INTRODUCTION
New approaches to food testing are becoming
increasingly urgent today, in view of continuously
growing production and consumption of various foods.
These approaches are primarily meant to ensure food
safety by identifying possible toxic effects that food
products and related additives may have on human
health [1–4]. Any food component can have a negative
effect on the human body. Excessive consumption can
lead to the accumulation of toxic metabolic products.
Some components can cause allergic reactions and
modulate adaptation reactions [5]. Such studies are
primarily based on in silico and in vivo methods of
testing various types of food products.
Fruit juice is an integral part of the human diet
and, undoubtedly, a complex food system. It contains
physiologically active substances (vitamins, minerals,
antioxidants, enzymes, and amino acids) that regulate a
variety of metabolic processes and increase the body’s
resistance to infections. In addition, epidemiological
studies have proved that fruits and vegetables reduce
the risk of chronic diseases [6, 7]. Clinical studies also confirm that fruit juice can have beneficial effects on
blood parameters, cholesterol, and heart function, as
well as prevent cancer and Alzheimer’s disease [8–11].
However, the benefits of fruit juice are not as
evident as they may seem [12]. As we know, natural
mutagens, such as pyrolysidine alkaloids and some
flavonoids, account for about 1% of dry matter in almost
all higher plants. Moreover, vitamins C, E, and A can
have mutagen-potentiating effects [13]. Recent studies
have shown that fruits and juices can contribute to the
development of cancer and asthma in children [7, 14–16].
Sugars contained in fruit juice and their potentially
adverse metabolic effects have long been in the center
of scientific debates. Fructose, in particular, is one of the
main carbohydrate components of fruit juice. As early
as the 1980s, it was considered responsible for several
metabolic abnormalities [17, 18]. This carbohydrate can
be “toxic”, especially when consumed with sweetened
drinks. Moreover, it can participate in the pathogenesis
of noncommunicable diseases such as obesity, diabetes,
or arthritis [19, 20]. Sucrose, another carbohydrate
component of fruit juice, has also shown negative
mutagenic effects [21]. In the USA and Europe, a half
of sugar consumption accounts for sweetened products
with a thick consistency (yogurt, candy and chocolate
bars, ice cream, etc.) and the other half, for sweetened
fizzy drinks and fruit juices. The negative health effects
of fructose have encouraged European countries to
impose taxes on sweetened drinks [22].
Quality control is an equally important aspect
of fruit juice safety. The past decades have seen a
significant increase in the demand for juice, partly
due to continuous improvement of its sensory (color,
smell, texture, and taste) and technological (convenient
packaging, long shelf life) characteristics. As a
result, juice composition has undergone a number
of changes, with added microelements and synthetic
substances (acidity regulators, stabilizers, thickeners,
and sweeteners). The technology of juice production
(e.g., heat treatment) also affects juice properties.
Although the use of these additives is strictly regulated,
scientists are increasingly emphasizing a need for
rigorous research into the mechanisms of their toxic
manifestations [23, 24].
Studies have shown that food additives can lead
to cancer and change the functioning of various
organs [25–27]. Children are especially vulnerable to
their toxic effects that can provoke allergies and other
diseases if manufacturers do not follow strict regulations
[28]. Although several types of food additives can
be used in juice in various combinations, there have
been no studies into their integrated toxic effect on the
human body. Moreover, as chemically active agents,
these additives or their oxidation products can interact
with natural organic or inorganic juice compounds and
cause especially dangerous mutagenic and carcinogenic
effects [29].
In this regard, in vivo studies of subchronic toxicity
of fruit juice components are becoming increasingly
urgent. Modern food scientists aim to develop models
in which the processes of detoxification and metabolism
of toxic compounds are similar to those in the human
body. At the same time, they strive not to use laboratory
animals [4].
We find biotesting quite effective when using
plants, in particular Allium cepa roots (Allium test).
This test has been successfully used to study toxicity,
cytotoxicity, and genotoxicity of various agents,
including food additives, as well as to determine
genotoxic effects of medicinal plant extracts [23,
30–32]. The Allium test is simple, economical, well
reproducible, highly sensitive, applicable in a wide pH
range (3.5–11.0), and just as efficient as other biotests.
We believe that this test can be reliably used to assess
subchronic toxic effects of various juice components,
both individually and in combination with each other.
Similar studies in animals may not produce objective
results. The components under study may be present in
the animals’ basic diet, compromising the results.
Our aim was a comparative study of subchronic
toxic effects that apple juice, its components, and
ethanol have on biomass growth, oxidative enzyme
activity at the cellular level, as well as the nature and
