The Freshwater Cyanobacterium Lyngbya

aerugineo-coerulea Produces Compounds Toxic

to Mice and to Mammalian and Fish Cells
Ivanka Teneva,1 Dafinka Asparuhova,2 Balik Dzhambazov,3 Rumen Mladenov,4
Kristin Schirmer1
1

Junior Research Group, Molecular Animal Cell Toxicology, UFZ Centre for Environmental
Research, Permoserstrasse 15, 04318 Leipzig, Germany
2

Cell Biology Laboratory, University of Plovdiv, 24 Tsar Assen Street, 4000 Plovdiv, Bulgaria

Section for Medical Inflammation Research, Department of Cell and Molecular Biology,
Lund University, I 11, BMC, 22184 Lund, Sweden
4

Department of Botany, University of Plovdiv, 24 Tsar Assen Street, 4000 Plovdiv, Bulgaria

Received 26 June 2002; revised 4 September 2002; accepted 26 September 2002

ABSTRACT: Despite a growing awareness of the presence of cyanobacterial toxins, knowledge about the
ability of specific species to produce toxic compounds is still rather limited. It was the overall goal of the current
work to investigate if probes derived from the freshwater species Lyngbya aerugineo-coerulea (Kutz.) Gomont,
a cyanobacterium frequently found in southern Europe and not previously investigated for the presence of
bioactive compounds, were capable of eliciting in vivo and in vitro toxicity. The cyanobacterial extract revealed
signs of neuro- as well as hepatotoxicity in mice, although these signs could not be explained by the
well-known respective cyanobacterial neuro- and hepatotoxins saxitoxin and microcystin. Cytotoxicity was
elicited by the cyanobacterial extract in all mammalian cell lines tested. As well, the rainbow trout liver cell line,
RTL-W1, was found to be susceptible to the cytotoxic effects of the extract, although the cytotoxicity was
dependent on temperature. In contrast, the cyanobacterial growth medium elicited cytotoxicity independent of
temperature, leading to morphological changes indicative of alterations to the cytoskeleton. Overall, the results
suggest that Lyngbya aerugineo-coerulea is an important cyanobacterium to be considered for its potential to
cause health risks on environmental exposure of it to mammals and fish. Applying a combination of mammalian and piscine cell line bioassays is a unique approach that, combined with chemical analysis, could
be used in the future to identify the structure and cellular mechanisms of the as-yet-unknown toxic Lyngbya aerugineo-coerulea metabolites in particular and to screen cyanobacterial extracts for their toxicity in
general. 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 9 20, 2003.
Keywords: cyanobacteria; Lyngbya aerugineo-coerulea; toxins; mouse bioassay; fish and mammalian cell
cultures; cytotoxicity

INTRODUCTION

Correspondence to: Dr. Kristin Schirmer; e-mail: Kristin.Schirmer@

uoe.ufz.de
Contract grant sponsor: European Commission Marie Curie Training
Site Fellowship (to Ivanka Teneva); contract grant number: HPMT-CT2000-00115.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/tox.10096

Many cyanobacteria (blue-green algae) have been found to

be producers of potent bioactive compounds. The incentive
for identifying these compounds and their biological effects
has been being able to find novel substances applicable to
biomedical research and gaining an understanding of the
exposure risks for humans as well as animals. Toxins produced by cyanobacteria have been reported in marine- as

2003 Wiley Periodicals, Inc.

10

TENEVA ET AL.

well as freshwater environments throughout the world (Van

Dolah, 2000). Acute exposure to cyanotoxins has been
shown to result in intoxication, sometimes leading to death
(Carmichael, 1994, 1997; Jochimsen et al., 1998; Falconer,
1999). Recent studies of the presence of cyanobacterial
toxins in drinking water in certain areas of China and the
possible link of these toxins to an increased rate of human
liver cancer led to the revelation that the risk from cyanobacterial toxins is not restricted to acute exposure but
may extend to chronic health effects (Ueno et al., 1996;
Carmichael, 1997; Daranas et al., 2001). Despite a growing
general awareness of the presence of cyanobacterial toxins,
knowledge about the ability of specific species to produce
compounds with biologically adverse effects is still rather
limited.
The filamentous mat-forming cyanobacteria of the genus
Lyngbya are a potentially rich source of cyanotoxins. The
marine species Lyngbya majuscula Gomont has been the
center of numerous investigations on the discovery of new
pharmaceuticals. For example, Koehn et al. (1992) isolated
the immunosuppressive peptides microcolins A and B. Recently, Luesch et al. (2000) discovered structurally similar
apramides. Other intriguing secondary metabolites of
Lyngbya majuscula are the cytotoxins lyngbyabellin A and
B (Luesch et al., 2000; Milligan et al., 2000). In addition,
lyngbyatoxin and aplysiatoxin were identified as the source
of the dermatitis called swimmers itch, which results
from skin contact with the cyanobacterium (Mynderse et al.,
1977; Cardellina et al., 1979). Although much less is known
about the toxins produced by the freshwater species Lyngbya wollei Farlow ex Gomont, one group of toxins recognized are saxitoxin-related neurotoxins, the compounds responsible for paralytic shellfish poisonings (Carmichael et
al., 1997; Yin et al., 1997). No other toxigenic species of the
genus Lyngbya has so far been described, despite nearly 100
species having been classified in the traditional way as
belonging to this genus (Geitler, 1932).
The toxicity of cyanobacterial extracts traditionally has
been investigated by exposing mice and monitoring their
survival, behavior, and histopathology. Aune and Berg
(1986) were among the first to suggest the use of isolated rat
hepatocytes to be able to more quickly and inexpensively
investigate cyanobacterial blooms for their toxicity. Recently, this assay was shown to correlate particularly well
with the content of microcystin, a common cyanobacterial
hepatotoxin (Heinze et al., 2001). However, inasmuch as the
hepatocyte assay still requires the sacrifice of animals and
the toxicity of many environmental samples cannot be explained by microcystin alone (Oberemm et al., 2001),
screening cyanobacterial extracts for their impact on cells
might be more easily done using established cell lines. The
use of such cell lines has become particularly attractive in
recent years because of the development of numerous colored and/or fluorescent cell-viability indicator dyes that are
applicable to multiwell plate readers and because of the

