Turkish Journal of Biology Turk J Biol

(2016) 40: 922-933

http://journals.tubitak.gov.tr/biology/
TBTAK
Research Article doi:10.3906/biy-1511-26

Apoptotic induction via membrane/DNA damage and metabolic inactivation by

synthetic food colorants in Allium cepa root meristem
Prajitha VAZHANGAT*, John Ernest THOPPIL
Cell and Molecular Biology Division, Department of Botany, University of Calicut, Kerala, India

Received: 09.11.2015 Accepted/Published Online: 21.01.2016 Final Version: 21.06.2016

Abstract: The present study investigates the cytotoxic and genotoxic effects of two frequently employed synthetic food colorants,
lemon yellow and orange red, using an Allium cepa assay. Uptake of food colorants by root cells at different concentrations and varied
exposures significantly altered the cytogenetic system, membrane integrity, mitochondrial function, root growth, and cell division. The
food colorants induced significant DNA damage, micronucleus formation, and other severe chromosomal aberrations at genetic level
and inhibited cell division as well as root growth. Severe DNA damage, membrane damage, and metabolic inactivation observed in the
result are indicative of apoptotic cell death. These results confirm the possible dose-dependent toxicity of these food colorants in plant
systems. The chromosomes of plants and animals are morphologically similar and appear to respond towards mutagens in a similar way
like mammals, indicating possible damage to the DNA of humans when these colorants are used indiscriminately.

Key words: Allium cepa, cytotoxicity, micronucleus, risk assessment, synthetic food colorants

