EVALUATING ENVIRONMENTAL ANTAGONISM BETWEEN
HAZARDOUS AL3+ IONS AND CA2+ IONS BY USING A PLANT ASSAY
Ditika Kopliku*, Anila Mesi (Dizdari)
Department of Biology and Chemistry, Faculty of Natural Sciences, University of Shkodra
“Luigj Gurakuqi”, L. “Vasil Shanto”, Rr. “Jeronim de Rada”, Shkodër, Albania.
*E-mail: ditika_kopliku@yahoo.com
Abstract. Nowadays outcomes in the study of the toxic effects caused by metals and their
interactions with essential elements have greatly increased the understanding of the toxicity
mechanisms at the biochemical level. The present investigation aimed to clarify the
environmental antagonism between hazardous aluminum ions and calcium by using Allium
cepa L. assay. The roots of onion bulbs were treated with ¼ EC50, EC50 and 4 EC50
concentration solutions of AlCl3 salt, firstly alone and then combined with four CaCl2 salt
concentrations (1-1000 mg/L). Experiments employing the observation of root morphological
aberrations and the evaluation of some cytogenetic endpoints in root meristem, as: mitotic and
phase indexes, interphase nuclear volume and DNA content, micronuclei and chromosomal
aberration frequency and types, were done. Al3+ treatments induced remarkable root growth
inhibition, reduction of mitotic activity and an increase of different chromosomal
abnormalities, especially c-mitosis and stickiness, as the concentration increased,
demonstrating high phyto- and genotoxic effects in A. cepa roots. Otherwise all Al3+/Ca2 cotreatments generally diminished the toxic activity of aluminum, resulting more effective at
EC50 cc. This study also discussed some recent data related to the possible mechanisms of Alinduced toxicity in plants in case of environmental contamination by this metal.
Key words: ion antagonism, aluminum, calcium, heavy metal toxicity, Allium cepa assay
Aims and background
The assessment of environmental quality is closely related to the application of monitoring
and remediation projects, which aim to reduce the risk that ecosystems incur by hazardous
substances. Metal contamination has become a serious problem in many developing countries
as Albania, due to the rapid industrial development and urbanization and the discharge of
different untreated or/and inadequately treated wastes directly to the environment. Aluminum
constitutes one of the major heavy metal pollutants known to contaminate soil, water and food
chains, posing threat to human and ecosystem health. Anthropogenic sources of Al pollution
include industrial mining and smelting of bauxite ore, kitchen utensils and appliances,
wrapping materials, drinking water (due to its use in municipal treatment), food additives,
cosmetics and medicines1. Meanwhile, Al bio-accumulation tendency, bioavailability and in
consequence, toxicity, have been reported mainly restricted to acid environments due to high
solubility. High availability of aluminum in acid soils (with a pH of 5.5 or lower) is among
the most important limitations factors to agricultural production and quality2. A significant
correlation between low pH and high Al concentration has also been shown in acidified
freshwater, where this metal may cause serious metabolic derangement in some hydrophytes3.
Aluminum ions (A3+) have been known for a long time to be hazardous to plants. Roots are
the most sensitive and accessible part to Al toxicity (with the root apex as the primary site in
most plants). Growth inhibition upon exposure to this metal has been used extensively as one
of the most distinct and earliest symptoms of Al rhizotoxicity4.
Al toxicity and tolerance mechanisms differ strikingly with its chemical form. The cellular
components and processes which have been proposed to be affected by Al are wide ranging
and some of the most important include: cell nuclei and DNA, mitosis and cell division,
composition, physical properties and structure of the plasma membrane, uptake of Ca2+ and
other ions, cytoplasmic calcium homeostasis, oxidative stress, cytoskeletal dynamics and the
cell wall-plasma membrane-cytoskeleton continuum. Disturbance of cytoplasmic Ca2+
homeostasis is suggested as the primary target of Al toxicity and may be involved in the
inhibition of the root elongation or cell division. The source of calcium for the increase of
cytosolic Ca2+ activity could be extracellular and/or intracellular, but it remains still
insufficiently documented, as well the effects on increased cytosolic Ca2+ ions. However the
beneficial effects of calcium on plants grown under conditions of Al toxicity have been
recognized for a long time, but the Al/Ca antagonism on growth, cell division, nuclei and
DNA is still in discussion5.