increased availability of cultures originating from nonmammalian animals, such as fish, which allow cyanobacterial
extracts to be evaluated with respect to environmental
health.
It was the overall goal of the current work to investigate
whether the freshwater species Lyngbya aerugineo-coerulea
(Kutz.) Gomont, a cyanobacterium frequently found in
Southern Europe (Vodenicharov, 2000), is capable of eliciting toxicity. To study this, the traditional in vivo mouse
bioassay as well as in vitro assays using mammalian and a
fish liver cell line were applied to extra- as well as intracellular samples obtained from L. aerugineo-coerulea
grown in culture. The results suggest that Lyngbya aerugineo-coerulea is an important cyanobacterium to be considered as a risk to mammals and fish in the freshwater
environment.

MATERIALS AND METHODS

Culture of Lyngbya aerugineo-coerulea and
Preparation of Extracts
Cultivation
The cyanobacterium Lyngbya aerugineo-coerulea (Kutz.)
Gomont, PACC (Plovdiv Algal Culture Collection) No.
8601, was grown intensively under sterile conditions, as
described by Dilov et al. (1972.) A Z-nutrient medium was
used for the culture (Staub, 1961). Cultures were synchronized by alternating light and dark periods of 16:8 h. The
temperature was 33C and 22C during the light and dark
periods, respectively. This culture regime was established in
order to closely mimic the conditions for optimal growth of
Lyngbya in its natural habitat in the summer months. The
intensity of light during the light period was 12 000 lux. The
culture medium was aerated with 100 L of air per hour per
liter of medium, adding 1% CO2 during the light cycle. The
cultivation period lasted 14 days.

Extraction
Extracts of the cyanobacterium were obtained using a
slightly modified version of the method of Krishnamurthy et
al. (1986). Briefly, cyanobacteria were removed from the Z
medium and weighed, then frozen and thawed, and extracted twice (3 h and overnight) with a watermethanol
butanol solution (15:4:1 v/v/v, analytical grade) at 22C
while stirring. The extracts were centrifuged at 10 000 rpm
(9500g) for 30 min. The supernatants of the two extracts
were pooled and organic solvents removed via speed-vac
centrifugation (SAVANT Instruments Inc., Farmingdale,
NY, USA) at 37C for 2 h. The resulting extract was
sterilized by filtration through a 0.22-m Millipore filter
(GS filter typemodified acrylic polypropylene) and pre-

TOXICITY OF L. AERUGINEO-COERULEA

pared to give equivalent final concentrations of 150 mg/mL

(wet weight/volume) of suspended cyanobacterial matter.
To investigate whether Lyngbya aerugineo-coerulea released toxic products into their culture environment, the
nutrient solution in which the cyanobacteria were cultivated
for 14 days was filtered through a 0.22-m Millipore filter.
The final equivalent concentration of suspended cyanobacterial matter per milliliter of culture medium was 20 mg/mL
(wet weight/volume). This cyanobacterial medium was
tested for cytotoxicity in the fish cell bioassays.

11

Continuous Mammalian Cell Lines. FL, A2058, RD,

and 3T3 cells were cultured in 75-cm2 flasks in Dulbeccos
Modified Eagles Medium (DMEM; Gibco, Paisley,
Scotland, UK), supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS; PAA Laboratories GmbH,
Linz, Austria), 100 U/mL of penicillin, and 100 g/mL of
streptomycin (Sigma, Steinheim, Germany), at 37C with
5% CO2 in air and high humidity. Trypsin treatment and
subculturing were done according to the Invittox protocols
(The Ergatt/Frame, 1990, 1992). Cell viability was measured with the trypan blue exclusion test (Berg et al., 1972)
prior to seeding.