1. Introduction
Nowadays, everything can be marketed effortlessly
when presented in an aesthetic way. This finds major
application in the field of food, beverages, medicines etc.
and food colorants plays a major role in it. In addition,
attractive colors can enhance the appetizing value and the
deliciousness of foods and drinks for consumers. Studies
on cytotoxic activities of food colorants have immense
relevance since adulteration can be found everywhere and
its aftereffects can result in dreadful diseases.
Synthetic colors are a major source of food intoxication
and surveys have been conducted to determine the
presence of nonpermitted food colors in different food
products. These nonpermitted colors are known to cause
adverse effects in experimental animals (Rus et al., 2010)
and in humans (Mpountoukas et al., 2010; Chequer et
al., 2012). Most of the synthetic dyes originally derived
from coal tar, commonly called coal-tar dyes, contain
the azo group. Azo dyes are compounds characterized
by the presence of one or more azo groups (N=N)
and constitute the most important class of dyes in the
textile industry (Kunz et al., 2002). According to several
authors, tests with microorganisms and mammalian cells
indicate that azo dyes are toxic compounds (Michaels and
Lewis, 1985; Amin et al., 2010), exhibiting genotoxic and
mutagenic activities (Chequer et al., 2009; Oliveira et al.,
* Correspondence: prajithav1@gmail.com
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2010). All the cytotoxic effects observed for azo dyes might
be due to the direct action of dyes on the cells, especially
to the formation of metabolites resulting from azo bond
reduction (Lin and Leu, 2008). These metabolites can react
with the DNA molecule, damaging both its structure and
function (Oliveira et al., 2010).
Some European countries have prevented the use of
many synthetic food colors as they led to the formation of
cancer. The use of these artificial colorants would be safe
if their consumption was below the acceptable daily intake
(ADI) limit. However, repeated exposure to even the
permitted synthetic colors may be hazardous (Agarwal,
1990). Food colorants, such as tartrazine, carmoisine, and
sunset yellow, are synthetic azo dyes. During the fifties,
people found that these dyes were somewhat harmful to
human health, such that they and their metabolic products
in the body could even cause cancer, deformation etc.
Thus, in most countries they are strictly limited in use.
The present study was designed to comprehend the
toxic potential of synthetic food colorants, namely lemon
yellow containing tartrazine and orange red containing
carmoisine and sunset yellow, in the plant system with
major emphasis towards their cytotoxicity, membrane
damage, mitochondrial function, cell division, and
root growth. Staining with Evans blue dye was used as a
marker of cell death. The selective staining of dead cells
 VAZHANGAT and THOPPIL / Turk J Biol
with Evans blue in the cell viability assay depends upon
exclusion of the dye from living cells at plasma membrane,
whereas the dye passes through the damaged membrane
of dead cells and accumulates intracellularly as a blue
protoplasmic stain (Turner and Novacky, 1974). In the
triphenyltetrazolium chloride (TTC) assay the cells having
active mitochondria were able to reduce TTC to insoluble
red-colored triphenyl formazan (TF), indicating increased
activity of the mitochondrial respiratory chain (Vargas et
al., 2006). The cells of multicellular organisms can die by
either of the two major mechanisms: accidental death or
programmed cell death (PCD), also referred to as necrosis
or apoptosis, respectively. Morphological hallmarks
ascribed to PCD include mainly cytoplasm shrinkage,
membrane blebbing, and chromatin condensation (Wyllie
et al., 1980).
Higher plants are currently recognized as excellent
bioindicators of cytotoxic, genotoxic, and mutagenic effects
of environments contaminated by toxic substances (Grant,
1994; Yi and Meng, 2003). However, this feature is due to
the possibility of assessing several genetic endpoints that
range from point mutation to chromosomal aberrations
in cells (Khanna and Sharma, 2013). The species Allium
cepa has been used as an efficient standard organism to
run genetic tests for cytoxicity, especially cytogenetic and
chromosome aberration tests (Leme et al., 2008; Leme and
Marin-Morales, 2009).
2. Materials and methods
2.1. Collection of test materials
The synthetic food colorants, namely lemon yellow
containing tartrazine (CAS No. 1934-21-0) and orange
red containing both carmoisine (CAS No. 3567-69-9) and
sunset yellow (CAS No. 2783-94-0), were purchased in pure
form from the local market. Certified bulbs of A. cepa were
purchased from an agricultural vendor and were stored in
dry and well-aerated conditions. Poorly preserved bulbs,
moldy ones, and those that had started shooting green
leaves were all discarded. Evans blue (CAS No. 314-13-
6) and TTC (CAS No. 298-96-4) were purchased in pure
form from the HiMedia chemical laboratory.
2.2. Preparation of test solution and control for
cytotoxicity assay
To make the stock solution, 1 g of both colorants was
weighed and dissolved in 100 mL of distilled water. The
lowest concentrations of the dye solutions, i.e. 0.005%,
0.01%, 0.05%, 0.1% (LX1, LX2, LX3, LX4 and OX1, OX2,
OX3, OX4) were chosen after preliminary analysis and
prepared for toxicity analysis by diluting the stock solution
with distilled water (Prajitha and Thoppil, 2016).
Distilled water and an organophosphorus pesticide,
methyl parathion (0.01%), were taken as the negative
control (NC) and the positive control (PC), respectively.
The parameters studied included mitotic index and
percentage of abnormal cells induced.
2.3. Evaluation of cytotoxicity
Uniformly sized bulbs of A. cepa were sorted and planted
in sterilized sandy soil without manure to prevent cellular
alterations. Germinated bulbs with healthy roots (12
cm) were collected at the peak mitotic period (0900
1000), washed in distilled water, and kept in different
concentrations of the test solutions. Root tips cut from the
samples at different time intervals of 30 min, 1 h, 2 h, and 3
h were washed in distilled water and immediately fixed in
modified Carnoys fluid for 1 h. Mitotic squash preparation
was done with the help of improved techniques (Sharma
and Sharma, 1990). Hydrolysis with 1 N HCl and staining
with 2% acetocarmine was carried out. Mitotic index
and abnormality percentage were calculated by counting
the normal mitotic cells and aberrant cells, respectively,
out of the total cells scored. All the slides were scanned
and tabulated and photomicrographs were taken with an
AmScope MU Series digital camera attached to a Magnus
microscope.
2.4. Evaluation of root growth inhibition
The synthetic food dyes, lemon yellow and orange red, were
dissolved in distilled water and different concentrations
were made as described above. Young and healthy bulbs
of Allium cepa having uniform size were selected and
