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Protoanemonin-inducedcytotoxiceffectsin
EuglenaGracilis
ArticleinCellBiologyInternational·August1997
DOI:10.1006/cbir.1997.0160·Source:PubMed
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Cell Biology International, 1997, Vol. 21, No. 7, 397–404
PROTOANEMONIN-INDUCED CYTOTOXIC EFFECTS IN EUGLENA GRACILIS
D. MARES1*, A. BONORA1, G. SACCHETTI1, M. RUBINI2 and C. ROMAGNOLI1
Department of Biology, Section of Botany, University of Ferrara and 2Institute of Medical Genetics,
University of Ferrara, Italy
1
Accepted 21 May 1997
The changes that protoanemonin, an antimicrobial agent of natural origin, brought about in the
alga Euglena gracilis were studied with light and electron microscopy and with flow cytometry.
The compound proved lethal for the alga at a dose of 5#10 "5 . At the sublethal dose of
3.5#10 "5  it caused: marked inhibition of growth; increase in the average cell volume;
inhibition of cytokinesis and induction of coenocytic organisms; loss of the flagellum and
stigma. Most nuclei were arrested in the G2/M phase of the cellular cycle. The chloroplasts were
still well organized, but there was a conspicuous decrease in carotenoids and chlorophylls a and
b, with a corresponding increase in pheophytins. The multifarious alterations seen in Euglena
are discussed on the basis of a possible interaction between this lactone and several enzymatic
? 1997 Academic Press Limited
systems.
K: Euglena gracilis; protoanemonin
INTRODUCTION
MATERIALS AND METHODS
Protoanemonin (PrA), a gamma-hydroxyvinylacrylic acid lactone, an acrid substance capable of
irritating the mucosae, is a secondary metabolite
produced by numerous species of Ranunculaceae
(Misra and Dixit, 1978; Bonora et al., 1987). It
originates from inactive glycoside ranuncolin by
enzymatic hydrolysis as a consequence of tissue
impairment.
Although the molecule’s cytotoxicity has been
confirmed, there are little data regarding its action
mechanism at the cellular level and what is available is rather dated (Erickson and Rosen, 1949;
Thimann and Bonner, 1949; Caltrider, 1967).
On the basis of the morpho-physiological effects
induced in yeasts and pathogenic hyphomycetes
(Mares, 1987; Mares, 1989; Mares et al., 1992), it
was previously hypothesized that the antifungal
activity could be due to an interaction between
PrA and thiol groups, in general, and with the
microtubules, in particular.
To further study the PrA action, Euglena gracilis,
a unicellular alga, was used as it revealed characteristics particularly useful in the preliminary study
of the action mechanisms of cytotoxic substances.
Chemicals
*To whom correspondence should be addressed.
1065–6995/97/070397+08 $25.00/0/cb970160
Aqueous fractions of Ranunculus bulbosus,
collected by steam distillation of fresh leaves
(purity=98%) were used as stock solutions and
added to the Euglena cultures after Millipore
0.22 ìm filtration. The amount was assessed and
the active principle characterized by ultraviolet
spectrophotometry, using a Perkin-Elmer 554 UV
recording spectrophotometer (Bonora et al., 1985).
Culture conditions
Asynchronous green cultures of Euglena gracilis
Klebs, strain ‘Z’ grown in our laboratories, were
cultured in the Hutner and Provasoli liquid growth
medium (Hutner and Provasoli, 1955). These
cultures were kept under the following conditions:
constant agitation; 25&1)C thermostatic chamber;
cycles of 16 h light (10 W m "2) and 8 h dark.