Toxicity of Lyngbya Extract in vivo

Mouse Bioassay
Six male DBA/1 mice (19 22 g) were used for the experiment (three mice per group). All mice were kept in a
climate-controlled environment with 12-h light:dark cycles
in polystyrene cages containing wood shavings. Mice were
fed standard rodent chow and water ad libitum in a specific
pathogen-free environment (as defined in http://net.inflam.
lu.se). Mice were injected ip with 0.5 mL of the test solution
containing equivalent final concentrations per mouse of 15
mg of suspended cyanobacterial matter (682790 mg/kg
mouse). To obtain this test solution, the algal extract was
diluted 1:4 with phosphate-buffered saline (PBS). Control
mice were injected with 0.5 mL of PBS. The animals were
observed for 24 h after treatment. Behavioral symptoms,
weight, and survival times were recorded.

Liver Histology
All animals were subjected to histological examination of
the liver for pathology. After termination of the experiment,
the liver slices were processed for light microscopy according to standard procedures. Briefly, the tissue samples were
fixed in 4% buffered formalin for 24 h, dehydrated in a
graded series of alcohols, cleared in xylene, and embedded
in paraffin wax. Multiple sections from each block were
prepared 5 m thick and stained with hematoxylin and
eosin (McManus and Mowry, 1965).

Toxicity of Lyngbya Extracts in vitro

Animal Cell Culture
Six cell cultures were used: one freshly established cell
culture of mouse thymus fibroblasts; four commercially
available mammalian cell lines, FL (normal amniotic cells,
human, ATCC CCL 62), A2058 (human metastatic melanoma, ECACC 91100402), RD (rhabdomyosarcoma, embryonic, human, ATCC CCL 136), and 3T3 (fibroblasts,
embryonic, mouse, ATCC CCL 92); and one cell line derived from a normal rainbow trout (Oncorhynchus mykiss)
liver (RTL-W1; Lee et al., 1993).

Primary Mouse Thymus Fibroblast Cell Culture. This

cell culture was prepared from three B10.Q mice. The
thymus of each mouse was removed and stored immediately
in sterile PBS containing 100 U/mL penicillin, 100 g/mL
streptomycin, and 2.5 g/mL amphotericin B (Serva, Heidelberg, Germany). All samples were finely minced with
scissors, digested for 45 min at 37C in 5 mL of PBS
containing 0.1% trypsin (Gibco BRL, Paisley, Scotland,
UK), and then digested in 0.1% collagenase D (Roche
Diagnostics GmbH, Mannheim, Germany) in DMEM containing 10% fetal calf serum (FCS, heat inactivated) for 90
min at 37C in an atmosphere with 5% CO2. After passage
of the cells through a 40-m cell strainer (FALCON,
Becton Dickinson, Le Pont De Claix, France), cells were
collected by centrifugation, washed twice with serumfree DMEM, and cultured in complete DMEM, as described above for the continuous mammalian cell lines.
After three passages the culture consisted of fibroblastlike cells only, as judged by cell morphology and recognition by the cells of the (surface) antiglycoproteins
VCAM-1, CD68, and CD11b, determined using a fluorescence-activated cell sorter (FACS; Becton Dickinson,
San Jose, CA, USA).
Continuous Fish Cell Line. RTL-W1 cells were cultured
as originally described by Lee et al. (1993) in an atmosphere
of air in 75cm2 Nunc culture flasks at 19C in Leibovitzs
L-15 medium without phenol red (Invitrogen, Karlsruhe,
Germany), supplemented with 5% fetal bovine serum, FBS
(Biochrom, Berlin, Germany), and penicillinstreptomycin
(20 U/mL and 20 g/mL, respectively; Biochrom, Berlin,
Germany), using previously described subcultivation procedures (Bols and Lee, 1994; Schirmer et al., 1994).

Exposure Conditions
Prior to exposure, cells were plated in 96-well tissue culture
plates at a density of 1.5 104 per 200 L of DMEM
medium with 10% FCS for mammalian cells and 5 104
cells per 200 L of L-15 medium with 5% FBS for the
piscine cell line. After 24 h of attachment, the medium was

12

TENEVA ET AL.

removed and replaced by the exposure medium as described

below.
Exposure to the Cyanobacterial Extract. Mammalian
cells were exposed to one concentration of the cyanobacterial extract. This concentration was equivalent to 15 mg/mL
(w/v) suspended cyanobacterial matter, the same concentration used in the in vivo experiments in mice, and was
obtained by adding 10 L of the extract to 190 L of
DMEM culture medium with 10% FBS. Control wells were
prepared by adding 10 L of Millipore water to 190 L of
culture medium. The cells were exposed to the cell extract
for 4 or 24 h prior to analysis of cytotoxicity by the MTT
assay or of effects on proliferation by the [3H]-thymidine
incorporation assay as described below.
The piscine cell line was exposed to varying concentrations of the cyanobacterial extract, with the highest concentration equivalent to an extract of 15 mg/mL of suspended
cyanobacterial matter. Exposure was done in L-15 medium
in the absence of serum or, in some cases, in the presence of
a 5% FBS supplement. In most cases the exposure temperature was 19C, as for routine maintenance. However, in
some experiments fish cells were exposed at 4C or 25C in
order to investigate if varying temperatures had an effect on
the toxicity of the extract. Cytotoxicity of the cyanobacterial
extract was assessed after 24 h of exposure by a combination of the alamarBlue/CFDA-AM cytotoxicity assays, as
described below.
Exposure to the Cyanobacterial Medium. In addition to
the cyanobacterial extract, the fish cell line was also exposed to varying concentrations of the medium in which
Lyngbya had been grown for 14 days. The highest concentration was 50% of the cyanobacterial medium, which was
obtained by adding 100 L of the cyanobacterial Z medium
to 100 L of L-15 medium without serum. An appropriate
control was prepared by adding L-15 to the fish cells with
up to 50% Z medium. The cytotoxicity of the cyanobacterial
growth medium was assessed after 24 h of exposure by a
combination of the alamarBlue/CFDA-AM cytotoxicity
assays as described below.

