti-frutti, at doses of 0.3; 0.6
and 0.9 mL, assessed alone and in combination at the exposure times
of 24 and 48 hours.

                                 Cookie Flavoring

Dose (mL)             ET    TCII    P     M     A     T    TCD

                      CO    4090   596   155   73    86    910
0.3                  24h    4572   161   109   64    94    428
                     48h    4712   113   73    43    59    288
                      CO    4258   556   87    47    51    741
0.6                  24h    4577   249   83    46    45    423
                     48h    4679   136   96    48    41    321
                      CO    4291   482   105   50    72    709
0.9                  24h    4420   75    72    28    13    188
                     48h    4463   17    06    08    06    37

                              Tutti-Frutti Flavoring

Dose (mL)             ET    TCII    P     M     A     T    TCD

                      CO    4508   279   110   54    49    492
0.3                  24h    4651   69    154   65    61    349
                     48h    4606   88    165   101   40    394
                      CO    4463   304   110   72    51    537
0.6                  24h    4684   51    147   62    56    316
                     48h    4664   74    128   78    56    336
                      CO    4208   323   137   159   73    692
0.9                  24h    4494   179   160   85    82    506
                     48h    4663   162   156   101   68    487

                     Cookie Flavoring + Tutti-Frutti Flavoring

Combined doses(mL)    ET    TCII    P     M     A     T    TCD

                      CO    4165   558   153   75    69    855
0.3 + 0.3            24h    4130   567   139   82    82    870
                     48h    4726   101   83    51    39    274
                      CO    4038   567   183   114   108   972
0.6 + 0.6            24h    4654   104   153   51    38    346
                     48h    4924   34    18    17    07    76
                      CO    4236   410   158   94    102   764
0.9 + 0.9            24h    4727   84    81    69    39    273
                     48h    4952   13    17    05    03    38

                    Cookie Flavoring

Dose (mL)             ET     MI (%)

                      CO    18.2 (a)
0.3                  24h    8.6 (b)
                     48h    7.8 (b)
                      CO    14.8 (a)
0.6                  24h    8.5 (b)
                     48h    6.4 (b)
                      CO    14.2 (a)
0.9                  24h    3.8 (b)
                     48h    0.7 (c)

                  Tutti-Frutti Flavoring

Dose (mL)             ET     MI (%)

                      CO    9.8 (a)
0.3                  24h    7.0 (a)
                     48h    7.9 (a)
                      CO    10.7 (a)
0.6                  24h    6.3 (a)
                     48h    6.7 (a)
                      CO    11.8 (a)
0.9                  24h    10.1 (a)
                     48h    9.7 (a)

Cookie Flavoring + Tutti-Frutti Flavoring

Combined doses(mL)    ET     MI (%)

                      CO    17.1 (a)
0.3 + 0.3            24h    17.4 (a)
                     48h    5.5 (b)
                      CO    19.4 (a)
0.6 + 0.6            24h    7.0 (b)
                     48h    1.5 (c)
                      CO    15.3 (a)
0.9 + 0.9            24h    5.5 (b)
                     48h    0.8 (c)

TCII--Total number of cells in interfase and of undifferentiated
cells; 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. Number and types of cellular abnormalities found in root
meristem cells of Allium cepa treated with water and synthetic food
flavorings cookie and tutti-frutti at doses of 0.3; 0.6 and 0.9 mL,
at the exposure times of 24 and 48 hours.

                               Cookie Flavoring

Dose (mL)             ET         Colchicine           Anaphase
                                 metaphase             bridge

                      CO             01                  00
0.3                   24h            25                  27
                      48h            19                  39
                      CO             00                  00
0.6                   24h            22                  15
                      48h            18                  22
                      CO             00                  00
0.9                   24h            13                  47
                      48h            00                  00

                              Tutti-Frutti Flavoring

Dose (mL)             ET         Colchicine           Anaphase
                                 metaphase             bridge

                      CO             00                  00
0.3                   24h            54                  39
                      48h            43                  37
                      CO             00                  00
0.6                   24h            22                  30
                      48h            23                  31
                      CO             00                  00
0.9                   24h            20                  49
                      48h            19                  42

                      Cookie Flavoring + Tutti-Frutti Flavoring

Combined doses (mL)   ET    Colchicine metaphase   Anaphase bridge

                      CO             00                  00
0.3+0.3               24h            29                  59
                      48h            21                  48
                      CO             00                  00
0.6+0.6               24h            25                  17
                      48h            00                  01
                      CO             00                  00
0.9+0.9               24h            23                  29
                      48h            00                  03

                                Cookie Flavoring

Dose (mL)             ET       Telophase       Micronuclei
                                 bridge

                      CO           00              00
0.3                   24h          13              51
                      48h          11              48
                      CO           00              01
0.6                   24h          29              43
                      48h          33              37
                      CO           00              00
0.9                   24h          19              11
                      48h          00              13

                              Tutti-Frutti Flavoring

Dose (mL)             ET       Telophase       Micronuclei
                                 bridge

                      CO           00              01
0.3                   24h          11              59
                      48h          19              74
                      CO           00              01
0.6                   24h          27              43
                      48h          21              48
                      CO           00              01
0.9                   24h          33              52
                      48h          38              62

                  Cookie Flavoring + Tutti-Frutti Flavoring

Combined doses (mL)   ET    Telophase bridge   Micronuclei

                      CO           00              01
0.3+0.3               24h          27              78
                      48h          02              21
                      CO           00              01
0.6+0.6               24h          13              47
                      48h          01              21
                      CO           00              01
0.9+0.9               24h          20              31
                      48h          00              09

                            Cookie Flavoring

Dose (mL)             ET      Binucleate      TCA
                                 cell

                      CO          00          01a
0.3                   24h         00          116b
                      48h         11          128b
                      CO          00          01a
0.6                   24h         18          127b
                      48h         12          122b
                      CO          01          01a
0.9                   24h         00          90b
                      48h         00          13c

                          Tutti-Frutti Flavoring

Dose (mL)             ET      Binucleate      TCA
                                 cell

                      CO          00          01a
0.3                   24h         02          165b
                      48h         00          173b
                      CO          00          01a
0.6                   24h         00          122b
                      48h         00          123c
                      CO          00          01a
0.9                   24h         00          154b
                      48h         00          161b

           Cookie Flavoring + Tutti-Frutti Flavoring

Combined doses (mL)   ET    Binucleate cell   TCA

                      CO          00          01a
0.3+0.3               24h         16          209b
                      48h         02          92c
                      CO          00          01a
0.6+0.6               24h         00          102b
                      48h         00          23c
                      CO          00          01a
0.9+0.9               24h         00          103b
                      48h         00          12c

ET--Exposure Time; CO--Control; TCA--Total Cell Abnormalities. Within
the same treatment, TAC values followed by diffe rent letters are
significantly different at 5% by [chi square] test.
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Author:Sales, Ila Monize Sousa; de Oliveira, Jussara Damascena; Santos, Fabelina Karollyne Silva dos; de Li
Publication:Acta Scientiarum. Biological Sciences (UEM)
Article Type:Ensayo
Date:Jul 1, 2016
Words:4425
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