When the cells reached the logarithmic phase of
growth, increasing concentrations of PrA were
added to the Erlenmeyer flasks to yield concentrations between 1#10 "6  and 1#10 "4 
(1#10 "6, 1#10 "5, 2#10 "5, 3.5#10 "5,
? 1997 Academic Press Limited
398
Growth was monitored by measuring the optical
density of the cultures at 750 nm with a Perkin
Elmer 554 spectrophotometer. From the optical
density was yielded the number of cells/ml, according to a calibration straight line, previously made
for Euglena. Cellular viability was assessed using
the methylene blue method, as described in Bonora
and Mares (1982). Samples for both these parameters were collected after 24 h from the moment
of treatment. The data were the average of at least
four experiments.
Light microscopy
In vivo microscopic examinations were performed
at 24 h on both PrA-treated (3.5#10 "5 ) and
control cultures. A Zeiss Axiophot microscope
equipped with conventional or fluorescence attachments was employed. A Zeiss filter combination
487908 (BP 436, FT 460, LP 470) was used to
observe the chlorophyll autofluorescence.
To visualize DNA, the algae were stained with
1 ìg ml "1 of the fluorescent probe 4*,6-diamidino2-phenylindole (DAPI) (Serva, Heidelberg) for
10 min at room temperature, then rinsed in several
changes of distilled water and observed under the
Zeiss filter combination 487901 (BP 365, FT 395,
LP 397).
Photographs were taken on AGFA Agfachrome 200 film, using the microscope’s automatic
exposure control.
Electron microscopy
Ultrastructural analyses were carried out at 24 h on
cells treated with PrA 3.5#10 "5  and on the
Euglena control cells. The cells were harvested by
gentle centrifugation, fixed in 3% phosphatebuffered glutaraldehyde, pH 7.2, postfixed in 1%
aqueous osmium tetroxide, dehydrated in an
ethanol series and embedded in Epon-Araldite.
Ultrathin sections were post-stained with uranyl
acetate and lead citrate, and examined with a Zeiss
EM 109 electron microscope at 80 kV.
Flow cytometry
After propidium iodide staining, flow cytometric
analyses of cellular DNA content were carried out
9
80
7
60
5
40
3
1
0.1 1
Mortality %
Growth kinetics and cellular viability
100
Cells/ml (× 100,000)
4.2#10 "5, 5#10 "5, 6.5#10 "5, 1#10 "4 ).
Controls, without PrA, were cultured in parallel. Three replicate cultures were used for each
concentration of PrA.
Cell Biology International, Vol. 21, No. 7, 1997
20
2
3.5 4.2 5
6.5
8
Concentration PrA (× 10–5 M)
0
10
Fig. 1. Growth of Euglena gracilis (solid line) and % mortality
(dotted line) after 24 h of treatment with PrA at doses between
1#10 "4 and 1#10 "6 . The data are an average of four
samples&SD.
at 24 h on Euglena cells treated with 3.5#10 "5 
PrA and on untreated control cells. Cells were
washed twice in ice-cold phosphate-buffered saline
(PBS), fixed in 70% ethanol at "20)C for 6 h, and
stained with 10 ìg ml "1 propidium iodide in PBS
containing 100 kU ml "1 RNAse A. Fluorescence
emission was monitored using a Vantage flow
cytometer equipped with a 488 nm argon laser
(Becton Dickinson) and a doublets detector. Ten
thousand cells were analysed for each sample.
Determination of pigments
For the determination of chlorophyll pigments,
their catabolites and carotenoids, the controls and
PrA-treated cells were exhaustively extracted with
known volumes of 90% aqueous methanol in a cold
bath ("20)C). The samples were centrifuged at
2000 g for 5 min and the resulting supernatants
were put together. Using this methanolic solution
the following absorbances were assessed using a
Perkin-Elmer 554 UV spectrophotometer: (a) chlorophyll a absorbance at 665.5 nm, (b) chlorophyll b
absorbance at 652.4 nm and (c) total carotenoids at
470 nm. These values correspond to the maxima of
absorbance of the principal plastid pigments.
