Protoplasma (2007) 231: 99–111
DOI 10.1007/s00709-006-0227-6
PROTOPLASMA
Printed in Austria
Comparison of the toxicity and distribution of cadmium and lead in plant cells
M. H. Wierzbicka1,*, E. Przedpel⁄ ska1, R. Ruzik 2, L. Ouerdane 3, K. Pol⁄ eć-Pawlak 2,
M. Jarosz 2, J. Szpunar 3, and A. Szakiel 4
1
Department of Ecotoxicology, Institute of Experimental Plant Biology, Faculty of Biology, University of Warsaw, Warsaw
Chair of Analytical Chemistry, Faculty of Chemistry, Warsaw University of Technology, Warsaw
3
Laboratory of Bioinorganic Analytical Chemistry, Unité Mixte Recherche 5034, Centre National de la Recherche Scientifique,
Université de Pau et des Pays de l’Adour, Pau
4
Department of Plant Biochemistry, Faculty of Biology, University of Warsaw, Warsaw
2
Received September 28, 2005; accepted April 25, 2006; published online March 20, 2007
© Springer-Verlag 2007
Summary. The toxicity of heavy metals (Cd, Zn, and Pb) was assessed
by in vivo observations of their effect on cytoplasmic streaming in
Allium cepa L. bulb scale epidermal cells. On the basis of our results, the
order of toxicity of the studied cations is Zn Pb Cd. The difference
in toxicity between cadmium and lead was found to be very large. When
cytoplasmic streaming was assessed, this difference was threefold. When
the total content of cadmium and lead (determined by inductively coupled plasma mass spectrometry) was the criterion, the difference in toxicity was 15-fold. Fractionation of the tissue and enzymatic digestion of
the cells revealed that the largest proportion of cadmium was located in
the cell walls (56%), whereas almost all of the lead (97.6%) was accumulated in an insoluble form. The speciation of water-soluble Pb and Cd
fractions is discussed on the basis of analysis by capillary zone electrophoresis interfaced with inductively coupled plasma mass spectrometry of water extracts from epidermal cells. Lead and cadmium appeared
to be bound mainly to salts, which explains their toxicity. Cadmium was
complexed (detoxified) by organic acids, while thiols were the metalcomplexing species for lead. Histidine formed complexes with both
cadmium and lead. Ultrastructural analyses showed that lead was
encapsulated in small vesicles in the cytoplasm. Fluorescence studies of
the endoplasmic reticulum (ER) revealed that it underwent extensive
fragmentation under the influence of lead, with numerous ER vesicles
appearing in the cells. In other words, the lead deposits in the cytoplasm
were contained in vesicles arising from fragmentation of the ER. These
observations indicate that epidermal cells have a rapid and effective
mechanism for detoxifying lead involving the ER, and this may be one
of the mechanisms accounting for the lower toxicity of lead in comparison with cadmium. The suitability of Allium cepa bulb scale epidermal
cells for use in ecotoxicological studies is also discussed. Step-by-step
directions for this test are given.
* Correspondence and reprints: Department of Ecotoxicology, Institute
of Experimental Plant Biology, Faculty of Biology, University of
Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.
E-mail: [email protected]
Keywords: Allium cepa; Epidermis; Cytoplasmic streaming; Endoplasmic reticulum; 3,3-Dihexyloxacarbocyanine iodide; Inductively coupled
plasma mass spectrometry.
Abbreviations: CZE-ICP-MS capillary zone electrophoresis interfaced
with inductively coupled plasma mass spectrometry; DiOC6 3,3-dihexyloxacarbocyanine iodide; ER endoplasmic reticulum; ICP-MS inductively
coupled plasma mass spectrometry.
Introduction
The toxicity of nonessential elements, such as cadmium
and lead, has been widely studied and discussed in recent
years (Hall 2002), prompted mainly by the steadily increasing pollution of the environment with these metals.
Plants have mechanisms that effectively protect them
from the stress induced by heavy metals. The many intrinsic tolerance mechanisms described to date allow
plants to grow and develop in environments rich in these
metals (Ernst 1998, Hall 2002, Wierzbicka and Rostański
2002).
The fact that heavy metals are channeled into the food
chain by plants (Wierzbicka and Antosiewicz 1993)
makes the exact determination of their toxicity particularly important. In ecotoxicology, the degree of toxicity of
a given compound is inextricably associated with the concept of toxic dose and represents the degree of hazard and
risk that it poses to living systems (Walker et al. 2002).
Lethal dose (LD or LD50) is a parameter commonly
used to assess toxicity. It also often pertains to effects other than death, e.g., it can be the concentration or dose that
causes a measurable effect in 50% of a population (Walker
100
et al. 2002). Tests used in assessing pollution include the
pollen tube growth test, the seedling growth test, and tests
on cell suspensions, e.g., of Galium mollugo (Galium cell
culture test) (Kristen 1997). The Allium cepa test is also
frequently used (Wierzbicka 1988, 1999a), as is the root
growth test (Wierzbicka and Pielichowska 2004).
In this study, we used a more precise method of determining the degree of toxicity of heavy metals by combining modern analytical techniques and microscopic methods
(electron and confocal microscopy).
