Environment International 39 (2012) 134–140
Contents lists available at SciVerse ScienceDirect
Environment International
journal homepage: www.elsevier.com/locate/envint
Estrogenic activity in extracts and exudates of cyanobacteria and green algae
E. Sychrová a, T. Štěpánková a, K. Nováková a, L. Bláha a, b, J.P. Giesy c, d, e, f, K. Hilscherová a,⁎
a
Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Kamenice 126/3, 625 00 Brno, Czech Republic
Centre for Cyanobacteria and their Toxins, Institute of Botany, Czech Academy of Sciences, Lidická 25/27, 602 00 Brno, Czech Republic
c
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
d
Zoology Dept. and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824, USA
e
Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, PR China
f
Zoology Department, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia
b
a r t i c l e
i n f o
Article history:
Received 28 April 2011
Accepted 12 October 2011
Available online 24 November 2011
Keywords:
Cyanobacteria
Eutrophication
Endocrine disruption
Estrogenicity
Algae
Phytoplankton
a b s t r a c t
Here is presented some of the first information on interactions of compounds produced by cyanobacteria and
green algae with estrogen receptor signaling. Estrogenic potency of aqueous extracts and exudates (culture
spent media with extracellular products) of seven species of cyanobacteria (10 different laboratory strains)
and two algal species were assessed by use of in vitro trans-activation assays. Compounds produced by cyanobacteria and algae, and in particular those excreted from the cells, were estrogenic. Most exudates were
estrogenic with potencies expressed at 50% of the maximum response under control of the estrogen receptor
ranging from 0.2 to 7.2 ng 17β-estradiol (E2) equivalents (EEQ)/L. The greatest estrogenic potency was observed for exudates of Microcystis aerigunosa, a common species that forms water blooms. Aqueous extracts
of both green algae, but only one species of cyanobacteria (Aphanizomenon gracile) elicited significant estrogenicity with EEQ ranging from 15 to 280 ng 17β-estradiol (E2)/g dry weight. Scenedesmus quadricauda exudates and extracts of Aphanizomenon flos-aquae were antagonistic to the ER when coexposed to E2. The EEQ
potency was not correlated with concentrations of cyanotoxins, such as microcystin and cylindrospermopsin,
which suggests that the EEQ was comprised of other compounds. The study demonstrates some differences
between the estrogenic potency of aqueous extracts prepared from the same species, but of different origin,
while the effects of exudates were comparable within species. The observed estrogenic potencies are important namely in relation to the possible mass expansion of cyanobacteria and release of the active compounds
into surrounding water.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Cyanobacteria are wide-spread organisms, which draw attention in
particular due to their mass expansion in aquatic reservoirs linked with
eutrophication. This leads to impairment of water quality, possibility for
its use for drinking water, recreation or protection of aquatic life. Most
obvious of the effects of cyanobacteria are decreased oxygen content,
shading of green algae, toxicity to zooplankton and changes in water
chemistry (Kopp and Hetesa, 2000). Intracellular and extracellular
products of cyanobacteria can be toxic and impart taste and odor to
water. Number of studies have documented hazardous potential of
these compounds to aquatic as well as terrestrial organisms (Chorus
and Bartram, 1999). Some species of cyanobacteria produce known cyanotoxins, among which the most thoroughly studied are the potent
liver toxicants microcystins (MC). Another product of cyanobacteria,
cylindrospermopsin, which was first isolated from Cylindrospermopsis
raciborskii, is mutagenic and cytotoxic, primarily to liver, kidney and
⁎ Corresponding author at: RECETOX, Kamenice 126/3, CZ62500, Brno, Czech Republic.
Tel.: +420 776741900; fax: +420 54949 2840.
E-mail address: hilscherova@recetox.muni.cz (K. Hilscherová).
0160-4120/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envint.2011.10.004
blood cells (Rastogi and Sinha, 2009; Sukenik et al., 2006). Although
originally identified in a tropical species, cylindrospermopsin has been
reported to occur in many countries including Central Europe (Bláhová
et al., 2009). There are also unidentified, biologically active compounds
in cyanobacteria (Oberemm et al., 1999; Okumura et al., 2007). Cyanobacteria are a source of a spectrum of novel, bioactive natural substances
with the potential biotechnological or biomedical use (Rastogi and
Sinha, 2009). The content of bioactive substances differs among cyanobacteria species and can also change as a function of life stage and environmental conditions. Extracts of cyanobacterial biomass can have
much greater effects than would be expected from the concentration
of known cyanotoxins (Oberemm et al., 1999; Tarczynska et al., 2001).
This could be due to unidentified substances and/or interactions
among the constituents of the mixtures. For example, some products
of cyanobacteria can increase uptake of toxins (Oberemm et al., 1999),
and inhibit detoxification enzymes (Best et al., 2002), or have synergistic effects (Leao et al., 2010).