After extraction with n-hexane to remove chlorophylls, pheophytins and lipophilic-carotenoids,
the absorbance at 470 nm was measured on the
methanolic solution to assess the amount of
hydrophilic-carotenoids. Values of lipophiliccarotenoids were determined by subtracting the
hydrophilic-carotenoids from the total carotenoids.
Cell Biology International, Vol. 21, No. 7, 1997
399
Fig. 2. (A) Typical appearance of control E. gracilis under light microscopy showing a normal elongated shape, the emergent
flagellum, the stigma (arrow-head) and many plastids. (B) Photomicrograph of a DAPI-stained control cell under fluorescence
microscopy: many red chloroplasts and a deep blue fluorescent nucleus are evident. (C) Coenocytic organism from a PrA-treated
culture, apparently made up of six or seven globular cells, in which the stigma is never visible. (D) Same as Fig. 2C. Under
fluorescence microscopy the organism shows two or three nuclei, fluorescent with DAPI and among a set of united chloroplasts.
(E) UV photomicrograph of DAPI-stained cells of PrA-treated Euglena showing larger nuclei with a non-uniform fluorescence.
Scale bars=10 ìm.
The hexanic phase was re-evaporated to dryness
at 35)C under a vacuum using a Rotavapor. The
resulting material was subsequently redissolved in
diethyl ether and the absorbance at the absorption
maxima of chlorophyll a and of chlorophyll b were
measured at 660 nm and 642.5 nm, respectively.
The amounts of chlorophyll a and b thus deter-
mined coincided with those obtained from the
spectrophotometric analysis of the methanolic
solution, proving the absence of phytol-depleted
chlorophyll derivatives.
To detect pheophytins, concentrated HCl was
added to the ether solution to convert chlorophylls
to pheophytins; subsequently, granular anhydrous
400
Cell Biology International, Vol. 21, No. 7, 1997
Cell Biology International, Vol. 21, No. 7, 1997
Na2SO4 was added to clarify and dry the solution
and its absorbance was then measured at 665.5 nm
and 653 nm (the respective absorption maxima of
pheophytin a and pheophytin b).
The concentration of each pigment was calculated as nanomoles/106 cells: the equation suggested
by Lichtenthaler (1987) was used for carotenoids;
that of White et al. (1963) for chlorophylls and
pheophytins. The values are the average of seven
different determinations&standard error.
RESULTS
PrA has proved particularly toxic for the alga used
in this study and in a dose-dependent manner. In
Fig. 1, the solid line shows the number of cells/ml,
read as OD at 750 nm, of each culture, after 24 h
of treatment with a single dose of PrA in the
1#10 "4–1#10 "6  range, while the dotted line
refers to the percentage of mortality after 24 h of
treatment, as compared with the controls.
Concentrations from 1#10 "4  to 5#10 "5 
were lethal for the algae: after 24 h of treatment at
these doses, culture mortality reached 95%. A
concentration of 3.5#10 "5  gave a 67% reduction in growth as compared to the controls, but
without compromising cellular viability (90%).
In fact, at the dose of 3.5#10 "5 , when protoanemonin was washed out after 24 h of contact
and the Euglena cells were resuspended in fresh
medium, the culture began growing again. Hence
the dose of 3.5#10 "5  was chosen to study the
morpho-physiological effect PrA has on Euglena.
Light microscopy revealed the control cells to be
elongated, highly motile through the long flagellum, with a visible orange stigma. They were green
from the presence of 10–12 plastids (Fig. 2A).
Under fluorescence microscopy, the chloroplasts
emitted a bright red auto-fluorescence while
nuclear DNA, stained with DAPI fluorochrome,
gave bright blue fluorescence (Fig. 2B).
After treatment with PrA, 90% of the cells
assumed a globular shape, increased in volume and
lost their flagellum and stigma. Furthermore, the
401
induction of coenocytic organisms, with globular
or multilobate shapes, was observed (Fig. 2C). In
these organisms (Fig. 2D) fluorescence microscopy
revealed numerous chloroplasts united to form an
anastomosing network. Finally, the treated organisms showed larger nuclei with non-uniform DAPI
fluorescence (Fig. 2E).