frequency of proliferative and cytogenetic disorders in
Allium cepa roots.
STUDY OBJECTS AND METHODS
To model the composition of apple juice, we used the
following materials: glucose (SIGMA-ALDRICH, lot.
SLBZ9363, Germany), fructose (SIGMA-ALDRICH, lot.
SLCC1647, Germany), sucrose (SIGMA-ALDRICH, lot.
BCCB2955, Germany), D-sorbitol (SIGMA-ALDRICH,
lot.BCBT4918, Germany), malic acid (SIGMAALDRICH,
lot.MKBS7851, Germany), and clarified
apple juice (10.5% carbohydrates) purchased from a
retail outlet.
For biotesting, we used small 5–7 g Allium cepa L.
onions of Stuttgart variety with a diameter of 2.5–3 cm,
with their dry scaly outer layers removed. The roots
were preliminarily germinated in 15 cm3 test tubes with
bottled water in a thermostat (23–25°C) for two or three
days in complete darkness. The bulbs with a sprouted
root length of at least 1 cm were selected for further
experiments. Prior to treating them with juice solutions
and other compounds listed above, we measured the
average root mass in the control group.
Then, the control samples were incubated in water,
while the test samples were incubated in aqueous
solutions in a thermostat (23–25°С) in complete
darkness for 1, 2, or 3 days, depending on the purpose
of the experiment. After incubation, the roots were cut
off, wiped with filter paper, and weighed [33]. EC50 was
determined as a concentration of material that reduced
the test function (growth in root mass) by 50% compared
323
Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 321–328
to the control, taking into account the average mass
of the roots before treatment (except when they were
treated with fructose).
For cytogenetic analysis, the cells of the root apical
meristem were stained with a 2% aceto-orcein solution
(1 g of orcein diluted in 50 cm3 o f 4 5% C H3COOH).
For long-term storage, the roots were placed in a 70%
ethanol solution used as a preservative. Instant squash
preparations were obtained to analyze the division
of apical meristem cells, using an Axioskop 40 light
microscope (Zeiss). In particular, we determined the
mitotic index (ratio of dividing cells to total cells)
and the chromosome aberration index (number of
chromosomal aberrations related to total cells).
The intensity of lipid peroxidation in root tissues was
determined based on the amount of malondialdehyde
(MDA) interacting with 2-thiobarbituric acid and
expressed in μmol/g (MDA in fresh mass) [34]. We
placed 0.2–0.9 (± 0.0001) g into a 15 cm3 polymer tube,
added 1 cm3 of trichloroacetic acid (Merck, Germany) at
a concentration of 200 g/dm3 and then another 3 cm3 of
the same solution after stirring the mixture. The tubes
were centrifuged at 1000 g and 4°C for 15 min. Then,
we transferred 1 cm3 of the upper liquid layer into
another tube and added 4 cm3 of thiobarbituric solution ‒
0.5 g thiobarbituric acid (Diaem, Russia) and 100 cm3 of
trichloroacetic acid (200 g/dm3). The tubes were tightly
closed and placed for 30 min in a water bath at 95°C,
followed by cooling in an ice bath. Next, the tubes were
centrifuged for 10 min at 1000 g and 20°C. The solutions
were spectrophotometrically detected on a Cary
WinUV 100 spectrophotometer (Varian, USA) at 600
and 532 nm.
Statistical processing was performed in Microsoft
Excel and Statistica (v. 12). The Student’s criterion
and Fisher transformation were used for comparative
analysis of percentages.
RESULTS AND DISCUSSION
After a three-day sprouting, the onion roots were
treated with apple juice diluted with water for 2 days
to determine the degree of juice dilution that causes
subchronic toxicity. According to the Allium test,
toxicity was determined by the changes in root mass
after exposure to juice solutions, compared to the
control. As we can see in Fig. 1, a decrease in root
mass was observed at ten times dilution and EC50 was
recorded at five times dilution (P ≤ 0.15).
The cytological analysis of the root meristem cells
showed that higher juice concentrations decreased
the mitotic index more intensively (Fig. 2) than the
growth in root mass (Fig. 1). As we can see, the level of
proliferation for meristematic cells, when treated with a
1:20 diluted solution of apple juice, was half the control
values, and their division almost stopped in the roots
with a 50% delay in mass growth (EC50).
As we know, plants grow due to two main processes,
cell division and extension. Like all eukaryotes,
plant cells enter the cell cycle in response to external
mitogenic stimuli. This process is regulated by a large
number of compounds, such as phytohormones, ARGOS
proteins, CLE peptides, transcription factors, cyclins,
and cyclin-dependent protein kinases. Decreased cell
proliferation during stress or after treatment with
abscisic acid may result from activated expression of
genes that encode protein inhibitors of cyclin-dependent
protein kinases, ICK/KRP. However, the mechanisms
that control differentiation can function independently
of the cell cycle [35]. It appears that the subchronic
amounts of apple juice triggered similar processes in our
study and, therefore, the inhibition of cell proliferation
did not significantly affect the growth in root mass.
The percentage of chromosomal aberrations in
dividing cells in relation to total stained cells was quite
low, about 0.4%, both in the control and the test samples
treated with 1:20 and 1:10 diluted juice. We found no
effect dependent on the amount. Neither could we