4 h in the dark. After incubation, the medium with the dye

was aspirated and plates inverted to drain unreduced MTT,
and 0.1 mL of 0.04 mol/L HCL in isopropanol was added to
each well in order to facilitate solubilization of the formazan
product. The plates were shaken, and absorbance was read
at 570 nm.

alamarBlue and CFDA-AM

Two fluorescent indicator dyesalamarBlue (BioSource,
Solingen, Germany) and 5-carboxyfluorescein diacetate
acetoxymethyl ester (CFDA-AM, Molecular Probes, Eugene, OR, USA)were used in combination, as previously
described, using L-15/ex as a simplified culture medium
(Schirmer et al., 1997). The alamarBlue dye measures,
similar to MTT, the redox potential of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester measures cell
membrane integrity. After exposure of the cells to the cyanobacterial extract or medium, the wells were emptied and
filled with 100 L of a mixuture of 5% (v/v) alamarBlue
and 4 M CFDA-AM in L-15/ex and incubated in the dark
for 30 min prior to fluorescent measurement. Fluorescence
was analyzed using a SPECTRAmax Gemini spectrofluorometer (Molecular Devices, Munich, Germany) at optimized excitation/emission wavelengths for alamarBlue
and CFDA-AM of 530/595 nm and 493/541 nm, respectively.

Proliferation Assay
The cells were plated and exposed to a final equivalent
concentration of cyanobacterial extract of 15 mg/mL as
described above. During the last 18 h of exposure, cells
were pulsed with 1 Ci [3H]-thymidine per well (Amersham Labs, Buckinghamshire, England). After the completion of 24 h of exposure, the cultures were harvested in a
Filtermate cell harvester (Packard Instrument, Meriden,
CT, USA). Incorporation of [3H]-thymidine was measured
in a Matrix 96 Direct beta counter (Packard). The mean cpm
values of triplicates were determined.

Cytotoxicity Assays
MTT
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO, USA] assay was
carried out in accordance with Edmondson et al. (1988).
This assay is based on the capacity of mitochondrial succinyl dehydrogenase to convert the soluble yellow tetrazolium
salt into an insoluble purple-blue formazan product. After
the desired time of contact with the cyanobacterial extract (4
or 24 h), 20 L of a 0.5% (w/v) solution of MTT in PBS
was added directly to each well and incubated at 37C for

Analysis of Toxins Based on ELISA

Saxitoxins
The samples were analyzed with a Ridascreen saxitoxin
ELISA kit (r-biopharm, Darmstadt, Germany). This is a
competitive enzyme-linked immunosorbent assay (ELISA)
for the quantitative analysis of saxitoxin and related toxins
based on the competition between the free toxins from
samples or standards and an enzyme-conjugated saxitoxin
for the same antibody. The mean lower detection limit of the
Ridascreen saxitoxin assay is about 0.010 ppb.

TOXICITY OF L. AERUGINEO-COERULEA

13

Microcystins
Analysis of samples was performed using a Microcystin
Plate kit (EnviroLogix Inc., Portland, OR, USA). As for the
saxitoxin ELISA, this is a quantitative competitive immunosorbent assay. The limit of detection of the EnviroLogix
Microcystin Plate kit is 0.05 ppb.

Statistical Analysis
The results are reported as means SDs from individual
determinations. Statistical differences were analyzed with a
t test using the Sigma Plot (Jandel Scientific) program.
Values of p 0.05 were regarded as significant.

RESULTS
Toxicity of Lyngbya Extract in vivo
Injection of Lyngbya aerugineo-coerulea extract into
DBA/1 mice caused symptoms typical of neurotoxins. Immediately after injection of the Lyngbya extract, mice
showed reduced activity and had convulsions and spasms as
well as respiratory difficulties. These symptoms lasted for
about 2 h. When survival was recorded 24 h after injection,
no lethality was observed. However, mice treated with the
Lyngbya extract showed an average weight loss of 1.5
0.3 g (n 3), whereas control mice gained an average of
0.18 0.16 g (n 3).
Histological examination of liver slices of treated mice
revealed signs of hepatotoxicity. Most notable was a granulovacuolar degeneration with perinuclear clumping of cytoplasm and an increased number of cells undergoing mitosis
(Fig. 1). Other signs of hepatotoxicity were minor inflammations and sinusoid congestions.