The assessment of contamination degree should not be based only on its physical and chemical
characterization but it should be combined with biological assays. Bio-assays can assess the
potential toxic effects from all the environmental components (including those due to unknown
substances and their synergic, antagonistic or additive effects) and allow an integrated
evaluation of the impact in biota. Higher plants are recognized as excellent genetic models to
detect environmental mutagens and are frequently used in monitoring studies6-9. Because of
the good correlation with mammalian bio-tests, the reported satisfactory results have helped
even in the research of cancer and other diseases. Nowadays outcomes in the study of the toxic
effects caused by metals and their interactions with essential elements have greatly increased
the understanding of the toxicity mechanisms at the biochemical level. The present
investigation aimed to clarify the environmental antagonism between hazardous aluminum
ions (Al3+) and calcium by using Allium cepa L. assay.
Experimental
Biological material and test procedure to assess the effective concentration EC50 of aluminum.
The biological materials used in the present investigation were equal-sized bulbs of common
onion (Allium cepa L., 2n=16). A. cepa assay was carried out based on the method of Fiskesjö6.
All experiments were set up in a completely randomized design with twelve test tubes per
sample, on top of which one onion bulb has been put with the root primordia downward in the
solution. A 96 h growth inhibition test of onion root was used to determine the corresponding
EC50 value of aluminum as AlCl3 salt. Eight definitive concentrations (1.5-6000 mg/L) were
chosen. Tap water was used as negative control (NC). EC50 endpoint was statistically evaluated
by plotting on graph root length values as percentage to NC against treatment concentrations
(Fig. 1). The trend-line equation with the biggest R2 value was used for this evaluation. Three
replications were done and EC50 value of AlCl3 salt in onion roots resulted 132.6 mg/L.
Morphological and cytogenetic analysis of A. cepa roots. The assessment of toxic potency of
Al3+ ions on A.cepa roots was done by testing the phytotoxic and genotoxic effects of three doses
of AlCl3 salt in tap water, representing ¼ EC50, EC50 and 4 EC50. These Al concentrations were
used as positive control samples. To detect the antagonist role of calcium each positive control
sample was mixed with 1, 10, 100 and 1000 mg/L concentrations of CaCl2 salt. The pH of all
treatment solutions was adjusted to 4.5 using HCl. Onion bulbs were grown in each Al treatment
and Al/Ca co-treatment solution. Macro- and microscopic endpoints, such as: root morphological
aberrations, mitotic and phase indexes (MI and PI), interphase nuclear volume (INV) and DNA
content (DNAC), frequencies of micronuclei formation (MNC), aberrant cells (FAC) and
chromosome aberration types (CA) in root meristematic tissue, were observed and evaluated.
Microscopy slides were prepared after 48 h. Root tips (10 mm) taken from 5 bulbs randomly
chosen in each series, were placed on slides and the terminal root tips (1-2 mm) were cut off and
MRL percentage to NC
used for further preparation. Slides were prepared in accordance with the standard procedure for
Feulgen staining of squashed material10. 1000 cells (in the field of view) per slide were examined.
MI was scored as percent ratio of mitotic cells, while PI values (of pro-, meta- and ana/telophase)
were expressed as percentage to MI. For INV investigation it was obtained the mean of two
diameters of each selected stained nucleus, observed at right angles to each-other and measured
under oil-immersion objectives. INV was evaluated using the formula 4/3r3, where r is the radius
of the nuclei11. DNAC was estimated by cytophotometric measurement of 2C telophase nuclei,
using a micro-spectrophotometer at a wavelength of 550 nm. The Feulgen stained root-tips were
washed in three changes of SO2 water for 10 min. each and dried briefly on absorbent paper.