determine this indicator in the test samples treated with
a higher concentration of juice (1:5 and 1:2 dilution) due
to the absence of dividing cells.
The abnormalities detected in both the control and
the test samples included the adhesion of chromosomes
to each other, their leading during anaphase, as well
Figure 1 Growth in root mass after treatment with apple juice
in different concentrations (n = 10). *P ≤ 0.15
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
2
4
6
8
10
Mitotic index, %
0
20
40
60
80
100
120
Growth in root mass,
% of control
control
fructose
2 %
fructose
5 %
4
5
6
7
8 index, %
10
15
20
25
MDA concentration,
μmol/g of fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
2
4
6
8
10
Mitotic index, %
0
20
40
60
80
100
120
Growth in root mass,
% of control
control
fructose
2 %
fructose
5 %
3
4
5
6
7
8 index, %
5
10
15
20
25
MDA concentration,
μmol/g of fresh mass
Figure 2 Mitotic index of root meristem cells after treatment
with apple juice in different concentrations (n = 10). *P ≤ 0.05
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
0
2
4
6
8
10
Mitotic index, %
control
fructose
2 %
fructose
5 %
3
4
5
6
7
8
Mitotic index, %
5
10
15
20
25
MDA concentration,
μmol/g of fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
control
juice 1:20
0
2
4
6
8
10
Mitotic index, %
control
fructose
2 %
fructose
5 %
5
6
7
8
index, %
10
15
20
25
MDA concentration,
μmol/g of fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
control
juice 0
2
4
6
8
10
Mitotic index, %
control
fructose
6
7
8
%
15
20
25
concentration,
fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
control
0
2
4
6
8
10
Mitotic index, %
7
8
15
20
25
concentration,
fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
10
Mitotic index, %
7
8
concentration,
fresh mass
324
Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 321–328
control
fructose
2 %
fructose
5 %
fructose
10 %
fructose
15 %
0
1
2
3
4
5
6
7
Mitotic index, %
as disorganization and disordered separation during
metaphase and anaphase. However, these abnormalities
were not distributed evenly among the samples. For
example, aberrations (Fig. 3) and anaphase leading
were almost ten times as high in the test samples.
Also, micronuclei were detected during telophase
and interphase in the samples treated with tenfold
diluted juice.
With the data at hand, we had to understand which of
the juice components was responsible for the identified
toxic effects and to what extent. Carbohydrates are
a major component of apple juice, with up to 10% of
fructose, glucose, and sucrose (in 100 g juice). Taking
into account published data on the negative effects of
glucose on plant growth and development, we conducted
several experiments to determine their toxicity for onion
roots [36, 37]. We started with fructose, as its content in
apple juice is two times as high as that of glucose and
sucrose.
As we can see in Fig. 4, higher concentrations of
fructose delayed the growth in root mass, but only
a 10% concentration of this carbohydrate revealed a
significant difference. After treatment with 10 and 15%
fructose solutions, the roots died, becoming thin, soft,
and slightly mucous. In the Allium test, this finding
probably indicated acute toxicity of fructose in the given
concentrations. Thus, the concentration of fructose
should not exceed 10%.
Fructose at concentrations of 2 and 5% decreased
the mitotic index in the test samples by only 17 and
33%, respectively, compared to the control (Fig. 5).
Like in the previous test, the comparative cytogenetic
analysis did not reveal a significant increase in the
number of chromosomal aberrations, compared to the
control. However, we observed some redistribution in
their spectrum. For example, higher concentrations of
fructose in the test samples caused more disorders such
as chromosomal bridges, fragmentation, and segregation
(up to 20%), compared to the control.
According to the results, the subchronic toxicity of
fructose, one of the main components of apple juice, is
mainly associated with a weak mitosuppressive effect in
the root meristem cells.
Then, we prepared a model aqueous solution from
the main chemical components of apple juice. Their
concentration ratios corresponded to those in juice [38].
In particular, 100 mL of the model solution contained
7 g fructose, 2 g glucose, 1 g sucrose, 0.5 g D-sorbitol,
and 0.3 g malic acid. Prior to that, we had measured
the pH of the study objects to make sure that its range
was acceptable for the Allium test (Table 1). Next, we
analyzed the subchronic toxicity of the resulting model
solution and apple juice in Allium cepa roots after two
days of germination and two days of treatment.
According to the results, the growth in root mass
after treatment with juice was 40% lower than after
using the model solution (Table 2), despite the same
degree of dilution (1:5, P < 0.05). The cytological
analysis showed that the mitotic index of the root
meristem cells after treatment with the 1:10 and 1:5
model solutions did not differ much from the control.
However, treatment with the 1:10 and 1:5 diluted juice,
just like in the previous experiment (Fig. 2), reduced
the mitotic index ten times and led to an almost
complete halt in cell division. Thus, the chemical
0
5
10
15
20
Control 1 %
ethanol
2 %
ethanol
Juice 1:2 Juice
1:2+1%
ethanol
Juice
1:2+2%
ethanol
MDA concentration,
μmol/g of fresh mass
Figure 3 Chromosomal aberrations in onion root meristem