rooting was initiated in small containers with different
concentrations of dye solutions. Sprouted roots were
treated with dye solutions for 24, 48, and 72 h for each
concentration. The experiments were done in triplicate.
The test samples were changed every 24 h with fresh test
samples. After the completion of the exposure period (24,
48, and 72 h) the roots were cut from the base with a sharp
razor blade, the number of roots were counted, and root
lengths were measured for each bulb with a meter rule
and used as an index of general toxicity. The parameters
studied included inhibition of root growth [mean root
length (MRL) and mean root number (MRN)].
Onion bulbs allowed to germinate in distilled water
and methyl parathion (0.01%) for 24, 48, and 72 h were
used as negative control (NC) and positive control (PC),
respectively.
2.5. Evaluation of apoptotic activity using Evans blue
staining
The loss of cell viability was studied using the Evans blue
staining method. The germinated bulbs were initially
treated with four concentrations (same as mentioned
previously) of both food colorants for 24 h. The root
tips treated with distilled water and methyl parathion
(0.01%) for 24 h was used as negative and positive control,
respectively. Control and treated roots were then stained
with 0.25% (w/v) aqueous solution of Evans blue for 15
min and subsequently washed with distilled water for 30
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min. The roots were then macro-imaged for a qualitative major nonclastogenic or physiological abnormalities
estimation of cell death. For a quantitative estimation, 10 observed included binucleate cells, tetranucleate cells,
root tips of equal length were excised and soaked with 3 mL micronucleus, ball metaphase, ball anaphase, hypoploid
of N,N-dimethylformamide for 1 h at room temperature. cells, polyploid cells, cytostasis, pole to pole arrangement
The absorbance of Evans blue released was measured at of chromosomes, vagrant chromosomes, stellate
600 nm. arrangement of chromosomes, scattering of chromosomes,
2.6. Evaluation of metabolic activity using TTC staining somatic pairing, tropokinesis, chromosome clumping,
Following exposure to the treatment and control as aberrant grouping at anaphase, diagonal anaphase,
mentioned above, 10 root tips were excised and immersed laggard formation, displaced chromosomes, early cell
in 0.5% 2,3,5-triphenyl tetrazolium chloride (TTC) and plate formation, equatorial separation of chromosomes,
kept at 35 1 C for 15 min in the dark. Subsequently, the stathmo-anaphase, unequal separation of chromosomes,
root tips were rinsed with distilled water and imaged. In shift in MTOC, diagonal cell plate formation, and macro-
addition, the colored complex TF was extracted from roots and microcell formation. The clastogenic abnormalities
in 95% ethanol and the absorbance was read at 490 nm. observed were giant cells, chromatin extrusion, nuclear
budding, nuclear lesion, nuclear erosion, nuclear peaks,
2.7. Statistical analysis
chromatin fragmentation, giant nucleus, chromosome
Data obtained after all the analyses were subjected to
bridges, chromosome coagulation, ring chromosome,
statistical analysis. Duncans multiple range tests and
exposure of chromosome scaffold, chromosome
one-way ANOVA were performed to determine mean
pulverization, and chromosome stickiness. Some rare
separation and significance of treatments using SPSS 20
abnormalities like unipolar anaphase, chromosome
(SPSS Inc., Chicago, IL, USA).
rosette, hyperchromasia, nuclear emergence, chromosome
gaps, and C-anaphase could also be observed.
3. Results
High levels of chromosomal abnormalities (P <
The present results show that both colorants induce
0.001) with increasing concentration and time duration
apoptotic mediated cytotoxicity, DNA damage, membrane
were found [LX4 - 3 h (52.60 0.09), OX4 - 3 h (60.19
damage, and reduced mitochondrial activity in root cells
0.08)], indicating the toxic effects of both dye solutions.
of A. cepa in a dose-dependent manner, indicating their
The treatments with orange red synthetic food colorant
severe toxicity. Cytological analysis of cells treated with
showed extremely significant chromosome abnormality
different concentrations of lemon yellow and orange red
when compared to the control and the lemon yellow dye
dye solutions showed that cell division was inhibited, as
solution. Thus it may be noted that the clastogenic and
confirmed by mitotic index values, which were lower than
nonclastogenic activity observed was mainly due to the
those of the negative control (97.07 0.01). A significant
effect of toxic chemicals present in both dye solutions.
reduction in mitotic index was observed mainly at higher
The mean root length and root number of Allium
concentrations (LX4 and OX4) during higher treatment
cepa grown in the food colorant solutions are shown in
period (3 h) of both dye solutions, of which orange red
Table 2. Significant root growth retardation and root
recorded a more severe reduction in mitotic index than
number reduction were observed in both of the tested dye
lemon yellow. In lemon yellow treatments, mitotic index
solutions, when compared to negative the control [LX4 - 72
reached 79.76 0.01 at the highest concentration (LX4)
h (MRL - 1.6 0.03, MRN - 7 0.88), OX4 - 72 h (MRL
after treatment for 3 h and it reached 83.86 0.01 at the
- 0.20 0.05, MRN - 6 0.57), PC - 72 h (MRL - 0.40
lowest concentration (LX1) after treatment for 30 min
0.10, MRN - 5 0.33), NC - 72 h (MRL - 2.40 0.05, MRN
(Table 1). In orange red treatments, mitotic index reached
- 25 0.66)]. The treatment with orange red dye solution
77.60 0.01 at the highest concentration (OX4) after
on root tips of A. cepa showed a slight reduction in root
treatment for 3 h and it reached 80.64 0.02 at the lowest
length at higher concentration (OX4) of 24, 48, and 72 h
concentration (OX1) after treatment for 30 min (Table 1).
than the positive control. In comparison, it was observed
The decrease in the mitotic index was positively correlated
that the retardation of root growth and root number was
with increasing concentration of the dye solutions.
much more severe in orange red food colorant solution
The cytogenetic assay done using two synthetic food
than in lemon yellow.
colorants also revealed that both possess significant
The Evans blue assay revealed that both food colorants
clastogenic and nonclastogenic activity when compared
have a negative effect on cell viability and induce significant
to the negative control (Table 1). The observed
cell death in a dose-dependent manner (Figure 5A). In
clastogenic and nonclastogenic aberrations are well
treatments, the apoptotic potential of both food colorants
represented in Figures 14. The dye solutions exhibited
was found to be dose-dependent, since maximum cell
high levels of nonclastogenicity, of which orange red
death of lemon yellow (0.45 0.05) and orange red (0.58
possess more nonclastogenicity than lemon yellow. The