Darkly stained ones were squashed in a drop of 45% glacial acetic acid. 100 2C telophase nuclei
per slide were measured12. The formation of micronuclei was examined in interphase cells and
their frequency (MNC) scored as percent ratio. FAC and CA types were observed in mitotic cells,
and then expressed as percent ratio. All parameter values have been calculated as means of 5
slides per sample. Root morphological aberrations were observed after 96 h.
100
90
80
70
60
50
40
30
20
10
0
0
1.5
6
15
60
150
600
1500 6000
AlCl3 concentration (mg/L)
Fig. 1. Assessment of EC50 endpoint of Al salt using a preliminary growth inhibition test in A.
cepa roots (MRL-mean root length, NC-negative control)
Statistical analysis. Analysis of Variance (One-way ANOVA) and post-hoc Student NewmanKeuls (SNK) tests were used to test for significant differences of all evaluated parameters in A.
cepa roots exposed to metals treatment. All the results were expressed as the mean of three
replicates per sample ± standard deviation (SD). Parameter differences to corresponding NC were
considered statistically significant at level 5%.
Results and discussion
In the present investigation the observation of onion roots treated with aluminum chloride
revealed a remarkable growth restriction and various morphological aberrations, positively
depending on the tested concentrations. At concentrations higher than 33.15 mg/L (¼ EC50) of
Al salt the toxic effect on root growth rapidly increased, showing cumulative inhibition activity
of aluminum. Since at ¼ EC50 treatment the root tips appeared curved and rigid, while
disorientated-bending and stubby roots with swollen, cracked and brownish tips occurred at
EC50 and abundantly at 4 EC50. Root growth reduction over 55% strongly indicates the presence
of phytotoxic substances and is a general phenomenon associated with most of heavy metals6,1315
. It has been proposed that the root growth inhibition and increase in root diameter observed in
roots exposed to Al are attributed to the disturbance of the cytoskeleton by altering microtubules
and actin microfilaments in their stability, organization, and polymerization2. Our data indicated
a high rhizototoxic effect of Al on A. cepa. On the other hand increased concentrations of basic
cations in solution of the root rhizosphere have been shown to diminish Al toxicity, depending
on pH and Al concentration3. The morphological symptoms caused by AlCl3 alone, observed in
this were moderately reduced with the addition of CaCl2, particularly at the medium dose
(EC50), demonstrating a distinct antagonistic effect of Ca2+ to Al3+ions.
Table 1 and figures 2 and 3 represent all data related to the cytological analysis of A. cepa root
meristem exposed to chosen AlCl3 treatments and AlCl3/CaCl2 co-treatments.
A
80
60
CaCl2
40
0 mg/L
20
1 mg/L
10 mg/L
0
1/4 EC50
EC50
100 mg/L
4 EC50
1000 mg/L
B
12
10
8
6
4
2
0
1/4 EC50
EC50
4 EC50
AlCl3 and AlCl3/CaCl2 treatments
Fig. 2. Effect of different AlCl3 treatments and AlCl3/CaCl2 co-treatments on (A) mitotic
index (MI-expressed as percentage to NC) and (B) frequency of aberrant cells with
chromosome aberrations (FAC) in A. cepa root tip meristem
The mitotic index reflects the frequency of cell division and is considered a confident endpoint
in identifying the presence of cytotoxic pollutants in the environment. Reported data have
suggested that a decrease of MI below 50% (of control) indicates sublethal effect, while below
22% denotes lethal effect on test organism. These effects are probably due to either disturbance
in the cell cycle or chromatin dysfunction induced by pollutant-DNA interactions. Only during
the last decade, researchers started to look at the cell cycle disturbance induced by aluminum,
focusing mostly on the misbalances of mitosis stage. Therefore, it is still unclear whereas
inhibition of cell division or cell elongation is the primary mechanism leading to root growth
inhibition2. The mitotic activity of Al-treated root cells evidently varied in a concentrationdependent manner: the detected inhibition came progressively increasing with addition of Al
salt (Fig. 2A). The significant decrease of MI as compared to NC started since at ¼ EC50 (35%
of NC, P<0.05, using One-way ANOVA test). The other doses induced a substantial depression
of cellular proliferation (47 and 73% of NC, respectively, P<0.001), demonstrating even
sublethal effect. As illustrated in Figure 2A, Ca2+ co-treatments significantly restricted the
mito-depressive activity of Al, demonstrating a certain dose-dependence to Ca salt
concentration (p<0.05, using post-hoc SNK test), as well. The Ca2+/Al3+ antagonistic effect was
mostly evident at Al ¼ EC50 and EC50: MI of treated roots increased respectively 10-14% and
11-12% across CaCl2 cc.