Modern analytical techniques, such as inductively coupled plasma mass spectrometry (ICP-MS), are used for
the determination of trace levels of elements in biological
tissues. The high sensitivity of this method enables quantification of metals not only in whole plants but also in
specific parts, tissues, and individual cells. More complex
hyphenated systems involving the coupling of high-performance separation techniques with ICP-MS detection,
e.g., high-performance liquid chromatography ICP-MS or
capillary zone electrophoresis interfaced with ICP-MS
(CZE-ICP-MS), are able to provide species-selective information on individual metal compounds involved in biochemical processes (Szpunar et al. 2003).
The objective of our study was thus to precisely assess
the degree of toxicity of cadmium and lead at the basic organizational level of plants, the cellular level.
In all of the experiments, we paid particular attention to
the doses of metals used. In ecotoxicological tests, it is always very important to use doses of the studied substance
that are not lethal but that are sufficiently high to enable
the observation and characterization of a specific reaction
of the biological system to the tested stressor. In the first
part of the study, doses of metals in equal concentration
ranges (chemical doses) were used. This is the most common approach in such studies. In the second part of our
study, however, doses causing comparable physiological
reactions of the cells were used. These were, therefore, biological doses, differing in concentration but evoking similar metabolic reactions of the cells. These doses were
adjusted to the differing degrees of toxicity of the particular metals. A premise of our study was to administer the
metals in the lowest possible concentrations, so as to
catch the first defensive reaction of the cells. For example,
when using sublethal doses of the studied factors, the
changes observed in the ultrastructure of cell organelles
are often destructive ones that appear in every stressful
situation regardless of the causative factor. In our study,
our aim was to treat the cells with a concentration of the
metal that made it possible to capture the first changes
that appear.
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
Material and methods
The experiments were performed on live bulb scale epidermal cells of
Allium cepa L. var. Bl/ońska. Fragments of the epidermis were stripped
from the inner side of the onion scale with tweezers and placed in the
appropriate solution.
Comparison of toxicities of Cd, Zn and Pb
To determine the degree of toxicity of lead, cadmium and zinc, solutions
of Pb(NO3)2, Cd(NO3)2, and Zn(NO3)2 were used in concentrations ranging from 1 to 100000 mg/dm3.
Tissue fragments (approximately 0.3 by 0.3 cm) were submerged in
solutions of the particular metals or in deionized water (control) and incubated for 4 h. Observations were made under a phase-contrast light
microscope. The number of cells in which cytoplasmic streaming was
visible was counted in six fields of vision (magnification, 400) in each
tissue fragment for each experimental combination using a Nikon Optiphot-2 microscope. Next, fragments of the epidermis from each experimental combination (i.e., from every concentration of each metal salt)
were transferred to deionized water for 48 h (postincubation) to determine if the changes were reversible. A total of 96 fragments of epidermis
were examined in this way.
Determination of biological doses
In order to determine the concentrations of metals causing similar metabolic reactions, solutions of Pb(NO3)2, Cd(NO3)2, and Zn(NO3)2 in 4%
sucrose (to ensure proper osmotic conditions) were used in an initial
concentration range of 0.001 to 100 g/dm3. Next, the range was narrowed
appropriately for each metal (2–25 mg/dm3). The duration of incubation
in metal salts was 24 h, after which the epidermis was transferred to an
aqueous solution of 4% sucrose for 24 h (postincubation). Cytoplasmic
streaming was again the criterion for assessing toxicity.
Measurement of the number of cells exhibiting cytoplasmic streaming
was conducted on 12 fragments of epidermis, 60 cells each (for all selected concentrations of the metals). Similar observations were made on
control cells (maintained in a 4% sucrose solution without added heavy
metals). A total of 300 epidermal fragments was examined.
Concentration and chemical forms of cadmium and lead in cells
On the basis of the above experiments, the following concentrations
were chosen for further study (biological doses): 20 mg of Pb(NO3)2
per dm3 (60.4 M) and 5 mg of Cd(NO3)2 per dm3 (21.1 M) (24 h of
incubation and 3 h postincubation). These concentrations caused similar
metabolic reactions in cells. Epidermal fragments treated in this way
were then subjected to chemical analysis by ICP-MS and by CZE-ICPMS. The analyses were performed in triplicate.
Standard solutions of the studied metal and ligand were prepared by
weighing an appropriate sample of the metal salt (10 g/ml) and powdered ligand to obtain a metal-to-ligand ratio of 4 : 1. Both samples were
then dissolved in 10 mM sodium acetate buffer (pH 7.4).
Epidermal cells were prepared for analysis as follows.
Extract 1: 1.0 g of an epidermal lyophilizate was treated with liquid
nitrogen for 15 min, after which it was ground in a mortar. The sample
was then transferred to a glass vessel, 2 ml of 10 mM sodium acetate
(pH 7.4) was added and the mixture was magnetically stirred for 1 h at
room temperature. The sample was then centrifuged at 15000 rpm (MPW120 centrifuge; MPW Med. Instruments, Warsaw, Poland) for 15 min. The
supernatant was filtered through a 0.45 m pore size filter (the first two
drops were discarded) and used for analysis.
Extract 2: The pellet from extract 1 was transferred to a glass vessel to
which 2 ml of sodium acetate (pH 7.4) containing a 5% solution of driselase (a multicomponent enzyme preparation from Basidiomycetes sp.
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
used in the isolation of protoplasts, containing cellulase, pectinase, laminarase, xylanase and amylase) was added, and magnetically stirred for
1 h. The mixture was left for 24 h and then centrifuged at 15000 rpm
(MPW-120) for 15 min. The supernatant was filtered through a 0.45 m
pore size filter (the first two drops were discarded) and used for analysis.