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Table 1. Mitotic index and abnormality percentage in control and various treatments.

Time Total Dividing Chromosomal aberrations Mitotic index Abnormality

Treatment
duration (h) cells cells Clastogenic Nonclastogenic SE % SE
820 715 0 0 87.19 0.02b 0.00 0.00a
1 821 710 0 0 86.47 0.01b 0.00 0.00a
2 702 660 0 0 94.01 0.02 b
0.00 0.00a
NC
3 719 698 0 0 97.07 0.01b 0.00 0.00a
788 307 110 211 38.95 0.04 a
40.73 0.12d
1 916 292 189 225 31.87 0.04a 45.19 0.09d
PC 2 832 208 199 227 24.99 0.05 a
51.20 0.10e
3 865 189 218 307 21.84 0.05 a
60.69 0.04j
843 707 135 255 83.86 0.01g 46.25 0.03f
1 1079 888 193 299 82.29 0.01 f
46.26 0.74e
LX1 2 926 770 117 189 83.15 0.11j 33.04 0.14b
3 892 750 161 188 84.07 0.01 j
39.12 0.02b
894 769 147 226 86.01 0.01i 41.71 0.10e
1 901 764 128 285 84.79 0.12 h
45.83 0.02de
LX2 2 900 746 188 207 82.88 0.01 i
43.88 0.10d
3 1127 920 246 271 81.62 0.01e 45.87 0.02c
968 820 106 239 84.70 0.01 h
35.64 0.09b
1 862 709 72 295 82.24 0.01f 42.57 0.10c
LX3 2 887 729 123 208 82.18 0.01 h
37.31 0.10c
3 1075 864 208 302 80.37 0.01b 47.43 0.08d
1023 827 150 220 80.83 0.10 d
36.16 0.09c
1 953 772 188 198 81.00 0.10 e
40.50 0.09b
LX4 2 887 725 157 231 81.73 0.01g 43.73 0.10d
3 865 690 139 316 79.76 0.01 h
52.60 0.09e
899 725 240 280 80.64 0.02j 57.84 0.09i
1 910 755 217 294 82.96 0.01 g
56.15 0.09f
OX1 2 883 713 238 280 80.74 0.01e 58.66 0.09i
3 956 788 230 336 82.42 0.01 g
59.20 0.08h
894 745 189 301 83.33 0.01f 54.80 0.03g
1 906 728 193 315 80.35 0.01 d
56.06 0.08f
OX2 2 819 658 149 307 80.33 0.01 d
55.67 0.04f
3 857 694 122 361 80.97 0.01c 56.35 0.09f
925 752 185 325 81.29 0.01 e
55.13 0.09h
1 898 746 218 289 83.07 0.01g 56.45 0.09f
OX3 2 932 760 227 310 81.54 0.01 f
57.72 0.08g
3 967 788 256 306 81.48 0.01d 58.11 0.08g
935 749 270 272 80.10 0.01 c
57.91 0.05i
1 964 762 271 298 79.04 0.01 c
59.02 0.08g
OX4 2 867 680 222 282 78.42 0.01c 58.12 0.09h
3 902 700 163 380 77.60 0.01 i
60.19 0.08i