Toxicity of Lyngbya Extract in vitro

Toxicity to Mammalian Cells
Exposure of the extract led to distinct responses depending
on the cell line and the exposure period. Both stimulatory
and cytotoxic effects were observed after 4 h of exposure,
whereas after 24 h cytotoxicity was revealed in all cells
(Fig. 2). The most pronounced response was seen in FL
cells. After 4 h of exposure, cells showed a stimulation of
MTT reduction above control levels of more than 40%,
whereas after 24 h of exposure, a decrease in cell viability
to 32% of control levels was observed. In contrast, 3T3 cells
seemed least sensitive, with 60% cell viability remaining
after 24 h of exposure. It was found that after a 24 h
exposure, most cells began to round up and detach from the
plates, floating into the medium.
The apparent cytotoxicity elicited by the Lyngbya extract
was confirmed by a significant inhibition of proliferation in

Fig. 1. Appearance of liver cells obtained from mice treated

in vivo with (A) the appropriate vehicle control or (B) the
Lyngbya aerugineo-coerulea extract. Mice were injected ip
with an equivalent final concentration of 15 mg suspended
cyanobacterial matter for 24 h prior to preparation of the liver
slices. Arrows indicate typical signs of hepatotoxicity
caused by the extract. These signs are (a) an increased
number of mitotic cells, (b) minor inflammations and sinusoid congestions, and (c) a granulovacuolar degeneration
with perinuclear clumping of cytoplasm. This is hematoxylineosin staining. The original magnification was 500.

the primary mouse thymus fibroblast cell culture and in the

3T3 cell line. Incorporation of [3H]-thymidine was inhibited
by 80% and 92% in the thymus- and 3T3-cell cultures,
respectively (Fig. 3).

Toxicity to the Fish Cell Line, RTL-W1

In contrast to the mammalian cell cultures, fish cells responded
much less clearly to exposure to the Lyngbya extract at 19C
under routine fish-cell-culture conditions. In both the presence
and absence of serum, cell viability was little affected by
exposure to the cyanobacterial extract for 24 h, as measured
with the alamarBlue/CFDA-AM assay (Fig. 4). In some
cases a slight increase in fluorescent unit readings was ob-

14

TENEVA ET AL.

cant loss of cell viability (Fig. 6). Cell response differed

depending on the presence or absence of serum. Without
serum in the culture medium, fluorescence unit readings
dropped to 20%30% of control levels, with no striking
differences in the readings for the different extract concentrations [Fig. 6(B)]. In contrast, a dose-dependent decrease
of cell viability was observed in the presence of serum [Fig.

Fig. 2. Viability of four mammalian cell lines on exposure to

a cell culture medium containing 10% L. aerugineo-coerulea
extract for 4 h (black bars) and 24 h (white bars) at 37C in
the presence of 10% serum. Cell viability was measured
with the MTT assay and absorbance readings expressed as
a percentage of the control, which received no extract but an
appropriate amount of tissue culture water. A volume of
10% Lyngbya extract is equivalent to a suspended cyanobacterial matter concentration of 15 mg/mL. One experiment is shown, for which each bar represents the average of
three culture wells, with vertical lines indicating the standard
deviation. Asterisks denote a significant response from the
cyanobacterial extract compared with the extract-free control, as determined by a t test ( 0.05).

served, but this was generally no more than 25% and was not
dose dependent. Likewise, fluorescent unit readings occasionally dropped below 100%, but, again, these readings either
were not significantly different from the control or did not
occur in a dose-dependent manner. Comparable results were
obtained when cells were exposed to the extract for 4 h at 19C
(data not shown). However, when cells were exposed to the
extract for 48 h, fluorescence unit readings for both alamarBlue and CFDA-AM increased significantly. For example,
at an equivalent concentration of 15 mg/mL suspended cyanobacterial matter, which was also the concentration applied to mammalian cells (Fig. 2), alamarBlue readings increased to 127%
11% and 136% 12% (average of three culture wells) in medium with and without serum, respectively. For CFDA-AM, these
readings were 120% 24% in the medium with serum and 178%
9% for the medium without serum. The dose dependence of
this increase in fluorescence unit readings was seen most clearly in
the medium without serum (Fig. 5).
Inasmuch as temperature could be a determinant of the
cytotoxic effects of the Lyngbya extract, RTL-W1 cells
were exposed for 24 h at 4C and 25C. At the lower
temperature cell viability was not affected (data not shown)
and was comparable to the cell viability data obtained at
19C (Fig. 4). However, exposure at 25C led to a signifi-

Fig. 3. [3H]-thymidine incorporation into 3T3 cells (top panel)

and mouse thymus fibroblasts (bottom panel) on exposure to
cell culture medium containing 10% L. aerugineo-coerulea extract or 10% tissue culture water as a control. Exposure was
done for 24 h at 37C in the presence of 10% serum. One
experiment is shown, in which each bar represents the average
of three culture wells, with vertical lines indicating the standard
deviation. Asterisks denote a significant response from the
cyanobacterial extract compared with the extract-free control,
as determined by a t test ( 0.05).