cells: adhesion in metaphase (a), leading in anaphase (b),
disorganization in anaphase (c), and a micronucleus in
telophase (d)
0
5
10
15
20
MDA concentration,
μmol/g of fresh mass
Control Juice 1:10 Juice 1:5 Model
solution 1:10
Model
solution 1:5
0
5
10
15
20
25
Control 1 %
ethanol
2 %
ethanol
Juice 1:2 Juice
1:2+1%
ethanol
Juice
1:2+2%
ethanol
MDA concentration,
μmol/g of fresh mass
(a) (b)
(c) (d)
Figure 4 Growth in root mass after fructose treatment
(n = 7). *P ≤ 0.1
juice 1:5
juice 1:2
*
*
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
2
4
6
8
10
Mitotic index, %
control fructose
2 %
fructose
5 %
fructose
10 %
fructose
15 %
0
20
40
60
80
100
120
Growth in root mass,
% of control
fructose
10 %
fructose
15 %
0
5
10
15
20
25
Control Juice 1:10 Juice 1:5 Model
solution 1:10
Model
solution 1:5
MDA concentration,
μmol/g of fresh mass
Juice 1:2 Juice
1:2+1%
ethanol
Juice
1:2+2%
ethanol
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
control
0
2
4
6
8
10
Mitotic index, %
control
fructose
2 %
fructose
5 %
fructose
10 %
fructose
15 %
-1
0
1
2
3
4
5
6
7
8
Mitotic index, %
0
5
10
15
20
25
MDA concentration,
μmol/g of fresh mass
0
5
10
15
20
25
Control 1 %
ethanol
2 %
ethanol
Juice 1:2 Juice
1:2+1%
ethanol
Juice
1:2+2%
ethanol
MDA concentration,
μmol/g of fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
0
2
4
6
8
10
Mitotic index, %
control
fructose
2 %
fructose
5 %
fructose
10 %
fructose
15 %
-1
0
1
2
3
4
5
6
7
8
Mitotic index, %
0
5
10
15
20
25
MDA concentration,
μmol/g of fresh mass
0
5
10
15
20
25
Control 1 %
ethanol
2 %
ethanol
Juice 1:2 Juice
1:2+1%
ethanol
Juice
1:2+2%
ethanol
MDA concentration,
μmol/g of fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
Mitotic index, %
control
fructose
2 %
fructose
5 %
fructose
10 %
fructose
15 %
-1
0
1
2
3
4
5
6
7
8
Mitotic index, %
MDA concentration,
0
5
10
15
20
25
Control 1 %
ethanol
2 %
ethanol
Juice 1:2 Juice
1:2+1%
ethanol
Juice
1:2+2%
ethanol
MDA concentration,
μmol/g of fresh mass
Figure 5 Mitotic index of root meristem cells after fructose
treatment (n = 7). *P ≤ 0.05
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
control
0
2
4
6
8
10
Mitotic index, %
control
fructose
2 %
fructose
5 %
4
5
6
7
8
index, %
5
10
15
20
25
MDA concentration,
μmol/g of fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
control
juice 1:20
juice 0
2
4
6
8
10
Mitotic index, %
control
fructose
2 %
fructose
5 %
fructose
10 %
fructose
1
2
3
4
5
6
7
8
Mitotic index, %
0
5
10
15
20
25
MDA concentration,
μmol/g of fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120 Growth in root mass,
% of control
*
*
control
juice 0
2
4
6
8
10
Mitotic index, %
control
fructose
2 %
fructose
5 %
3
4
5
6
7
8
Mitotic index, %
5
10
15
20
25
MDA concentration,
μmol/g of fresh mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
control
0
2
4
6
8
10
Mitotic index, %
8
20
25
concentration,
mass
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
100
120
Growth in root mass,
% of control
*
*
Mitotic index, %
8
concentration,
mass
325
Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 321–328
components of the model solution, which make up
the bulk of juice solids, were not responsible for the
subchronic toxicity associated with violation of mitosis
in the roots. Obviously, this effect was caused by other
juice compounds with antiproliferative activity of
natural origin.
Based on the data, we can conclude that these
compounds (one or more) are present in juice in small
quantities and have high biological activity. We need
further studies to identify these substances and better
understand the mechanisms of potential juice toxicity.
MDA is known to reflect the degree of lipid
peroxidation resulting from the oxidation process. The
higher its concentration, the more damaged are lipids
in the walls of plant cells. In our study, the treatment of
onion roots with the 1:10 and 1:5 diluted model solutions
produced a dose-dependent increase in MDA, with its
maximum levels twice as high as in the control samples
(Fig. 6). However, apple juice in the dilutions of 1:10 and
1:5 increased this indicator by only 11%. Apparently,
these results are indicative of the juice’s antioxidant
activity.
To study toxic effects, we treated the onion roots,
which had germinated for two days, with the 1:10 and
1:2 diluted juice for only one day. As we can see in
Table 3, a day of incubation brought about a slightly
higher (10%) decrease in mitotic indices in these test
samples than in those treated for two days, compared to
the control (Fig. 2). Thus, the toxic effect was recorded
as early as after the first cycle of cell division, while
the decrease in root mass growth was more likely to be
cumulative.
Food additives are commonly studied for toxicity
separately from those food products which they are
part of. We believe that such practice does not allow
scientists to objectively determine the patterns of toxic
manifestations. Therefore, our further experiments
attempted to evaluate the effect of ethanol on the
previously detected toxicity of Allium cepa roots, which
had been germinated for two days and then incubated
with apple juice for another two days. We chose this
food additive due to the fact that 1 and 2% aqueous
ethanol solutions delay the growth of Allium cepa roots
within EC50 [29, 32]. In addition, ethanol may be part of
some juice-containing products.
Table 4 shows that 1% ethanol increased the average