The analysis was carried out on an Agilent 3D CZE apparatus
(Agilent Technologies, Waldbronn, Federal Republic of Germany), using 90 cm long fused-silica capillaries with an internal diameter of
75 m. The separated species were detected by ICP-MS using an HP
Agilent 7500 (Yokogawa Analytical Systems, Hachioji, Tokyo, Japan)
fitted with an MCN-100 nebulizer (Cetac, Omaha, Nebr., U.S.A.).
Analysis was conducted under the following conditions: electrolyte,
20 mM Tris-HCl (pH 7.4); sheath liquid, 5 mM HNO3-NH4 (pH 7.4)
plus 20 ppb Ge; voltage, 25 kV (20 A). The isotopes 114Cd, 111Cd,
207
Pb, 208Pb, and 72Ge were monitored.
The total analysis was performed by ICP-MS using an HP Agilent 7500
instrument as follows. After adding 1 ml of demineralized water, samples of epidermis were centrifuged at 200 g. The supernatant was decanted and 1 ml of water was again added to the pellet; they were mixed
and centrifuged at a speed at which the efficiency of density fractionation was about 90%. The remaining pellet was lyophilized and then mineralized in nitric acid. Next, the solution was quantitatively transferred to
a volumetric flask (volume, 10 ml). The solution prepared in this way
was used to determine the total metal content, which was then expressed
in relation to dry matter. The total metal contents of extracts 1 and 2
were determined.
101
wavelength of 600 nm (green light). Several tens of cells from each of 15
fragments of epidermis were viewed per metal.
Statistical analysis
The results are presented as arithmetic means plus standard deviation.
All of the data and calculations were analyzed by Microsoft Excel. Statistical significance was tested by Student’s t test for 0.05.
Results
Comparison of toxicities of Cd, Zn, and Pb
Microscopic observations of live A. cepa epidermal cells
treated with various doses of cadmium, zinc, and lead salts
made it possible to follow the cells’ reactions to a given
compound. After 4 h of treatment, as the metal (Cd, Zn,
and Pb) concentration increased, the number of cells exhibiting cytoplasmic streaming decreased. The strongest
inhibitory effect was seen with cadmium, a moderate one
with zinc, and the weakest with lead (Fig. 1a).
Ultrastructural detection of lead in cells
Lead was located by a transmission electron microscopy after treating
the epidermal cells for 24 h with 20 mg of Pb(NO3)2 per dm3 (60.4 M)
in 4% sucrose and then postincubating them for 3 h in 4% sucrose.
Epidermal cells incubated in 4% sucrose with no lead salts served as
controls.
Tissue fragments prepared in this way were fixed for 1 h in 2.5% glutaraldehyde. They were then washed (3 times 10 min) in 0.1 M cacodylate buffer containing 1% caffeine, pH 7.3 (according to Dawson et al.
[1969]) and left overnight at room temperature in this buffer. Fixation
was continued the next day by treating the material with 1% OsO4 for
1 h. The samples were then dehydrated in a graded alcohol series and
embedded in epoxide resin (Epon-Spurr). Ultrathin sections were analyzed in an electron microscope without staining, and again after staining in a saturated solution of uranyl acetate (30 min) and with lead
citrate (30 min) (Reynolds 1963). Viewing of unstained sections permitted identification of lead deposits, as they are electron dense. Subsequent
staining of the same sections (using a standard method) enabled ultrastructural analysis since the cell organelles became clearly visible
(Wierzbicka 1987a, b; Antosiewicz and Wierzbicka 1999).
Fluorescence staining of intracellular membranes
DiOC6 (3,3-dihexyloxacarbocyanine iodide) has a positive charge
which allows it to react with cellular membranes in a dose-dependent
manner.
Epidermal fragments were incubated for 24 h in solutions of metals
(in 4% sucrose): 20 mg of Pb(NO3)2 per dm3 (60.4 M) or 5 mg of
Cd(NO3)2 per dm3 (21.1 M). After incubation, the A. cepa epidermal
cells were rinsed in deionized water and stained for 2 min in a solution
of 50 g of DiOC6 per ml (modified after Terasaki et al. [1984]). After
incubation in the stain, the epidermal sections were rinsed well in deionized water and viewed under a Zeiss fluorescence confocal microscope
(Axiovert 100M), equipped with an Fset filter and argon laser, using
40 and 63 lenses (both with immersion oil). The image was saved by
the LSM 510 (laser scanning microscope) computer program. DiOC6 has
an absorption maximum at 483 nm (blue light) and emits light with a
Fig. 1 a, b. Percentages of A. cepa epidermal cells with visible cytoplasmic streaming after treatment with heavy metals (lead, zinc, cadmium)
in concentrations ranging from 0.001 to 100 g/dm3. a 4 h incubation in
metal ion solution. b 4 h incubation in metal ion solution plus 48 h in
water (postincubation)
102
These observations were confirmed after the epidermal
fragments had been incubated for 48 h in water. The number of cells with visible cytoplasmic streaming decreased,
especially after exposure to cadmium and zinc (Fig. 1b).
These results indicate that cadmium has the greatest toxic
effect on epidermal cells, and that this effect is much
stronger than that of zinc or lead.
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
Comparison of metabolic reactions of cells to Cd,
Zn, and Pb
Further experiments were conducted to compare the metabolic reaction of cells to Cd, Zn, and Pb in more detail.