Some synthetic and natural compounds can alter normal functioning
of the endocrine system (Sumpter and Johnson, 2005). These endocrinedisruptive compounds can, among other adverse health effects, negatively affect normal reproduction or developmental processes. Modulation of
E. Sychrová et al. / Environment International 39 (2012) 134–140
reproduction-related parameters in both vertebrate and invertebrate
species after exposure to cyanobacteria has been observed. In vivo studies with rats documented negative effects of intra-peritoneal exposure
to microcystin and crude extracts of Microcystis aeruginosa on male reproductive organs, sperm quality and levels of hormones (Ding et al.,
2006; Li et al., 2008). Exposure to nodularin and microcystinproducing cyanobacteria species (Nodularia spumigena, M. aeruginosa,
Aphanizomenon flos-aquae) also adversely affected reproduction and
development of oocytes in aquatic invertebrates (Kozlowsky-Suzuki
et al., 2009; Trubetskova and Haney, 2006). In contrast, extensive liver
injury, but no effect on reproduction was observed in a chronic study
where adult mice were exposed for one year to extracts from a bloom
of the cyanobacterium M. aeruginosa (Falconer et al., 1988). Except for
the lesser weight of produced eggs and chicks, reproductive parameters, such as fertilization, egg viability, hatching, number of 14-day old
offspring were better in Japanese quail exposed to environmental cyanobacterial biomass containing microcystin in feed than in the control
group (Damkova et al., 2009).
Despite the observed effects on reproduction-related parameters,
growth and development, there is little information on the endocrine
disruptive potential of compounds from cyanobacteria. An in vitro
study with granullosa cells showed that progesterone production
was inhibited by the cyanotoxin cylindrospermopsin (Young et al.,
2008). A recent study reported weak estrogenic potency of the cyanotoxins microcystin and nodularin by use of an in vitro transactivation
assay in cells stably transfected with an estrogen-regulated luciferase
gene (Oziol and Bouaďcha, 2010). The observed effect was estrogen
receptor (ER)-mediated, since estrogenic effect of cyanotoxins was
inhibited by an ER antagonist. Another recent study focused on gene
expression profiling in larval zebrafish documented strong upregulation of ER-controlled vitellogenin genes in Microcystisexposed larvae, which indicates the presence of estrogenic substance(s) and suggests that Microcystis might be a natural source of
estrogens (Rogers et al., 2011).
Endocrine disruptive effects can be mediated through interference
with hormone functions at different levels of the endocrine system.
Some of these effects can occur via estrogen receptor (ER) signaling.
Estrogens are responsible for metabolic, behavioral and morphologic
changes that occur during various stages of reproduction. They influence cell proliferation and differentiation, development and activity
of tissues participating in process of reproduction. They also control
bone formation, regulation of organism homeostasis, cardiovascular
system and behavior. Estrogenic compounds are characterized by
their ability to bind to and activate the estrogen receptor, which is a
transcription factor belonging to the steroid receptor family (Gillesby
and Zacharewski, 1998). Compounds including natural products,
pharmaceuticals and industrial chemicals have been shown to be estrogen mimics. While there are structural similarities among some
compounds that are ER agonists, other ER-active compounds do not
share similar structures (Janosek et al., 2006).
In vitro transactivation assays based on recombinant cells, which
contain a reporter gene under the control of ER binding, are useful
for estimation of total receptor-mediated potency of samples. They
also account for possible interactions among compounds in mixtures.
In this way the anti/estrogenic potency of compounds and their mixtures, defined as the ability to interfere with estrogen receptor signaling, has been assessed in numerous studies (Campbell et al., 2006;
Hilscherova et al., 2000; Witters et al., 2010).
Our recent research has demonstrated estrogenic potential of
samples prepared from complex cyanobacteria biomasses collected
in the environment and also from laboratory cultured cyanobacterial
species (Stepankova et al., 2011). The study, the results of which are
reported here, was conducted to characterize the estrogenic potential
of compounds produced by laboratory cultures of seven species of
cyanobacteria and two green eukaryotic algal species. The biological
activities of cyanobacterial products can be caused by extracellular
135
compounds released into the environment, but also by substances
present inside cells, that can be eaten or released during degradation
of cyanobacteria. For this reason this study investigated potential effects of cyanobacterial exudates as well as of the compounds contained within the cyanobacterial cells (tested as aqueous extracts).
2. Material and methods
2.1. Preparation of samples of laboratory grown cyanobacteria and algae
for exposure
Extracts and exudates of ten pure cyanobacterial cultures (10 strains
of 7 species) and two algal species grown in the laboratory were tested
for their potency to interact with the ER (Table 1). These represent species commonly occurring in the environment and often forming cyanobacterial blooms. Three cyanobacteria were represented by cultures
from two different sources (collections) to compare the variability
within one species. All species were cultured in a mixture of Zehnder
medium (Schlosser, 1994), Bristol (modified Bold) medium (Stein,
1973) and distilled water in a ratio 1:1:2 (v/v/v). Cells were grown at
22± 2 °C under continuous light (cool white fluorescent tubes,
3000 lx) and aerated with air sterilized by passing through 0.22 μm filter (Labicom, Olomouc, Czech Republic). Cells were separated from extracellular products of laboratory cultures by centrifugation at 3000 g.