When observed under the electron microscope,
the nuclear features in the PrA-treated cells were
typical of the G2 phase of the cell cycle; in Euglena
gracilis, in this phase, the nucleus is characterized
by the high scattering of chromosomes, broken
down into small chromatinic masses (Fig. 3A). In
the control culture, G2-phase nuclei were rarely
observed, while, in a large portion of cells, nuclear
morphology corresponded to the G1 and S phases
of the cell cycle. Figure 3B shows a nucleus in the
G1 phase, with large electron-dense chromosomes,
adherent to the nuclear membrane in several
points. To further investigate the G2-like morphology of the PrA-treated nuclei, flow cytometric
analyses were carried out on the controls and on
3.5#10 "5  PrA-treated cells. The obtained propidium iodide-fluorescence histogram of control
Euglena cells (Fig. 4a) corresponds to a typical
bimodal curve, having two peaks relative to the G1
(average channel=250) and G2/M (average channel=435) phases, respectively. The observed 30%
of G2/M cells in Euglena culture fits well with
reported data (Bertaux et al., 1976) that indicate
the G2/M period requires around 1/3 of the entire
generation time to be completed.
Figure 4b presents the DNA content histogram
of the PrA-treated cells. Surprisingly, the peak
corresponding to the G1 phase disappeared completely, while the G2/M peak (average channel=430) increased to approximately the size of
the G1 peak in the control cells; moreover a
new smaller peak appeared at a DNA content
close to twice the initial G2/M value (average
channel=780).
Electron microscopy of the pellicle further confirmed that PrA does inhibit Euglena cell cytokinesis. In fact, most treated cells have the pellicle
blocked in an incipient state of replication, as is
Fig. 3. (A) Electron micrograph of a PrA-treated cell showing a nucleus in G2 phase with many small chromatinic masses. (B)
A typical nucleus in G1 phase from a control cell of Euglena. Also note a normal chloroplast with pyrenoid (p) and paramylum
granules (g). (C) Detail from a PrA-treated cell showing the pellicle in the state of replication, with alternating tall and small
ridges. (D) Cross section of the pellicle complex of E. gracilis control cell, showing a ridge and groove configuration, 4–5
microtubules and a subpellicular tubule of the endoplasmic reticulum. (E) PrA-treated cell showing an almost normal
chloroplast, revealing only wide interthylakoid spaces. Also note the double-folded pellicular complex. (F) Detail of a reservoir
region of a PrA-treated cell. Note the stigma reduced to few small globules (arrow) and scattered diffuse structures. (G) The
eyespot region of E. gracilis control. Note the well developed stigma consisting of numerous and large osmiophilic globules. Scale
bars=1 ìm.
402
Cell Biology International, Vol. 21, No. 7, 1997
(a)
60
Control cells
Number of cells
100
(b)
Treated cells
M3
M3
M2
M2
M1
M1
0
200
400
600
800
1000 0
200
Fluorescence intensity
400
600
800
1000
Fig. 4. (a) Distribution of DNA content (arbitrary fluorescence units) in a culture of control Euglena obtained by propidium
iodide-staining and flow cytometry. Note the presence in the asynchronous culture of cells in G1, S and G2 phases of the cell
cycle. (b) Histogram of DNA content (arbitrary fluorescence units) in a PrA-treated culture of Euglena. Note the disappearance
of the G1 peak, the increase in the G2 peak and the presence of cells with higher DNA content.
shown by the characteristic double folding in Fig.
3C. In fact, in dividing cells, new daughter ridges
appear as upwellings between parental ridges
(Buetow, 1968) whereas in control cells, the pellicle
was composed of ridges and grooves of fairly
constant dimension (Fig. 3D).