The results are presented in Fig. 2. It was found that the
concentrations of these metals that have similar degrees
of toxicity to cells were 20 mg of Pb(NO3)2 per dm3
(60.4 M) (Fig. 2a), 5 mg of Cd(NO3)2 per dm3 (21.1 M)
(Fig. 2b), and 15 mg of Zn(NO3)2 per dm3 (79.3 M)
(Fig. 2c). In all of these cases, about 80% of the cells had
visible cytoplasmic streaming after 24 h of treatment. This
number remained constant during the subsequent 24 h of
postincubation (in relation to the control, this difference
was not significant). These doses are also limiting doses
(the highest possible), since even a small increase in the
concentration of the metal ion caused a drastic decline in
the number of cells with visible cytoplasmic streaming,
especially during postincubation (a statistically significant
difference in relation to the control) (Fig. 2).
Our experiment made it possible to compare the toxicities of the studied metals by determining the dose causing
a similar metabolic reaction in the cells. Assuming that
concentrations expressed as micromolar concentrations are
the most comparable, the toxicities of these metals were in
the following order: Cd >> Pb Zn. The difference in toxicity was 3-fold between cadmium and lead (Cd >> Pb) and
4-fold between cadmium and zinc (Cd Zn).
Concentration of cadmium and lead in cells
Fig. 2. Percentages of A. cepa epidermal cells with visible cytoplasmic
streaming after incubation in solution of lead nitrate (a), cadmium nitrate
(b), or zinc nitrate (c) for 24 h () and after additional 24 h in water (postincubation) (■). Concentrations of 20 mg of Pb(NO3)2, 5 mg of Cd(NO3)2,
and 15 mg of Zn(NO3)2 per dm3 were chosen for further use (biological
doses). Statistically significant differences are marked with an asterisk
Further experiments were performed only on cadmium
and lead using biological doses, i.e., incubation for 24 h in
5 mg of Cd(NO3)2 per dm3 (21.1 M) and 20 mg of
Pb(NO3)2 per dm3 (60.4 M).
The total concentration of cadmium in cells was
0.5753 mg/kg of dry matter (5.1 M), whereas that of
lead was 16.5356 mg/kg of dry matter (78.7 M). The
levels of these metals in the aqueous fractions of the cells
were, however, many times lower. The amount of cadmium in the aqueous fraction was 0.0054 mg/kg of dry
matter (0.05 M), while that of lead was higher and
equaled 0.373 mg/kg of dry matter (1.8 M). The total
cadmium concentration in control cells was 0.0137 mg/kg
of dry matter; that of lead, 0.3915 mg/kg of dry matter.
The amount of cadmium and lead in cell walls was also
determined. The tissue was fractionated and enzymatically
digested (with driselase), making it possible to determine
the size of the cadmium pool released from cell walls. The
cell walls contained 0.3221 mg of cadmium per kg of dry
matter (2.9 M) and 0.3621 mg of lead per kg of dry matter
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
(1.7 M). The remaining part of the metals was in water-insoluble forms. The cells contained 0.2478 mg of insoluble
cadmium per kg of dry matter (2.2 M) and 15.8005 mg of
insoluble lead per kg of dry matter (75.2 M). These results
allow an approximation of the distribution of both metals in
cells: cadmium, total concentration of 0.5753 mg/kg of dry
matter (5.1 M), of which 56% was in cell walls, 1% in the
aqueous fraction, and 43% in water-insoluble form; lead, total concentration of 16.5356 mg/kg of dry matter (78.7 M),
of which 2.2% was in cell walls, 2.2% in the aqueous fraction, and 95.6% in water-insoluble form.
103
These results also enable an exact evaluation of the degree
of toxicity of cadmium and lead. A comparable reaction in
epidermal cells required 28 times more lead than cadmium
on a weight basis (milligram) and 15 times more if considered in molar terms. The difference in toxicity between cadmium and lead is, therefore, very high, at least 15-fold.
Chemical form of lead and cadmium – speciation analysis
The aqueous fraction of epidermal cells (Cd, 0.05 M; Pb,
1.8 M) contains metals in water-soluble chemical forms
Fig. 3. CZE-ICP-MS electropherogram profiles of the water extracts of Cd-containing
cells (bold line) and of cadmium nitrate
(dotted line) (a) and of Pb-containing cells
(bold line) and lead nitrate (dotted line) (b).
a A aqueous cadmium salts, B Cd and malic
acid and/or citric acid, C Cd and histidine. b
A aqueous lead salts, B Pb and histidine, Pb
and glutamic acid and/or glutathione, C not
identified
104
that may comprise various chemical species. This is the
fraction in which metals in the chemical forms responsible
for toxic effects on the cell can be expected to be found, as
well as those in forms indicative of cellular detoxification
processes (Hall 2002). Speciation analysis of water-extracted lyophilized epidermal cells provides the means to determine the chemical forms in which the metals are present.