TOXICITY OF L. AERUGINEO-COERULEA

Fig. 4. Viability of RTL-W1 cells on exposure to an extract of

L. aerugineo-coerulea for 24 h at 19C in the (A) presence or
(B) absence of serum. Cell viability was measured with
alamarBlue (black bars) and CFDA-AM (white bars), and
fluorescent units readings are expressed as a percentage of
the control, which received no extract but an appropriate
amount of tissue culture water. A volume of 10% Lyngbya
extract is equivalent to a suspended cyanobacterial matter
concentration of 15 mg/mL. One representative experiment
is shown. Each bar represents the average of three culture
wells, with vertical lines indicating the standard deviation.
Asterisks denote a significant response from the cyanobacterial extract compared with the extract-free control, as determined by a t test ( 0.05).

6(A)]. At the highest concentration applied in this study, the

average cell viability of three culture wells was 42% 4%
for alamarBlue and 35% 7% for CFDA-AM.

Fig. 5. Viability of RTL-W1 cells upon exposure to an extract

of L. aerugineo-coerulea for 48 h at 19C in the presence (A)
or absence (B) of serum. Cell viability was measured with
alamarBlue (black bars) and CFDA-AM (white bars) and
fluorescent unit readings were expressed as described in
Figure 4. One representative experiment is shown. Each bar
represents the average of three culture wells with vertical
lines indicating the standard deviation. Asterisks denote a
significant response from the cyanobacterial extract as
compared with the extract-free control, as determined by a
t test ( 0.05). In one instance, a significant difference was
found between the reading obtained with alamarBlue and
that with CFDA-AM, ( 0.05; indicated by $).

15

Fig. 6. Viability of RTL-W1 cells on exposure to an extract of

L. aerugineo-coerulea for 24 h at 25C in the (A) presence or
(B) absence of serum. Cell viability was measured with
alamarBlue (black bars) and CFDA-AM (white bars), and
fluorescent unit readings were expressed as described in
Figure 4. One representative experiment is shown. Each bar
represents the average of three culture wells, with vertical
lines indicating the standard deviation. Asterisks denote a
significant response from the cyanobacterial extract compared with the extract-free control, as determined by a t test
( 0.05).

Toxicity of the Lyngbya aerugineo-coerulea

Growth Medium to RTL-W1 Cells
The medium in which Lyngbya had been grown for 14 days
prior to extraction was applied to the fish liver cells in
concentrations of up to 50%. A similar concentration of Z
medium in L-15 had no detrimental effect on cell viability
compared with 100% L-15 culture medium (data not
shown). In contrast to the cyanobacterial extract, significant
cytotoxicity was observed with the cyanobacterial medium
at 19C, the temperature used for routine maintenance of the
fish cell line. This cytotoxicity was far greater in the absence
than in the presence of serum (Fig. 7). A change in exposure
temperature to 4C or 25C did not significantly alter these
results (data not shown).
The distinct cytotoxic responses observed with the cyanobacterial medium were also confirmed by characteristic
morphological changes, which were not observed with the
cyanobacterial extract. At concentrations of 20% cyanobacterial medium and above, cells acquired a more fibroblastlike appearance and formed circles around empty culture
surface areas [Fig. 8 B(a)]. In some areas cells were reduced
to long, thin extensions and started to detach [Fig. 8B(b)]. In
contrast to the measurements of cytotoxicity, these morphological changes were marginally more pronounced at the
higher exposure temperature, 25C.

Analysis of Toxins Based on ELISA

Although in the assay of saxitoxins slightly increased background levels were found in both the extract and the growth
medium, neither saxitoxin nor microcystin could be detected above the mean lower limits of detection of the

16

TENEVA ET AL.

Fig. 7. Viability of RTL-W1 cells on exposure to the medium

in which L. aerugineo-coerulea had been grown for 14 days.
Cells were exposed to the medium for 24 h at 19C in the (A)
presence or (B) absence of serum. Cell viability was measured with alamarBlue (black bars) and CFDA-AM (white
bars), and fluorescent unit readings are expressed as a
percentage of the control, which received an appropriate
amount of tissue culture water with Z medium. A volume of
50% cyanobacterial medium is equivalent to the volume in
which 10 mg of suspended cyanobacterial matter has been
grown. One representative experiment is shown. Each bar
represents the average of three culture wells, with vertical
lines indicating the standard deviation. Asterisks denote a
significant response from the growth medium compared
with the control, as determined by a t test ( 0.05).

adverse effects when applied to the mammalian and fish-cell

bioassays used in this report.
Injection of L. aerugineo-coerulea extracts into mice
yielded signs of neuro- as well as hepatotoxicity, but these
signs could not be attributed to either the neurotoxin saxitoxin or the hepatotoxin microcystin and their analogues
cross-reacting in the ELISA assays. The lack of microcystins and nodularin is in line with other reports on toxins
produced by the Lyngbya species, for which microcystins or
nodularin have not yet been noted. Analogues of saxitoxin,
which have previously been identified in L. wollei (Carmichael et al., 1997; Onodera et al., 1997; Yin et al., 1997)
and L. majuscula (Fujiki et al., 1981; Nakayasu et al., 1981),
are decarbamoyl gonyautoxin II and III (paralytic shellfish
poison toxins). Although their presence in the extract prepared from L. aerugineo-coerulea cannot entirely be ruled

ELISA assays. This result indicates that toxins capable of

cross-reacting in the ELISA tests were not present at significant amounts. These toxins include decarbamoyl saxitoxin and gonyautoxins II, III, B1, C1, and C2 for the assay
of saxitoxins. For the assay of microcystin, toxins capable
of cross-reacting but not apparently present in the Lyngbya
samples were the microcystin variants LR, LA, RR, and YR
as well as nodularin.