Figure 3 showed the variation of phase index (PI) in A. cepa root tip meristem exposed to all
Al3+ and Al3+/Ca2+ loaded samples. The results revealed significant changes in all mitotic
stages (P<0.05, using One-way ANOVA test), indicating notable irregularities in cell cycles.
EC50 and 4 EC50 of Al treatment strongly decreased the frequency of metaphase cells (38 and
78%, as compared to corresponding NC, respectively), while increased prophase rate (145 and
192%). The data demonstrated that aluminum ions at high concentrations caused an obvious
obstruction of metaphase stage, probably attributed to the blocking of cell division by this
metal at the end of prophase. This mito-depressive action on cell division may be a reason of
MI reduction previously observed in onion roots. Contrariwise, at the lowest tested dose of Al
(1/4 EC50) it was detected a certain but not significant decrease of prophase and increase of
metaphase rates (18 and 124%, respectively). The microscopic investigation related to the
addition of Ca2+ ions showed some improvement of PI, being simultaneously dose-dependent
to Al and Ca chlorides (p<0.05, using post-hoc SNK test). At Al EC50 and 4 EC50 cotreatments it was noticed a tendency of return to normal rates of prophase and metaphase,
being more evident at EC50 (Fig. 3). Meanwhile, the lowest Al co-treatments caused a further
accumulation of cells in metaphase, exciding by 35-51% the respective NC.
Prophase
Metaphase
Ana/Telophase
80
60
40
20
0
NC
0
1
10
100 1000
1/4 EC50
0
1
10
EC50
100 1000
0
1
10
100 1000
CaCl2
4 EC50
AlCl3 and AlCl3/CaCl2 treatments (mg/L)
Fig. 3. Effect of different AlCl3 treatments and AlCl3/CaCl2 co-treatments on phase index (PI)
of A. cepa root mitotic cells
The evaluation of interphase nuclear volume (INV) and DNA content (DNAC), summarized
in Table 1, showed a significant decline of nuclei size and DNA weight in onion root cells
exposed to all positive Al-doped samples (P<0.05, using ANOVA test). After 48 h treatment
INV and DNAC were reduced as high as: 21, 34 and 46%, and 16, 27 and 40% (compared to
NC), with the increase of Al-dose, respectively at ¼ EC50, EC50 and 4 EC50. Recorded nuclear
volume and DNA content reduction may be attributed to the mito-depressive action of
aluminum. The reduction could be due to blocking the G1 and suppressing DNA synthesis12.
Moreover pycnotic cells, having rather small condensed nuclei occurred depending on Al
dose, notably increased at 4 EC50. Pyknosis is the irreversible condensation of chromatin in
the nucleus of a cell undergoing necrosis or apoptosis. This phenomenon was detected even
during morphological observation of A. cepa roots (brownish tips). Findings from the
literature emphasized that a pronounced effect on plants, which are grown in acid soils having
present phytotoxic quantities of aluminum, is the reduction of DNA synthesis3,16. Al has been
reported to prefer binding to DNA compared to histone and non-histone proteins, decreasing
not only the rate of DNA synthesis, but also template activity. It has been suggested that the
binding of aluminum in DNA (phosphorus) prevents the double helix from separating and
serving as the template for RNA synthesis. The double strands of DNA are captured by the
aluminum (Al3+) and are unable to separate. In addition, chromatin fibers can be cross-linked
by the binding of the Al3+ to DNA-phosphate between fibers, which results in less active
transcription and possible interference with cell division3. As shown in Table 1, the increment
of Ca salt in all co-treatments evidently balanced the adverse effect of aluminum against
nuclei and DNA in onion root cells. INV and DNAC values of exposed root cells were
increased respectively of: 8-15% and 2-12% (at ¼ EC50), 7-18% and 6-15% (at EC50), 5-13%
and 2-8% (at 4 EC50), as compared to corresponding positive doses.