Speciation analysis of lead and cadmium (extract 1) was
performed by CZE-ICP-MS (Fig. 3). Three peaks of cadmium-containing eluting compounds could be observed in
the CZE-ICP-MS electropherogram profiles of the water
extracts of Cd-containing cells (Fig. 3a). After comparison
with the electropherogram of standards for metal complexes and salts, the first and main peak (7 min) seemed to
correspond to aqueous cadmium salts (low binding compounds), whereas the second one (8 min) could be complexes with malic and/or citric acids, and the third (9 min)
with histidine. Three peaks were also detected in the analysis of Pb-containing cells (Fig. 3b). After comparison with
cadmium, it is likely that the first peak (8.5 min) corresponded to aqueous lead salts, whereas the second (10 min)
could be complexes with histidine, glutamic acid, and/or
glutathione. The third, unidentified peak (22–26 min)
seemed to be specific for lead because it did not correspond
to any peak in the cadmium electropherogram.
Ultrastructural detection of lead in cells
The distribution of cadmium and lead in the particular
cellular compartments differed. The largest proportion of
cadmium (56%) was accumulated in cell walls and constituted the nontoxic pool of this element. The largest (97.6%)
fraction of lead, however, was in water-insoluble form,
which makes it nontoxic. Electron microscopy enabled
precise localization of the water-insoluble form of lead.
The ultrastructure of control cells was typical for plant
cells (Fig. 4). Cell nuclei (Fig. 4) with clearly visible euand heterochromatin and a double-layered nuclear membrane, mitochondria with well-defined cristae (Figs. 5 and
8), Golgi apparatus with characteristic dictyosome vesicles
(Fig. 4), and endoplasmic reticulum (ER) (Fig. 4), etc.,
were observed.
Electron microscopy revealed that the largest amounts
of lead were in the cytoplasm in the form of small, electron-dense deposits (Figs. 5–7). They were usually seen inside small vesicles (Figs. 5 and 7). Smaller amounts were
found in cell walls (Figs. 5 and 6). Lead deposits were also
found in the space between the cell wall and the plasma
membrane (Fig. 8). These deposits were in the apexes of
tubular protrusions of the plasma membrane (plasmo-
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
tubules) (Fig. 8). Their presence indicates that lead was
being transported between the cytoplasm and plasma membrane. Lead was also found to be present in plasmodesmata
(Fig. 7), which points to the possibility of transport between
neighboring cells. Viewing unstained sections (Fig. 8) permitted identification of lead deposits as they are electrondense.
No destructive changes in the ultrastructure of cellular
organelles (mitochondria, nucleus, and proplastids) were
found after treatment with selected doses of lead (Figs. 5–8)
in comparison to controls (Fig. 4). This confirms the basic
premise of this study that the applied dose did not disrupt
normal cell metabolism.
It can be concluded that the ultrastructural study made
it possible to locate the largest pool of lead in epidermal
cells. This pool was in the cytoplasm in the form of small
but encapsulated deposits. Elemental analysis showed that
as much as 97.6% of the lead was in water-insoluble form.
Therefore, the majority of the lead was located in the cytoplasm in small vesicles. If this is the case, then a significant increase in the number of tiny vesicles should be
expected in cells subjected to the influence of lead. We
tested this hypothesis by fluorescence methods.
Influence of cadmium and lead on intracellular
membrane system
The ER constitutes the largest membrane system in a cell.
Following in vivo fluorescence staining (DiOC6), it was
possible to view the intracellular system of membranes in
control cells and in cells treated with lead and cadmium.
The system of ER membranes visible in control cells
(Fig. 9A, B) corresponded to published data, which describes ER membranes as a network formed by interconnected tubules and cisternae (Loewy et al. 1991, Kl/ yszejko-Stefanowicz 1998, Alberts et al. 1999, Woźny et al.
2001). In control cells, the ER was distributed close to the
plasma membrane. A large vacuole was in the central part
of the cell (Fig. 9B). The ER network was polygonal, with
the nodes typically branched in three directions. Cisternae
were usually located in tubule nodes (Fig. 9A). ER tubules
dominated in the majority of control cells (Fig. 9A), although in some cells, the number of tubules was close to
that of cisternae.
The system of intracellular membranes in epidermal cells
treated with lead salts was visible in the form of numerous
small vesicles (Fig. 10A). The system of tubules and cisternae typical of the ER was usually not seen. This observation
suggests that the membrane system disintegrated into small
vesicles. This result supports the hypothesis that the number
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
105
Figs. 4–8. Electron micrographs of Allium cepa L. storage bulb scale epidermis fixed in glutaraldehyde and OsO4
Fig. 4. Control. Cytoplasm with visible organelles: Golgi apparatus (GA), endoplasmic reticulum (ER), mitochondria (M), nucleus (N) with visible
double membrane (PN), plasma membrane (P), cytoplasm (C), cell wall (CW). 10,000, stained section
Figs. 5–8. Distribution of lead in A. cepa epidermal cells after 24 h incubation in an aqueous solution of Pb(NO3)2 at a concentration of 20 mg/dm3
Fig. 5. Lead deposits in vesicles in the cytoplasm (C) (arrow) and cell wall (CW) (arrowheads). 14,000, unstained section
Fig. 6. Lead deposits (electron-dense areas) in the cytoplasm (C) (arrows) and cell wall (CW) (arrowheads). 14,000, unstained section
Fig. 7. Lead deposits in plasmodesmata (Pl) and cytoplasm (C) (arrow). 18,000, stained section
Fig. 8. Lead deposits over the plasma membrane (P) and the apexes of tubular protrusions of the plasma membrane (plasmotubules, Pt) (arrows).
8000, unstained section
of small vesicles in the cytoplasm, in which lead may be
stored, rises markedly in response to lead (Fig. 10A, B).