DISCUSSION
Several cyanobacterial species that belong to the order
Oscillatoriales have previously been reported to be capable
of producing potent toxins. Most notable are the investigations on species of the prominent genus Oscillatoria, including a number of hepatotoxin- as well as neurotoxinproducing strains (Mynderse et al., 1977; stensik et al.,
1981; Skulberg et al., 1992). In contrast to Oscillatoria,
only two toxigenic species have been reported for Lyngbya
so far. These are the marine species L. majuscula (Milligan
et al., 2000a, 2000b; Luesch et al., 2000a, 2000b, 2001a,
2001b; Nogle et al., 2001; Osborne et al., 2001) and the
freshwater species L. wollei (Carmichael et al., 1997; Onodera et al., 1997; Yin et al., 1997). The results of our study
have revealed for the first time the ability of another freshwater species of Lyngbya, namely, L. aerugineo-coerulea,
to be toxigenic. Both an intracellular extract and an extracellular sample of L. aerugineo-coerulea elicited significant

Fig. 8. Phase-contrast appearance of RTL-W1 cells on exposure to cell culture medium containing 30% of the cyanobacterial medium in which Lyngbya had been grown for
14 days (B) compared to the appropriate control (A). Arrows
indicate the formation (a) of circles around empty culture
surface areas because of the cyanobacterial medium and (B)
of long, thin extensions originating from the normally epithelial-like cells. The photographs were taken at 100.

TOXICITY OF L. AERUGINEO-COERULEA

out, they were below the level of detection in the saxitoxin

ELISA and thus unlikely to be the cause of the observed
neurological effects. Rather, recovery from signs of neurotoxicity after 2 h of injection into mice, as observed in our
study, has recently been described for another toxin identified in L. majuscula: lyngbyatoxin A (Ito et al., 2002). In
addition to causing transient signs of neuropathology, lyngbyatoxin A has been shown by the same authors to cause
increased mitosis and small granulomas (inflammatory
cells) in livers of mice upon oral administration (Ito et al.,
2002). Similar signs of hepatotoxicity were observed in the
current study. Thus, lyngbyatoxin A or a structurally similar
compound is a potential candidate for eliciting at least some
of the neuro- as well as hepatotoxicity by L. aerugineocoerulea.
The ability of the L. aerugineo-coerulea extract to cause
adverse effects in vivo was confirmed in vitro using mammalian cell lines. The extract was a strong inhibitor of
[3H]-thymidine incorporation, indicating a significant reduction in DNA synthesis. Inhibition of DNA synthesis
could have been the cause of the increase in mitosis found
in the livers of treated mice. A similar correlation between
the rate of DNA synthesis and mitosis has previously been
observed, for a cyanobacterial indolinone, welwistatin,
which caused a depletion of microtubules in human ovarian
carcinoma and vascular smooth-muscle cells (Zhang and
Smith, 1996). Yet, from the current in vitro data, a specific
mode of toxicity from the L. aerugineo-coerulea extract
could not be deduced. One reason for this is the cytotoxicity
that developed in a time-dependent manner to varying degrees in the different mammalian cell lines. This cytotoxicity, as measured with the MTT assay, could be both a cause
or a result of the inhibition of DNA synthesis. To elucidate
the mechanisms by which the L. aerugineo-coerulea extract
can inhibit DNA synthesis and cause cytotoxicity will be an
important future task.
One intriguing aspect of exposing a fish liver cell
culture along with those of mammalian cell lines was that
it revealed unique reactions of the two animal cell systems to the cyanobacterial extract. The rainbow trout
liver cell line, RTL-W1, was derived from a normal male
liver (Lee et al., 1993) and is the first fish cell line to be
investigated for toxicity caused by cyanobacteria. Normally, fish cells are grown at 19C, which is close to the
organisms temperature. At this temperature no cytotoxicity was observed within 24 h of exposure to the L.
aerugineo-coerulea extract. However, an increase in exposure temperature to 25C, generally the highest temperature at which salmonid cells can be maintained (Bols
et al., 1992), led to severe toxicity by the algal extract,
particularly when serum was absent. A greater sensitivity
of fish cells to toxic substances in the absence of serum
has been observed repeatedly in the past (Kocan et al.,
1983; Lyons-Alca ntara et al., 1996; Schirmer et al.,1997,
2000). In these previous studies the protective effect of