NC - negative control (distilled water); PC - positive control (0.01% methyl parathion); LX1 and OX1- 0.005% solution of lemon yellow (LY) and orange
red (OR); LX2 and OX2- 0.01% solutions of LY and OR; LX3 and OX3- 0.05% solutions of LY and OR; LX4 and OX4- 0.1% solutions of LY and OR. SE -
standard error. Means within a column followed by the same letters are not significantly different at P < 0.05 as determined by Duncans multiple range tests.

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Figure 1. Clastogenic and nonclastogenic aberrations in lemon yellow and orange red treated Allium cepa root tip cells.
a) Binucleate cell showing chromatin extrusion at interphase; b) Bridged binucleate cell; c) Hyperchromasia; d) Micronucleus; e) Nuclear
budding and micronucleus; f) Nuclear emergence; g) Nuclear lesions in a binucleate cell; h) Multiple nuclear erosion; i) Nuclear peaks; j)
Chromatin fragmentation in a giant nucleus; k) Nuclear erosions and lesions in a giant cell; l) Tetranucleate cell; m) Bridged binucleate
cell at prophase; n) Chromatin fragmentation in a giant nucleus; o) Chromatin fragments; p) Ball metaphase. Bar - 10 m.

0.03) was observed after 0.1% treatment. Severe apoptotic
activity was observed after treatment with the positive
control (0.78 0.04), whereas no cell death was observed
with the negative control. The roots exposed to different
concentrations of both colorants and the positive control
showed a dose-dependent uptake of blue dye compared to
negative control roots (Figure 5A), indicating the presence
of apoptosis-inducing chemicals in both synthetic
colorants.
The effect of the synthetic food colorants lemon yellow
and orange red in mitochondrial function tested by TTC
staining technique resulted in significant decreases in
mitochondrial activity in treated A. cepa root meristematic
cells. The inhibition of mitochondrial/metabolic activity
was found to be maximum in the positive control (0.001
0.00); hence it remained unstained. Dose-dependent
inhibition was observed in the treatment group, since it
shows a dose-dependent decrease in stainability (Figure
5B). In treatment the least mitochondrial activity for lemon
yellow (0.008 0.00) and orange red (0.004 0.00) was
observed at the highest concentration (0.1%). The negative
control having active mitochondria got stained maximum,
indicating the highest mitochondrial/metabolic activity
(0.86 0.06). Roots exposed to food colorants and the
positive control were unable to reduce TTC to insoluble
red-colored TF, indicating decreased activity of the
mitochondrial respiratory chain. Food colorants induced
severe mitochondrial dysfunction in the present study,
indicating the possible role of food colorants in metabolic
inactivation.