Table 1. Effect of different AlCl3 treatments and AlCl3/CaCl2 co-treatments on interphase
nuclear volume (INV), DNA content (DNAC) and frequencies of micronuclei (MNC) and
chromosome aberration types (CA) in A. cepa root tip meristem
Treatments cc
CA types (%)
INV± SD
DNAC± SD
MNC± SD
(mg/L)
3
(μm )
(pg)
(%)
AlCl3
CaCl2
c-M
Stc
Br/Fr
Lg
NC
508.37±0.46 a
95.13 ± 1.01 a
3.1±0.2a
0.7
1.7
bc
bc
c
0
401.61±0.41
79.91±0.52
9.4±0.3
2.1
1.2
0.6
0.4
1
447.38±0.08 b
89.42±0.36 a
6.5±0.7 b
1.4
0.7
0.6
0.2
¼ EC50
10
462.62±0.67 a
81.83±0.29 b
5.2±0.4 b
1.6
0.8
0.7
0.3
(33.15)
100
457.53±0.55a
86.57±0.45 a
5.6±0.1 b
1.3
1.0
0.7
0.1
1000
437.20±1.21 b
84.67±1.18 b
5.7±0.5 b
1.7
0.7
1.4
de
d
g
0
335.52±0.29
69.45±0.37
24.2±2.1
4.7
5.4
0.3
0.9
1
381.28±0.29 c
76.10±0.22 c
17.8±2.3 ef
3.0
3.5
0.8
0.4
EC50
10
396.53±0.64 bc
75.15±0.95 cd
12.7±1.1 d
2.5
3.8
0.3
0.5
(132.6)
100
360.94±0.39 cd
73.25±0.67 cd
11.6±0.9 d
2.9
3.4
0.9
0.2
1000
371.11±1.13 c
79.87±0.60 bc
11.8±0.6 d
2.3
2.9
1.1
0.4
f
f
g
0
274.52±0.17
57.08±0.34
21.7±1.4
2.7
4.1
1.1
1
299.93±0.26 ef
58.98±1.08 ef
18.3±0.9 f
2.1
3.3
0.3
0.7
EC30
10
389.77±0.43 c
60.88±0.51 e
18.5±1.2 f
2.4
3.2
0.7
0.5
(530.4)
100
305.02±0.46 e
58.03±0.86 ef
15.6±1.5 e
2.2
3.3
0.1
0.5
1000
310.12±0.55 e
61.83±0.82 e
15.2±0.8 e
1.8
3.5
0.2
0.4
Means with the different superscript letter along the column are significantly different (p<0.05) in the SNK test; CA
- chromosome aberrations: c-M - c-mitosis; Stc - stickiness; Br/Fr - bridges and fragments; Lg - laggard chromosomes;
SD - standard deviation.
The root meristematic cells are considered an appropriate indicator for the detection of
genotoxical and clastogenic potency of environmental pollutants and the suppression of their
mitotic activity is usually accompanied by an increase of chromosome aberrations. In the
present investigation the genotoxic potential of aluminum was assessed by evaluating the
frequencies of abnormal cells with chromosomal aberrations (FAC) and micronuclei (MNC)
in root meristem of A. cepa. Characterization of chromosome aberrations apparently showed a
positive dose-effect relationship between FAC endpoint and aluminum chloride salt (Fig. 2B).
The significant increase of FAC values started since at Al ¼ EC50 (179% of NC, P<0.05,
using One-way ANOVA test), while EC50 and 4 EC50 increased FAC about 5 and 3 folds
higher than NC (significant at P<0.001), demonstrating strong genotoxic activity of
aluminum. However, the frequency of chromosomal aberrations decreased at the high
concentration (4 EC50), with a corresponding decline previously observed at MI. This fact
confirmed that EC50 is an appropriate toxicity endpoint to select the test concentrations for
genotoxicity assays, because there are enough mitotic cells present to permit the microscopic
investigation of chromosomal aberrations.
