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 Environmental Pollution 120 (2002) 779–786
www.elsevier.com/locate/envpol

Euglena gracilis as a model for the study of Cu2+ and Zn2+ toxicity
and accumulation in eukaryotic cells
Marcelo Einicker-Lamasa,b,*, Gustavo Antunes Meziana, Thiago Benevides Fernandesa,
Fabio Leandro S. Silvaa, Flávio Guerrac, Kildare Mirandac, Marcia Attiasc,
Mecia M. Oliveiraa
a
Laboratório de Biomembranas, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
b
Laboratório de Fı´sico-Quı´mica Biológica Aı´da Hassón Voloch, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
21949-900 Ilha do Fundão, Rio de Janeiro, RJ, Brazil
c
Laboratório de Ultraestrura Hertha Meyer, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil

Received 23 October 2001; accepted 28 February 2002

‘‘Capsule’’: The proto209 Euglena gracilis is an effective model for studies of copper and zinc toxicity and responses at the
sub-cellular level.

Abstract
We have observed the effect of copper and zinc on the biology of Euglena gracilis. The cells displayed different sensitivities to
these metals, as the apparent LC50 for Cu2+ was 0.22 mM, and for Zn2+ it was 0.88 mM. While Zn2+ was able to increase cell
proliferation even at 0.1 mM, the minimal CuCl2 concentration tested (0.02 mM) was sufficient to impair cell division. Higher
concentrations of these metals not only inhibited cell division in a concentration-dependent manner, but also interfered with the
metabolism of E. gracilis. A higher accumulation of proteins and lipids per cell was observed at the DI50 concentration for metal-
treated cells. These results suggest that the test concentration of both metals leads to a failure in completing cell division. Ultra-
structural analysis indicated a chloroplast disorganization in copper-treated cells, as well as the presence of electron dense granules
with different shapes and sizes inside vacuoles. Microanalysis of these granules indicated an accumulation of copper, thus suggest-
ing a detoxification role played by the vacuoles. These results indicate that E. gracilis is an efficient biological model for the study of
metal poisoning in eukaryotic cells. They also indicate that copper and zinc (copper being more poisonous) had an overall toxic
effect on E. gracilis and that part of the effect can be ascribed to defects in the structure of chloroplast membranes. # 2002 Elsevier
Science Ltd. All rights reserved.
Keywords: Euglena gracilis; Heavy metals; Pollution; Cu2+; Zn2+