The chloroplasts were still relatively well organized even if a small increase in the interthylakoid
spaces was noted (Fig. 3E). On the other hand,
fluorescence microscopy revealed only a slight
decrease in plastidial fluorescence. Analysing the
pigment composition after 24 h of PrA treatment,
there was a conspicuous decrease in chlorophylls a
and b, respectively 51% and 20% (Table 1). A
Table 1.
Pigment composition of the alga Euglena gracilis, after
24 h of PrA treatment
Controls
6
Chl a
Chl b
Phy a
Phy b
Hy-Cars
Li-Cars
Treated
6
nmol/10 cells
nmol/10 cells
6.89&0.21
0.80&0.05
0.16&0.02
—
1.33&0.07
0.48&0.03
3.40&0.13
0.64&0.04
0.46&0.03
—
0.99&0.07
0.21&0.02
%
Change
"51
"20
+187
—
"26
"56
Chl a and b, chlorophylls a and b; Phy a and b, pheophytins
a and b; Hy-Cars, hydrophilic carotenoids; Li-Cars, lipophilic
carotenoids.
corresponding increase in chloropigment degradation products due to loss of magnesium was
recorded; in particular, among polar chlorophyll
catabolites, pheophytin a increased about threefold in comparison with what was found in the
control cells (187%). Only traces of pheophytin b
were evident, at the sensitivity limit for the method
used. Moreover, none of the possible polar catabolites of chlorophylls (i.e. the phytol-depletion
derivatives chlorophyllides and pheophorbides)
were found in the methanolic phase. The total
carotenoids were also greatly lowered, the
lipophilic-carotenoids decreasing more than the
hydrophilic ones ("56% vs "26%).
It is worth noting that our preliminary data, not
reported, obtained by HPLC analyses on control
Euglena cells, showed that the lipophilic-carotenoid
fraction consists chiefly of â-carotene, ketocarotenoids (cantaxanthin and echinenone) and of the
xanthophyll diatoxanthin. Instead the hydrophilic
fraction especially contained, above all, monoepoxidic xanthophylls such as diadinoxanthin and
neoxanthin. Because lipophilic-carotenoids constitute the majority of carotenoids in the stigma
(Goodwin, 1976) their spectrophotometrically
demonstrated decrease was in good agreement with
the morphological data, i.e. in the cells treated with
PrA, the stigma could either no longer be detected
or was extremely reduced in size (Fig. 3F) when
compared to a normal cell. In Euglena controls the
stigma is composed of grouped granules forming
Cell Biology International, Vol. 21, No. 7, 1997
an irregular body, partly surrounding the reservoir
wall, always facing the dorsal flagellum (Fig. 3G).
Another morphological alteration due to PrA
treatment was the presence of scattered structures
in the reservoir and in the cytoplasm surrounding
the reservoir (Fig. 3F).
DISCUSSION
The data presented in this study reveal the significant inhibition PrA exerts on Euglena gracilis: the
lactone inhibits the alga at doses comparable to
or lower than those used in previous studies on
bacteria (Didry et al., 1993) and fungi (Misra and
Dixit, 1978; Mares, 1987). Its wide spectrum of
activity may be related to the drug’s ability to
penetrate the microbial cell and to the presence of
an unsaturated lactone as a structural element.
In fact, there are many compounds of vegetable
origin whose cytotoxicity was related to the presence in the molecule of a lactone ring, such as
the antitumoral sesquiterpene lactones, the cardiac
glucosides, the coumarin derivatives.
In previous works (Mares, 1987), it was supposed that the mechanism of action of the
substances containing an
system (especially sesquiterpene
lactones)
or an
group (especially coumarin
and protoanemonin)
is based on the capacity of these substances
to interact with -SH groups by a Michael-type
addition (Hall, 1977) and that this relation determines the biological action of these substances
(Thimann and Bonner, 1949; Caltrider, 1967). The
numerous alterations observed in Euglena after
PrA treatment may therefore be considered the
result of such an interaction of this lactone with
-SH groups present in several alga systems.