The most frequently detected CA types (Table 1) were due to chromatin dysfunction
(stickiness) or spindle failure (c-mitosis). Sticky chromosomes were randomly dispersed
inside the cells. Their frequency markedly increased, especially at Al EC50 and 4 EC50
treatments (31.7 and 71.6% of corresponding FAC). Cells with chromosome bridges,
fragments, laggard and vagrant chromosomes were found only in small amount. Stickiness is
considered a common sign of highly toxic effects on chromosomes, an irreversible type
probably leading to cell death, reported in Allium root cells after treatment with various heavy
metals6,13-15. Concerning c-mitosis, Al treatment was shown to disorder the structure and
function of the mitotic and cytokinetic apparatus, mainly by disturbing microtubule
assembly/disassembly in the root tip cells, subsequently affecting chromosome movements
carried out by the mitotic spindle. Anyway the mechanism of its effects on plant microtubules
remain insufficiently investigated. It has been suggested that Al3+ inhibits the activity of
mitotic key enzymes by disturbing the cytoplasmic Ca2+ homeostasis. As a consequence, the
physiological activity and regulation of calmodulin (CaM), located in the mitotic spindle as an
activator of its key enzymes, is destroyed, leading in turn to disturbance or inhibition of
mitosis2,5,16,17. Bridges result from chromosome and/or chromatid breakage and fusion
whereas laggard chromosomes from weak c-mitosis, increasing the risk for aneuploidy.
As shown in Table 1, it was observed a significant increase of micronuclei frequency (MNC) in
interphase cells of onion root tips treated with the three doses of AlCl3 salt (about 3, 8 and 7 folds
higher than NC, respectively, P<0.05 using ANOVA test). Micronuclei formation (MNC) is
considered an important cytogenetic endpoint of observed toxicity by indicating the level of
accumulated genetic damage during the cells cycle. MNC often results from the acentric
fragments or laggard chromosomes that fail to incorporate into the daughter nuclei during
telophase stage of dividing cells and can cause cellular death due to the deletion of primary genes.
The data related to genotoxic effects of aluminum were in agreement with earlier studies in
plants4,6,14,16,17 and humans18. On the other hand, it was revealed a certain reduction of the
genotoxic effects on A. cepa root tip cells exposed to all Al/Ca co-treatments as high as of:
12-33 and 31-45% at ¼ EC50, 36-57 and 27-53% at EC50, and 16-27 and 15-30% at 4 EC50,
for FAC and MNC, respectively, as compared to respective NC-s. In addition the
condensation rate of chromosomes in observed stickiness was not as massive as in Al3+
treatments. Based on these data it may be noted that the attenuation of adverse genotoxic
effect of aluminum due to the addition of calcium appeared more effective at EC50, being
consistent with the previous cytological observations.
As Al toxicity is a significant limiting factor for global crop production, many plant species
and cultivars with inheritable tolerance to Al stress have become important resources for
investigating toxicity and tolerance. The use of Allium cepa test in the present investigation
demonstrated its capability in evaluating aluminum phytotoxicity and genotoxicity and the
antagonistic effects of Ca2+ ions to hazardous Al3+ ions. Being a short-term, inexpensive, a
standardized method in controlled laboratory conditions and sensitive bio-test, A. cepa is
recommended by international environmental protection agencies for toxic potency
assessment of pollutants9, especially in developing countries as Albania.
Conclusions
The present study provided evident data related to aluminum-induced toxicity and highlighted the
protective role of external calcium to hazardous Al3+ ions by using an in vivo plant assay. The
most severe cytological effect of Al treatment was c-mitosis, which bring to the suggestion that
spindle fibers are the main target of Al toxicity. The attenuation of adverse effect of aluminum by
calcium resulted generally more effective at EC50 of AlCl3 salt. The data confirmed the efficiency
of Allium cepa as a model plant system with multiple morphological and cytogenetic endpoints to
detect and assess the impact of environmental mutagens. This assay could be availably applied
as an integral tool in quality environmental monitoring, especially in developing countries as
Albania.
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