Vesicles were not observed in cells incubated with
cadmium. ER cisternae (Fig. 11A, B) were, however,
much more numerous than in control cells. ER tubules
were less numerous and did not form the regular network seen in the controls. The images shown in Figs. 9–11
are representative for the studied combinations (sev-
106
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
Figs. 9–11. ER in epidermal cells of A. cepa storage bulb scale stained with DiOC6. Panels A and B are optical cross sections
Fig. 9 A, B. Control cell. The ER forms a network composed of two elements: flat cisternae (asterisks) and thin ER tubules (arrows). Mitochondria
(M) are also visible. 520
Fig. 10 A, B. Cell incubated for 24 h in a solution of 20 mg Pb(NO3)2 per dm3. The ER is visible in the form of vesicles (arrowheads). 520
Fig. 11 A, B. Cell incubated for 24 h in a solution of 5 mg Cd(NO3)2 per dm3. The ER is visible mainly in the form of numerous cisternae. 520
eral tens of cells in each of 15 epidermal fragments
were observed). The most peripheral parts of the cell
where the ER was visible are shown. A large vacuole
(seemingly empty) was in the central part of the cell and
the ER formed a thin peripheral strip (Figs. 9B, 10B,
and 11B).
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
Discussion
Comparison of Cd, Zn, and Pb toxicity
The most commonly accepted toxicity sequence of metals is
Hg Cu Cd Ag Pb Zn (Shaw 1990). This order
may, however, be different for different organisms. Conflicting information on the toxicity of the three metals studied in
this experiment (Cd, Zn, and Pb) can be found in the literature. Niober and Richardson (1980) published a sequence of
heavy metals according to their toxicity to various organisms. Cadmium was found to be more toxic to algae than
zinc (Cd Zn). For fungi, the order of the ions was Cd Pb Zn; for barley, Pb Cd Zn; flatworms, Cd Zn
Pb; segmented worms, Zn Pb Cd; fish, Pb Cd Zn; and mammals (rats, mice, rabbits), Cd Pb Zn.
The experiments presented in this study have resulted
in the following classification of the metals in terms of
their toxicity: Cd Pb Zn, when molar concentrations
are used. In this evaluation, the way in which concentrations are expressed is very important, as Ivanov et al.
(2003) noted. Those authors investigated the effect of various metals on the growth of Zea mays L. roots and gave
two series of metal toxicity depending on the form in
which the metal concentration is expressed. When it was
given as grams per liter, the metals were in the following
order: Cd Zn Pb. When they were given in molar
concentrations, the toxicity of lead and zinc were found to
be very similar: Cd Zn Pb.
Similarly, the results of our study give two possible series of metal toxicities, depending on how their concentrations are expressed. When given in micrograms per cubic
decimeter (i.e., grams per liter), the order of toxicity is
Cd >> Zn Pb, since to achieve comparable metabolic
cellular reactions, it was necessary to use 5 mg of
Cd(NO3)2 per dm3, 15 mg of Zn(NO3)2 per dm3, and
20 mg of Pb(NO3)2 per dm3. If, however, the concentrations of these metals are expressed in moles, the series is
Cd >> Pb Zn, since to obtain comparable metabolic cellular reactions, it was necessary to use 21 M Cd(NO3)2,
60 M Pb(NO3)2, and 80 M Zn(NO3)2. The reason for
such marked differences in the two series lies in the large
differences in the molecular weights of these molecules. It
should be noted that it is much more appropriate to compare concentrations expressed in moles.
Our study showed that cadmium was three times more
toxic than lead because to cause a similar metabolic reaction, it was necessary to use a dose of cadmium three
times smaller than that of lead (21 and 60 M, respectively). After treatment at these doses, the amounts of
107
these metals in tissues differed even more, since the total
amount of cadmium was 15 times smaller than that of
lead (5 and 78 M, respectively). This points to a 15-fold
difference in toxicity between cadmium and lead.
In our study, we compared the toxicity of two nonnutritive metals (cadmium and lead) with zinc, a trace element,
classified as such because it is necessary in plant metabolism. Excesses of trace elements, e.g., zinc or copper, are
known to be harmful to plants (Mengel and Kirkby 1978).
In contrast to many reports, our study showed that zinc is
less toxic than lead and much less toxic (4 times) than
cadmium. This result becomes understandable in light of
the fact that an excess of trace elements has “always” accompanied plants on soils of various composition, so they
could have been expected to evolve effective and widespread defense mechanisms at both cellular and systemic
levels. In contrast, elements such as cadmium and lead
have been appearing in excessive amounts in the environment for only the last hundred years or so, as the result of
human activity. This is too short a time to evolve commonly occurring systems of protection from these elements, hence their greater toxicity.
Distribution of cadmium and lead in plant cells
CZE-ICP-MS analysis indicated that lead and cadmium in
water extracts of A. cepa epidermal cells are mainly bound
to salts, which could explain their toxicity, and to a lesser
extent, to the usual natural organic ligands present in the
cells (organic acids, amino acids, and thiols). The most interesting information is the presence of an unknown compound in the electropherogram of the water extract of leadcontaining cells (third peak [22–26 min] in Fig. 3b). It is
possible that this broad peak eluting late in the electropherogram corresponds to inorganic (e.g., salts) or organic (e.g.,
pectates) negatively charged compounds agglomerated with
lead, which could be precursor compounds involved in the
formation of the precipitated lead observed in the vesicles.