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Figure 2. Clastogenic and nonclastogenic aberrations in lemon yellow and orange red treated Allium cepa root tip cells.
a) Ball metaphase with fragments; b) Chromosome rosette; c) Coagulated chromosomes in a hypoploid cell; d) Cytostasis in a polyploid
cell; e) Cytostasis showing ring chromosome; f) Exposure of chromosome scaffold; g) Pole to pole arrangement and ring chromosome;
h) Pole to pole arrangement with vagrant chromosomes; i) Pulverized ball metaphase; j) Giant polyploid cell with vagrants; k) Giant cell
with sticky stellate metaphase; l) Ring chromosome; m) Ring chromosome in pole to pole metaphase; n) Scattered chromosomes in a
polyploid cell; o) Somatic pairing; p) Somatic pairing showing tropokinesis. Bar - 10 m.

4. Discussion et al., 2014; Prajitha and Thoppil, 2016). Numerous studies

Numerous and diverse food additives, such as colorants,
preservatives, sweeteners, and antioxidants, are consumed
by humans in their typical daily diet. One approach for their
safety is to assess the toxicity of such additives using A. cepa
test material. The Allium model has long been acknowledged
to possess the ability to interact with mutagenic agents
during its cell cycle; as a result the test has often been used
to determine the cytotoxic and genotoxic effects of harmful
chemicals (Fiskesjo, 1997; Nithyameenakshi et al., 2006).
The root growth assay using A. cepa indicated that there was
concentration-dependent inhibition in root growth and
root number, emphasizing its toxicity and corroborating
earlier reports on the cytotoxic effects of some chemicals
and extracts on after all the analyses root tips (Olorunfemi
and Ogunsanwo, 2011; Sreeranjini and Siril, 2011; Timothy
have shown that whenever there is root growth and root
number inhibition (macroscopic parameter) in the Allium
test, there is always a reduction in the number of dividing
cells or mitotic index (microscopic parameter) (Olorunfemi
et al., 2011a, 2011b, 2011c). Such a linear relationship
between macroscopic and microscopic parameters was also
observed in the present study. Inhibition of root growth at
high dose of chromium treatment in A. cepa was observed
by Patnaik et al. (2013). According to their observation,
such root growth inhibition was correlated with the dose-
dependent increase in generation of reactive oxygen species,
cell death, lipid peroxidation, repression of antioxidative
enzymes, induction of DNA damage, chromosome
aberrations, or micronuclei in root cells. Hence significant
mitotic inhibition, DNA damage, and cell death observed

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Figure 3 Clastogenic and nonclastogenic aberrations in lemon yellow and orange red treated Allium cepa root tip cells.
a) Sticky chromosomes showing fragments; b) Tropokinesis; c) Ball anaphase with a bridge; d) C-anaphase; e) Multiple chromosome
bridges; f) Chromosome clumping in a hypoploid cell; g) Chromosome fragments; h) Chromosome fragment and vagrant; i)
Chromosome gaps; j) Coagulated anaphase in a hypoploid cell; k) Aberrant grouping at anaphase in a giant cell; l) Coagulated anaphase
with vagrants; m) Diagonal anaphase with bridge and laggard; n) Diagonal scattered anaphase; o) Diagonal sticky anaphase with a
broken bridge; p) Displaced anaphase showing chromosome bridge; q) Early cell plate formation at late anaphase. Bar - 10 m.

this study might be the possible reason for root growth
inhibition.
The inhibition in cell division reflects the mitodepressive
potential of the tested food colorants. Reduction in mitotic
activity is accompanied by decreased amounts of DNA,
which could be due to inhibition of DNA synthesis or
blocking in the G2 phase of the cell cycle, preventing the
cell from entering mitosis (Turkoglu, 2009). In the light
of this observation, both dye solutions can be considered
toxic. These results bear a resemblance to those obtained by
Gomes et al. (2013), who reported that the mitotic index of
A. cepa root tips was successively decreased with the increase
in dye (sunset yellow, bordeaux red, and tartrazine yellow)
concentrations and duration of treatments. The carmoisine-
induced mitodepressive effects in A. cepa were also reported
by Mahfoz et al. (2010). The mitodepressive effects of
carmoisine may be attributed to its inhibitory effect on the
onset of mitosis, by lengthening the mitotic cycle and/or
delaying spindle formation (Polit et al., 2003). Mahfoz et al.
(2010) suggested that carmoisine inhibits DNA replication
by reducing oxidative phosphorylation, which results in
lower ATP levels. The significant decline in mitotic index
noted in the present study may be due to the interference by
the colorants in the cell cycle by inhibiting DNA synthesis
or blocking in the G2 phase of the cell cycle or due to the
increase in the incidence of chromosomal aberrations with
corresponding increase in the concentration of the colorants.
Chromosomal aberrations are either numerical or
structural, the latter resulting from a break or exchange
of chromosomal material (Khanna and Sharma, 2013;
Timothy et al., 2014). The chromosomal aberrations noted
in this study were mainly stickiness of chromosomes,