mass of the roots treated with the 1:2 diluted juice
by a factor of five. We believe that this effect can be
associated with the activity of lipid oxidation enzymes
and its regulation. Indeed, raising ethanol concentration
to 2% not only decreased their activity, but made it
lower than the control values (Fig. 4). However, in the
1:2 diluted juice samples, MDA was almost 1.7 times as
high as in the control (Fig. 7), which we had expected
from the previous results (Fig. 3). Thus, 1% ethanol
appeared to slow down the destruction of cell wall
lipids caused by the juice components, which had a
positive effect on the root growth. The question is, why
is it that a higher concentration of ethanol (2%) did not
cause a similar effect? Probably, despite lower lipid
oxidation, the total toxicity of 2% ethanol was so high
that it prevented the roots from growing and developing.
Table 1 pH of study objects
Study object pH
Juice 3.88
Model solution 2.62
Juice:water 1:5 4.23
Juice:water 1:10 5.11
Model solution:water 1:5 3.78
Model solution:water 1:10 4.63
Table 2 Root mass growth, mitotic activity, and chromosomal
aberrations of onion root meristem cells after two days
of treatment with apple juice and model solution (n = 10)
Test variant Growth in
root mass,
g/onion
Mitotic
index, %
Chromosomal
aberrations,
%
Control 0.273 ± 0.024а 10.30 ± 0.35a 0.21 ± 0.05a
Juice:water 1:10 0.164 ± 0.031b 1.25 ± 0.14b 0.18 ± 0.05a
Juice:water 1:5 0.091 ± 0.013c 0.99 ± 0.13b nd
Model solution:
water 1:10
0.184 ± 0.027d 7.44 ± 0.32a 1.10 ± 0.13b
Model solution:
water 1:5
0.158 ± 0.010d 9.15 ± 0.32a 0.40 ± 0.07b
a,b; a,c; a,d; c,d P ≤ 0.05; b,c P ≤ 0.1
nd ‒ not detected
Figure 6 MDA concentration in onion roots treated with apple
juice and model solutions
control
juice 1:10
juice 1:5
juice 1:2
0
20
40
60
80
Growth in root mass,
% of control
*
*
control
juice 1:20
juice 1:10
juice 1:5
juice 1:2
0
2
4
6
8
Mitotic index, control fructose
2 %
0
20
40
60
80
100
Growth in root mass,
% of control
control
fructose
2 %
fructose
5 %
fructose
10 %
fructose
15 %
-1
0
1
2
3
4
5
6
7
8
Mitotic index, %
0
5
10
15
20
25
Control Juice 1:10 Juice 1:5 Model
solution 1:10
Model
solution 1:5
MDA concentration,
μmol/g of fresh mass
0
5
10
15
20
25
Control 1 %
ethanol
2 %
ethanol
Juice 1:2 Juice
1:2+1%
ethanol
Juice
1:2+2%
ethanol
MDA concentration,
μmol/g of fresh mass
Table 3 Root mass growth, mitotic activity, and the frequency
of chromosomal aberrations of onion root meristem cells after
a day of treatment with apple juice (n = 5)
Sample Growth in
root mass,
g/onion
Mitotic
index, %
Chromosomal
aberrations,
%
Control 0.113 ± 0.018a 8.31 ± 0.31a 0.42 ± 0.07a
Juice:water 1:10 0.099 ± 0.018a 4.57 ± 0.25b 0.17 ± 0.05b
Juice:water 1:2 0.057 ± 0.011a 1.25 ± 0.12c 0.13 ± 0.04b
a,b; a,c; b,c P ≤ 0.05
326
Samoylov A.V. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 321–328
Another observation we made was that adding 1 and
2% ethanol to the juice did not increase the proliferative
activity of the meristem cells. This result was quite
predictable since treating roots with ethanol solutions
decreased the mitotic index of the meristem cells,
compared to the control.
CONCLUSION
Our study showed that apple juice manifested
subchronic toxicity when it was diluted with water in a
ratio of 1:5 (~ 2% soluble solids). The toxicity caused a
50% delay in the growth of Allium cepa roots, compared
to the control. At the same time, it sharply inhibited
the division of meristem cells, with their mitotic index
decreasing by a factor of 18 and the MDA concentration
increasing by 11%. To identify the mechanisms of
these disorders, we treated the roots with the main
compounds of juice dry solids – fructose, glucose,
sucrose, D-sorbitol, and malic acid – and compared the
above indicators. We found that in contrast to the 1:5
diluted juice, 2% fructose decreased the mitotic index by
only 17%, compared to the control. The model solution
containing 1.4% fructose, 0.4% glucose, 0.2% sucrose,
0.1% D-sorbitol, and 0.06% malic acid showed a 40%
higher growth in root mass compared to the 1:5 diluted
juice (P < 0.05), the same mitotic index of meristem
cells as the control, and a doubled concentration of MDA
compared to the control.
Thus, the subchronic toxicity of apple juice primarily
manifested through its antiproliferative activity in the
meristem cells. However, the above juice components
were not involved in that activity. What they were
responsible for was an increased level of lipid oxidation
in the root tissues, which was restrained by the natural
antioxidants present in the juice.
In addition, we analyzed the contribution of a food
additive (ethanol) to the potential toxicity of apple juice,
using the Allium test. We found that 1% ethanol in the
1:2 diluted juice reduced the concentration of MDA
in the roots by 30%, with no effect of acute toxicity in
relation to their growth.
The above effects of, and relationships between,
various biomarkers of apple juice and its components
can form a basis for more detailed large-scale research
into its safety. Our findings can also be used to study
the toxic potential of juice depending on manufacturing
technology or food additives, as well as to create new
juice-based products.
CONTRIBUTION
All the authors were equally involved in writing the
manuscript and are equally responsible for plagiarism.
CONFLICT OF INTEREST
The authors declare that they have no conflict of
interest.