1. Introduction cells constitute critical steps in cell survival. The study

Heavy metal pollution is a world-wide problem, due
to the industrial discharges. Considerable efforts have
therefore been made to identify polluted areas and
develop efficient methods for their quantitative assess-
ment (Anton et al., 2000; Carvan et al., 2000; Fernandez
et al., 2000; Girling et al., 2000).
In natural and polluted environments, the uptake
and handling of essential and toxic metals by different
* Corresponding author. Tel.: +55-21-2562-6520.
E-mail address: einicker@biof.ufrj.br (M. Einicker-Lamas).
0269-7491/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0269-7491(02)00170-7
of these reactions is very important in understanding
the essential metal metabolism as well as the cell
defense machinery involved during environmental
metal intoxication (Gingrich et al., 1984, 1988). Most
eukaryotic cells are able to control the internal con-
centration of metals that have a physiological role in
cell metabolism. These essential metals function as
catalysts for biochemical reactions, are stabilizers of
protein structures, and play a role in the maintenance
of the osmotic balance (Bruins et al., 2000). But the
metals without a physiological role, or even the essen-
tial metals in excess, are commonly accumulated,
780 M. Einicker-Lamas et al. / Environmental Pollution 120 (2002) 779–786
causing serious disturbances in vital metabolic path-
ways (Albergoni et al., 1980). Selective pressures from
a metal-containing environment have led to the devel-
opment of resistance systems to virtually all toxic
metals (Rouch et al., 1995).
Mercury, cadmium, zinc, aluminum and copper are
the principal heavy metal pollutants found in the envir-
onment, with mercury and cadmium being the most
serious toxicants in the aquatic environment, and
responsible for episodes of chronic poisoning in humans
(Jones et al., 1987; Moreira, 2001). Copper and zinc are
essential for the activity of several enzymatic systems,
and their concentration in the cells must be controlled
(Albergoni et al., 1980). Zinc deficiency in Euglena gra-
cilis has been shown to affect growth, morphology, cell
cycle and mitosis. These observations are best explained
by a role for zinc in gene regulation, through zinc-
dependent enzymes (Vallee, 1983). However, when the
external concentration of Zn2+ and Cu2+ is beyond a
limited value, it causes harmful effects.
The exposure of a variety of organisms to toxic
metals induces the synthesis of low molecular weight,
thiol-rich proteins called metallothioneins or chelatins
(Coppellotti, 1989). These proteins play a role in
detoxification due to their ability to sequester heavy
metals by binding them with clusters of thiolate bonds
(Hamer, 1986). This protects the cells by blocking the
linkage of the heavy metals to functionally important
sulfhydryl groups of cellular enzymes, proteins and
amino-acids (Wong and Klaassen, 1981, Bruins et al.,
2000). There are other proteins involved in cellular
protection against heavy metal intoxication like the
glutathione reductase, trypanothione reductase (Mon-
trichard et al., 1999) and the P-glycoprotein (Einicker-
Lamas et al., 1995).
E. gracilis, a freshwater protozoon, has been used as a
biological model in different studies due to its remark-
able metabolic plasticity. When grown in the presence
of light, it is green, autotrophic and photosynthetic;
when grown in the dark it is colorless and heterotrophic.
This interesting adaptability makes E. gracilis an excel-
lent model for research in eukaryotic cell biology (Lee-
dale, 1982; Price, 1989; Foltinova and Grones, 1997).
Our group had demonstrated the toxic effects of cad-
mium, which had been considered as a traditional non-
physiological metal, on E. gracilis membrane lipids
(Einicker-Lamas et al., 1996), pointing E. gracilis as a
possible bio-indicator in polluted areas.
In the present study we tested the effects of two physi-
ological metals, Cu2+ and Zn2+, on E. gracilis biology.
These two metals are frequently present in industrial
residues, and both are among the principal pollutants of
the watershed in the State of Rio de Janeiro, Brazil
(Dornelles, 1998; Moreira, 2001). We tested the effects of
such metals on the cell proliferation, protein metabo-
lism, lipid metabolism and ultrastructural damage in
order to evaluate the metabolical alterations during
Zn2+ and Cu2+ exposure.
2. Materials and methods
2.1. Cell cultures
E. gracilis wild strain, was a gift from Dr. Silvio Celso
G. Costa (Protozoology Department, Instituto Oswaldo
Cruz/RJ). Cells were grown at room temperature in a
composite medium under constant illumination as pre-
viously described (Einicker-Lamas et al., 1996). Ali-
quots of cell suspension were transferred to fresh
medium every three days for the different experiments.
In test cultures, copper was added as CuCl2 and zinc
was added as ZnCl2. Cells were counted in a hemocyt-
ometer, and Trypan blue was used to determine the cell
viability in the different experimental conditions. In all
experiments, cells were collected and processed after 72
h of growth.
2.2. Protein determination
Aliquots (1 ml) were taken from the cell cultures for
protein determination. Cells were centrifuged in a
Eppendorf Centrifuge 5415C at 14,000 rpm for 1 min
and the cells were washed three times with phosphate
buffered saline (PBS). To solubilize the proteins, 1.0 ml
0.1 N NaOH was added and an aliquot (100 ml) was
taken for protein determination. The protein content
was determined by the method of Bradford (1976),
using bovine serum albumin as standard.
2.3. Lipid extraction and quantification
The cultures were harvested in a IEC Centra MP4R
centrifuge for 10 min at 2000 rpm. The medium was
removed and cells were washed three times with PBS.
Total lipids were extracted using Chlor-
oform:Methanol:HCl (2:1:0.075 v/v) as previously
described (Oliveira et al., 1990). After the extraction,
lipids were dry under N2 and quantified gravimetrically.
2.4. Transmission electron microscopy
Cells in culture medium were fixed for 10 min in 1%
glutaraldehyde at room temperature, centrifuged
(1000g) and fixed in 2.5% glutaraldehyde in 0.1 M,
pH 7.2 cacodylate buffer overnight at 4  C. They were
rinsed three times in the same buffer, post-fixed for 40
min in 1% OsO4 in 0.1 M cacodylate buffer, pH 7.2,
rinsed in the same buffer, dehydrated in acetone and
embedded in epoxy resin (Polybed-Polysciences). Ultra-
thin sections were stained in uranyl acetate 5% (w/v)
and Reynolds lead citrate, and observed in a ZEISS 900
 M. Einicker-Lamas et al. / Environmental Pollution 120 (2002) 779–786 781