The chemical analysis of lead and cadmium in fractions
of A. cepa epidermal cells was performed by ICP-MS,
providing data on their distribution in cells. It was shown
that lead was accumulated mainly in a water-insoluble
form (97% of the total amount of lead). Cadmium, on the
other hand, was accumulated mainly in cell walls (56% of
the total amount of cadmium). This indicates completely
different distributions of the metals in the cells. It may
also signify completely different cellular strategies in relation to these two metals.
Electron microscopy was used to more precisely locate
lead in epidermal cells, in particular, the water-insoluble
108
97% of the total pool. Transmission electron microscopy
is frequently used in studies locating heavy metals
(Wierzbicka 1987a, b, 1995; Neumann et al. 1995; Antosiewicz and Wierzbicka 1999; Glińska and Gabara 2002;
Baranowska-Morek and Wierzbicka 2004). The advantage
of this method is that it makes it possible to precisely
determine the distribution at the ultrastructural level.
Antosiewicz and Wierzbicka (1999) showed that the electron-dense deposits contain lead (X-ray microanalysis) and
that lead does not wash out of plant tissues during fixation
and processing for electron microscopy, making this
method well suited for ultrastructural observations of lead,
especially of its water-insoluble forms.
Our ultrastructural studies showed that lead deposits are
found in small vesicles in the cytoplasm of onion epidermal cells. Their number and the frequency with which we
observed them indicated that this is where the largest pool
of lead is found. In other words, this is where the 97%
pool detected by ICP-MS is located.
Smaller deposits of lead were also found in the cell wall.
These observations are in agreement with published reports
that name the cell wall as the main site of accumulation, not
only of lead but also of other metals (Neumann et al. 1995,
Wierzbicka 1998, MacFarlane and Burchett 2000, Jarvis
and Leung 2001, Seregin and Ivanov 2001, Hall 2002,
Heumann 2002, Baranowska-Morek and Wierzbicka 2004).
Moreover, lead was found in plasmodesmata. Jarvis and
Leung (2001) also described small deposits of this metal
in plasmodesmata of Chamaecytisus palmensis, while
Heumann (2002) reported the presence of zinc in them in
Armeria maritima. These observations suggest that heavy
metals are transported between cells.
The presence of lead in the space between the plasma
membrane and cell wall and the presence of plasmotubules
point to the existence of active defense mechanisms in
cells. Plasmotubules were first described by Harris et al.
(1982). Three years later, Harris and Chaffey (1985) described the role of plasmotubules in the transport of dissolved substances between xylem and phloem cells from
the apoplast to the symplast. Wierzbicka (1998) reported
for the first time that plasmotubules play a role in moving
lead from the symplast to the apoplast in A. cepa root cells.
Intracellular membrane network after treatment
with Cd and Pb
The role of the ER in plant cells contaminated with heavy
metals has not yet been unequivocally determined. It is
known that the ER can be a site of lead accumulation in
cells (Woźny et al. 1982).
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
The results of our study point to a very important role of
the ER in detoxification of lead. Our observations indicate
that it disintegrates into small vesicles containing lead deposits. The intracellular membrane system appeared very
different following cadmium treatment. Numerous cisternae were observed in the cells, tubules were less numerous
and disrupted, and they did not form a regular network.
It seems that the difference in the reaction of cells to Pb
and Cd stems from their ability to inactivate a major part
of the lead pool (95%) in the form of deposits enclosed
in vesicles in the cytoplasm. The concentration of lead in
insoluble deposits was 75.2 M, while that of cadmium
was only 2.2 M. The concentrations of both metals in the
remaining parts of the cell (cell wall and aqueous fraction)
were, however, on a similar level and equaled about 3 M.
Isolating lead from the cytoplasm with a membrane
seems to be one of the basic detoxification strategies that
plants are capable of. It therefore seems that the basic difference in the toxicity of the two metals lies in the ability
of the protoplast to react to lead and cadmium. Both of
these metals enter the cells, although lead does so to a
higher degree. In the symplast, lead is rapidly isolated by
plasma membranes from the rest of the cell and is expelled to the apoplast by plasmotubules (Wierzbicka
1988). Very rapid and effective detoxification, therefore,
explains the much lower toxicity of lead compared with
that of cadmium.
Chemical form of cadmium and lead
Speciation analysis of the aqueous fraction of A. cepa epidermal cells indicated the presence of further detoxification pathways. Ligands forming complexes with cadmium
and lead were found. Forming complexes is one of the basic mechanisms of heavy-metal tolerance and detoxification (Hall 2002).
CZE-ICP-MS showed that both metals (Cd and Pb) occur mainly as water-soluble salts. In this chemical form,
they are toxic and poison epidermal cells. The presence of
organic compounds was also found. These were cadmium
complexes with organic acids (Cd plus malic and/or citric
acid), lead complexes with organic acids and thiols (Pb
plus glutamic acid and/or glutathione), and cadmium and
lead complexes with histidine (Fig. 3).
The metals were made harmless through chemical reactions and formation of complexes with organic acids. It is
known that organic acids, such as citric and malic acids,
play an important role as heavy-metal ligands and a major
one in the detoxification of metals such as zinc (Hall
2002). The role of histidine as a metal-binding ligand is
M. H. Wierzbicka et al.: Cadmium and lead in onion cells
also known, but mainly as a complexing agent for nickel.