The induction of syncytium-like structures can
be considered the result of an inability of the
cells to accomplish the cytokinesis. The possible
formation of PrA-SH complexes, blocking the free
sulphydryl groups required for cytokinesis (Dan,
1966) might not allow the cellular cycle to proceed,
thus arresting cell division at a premature stage,
and leaving the pellicle in duplication stage. Similar
features were observed in Euglena when free
403
sulphydryl groups were missing (Vannini et al.,
1982; Fasulo et al., 1983).
Flow cytometric evidence indicates that PrAtreated cells have approximately twice the DNA
content of culture prior to treatment. The 24 h PrA
treatment completely empties the pool of cells with
G1 DNA content, while strongly increasing the
G2/M pool, and inducing the appearance of a new
pool of cells having twice the G2/M DNA content.
Longer PrA exposures (data not shown here) do
not change the DNA content profile, and lead to an
arrest of cell growth. This phenomenon cannot
simply be explained as a G2 block of the cell cycle,
but rather as a defect in completing the cytokinesis
process. We suggest that under PrA treatment
Euglena cells skip cell division and enter a postmitotic phase still maintaining the G2/M DNA
content and the G2-like morphology features, followed in some cells at least by a further round of
nuclear DNA synthesis as indicated by events in
the zone M3 (Fig. 4b).
In fact, optical and electron microscopy of
treated cells shows that most of the nuclei actually
have the characteristic features of the G2 phase of
the cell cycle (Bertaux et al., 1989). The maintenance of G2-like morphology is consistent with the
hypothesis of incompleted cytokinesis.
In fungi it has been reported that PrA interacts with the -SH groups of microtubules
(Mares, 1989). It is possible that, affecting microtubules physiology, PrA treatment might cause the
observed cell division impairment in the alga.
Furthermore, the spectrophotometric analyses of
pigment composition reveal that a 3.5#10 "5 
concentration of PrA is most likely a critical
dose, corresponding to a generalized decrease in
both chlorophylls and carotenoids, in agreement
with the morphological and cellular viability data
(Table 1).
Since carotenoids protect chlorophyll a
from photooxidation (Lichtenthaler, 1987), their
decrease can lead to the destruction of chlorophyll
pigments, as confirmed by the concomitant
decrease in plastid fluorescence observed after PrA
treatment. Chlorophyll a turns into its lipophilic
catabolite pheophytine a, with consequent release
of magnesium ions. It is known that the Mg2+ has
a stabilizing effect on thylakoid stacking (Raval
and Biswal, 1984) and thus it has been hypothesized that the magnesium ions, released after
PrA treatment, may prevent plastidial lamellae
disorganization. The disappearance of the stigma
granules after treatment with PrA is in very good
agreement with the present spectrophotometric
data on the reduction of lipophilic-carotenoids. In
404
fact, the euglenoid eyespot consists of a cluster of
orange–red granules containing â-carotene and the
red ketocarotenoids cantaxanthin and echinenone
(Goodwin, 1976; Gualtieri, 1986); moreover, the
formation of the stigma requires the presence
of carotenoids (Osafune and Schiff, 1980). Any
substance affecting carotenoid biosynthesis or
catabolism might inhibit stigma development.
The data reported here, even if not conclusive,
indicates that PrA strongly alters Euglena cell
cytology, particularly affecting the cell division.
Further investigations are required to better understand how PrA exerts its effects on the physiology
of the cell, possibly extending the study to the
analysis of the effects on the cell cycle of metazoans.
ACKNOWLEDGEMENTS
This research was supported by grants from
the Italian Consiglio Nazionale delle Ricerche
(CNR) and Ministero dell’Università e della
Ricerca Scientifica e Tecnologica (MURST).
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