Fig. 1. Cell proliferation is inhibited by the metals in a time- and dose-dependent manner. Cell cultures were prepared as described in Section 2, and
different concentrations of Zn2+ (A and B) and Cu2+ (C and D) were tested. The initial cell concentration was 0.5105 cells/ml. Cells were counted
in a hemocytometer. Each point represent the means SD of five different experiments (n=5).

transmission electron microscope as described (Attias 2.6. Statistical analysis

et al., 1996).
2.5. Electron-probe X-ray microanalysis
Cells were fixed as described for Transmission Elec-
tron Microscopy, except that the steps of post-fixation
and section staining were omitted and 100 nm sections
were collected on nylon grids. Energy-dispersive X-ray
spectra were recorded from different cytoplasmic struc-
tures, specially from the electron dense inclusions found
in large vacuoles of Cu2+- treated cells. As a control,
spectra from different regions of the cytoplasm and
from the epoxide resin were collected. Specimens were
analyzed in a Zeiss LEO 912 transmission electron
microscope operating at 80 kV. X-rays were collected
for 300 s using a Si(Li) detector with a ultra-thin win-
dow on a 0–10 keV energy range with a resolution of 10
eV/channel as previously described (Miranda et al.,
2000). Analyses were performed using an Oxford Link
ISIS system attached to the microscope.
Data were analyzed by two-way analysis of variance
(ANOVA), considering as factors the different treat-
ments. In all the cases, the considered level of sig-
nificance was less than 0.05. Statistical comparisons for
each experimental group are shown in the legends of the
figures. All the experiments were done in duplicates
using different cell cultures.
3. Results and discussion
Copper occurs in a variety of mineral forms as sul-
fides (chalcopyrite, chalcocite, covellite), oxides
(cuprite) and carbonates (malachite, azurite) and is
associated with aqueous discharges from mining, metal
plating, power generation, and the manufacture of elec-
trical equipment. Copper is also used in its sulfate or
oxide form in the formulation of pesticides and algicides
(Girling et al., 2000). A major factor complicating
782 M. Einicker-Lamas et al. / Environmental Pollution 120 (2002) 779–786
Fig. 2. Higher concentration of proteins in metal exposed cells. Cells
were exposed to 0.22 mM CuCl2 or 0.88 mM ZnCl2. After 3 days of
exposure, cells were counted and processed for protein determination
as described in Section 2. Results are expressed as meansSD of four
different experiments (n=4).
studies on the toxicity of copper is the complexity of its
aquatic chemistry, as it is affected by water parameters
which alter speciation and bioavailability, particularly
alkalinity, pH, and the amount of suspended or dis-
solved organics (Girling et al., 2000). In our experiments
either copper or zinc were used as chloride salts and
were free-soluble in the culture medium, therefore the
observed effects can be ascribed to the presence of these
chemical forms within E. gracilis cells.
Fig. 1 shows that Zn2+ and Cu2+ inhibited cell pro-
liferation in a time- and dose-dependent manner. It is
important to note that 0.2 mM Zn2+-treated cells had
an increase in cell proliferation in the first 24 h exposure
(Fig. 1A). This finding must be explained due to the
zinc-requirement of some important enzymes involved
in the DNA synthesis (Vallee, 1983). In contrast, lower
concentrations of CuCl2 were enough to provoke a sig-
nificant inhibition in cell proliferation after 24 h
(Fig. 1C). The toxic effects were time-dependent and
easily observed after 72 h of exposure, where sub-
micromolar concentrations strongly affected cell pro-
liferation, while only milimolar concentrations of Zn2+
affected E. gracilis survival (Fig. 1A and C). Fig. 1B and
D shows that the effect is dose-dependent with a LC50 of
0.88 mM for Zn2+ and 0.22 mM for Cu2+, respectively,
after 72 h of exposure. These values were higher than
the allowed range determined by the Brazilian Council
of Environment Protection (CONAMA), but similar to
the concentrations of these metals found in some pol-
luted areas in Rio de Janeiro State, Brazil (Lacerda et
al., 1987; Dornelles, 1998; Moreira, 2001). When cells
exposed for 72 h to these metals were washed and put in
a heavy metal-free fresh medium, they were able to
proliferate again like the control cells, after 48 h in the
new metal-free medium (data not shown), pointing to a
reversible poisoning effect, suggesting that E. gracilis
Fig. 3. Higher total lipid content in metal-exposed cells. Cells were
exposed to 0.22 mM CuCl2 or 0.88 mM ZnCl2. After 3 days of expo-
sure, cells were counted and processed for lipid extraction and deter-
mination as described in Section 2. Results are expressed as
meansSD of three different experiments (n=3).
cells have an active detoxification system which enable
these cells to remove the accumulated metal.
We started to study the effect of the metals on differ-
ent biochemical pathways of the protozoa, using 0.22
and 0.88 mM for CuCl2 and ZnCl2-treated cells,
respectively. Fig. 2 shows that both Cu2+ and Zn2+-
exposed cells present higher protein content than the
control cells. This finding indicates that cell division
seems to be impaired in heavy metal-exposed cells.
Similar phenomenon was also observed in cadmium-
exposed E. gracilis, where the total lipids and chlor-
ophyll contents were also higher in Cd2+-treated than in
control cells (Einicker-Lamas et al., 1996).
When the lipids were analyzed a marked increase in
the total lipid content of metal-treated cells was detected
(Fig. 3). It can be observed that copper lead to a 2-
fold increase in lipids, whereas zinc caused a 2.6-fold
increase. In Cd2+-treated E. gracilis the increase of total
lipid was due to a higher concentration of cholesterol
and the phospholipid phosphatidylglycerol, which is a
chloroplast membrane marker lipid (Einicker-Lamas et
al., 1996). In the present experiments we do not know
whether any particular lipid is augmented or a general
effect is present. The damage caused by cadmium on E.
gracilis chloroplasts, was already reported as well as
alterations in the chlorophyll content in Cd2+-exposed
cells (Duret et al., 1986; Einicker-Lamas et al., 1996)
indicating that this organelle may be the primary target
for the heavy metal toxic effects.
In the present study, it was observed by light-
microscopy alterations in cell motility and shape when
E. gracilis cells were grown for 24 h in the presence of
0.22 mM CuCl2. At this time and metal concentration
exposure, cells were still viable and the poisoning effects
of Cu2+ were reversible. These observations lead us
to investigate the ultrastructure of the cells exposed to
Cu2+. Although cells looked more spherical and dense
 M. Einicker-Lamas et al. / Environmental Pollution 120 (2002) 779–786 783