1. Khanferyan RA, Vybornaya KV, Radzhabkadiev RM, Evstratova VS, Nalivayko NV, Semin VB, et al. Frequency of consumption of sweet carbonated drinks by the population of different age groups of the Russian Federation. Problems of Nutrition. 2017;86(3):55-58. (In Russ.).

2. Petrov AN, Galstyan AG, Radaeva IA, Turovskaya SN, Illarionov EE, Semipyatniy VK, et al. Indicators of quality of canned milk: Russian and international priorities. Foods and Raw Materials. 2017;5(2):151-161. DOI: https://doi.org/10.21603/2308-4057-2017-2-151-161.

3. Bessonov VV, Khanferyan RA, Galstyan AG, Kucherov YuN. Potential side effects of caffeine consumption in healthy adults, pregnant women, adolescents and children (review of foreign literature). Problems of Nutrition. 2017;86(6):21-28. (In Russ.).

4. Blaauboer BJ, Boobis AR, Bradford B, Cockburn A, Constable A, Daneshian M, et al. Considering new methodologies in strategies for safety assessment of foods and food ingredients. Food and Chemical Toxicology. 2016;91:19-35. DOI: https://doi.org/10.1016/j.fct.2016.02.019.

5. Yu R, Yang B, Cai L, Lu X, Wang X. Excess free fructose beverages and allergy in children and adolescents: results from NHANES 2005-2006. Annals of Family Medicine. 2018;16(5):408-418. DOI: https://doi.org/10.1370/afm.2292.