Glutathione is a substrate for the synthesis of phytochelatins, which are metal-complexing peptides. They
play a key role in heavy-metal tolerance, particularly to
cadmium (Hall 2002).
In this study, we showed for the first time that histidine
is a ligand for cadmium and lead in the epidermal cells of
A. cepa. Organic acids (malic and/or citric) form complexes only with cadmium, whereas thiols are the ligands
for lead (glutamic acid and/or glutathione). Interestingly,
phytochelatins were not found.
As mentioned earlier, an interesting result is the presence of an unknown peak in the electropherogram of the
water extract of lead-containing cells (third peak
[22–26 min] in Fig. 3b) that could correspond to inorganic
(e.g., salts) or organic (e.g., pectates) negatively charged
compounds agglomerated with lead. These may be precursor compounds involved in the formation of the precipitated lead observed in the vesicles.
A comparison of our results with known heavy-metal
detoxification mechanisms (see review by Hall 2002)
shows that our study demonstrated for the first time that
the ER plays a role in the detoxification of lead. This
study also identified new complexing agents for cadmium
and lead and, therefore, chemical species participating in
the detoxification of these metals.
Use of A. cepa bulb scale epidermal cells
in ecotoxicological tests
One of the key and difficult matters in all types of studies
of the reaction of plants to heavy metals is the choice of an
appropriate test and the use of suitable doses in the experiments (Wierzbicka 1989, 1994, 1995, 1999b). The relationship between cytoplasmic streaming and the factors
studied by us in this study seems to be a very sensitive and
good tool for the evaluation of the dependence between a
stressor and the condition of the cell. Cytoplasmic streaming is the net outcome of many factors. The cytoskeleton,
the general energy level of the cell, and the physicochemical properties of the cytoplasm itself, all play a role
(Woźny et al. 2001, Kopcewicz and Lewak 2002). Observation of cytoplasmic streaming, therefore, makes it possible to register the reaction (of the action–reaction type) of
living cells to a stress factor and to chart the simple relationship of dose to stress. In our opinion, observations of
cytoplasmic streaming are a very good indicator for the assessment of the toxicity of a given substance.
The use of A. cepa bulb scale epidermal cells in ecotoxicological studies also seems very advantageous. This is a
109
single layer of uniform cells that are very easy to isolate.
These cells have already been used in numerous other
studies, e.g., of the dynamics, shape, and organization of
the ER (Quader and Schnepf 1986, Quader et al. 1987,
Lichtscheidl and Url 1990), cell wall properties (Wilson et
al. 2000), and the thermophysical and physicochemical
properties of a monolayer tissue (Ng et al. 2000, Ferrando
et al. 2002). The epidermis of A. cepa bulb scales was also
used in studies of plasmolysis (Lang-Pauluzzi 2000), crossmembrane transportation (Gens et al. 1996, Reuzeau et al.
1997), and the structure and dynamics of cell organelles
in vivo (Scott et al. 1999). To date, they have not been
used in ecotoxicological studies.
An important trait of the test used by us is its high sensitivity. It was possible to test the reaction of cells to the
range of concentrations that actually occurs in the environment. For example, the heavy-metal content of fodder
plants cultivated in unpolluted regions ranges from 0.05 to
0.5 mg of cadmium per dm3 and 30 mg of lead per dm3
(Kabata-Pendias and Pendias 1999). Phytotoxic concentrations of these elements in plants are 5–50 mg of Cd per
dm3 and 30 mg of Pb per dm3 (Kabata-Pendias and Pendias 1999). In the natural environment, when the geochemical background of the analyzed elements was 0.27 mg of
Cd per kg and 10.3 mg of Pb per kg, their content in, for
example, A. cepa plants from areas not subject to industrial
pressure was within 0.17–0.31 mg of Cd per kg and 14.1–
49.2 mg of Pb per kg (Michalak and Wierzbicka 1998).
The concentration range used in our study was therefore relatively low and comparable to environmental conditions.
It is worth noting that the metal doses used in this study
were chosen very precisely because they were selected according to the metabolic reaction of cells. In addition,
these were the lowest possible doses, that is, at borderline
levels that did not poison normal cell metabolism. As a result, it was possible to conduct an exact comparative
analysis in the range of naturally occurring concentrations. This is why we recommend using the epidermis test
for the study of other pollutants.
We suggest conducting the tests using the following procedure. The onion (A. cepa) bulbs should be of the same variety and stored under identical conditions. Ideally, they
should always be from the same grower. Incubate fragments
of the epidermis for 4 to 24 h in a solution of the studied
factor in 4% sucrose. Then conduct microscopic observations and count cells with visible cytoplasmic streaming.
The test should be conducted with a control. A phase-contrast light microscope is recommended. In order to obtain
statistically reliable results, observations of at least 360 cells
(counted in randomly selected fields of vision, e.g., 6) from
110
6 fragments of epidermis per experimental combination
should be made. We also suggest using the lowest possible
doses of the studied factors and to select the doses according
to the biological reaction of the cells. Cadmium nitrate at a
dose of 5 mg/dm3 in 4% sucrose for 24 h can be used as a
reference. This is the threshold dose above which cadmium
becomes too toxic. This is then a good reference to which
all other studied compounds can be compared. In this test,
the toxicity of the studied compounds is compared by using
those (molar) concentrations that cause identical metabolic
reactions in the cells.
We suggest that A. cepa bulb scale epidermal cells be
considered a model system for ecotoxicological tests.
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