Fig. 4. Transmission electron microscopy of metal-exposed cells. Euglena gracilis cells were prepared for transmission electron microscopy as
described in Section 2. A and B are control cells. A and C are general views of the cells. Mitochondria (m), chloroplast (c), Golgi complex (G),
vacuoles (V), and nucleus (N), are readily recognizable. C and D are Cu+2 treated cells. At higher magnifications alterations in tylacoid membranes
(arrow), and mitochondrial cristae (arrowheads) are noticed. Vacuoles with different shapes and densities appeared in Cu2+-treated cells: larger and
less dense (white asterisk), and small and denser (arrowhead). Bars=1.2 mM (A and C); 0.4 mM (B and D).

at light-microscopy, no relevant alterations of cell shape
were observed by scanning electron microscopy (data
not shown).
Ultrathin sections of both control and Cu+2-treated
cells were observed at the transmission electron micro-
scope, where all cell structures were readily recognizable
(Fig. 4). Most prominent, besides the nucleus, were the
mitochondrion, chloroplasts, Golgi complex, vacuoles
and both cytoplasmic and vacuolar dense inclusions
(Fig. 4C and D). In Cu+2 treated cells mitochondria
seemed to have less cristae, and the tylacoidal mem-
branes of chloroplasts were less electrondense (Fig. 4B
and D). Pyrenoids were also frequently seen in the
stromal space of Cu2+-exposed cells. Photoautotrophic
E. gracilis chloroplasts are usually elongated or spindle
shaped (Fig. 4A and B). Pyrenoids, a denser and finely
granular area in the stroma are present more often dur-
ing the first half of the growth phase of E. gracilis,
during which chloroplasts grow in order to divide, to
provide the same number of chloroplasts to the daugh-
ter cells after cell division (Pellegrini, 1980). In dividing
cells, few pyrenoids are present. We observed that
Cu2+-treated cells had more pyrenoids than control
ones, supporting the hypothesis that this metal inter-
feres with the normal cell life cycle of E. gracilis,
impairing the cell proliferation, as observed in Fig. 1.
This is also in agreement with the significantly rise in
proteins (Fig. 2) and lipids (Fig. 3).
784 M. Einicker-Lamas et al. / Environmental Pollution 120 (2002) 779–786