6. Hung H-C, Joshipura KJ, Jiang R, Hu FB, Hunter D, Smith-Warner SA, et al. Fruit and vegetables intakeand risk of major chronic disease. Journal of the National Cancer Institute. 2004;96(21):1577-1584. DOI: https://doi.org/10.1093/jnci/djh296.

7. Farvid MS, Chen WY, Michels KB, Cho E, Willett WC, Eliassen AH. Fruit and vegetable consumption in adolescence and early adulthood and risk of breast cancer: population based cohort study. BMJ. 2016;353.

8. Khan N, Afaq F, Kweon M-H, Kim K, Mukhtar H. Oral consumption of pomegranate fruit extract inhibits growth and progression of primary lung tumors in mice. Cancer Research. 2007;67(7):3475-3482. DOI: https://doi.org/10.1158/0008-5472.CAN-06-3941.

9. Stowe CB. The effects of pomegranate juice consumption on blood pressure and cardiovascular health. Complementary Therapies in Clinical Practice. 2011;17(2):113-115 DOI: https://doi.org/10.1016/j.ctcp.2010.09.004.

10. Singh M, Arseneault M, Sanderson T, Murthy V, Ramassamy C. Challenges for research on polyphenols from foods in Alzheimer’s disease: bioavailability, metabolism, and cellular and molecular mechanisms. Journal of Agricultural and Food Chemistry.2008;56(13):4855-4873.

11. Gil MI, Tomás-Barberán FA, Hess-Pierce B, Holcroft DM, Kader AA. Antioxidant activity and total phenolic, organic acid and sugar content in commercial pomegranate juice. Journal of Agricultural and Food Chemistry. 2000;48(10):4581-4589.

12. Pepin A, Stanhope KL, Imbeault P. Are fruit juices healthier than sugar-sweetened beverages? A review. Nutrients. 2019;11(5). DOI: https://doi.org/10.3390/nu11051006.

13. Durnev AD, Oreshchenko AV. Pishcha: mutagenez i antimutagenez [Food: mutagenesis and antimutagenesis]. Storage and Processing of Farm Products. 1996;(3):14-18. (In Russ.).

14. Büchner FL, Bueno-de-Mesquita HB, Linseisen J, Boshuizen HC, Kiemeney LALM, Ros MM, et al. Fruits and vegetables consumption and the risk of histological subtypes of lung cancer in the European prospective investigation into cancer and nutrition (EPIC). Cancer Causes and Control. 2010;21(3):357-371. DOI: https://doi.org/10.1007/s10552-009-9468-y.

15. Rietjens IMCM, Martena MJ, Boersma MG, Spiegelenberg W, Alink GM. Molecular mechanisms of toxicity of important food-borne phytotoxins. Molecular Nutrition and Food Research. 2005;49(2):131-158. DOI: https://doi.org/10.1002/mnfr.200400078.

16. DeChristopher LR, Uribarri J, Tucker KL. Intakes of apple juice, fruit drinks and soda are associated with prevalent asthma in US children aged 2-9 years. Public Health Nutrition. 2016;19(1):123-130. DOI: https://doi.org/10.1017/S1368980015000865.

17. Hallfrisch J, Ellwood K, Michaelis OE, Reiser S, Prather ES. Plasma fructose, uric acid, and inorganic phosphorus responses of hyperinsulinemic men fed fructose. Journal of the American College Nutrition. 1986;5(1):61-68.

18. Reiser S, Smith Jr JC, Mertz W, Holbrook JT, Scholfield DJ, Powell AS, et al. Indices of copper status in humans consuming a typical American diet containing either fructose or starch. The American Journal of Clinical Nutrition. 1985;42(2):242-251. DOI: https://doi.org/10.1093/ajcn/42.2.242.

19. Bray GA, Nielsen SJ, Popkin BM. Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity. The American Journal of Clinical Nutrition. 2004;79(4):537-543.

20. DeChristopher LR, Uribarri J, Tucker KL. Intake of high-fructose corn syrup sweetened soft drinks, fruit drinks and apple juice is associated with prevalent arthritis in US adults, aged 20-30 years. Nutrition and Diabetes. 2016;6(3). DOI: https://doi.org/10.1038/nutd.2016.7

21. Dragsted LO, Daneshvar B, Vogel U, Autrup HN, Wallin H, Risom L, et al. A sucrose-rich diet induces mutations in the rat colon. Cancer Research. 2002;62(15):4339-4345.