17

serum was attributed to an affinity of the toxic substances

to serum components or to the provision by the serum of
protective molecules, such as antioxidants (Schirmer et
al., 2000). In light of the unknown identity of the cytotoxic compounds produced by L. aerugineo-coerulea, the
exact mechanism of protection by serum cannot be deciphered at this point. However, knowledge about the increased sensitivity of the fish cells and their ability to be
sustained in a serum-free medium should give rise to an
experimental system in which protective agents can be
studied without interference by serum components.
As it did for mammalian cells, time appeared to be one
determinant of whether the Lyngbya extract yielded measurable effects in the fish cells. This was seen when
RTL-W1 cells were exposed at 19C for 48 h instead of 4
or 24 h. Surprisingly, this exposure regime resulted in a
dose-dependent increase in both alamarBlue and
CFDA-AM readings. Inasmuch as this phenomenon occurred both in the presence and in the absence of serum, it
is unlikely to be a result of cell proliferation because fish
cells show very little growth without serum and within 48 h
(Lee et al., 1993). Thus, this increase could be from a
modulation of cellular membrane function. In support of
this hypothesis, pendolmycin, an indole alkaloid which is
structurally similar to lyngbyatoxin A, has previously been
shown to modify membrane function by inhibiting phosphatidylinositol turnover in A431 cells and by activating arachidonic acid release and hexose transport in C3H10T1/2
cells (Umezawa et al., 1989). The alamarBlue is acted on
by oxidoreductases (Goegan et al., 1995; OBrien et al.,
2000) and the CFDA-AM by cellular esterases, both of
which can be membrane-bound, and alteration of membrane
composition or function potentially could increase the availability of these enzymes for the fluorescent dyes. A similar
mechanism might have led to the increase in the conversion
of MTT in the FL cell line after 4 h of exposure to the
cyanobacterial extract. Exposure to the extract of the same
cell line led to a significant reduction in cell viability 20 h
later. Thus, the stimulation of MTT, alamarBlue, and
CFDA-AM conversion may reflect subtle changes to cellular membranes that lead after prolonged exposure to cytotoxicity.
In contrast to the L. aerugineo-coerulea extract, the
medium in which Lyngbya had been grown for 14 days prior
to extraction showed significant cytotoxic effects in
RTL-W1 cells at 19C within 24 h of exposure. Several
lines of evidence suggest that different cytotoxic compounds with different modes of action must have been
involved in the effects elicited by the medium and the
extract, respectively. First, susceptibility of the fish cells to
the cytotoxicity of the L. aerugineo-coerulea extract was
dependent on temperature, whereas sensitivity to the cyanobacterial medium was not. Second, the L. aerugineocoerulea medium led to morphological changes in the fish
liver cells that were not observed with the cyanobacterial

18

TENEVA ET AL.

extract. The morphological changes elicited by the cyanobacterial medium suggest alterations to the cytoskeleton.
One toxin of the marine species Lyngbya majuscula that has
been described as having disrupted a cellular microfilament
network is Lyngbyabellin A (Luesch et al., 2000). This
toxin caused the normally fibroblastic A-10 cells to exhibit
a neuronlike morphology (Luesch et al., 2000). A similar
morphology was seen in RTL-W1 cells, which normally are
epithelial-like, on exposure to the L. aerugineo-coerulea
medium.
Despite the cyanobacterial extracts investigated in this
study having originated from L. aerugineo-coerulea grown
in culture, several environmentally relevant conclusions can
be drawn about the Lyngbya species in specific and cyanobacterial toxins in general. First, L. aerugineo-coerulea is
the second freshwater species of Lyngbya shown to be
capable of producing toxic substances. The culture conditions used were adopted to reflect temperatures and light
exposures typical of the summer months in southern Europe, where L. aerugineo-coerulea can be found and where
it is involved in the formation of cyanobacterial blooms
(Vodenicharov, 2000). Thus, the data obtained from cultured strains can serve as a guide for the toxic potential to be
expected in nature. Second, L. aerugineo-coerulea appears
to release substantial amounts of cytotoxic compounds into
the surrounding medium. This release is unlikely to be a
result of cell lysis alone because different toxic effects were
seen in the cells and the medium. Extracellular concentrations of cyanotoxins in water have only recently been included in some surveys and have been found to be rather
low (Chorus, 2001). However, given the toxic potency of
the cyanobacterial medium in the current study and considering the general lack of knowledge about toxin release,
fate, and transport, the possibility of human and wildlife
exposure to extracellular toxic cyanobacterial metabolites in
water should not be ignored. Finally, as previously observed
with other cyanobacterial samples, the toxicity elicited by L.
aerugineo-coerulea could not be explained by the prominent cyanobacterial toxin microcystin or by saxitoxin. This
highlights the importance of an overall toxicological analysis in the environmental assessment of risk of exposure to
cyanobacteria. Applying a combination of mammalian and
piscine in vitro bioassays is a promising approach that,
combined with an effect-directed chemical analysis, could
be used in the future to identify the structure and cellular
mechanisms of the as-yet-unknown toxic Lyngbya aerugineo-coerulea metabolites, in particular, and to screen cyanobacterial extracts for their toxicity, in general.

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