Fig. 5. Metal identification in ultra-thin sections of E. gracilis. Microanalysis X-ray spectrum. A, B—Energy dispersive X-ray spectra collected from
the larger electron dense inclusions showing its characteristic peaks in a control cell (A) and Cu2+-exposed cell (B). Note that exposure to Cu2+
caused an accumulation of the metal (B, arrow). C, D—Ultrathin sections of CuCl2 treated cells showing the cytoplasmic vacuoles (V) in which X-
ray microanalysis were performed. Larger and lighter inclusions (arrows) are found projecting from the vacuolar membrane while smaller and dar-
ker inclusions are seen alongside this structure (arrowhead). E—X-ray spectrum of a smaller electron dense inclusion showing oxygen, phosphorus
and calcium peaks. F—Control X-ray spectrum collected from the epoxide resin. Electron micrograph of a treated cell for reference on the cell sites
that were used to generate the elemental spectra. Inset: arrow points to the large cooper-rich proteinaceous inclusions, while the arrowhead points to
the small inclusions. Bars=1.1 mM (C) and 0.3 mM (D).

Dense inclusions were observed both in control and
treated cells, but were much larger and numerous in the
latter (Fig. 4A and C). A closer observation of these
inclusions showed that in control cells they were small
and very electrondense, while in Cu+2 treated cells they
were composed of a large amorphous core surrounded
by small and very dense globets (Figs. 4C, D and 5D).
The presence of numerous vacuoles, and the increase
number of electrondense inclusions in metal-exposed
cells suggested that it could be a detoxification pathway
being used by E. gracilis in order to prevent cell damage,
keeping the metal in specific vacuoles. To confirm this
hypothesis was determined the metal content of those
dense inclusions.
Electron probe X-ray microanalysis was performed in
different regions of the cells, and our data showed that
the only structures which presented characteristic peaks
of interest were the electron dense inclusions found
within cytoplasmic vacuoles (Fig. 5B and D). Larger
and less electron dense inclusions, projecting from the
inner face of the vacuolar membrane, showed an
appreciable amount of oxygen, phosphorus, sulfur, cal-
cium and zinc (Fig. 5A). Exposure to CuCl2, besides
inducing morphological alterations in the vacuoles as
 M. Einicker-Lamas et al. / Environmental Pollution 120 (2002) 779–786
well as their electron dense inclusions—increasing in
number and volume (data not shown)—and also resul-
ted in an accumulation of the metal in the larger inclu-
sions (Fig. 5B). The smaller and denser inclusions, with
no apparent organic mass, were found to consist mainly
of oxygen, phosphorus and calcium, probably in the
form of calcium phosphate, both in control and CuCl2
treated cells (Fig. 5E). Cu2+ could not be detected in
other intracellular compartments such as chloroplasts,
mitochondria or starch granules. As a control, we also
made microanalysis in the supporting epoxide resin,
which showed no characteristic peaks of interest
(Fig. 5F).
The detection of Cu2+ in the larger inclusions but not
in the smaller ones led to some speculations that could
be confirmed with the results obtained both on protein
content and ultrastructural analysis. The divalent cation
could bind to both dense inclusions present in the
vacuoles due to the negative net charge of their phos-
phates. However, detection of Cu2+ only in the larger
inclusions indicates that the metal accumulates only in a
sulphur-rich structure or that, in the smaller inclusion, it
is present in concentrations beyond the detection limit of
the X-ray system. It is well known that several proteins
such as metallothioneins and chelatins (Hamer, 1986;
Coppelloti, 1989), which are involved in mechanisms
of detoxification to heavy metals by a great variety of
organisms, are sulfur-rich in thiol groups proteins. In the
case of E. gracilis, increase in protein content after CuCl2
exposure, together with an increase in the number and
volume of the vacuoles within the cells and the detection
of Cu2+ in a sulfur-rich structure inside these vacuoles,
strongly suggests an increase in the expression and
synthesis of metallo-proteins, pointing E. gracilis
vacuoles as organelles involved in the mechanism of
detoxification by these cells. It can be also postulated the
presence of metal-ATPases in the detoxification process.
These metal-ATPases were identified in bacteria and
eukaryotic cells (Payne and Gitlin, 1998; Eide, 2000),
and would be responsible to remove the citoplasmatic
excess of the tested metals to the interior of the vacuoles.
The results obtained here present this free-living uni-
cellular E. gracilis as an excellent model for the study of
heavy metal intoxication in eukaryotic cells. It would be
very interesting to develop a bio-monitoring system in
the effluents from the industries in order to minimize the
aggressions to the environment, and E. gracilis could be
one of the bio-indicators.
Acknowledgements
This work was supported by MCT-CNPq, FAPERJ
and FINEP. G.A.M., T.B.F., F.L.S.S. and F.G. were
recipients of ‘‘Undergraduate Scientific Training’’ fel-
lowships from MCT-CNPq.
785
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