22. Tappy L, Mittendorfer B. Fructose toxicity: is the science ready for public health actions? Current Opinion in Clinical Nutrition and Metabolic Care. 2012;15(4):357-361. DOI: https://doi.org/10.1097/MCO.0b013e328354727e.

23. Samoilov AV, Suraeva NM, Zaitseva MV, Kurbanova MN, Stolbova VV. Comparative assessment of artificial sweeteners toxicity via express biotest. Health Risk Analysis. 2019;(2):83-90. (In Russ.).

24. Shahmohammadi M, Javadi M, Nassiri-Asl M. An overview on the effects of sodium benzoate as a preservative in food products. Biotechnology and Health Sciences. 2016;3(3).

25. Golka K, Kopps S, Myslak ZW. Carcinogenicity of azo colorants: influence of solubility and bioavailability. Toxicology Letters. 2004;151(1):203-210. DOI: https://doi.org/10.1016/j.toxlet.2003.11.016.

26. Amin KA, Abdel Hameid H, Abd Elsttar AH. Effect of food azo dyes tartrazine and carmoisine on biochemical parameters related to renal, hepatic function and oxidative stress biomarkers in young male rats. Food and Chemical Toxicology. 2010;48(10):2994-2999. DOI: https://doi.org/10.1016/j.fct.2010.07.039.

27. Eberechukwu SE, Amadikwa AA, Okechukwu MO. Effect of oral intake of sodium benzoate on some haematological parameters of wistar albino rats. Scientific Research and Essays. 2007;2(1):6-9.

28. Jacob SE, Hill H, Lucero H, Nedorost S. Benzoate allergy in children - From foods to personal hygiene products. Pediatric Dermatology. 2016;33(2):213-215. DOI: https://doi.org/10.1111/pde.12759.

29. Kitano K, Fukukawa T, Ohtsuji Y, Masuda T, Yamaguchi H. Mutagenicity and DNA-damaging activity caused by decomposed products of potassium sorbate reacting with ascorbic acid in the presence of Fe salt. Food and Chemical Toxicology. 2002;40(11):1589-1594. DOI: https://doi.org/10.1016/s0278-6915(02)00119-9.

30. Moura AG, Santana GM, Ferreira PMP, Sousa JMC, Peron AP. Cytotoxicity of cheese and cheddar cheese food flavorings on Allim cepa L. root meristems. Brazilian Journal of Biology. 2016;76(2):439-443. DOI: https://doi.org/10.1590/1519-6984.20514.

31. Pandey H, Kumar V, Roy BK. Assessment of genotoxicity of some common food preservatives using Allium cepa L. as a test plant. Toxicology Reports. 2014;1:300-308. DOI: https://doi.org/10.1016/j.toxrep.2014.06.002.

32. Almeida PM, Araújo SS, Santos IRMR, Marin-Morales MA, Benko-Iseppon AM, Santos AV, et al. Genotoxic potential of leaf extracts of Jatropha gossypiifolia L. Genetics and Molecular Research. 2016;15(1). DOI: https://doi.org/10.4238/gmr.15017415.

33. Kurbanova MN, Suraeva NM, Rachkova VP, Samoylov AV. Comparative study of indicators of toxic activity in the Allium-test. Agrarian Bulletin of the Urals. 2018;171(4):25-30. (In Russ.).

34. Zhang H, Jiang Y, He Z, Ma M. Cadmium accumulation and oxidative burst in garlic (Allium sativum). Journal of Plant Physiology. 2005;162(9):977-984. DOI: https://doi.org/10.1016/j.jplph.2004.10.001.

35. Kuluev BR. Regulators of cell division and proliferation in plants. Biomics. 2017;9(2):119-135. (In Russ.).

36. Lastdrager J, Hanson J, Smeekens S. Sugar signals and the control of plant growth and development. Journal of Experimental Botany. 2014;65(3):799-807. DOI: https://doi.org/10.1093/jxb/ert474.

37. Ma QX, Cao XC, Xie YA, Xiao H, Tan XL, Wu LH. Effects of glucose on the uptake and metabolism of glycine in pakchoi (Brassica chinensis L.) exposed to various nitrogen sources. BMC Plant Biology. 2017;17. DOI: https://doi.org/10.1186/s12870-017-1006-6.

38. Ivanova NN, Khomich LM, Perova IB. Apple juice nutritional profile. Problems of Nutrition. 2017;86(4):125-136. (In Russ.).