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Figure 4. Clastogenic and nonclastogenic aberrations in lemon yellow and orange red treated Allium cepa root tip cells.
a) Equatorial separation of sticky chromosomes; b) Laggards; c) Pulverized chromosomes with multiple bridges; d) Ring chromosome;
e) Stathmo-anaphase showing exposure of chromosome scaffold; f) Unequal separation; g) Unipolar anaphase; h) Abnormal telophase
after equatorial separation; i) Chromosome bridges; j) Coagulated chromosomes; k) Sticky laggards; l) Pulverized chromosomes with
bridge; m) Shift in MTOC and vagrant chromosome; n) Chromatin bridge at cytokinesis; o) Broken chromatin bridge; p) Diagonal cell
plate formation; q) Macro- and microcell formation; r) Multiple nuclear lesion. Bar - 10 m.

chromosome bridges, formation of micronuclei,
chromosome fragmentation, chromosome laggards, ring
chromosomes, and giant cells. Stickiness resulted from
increased chromosomal contraction and condensation or
might be from the depolymerization of DNA and partial
dissolution of nucleoproteins. Chromosome stickiness
reflects toxic effects, usually of an irreversible type and
probably leading to cell death (Turkoglu, 2007; Khanna
and Sharma, 2013). This study observed ana/telophase
bridges that result most possibly from sticky chromosomes,
as well as impaired chromosome segregation, which may
indicate mitotic spindle disturbances. Bridges probably
occur by the interruption and joining of chromosomes or
chromatids (Turkoglu, 2007), as a result of chromosome
stickiness or due to unequal translocation and cause
structural chromosome mutation. This type of anomaly
was also observed in the mitosis of Vicia faba and A.
cepa after treatments with food additives (Gomurgen,
2005; Turkoglu, 2007). Micronuclei can be spontaneously
originated due to the development of an isolated
chromosome that results from an unequal distribution of
genetic material or it can be formed as a result of acentric
fragments or entire chromosomes not incorporated into
the main nucleus during the cell cycle. Therefore, any
substance that is able to promote micronuclei formation
is said to be clastogenic or aneugenic (Meng and Zhang,
1992). Lagging chromosomes resulted due to failure of
the chromosomes to become attached to the spindle fiber
and to move to either of the two poles (Albertini et al.,
2000). Turkoglu (2007) reported the induction of lagging
chromosomal aberration, also called laggard/s following
treatment with food additives. Ring chromosomes are

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Table 2. Mean root length of Allium cepa L. germinated in different concentrations of lemon yellow and orange red solutions.

24 h 48 h 72 h
Treatment Mean root length Mean root Mean root length Mean root Mean root length Mean root
(cm) SE number SE (cm) SE number SE (cm) SE number SE
NC 1.06 0.08c 12 1.15d 1.66 0.12h 19 0.57f 2.40 0.05g 25 0.66f
PC 0.06 0.03ab 1 0.88ab 0.20 0.05ab 3 0.33ab 0.40 0.10b 5 0.33a
LX1 0.20 0.05b 3 0.57b 1.20 0.05g 9 0.57d 1.90 0.05f 18 0.57e
LX2 0.10 0.00ab 6 0.57c 0.90 0.05f 11 0.57e 1.50 0.05e 16 0.57d
LX3 0.10 0.00ab 1 0.33ab 0.70 0.05e 8 0.57d 1.30 0.05d 12 0.57c
LX4 0.06 0.03ab 1 0.57a 0.5 0.05d 2 0.33a 1.6 0.03c 7 0.88ab
OX1 0.20 0.05b 2 0.57ab 0.40 0.05cd 5 0.57bc 1.30 0.05d 10 0.33c
OX2 0.10 0.00ab 1 0.00a 0.30 0.05bc 5 0.57bc 1.06 0.06c 11 0.57c
OX3 0.06 0.33ab 0.00 0.00a 0.13 0.03ab 5 0.33c 0.90 0.05c 8 0.57b
OX4 0.00 0.00a 0.00 0.00a 0.10 0.00a 3 0.57a 0.20 0.05a 6 0.57a

NC - negative control (distilled water); PC - positive control (0.01% methyl parathion); LX1 and OX1 - 0.005% solution of lemon yellow
(LY) and orange red (OR); LX2 and OX2- 0.01% solutions of LY and OR; LX3 and OX3 - 0.05% solutions of LY and OR; LX4 and OX4
- 0.1% solutions of LY and OR. SE - standard error. Means within a column followed by the same letters are not significantly different at
P < 0.05 as determined by Duncans multiple range tests.

Figure 5. Effect of food colorants (lemon yellow and orange red) on cell viability and mitochondrial function; OD - Optical density.
(A) Evans blue dye exclusion assay: Effect of food colorants (lemon yellow and orange red) on cell viability; graph and figures in the inset
showing a significant dose-dependent increase in cell death. (B) Effect of mitochondrial activity after TTC staining; graph and figures in
the inset showing dose-dependent decrease in mitochondrial activity.

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 VAZHANGAT and THOPPIL / Turk J Biol
the result of loss of chromosomes from the telomeric side
(Khanna and Sharma, 2013). The fragments resulting from
chromosomal and chromatid break indicate its mutagenic
events in the cell. An earlier study (Prajitha and Thoppil,
2016) had reported the induction of giant cells with
different chromosomal aberrations by food colorants.
Tartrazine was reported to induce DNA damage in
the liver and kidney in mice (Hassan, 2010). The toxic
effects of tartrazine on the condensation of chromosome
in mitosis and its DNA binding ability were reported
by Mpountoukas et al. (2010). Tartrazine was also
reported to induce chromosomal aberrations in A. cepa
(Roychoudhury and Giri, 1989). A study of tartrazine in
Wistar rats indicated its histological and hematological
toxicity (Himri et al., 2011). Tartrazine is transformed into
aromatic amine sulfanilic acid after being metabolized by
the gastrointestinal microflora (Moutinho et al., 2007).
The formed aromatic amines can generate reactive oxygen
species as part of their metabolism by interaction of these
amino groups with nitrite- or nitrate-containing foods
or in the stomach. The reactive oxygen species could be
produced in the metabolism of nitrosamines and increase
oxidative stress (Bansal, 2005).
Abdelmigid (2009) studied different types of DNA damage
induced by the activity of various synthetic food colorants
including tartrazine, carmoisine, and sunset yellow, and they
were detected by changes in RAPD profile. The study by
Amin et al. (2010) concluded that tartrazine and carmoisine
affect adversely and alter biochemical markers in vital organs
such as the liver and kidney even at lower doses. Hence they
have been reported as renowned mutagenic and clastogenic
agents. Previous studies suggested food color as one of the
best known chemical mutagens as it reacts with biomolecules
including DNA and damages their structure and biological
activity (Tsuda et al., 2001; Das and Mukherjee, 2004),
leading to genetic alterations in DNA molecules. According
to Rus et al. (2010), tartrazine and carmoisine were found to
have hepatotoxic and nephrotoxic action, causing congestion,
stasis, and edema in the liver and kidney, hepatocyte and
kidney apoptosis, and atrophy of some renal corpuscles.
The carmoisine-induced chromosomal as well as spindle
aberration and other cellular-nuclear abnormalities on root
meristems of Allium cepaandHippeastrum reginae were also
analyzed by Abraham et al. (2007), indicating its genotoxicity.
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