Both the concentrated biomass and remaining exudates in media
were stored frozen. The concentration of dry matter was determined
gravimetrically after lyophilization. Concentrations of samples were adjusted with distilled water to 4 g dw/L.
The extracellular organic compounds were concentrated from medium by SPE by use of two columns in tandem. The first was Oasis HLB
Cartridge 500 mg (Waters, Milford Massachusetts, USA), conditioned
with 5 mL methanol, equilibrated by 10 mL distilled H2O. The eluent
from the Oasis HLB column was passed through the second Carbograph
Extract-Clean™ Column 500 mg (Alltech, Deerfield, Illinois, USA),
which had been conditioned with 10 mL methanol and then equilibrated with 10 mL distilled H2O. After loading the organic materials onto
the columns, the columns were eluted with 10 mL methanol. The eluate
was evaporated by vacuum-evaporation to near dryness and washed
out twice with 500 μL MeOH. The eluates were then sonicated and
transferred to Eppendorf® vials where they were evaporated to dryness
and stored frozen. To obtain a 2000-fold concentrated sample, the content was dissolved in 50% methanol in H2O or dimethylsulfoxide
(DMSO) and sonicated on ice.
Thawed samples of collected cyanobacterial biomass were homogenized by sonication with ultrasound Dezintegrator® (Bandelin
Sonoplus HD 2070, Berlin, Germany, 95–100% power, cycle 0.9)
three times for 8 min in a cooling bath. The algal biomass was homogenized the same way with the addition of ballotine to facilitate disruption of the cell walls. A portion of the homogenized biomass was
centrifuged at 3000 g for 15 min, which separated the aqueous extract from the cellular debris. The aqueous extract was supplemented
with distilled water to the original volume of the biomass prior to
centrifugation to keep the original concentration of the compounds.
This extract was filtered through 0.2 μm filter to obtain the sterility
necessary for in vitro testing.
Concentrations of microcystins were determined as previously described (Blahova et al., 2008). Briefly, biomasses extracted with 50%
methanol with sonication were analyzed by use of an Agilent 1100 Series
HPLC equipped with a PDA detector (Agilent Technologies, Waldbronn,
Germany) and Supelcosil® ABZ+ Plus column 15 cm× 4.6 mm ×5 μm
using gradient elution with acetonitrile. The microcystins were identified
based on retention time and UV spectra. Their quantification was based
on external calibrations, with a limit of detection of 5 μg/g dw for
microcystin.
The detailed method for the analysis of cylindrospermopsin (CYN)
has been published previously (Blahova et al., 2009). Samples were
136
E. Sychrová et al. / Environment International 39 (2012) 134–140
Table 1
Characterization of the tested cyanobacterial and algal samples, their cyanotoxin content, and determined estrogen equivalent values (EEQ) for exudates. The table presents the EEQ
values determined at the EC20 and EC50 levels of response along with EEQ value derived from a response reached at 1 × concentration (equal to the original concentration in the
cultivation).
Sample
Species
Cyanobacteria
1
Anabaena flos-aquae
2
Aphanizomenon flos-aquae
Collectiona
Identification
code in
collection
Place of origin
Country
Water body
UTEX
PCC
1444
7905
USA
Netherland
Mississippi
Lake Brielse Meer
31.87
06
31.79
009
1.97
7806
Canada
Ireland
Germany
Great Britain
Hungary
Netherland
unspecified
Lake LoughNeagh
Lake Plussee
reservoir Queen Elizabeth
Lake Balaton
reservoir Braakman
Germany
Lake Plussee
France
Rendeau
USA
Germany
Greifswald
3
4
5
6
7
8
Aphanizomenon flos-aquae
Aphanizomenon gracile e
Aphanizomenon gracile
Aphanizomenon klebahnii
Cylindrospermopsis raciborskii
Microcystis aeruginosa
SAG
RCX
SAG
CCALA
SAG
PCC
9
Planktothrix agardhii
CCALA
10
Planktothrix agardhii
SAG
Algae
11
12
Chlorella kessleri
Scenedesmus quadricauda
CCALA
CCALA
159
32.79
250
463
Toxin
productionb
(μg/g dw)
Estrogen equivalent
(EEQ, ng E2/L )
1×
EC20
EC50
n.d.c
MC n.d.
CYN 3100
n.d.
n.d.
n.d.
n.d.
n.d.
MC 2500
CYN n.d.
MC 170
CYN n.d.
MC 200
CYN n.d.
0.67
n.s.d
0.58
n.s.
0.19
n.s.
n.s.
1.5
0.67
n.i.f
1.7
11.8
n.s.
1.82
0.74
0.69
1.6
6.2
n.s.
0.79
0.61
0.66
1.7
7.2
0.48
0.51
0.54
n.i.
0.75
0.81
1.9
3.88
1.2
4.5
0.69
2.7
CCALA: Culture Collection of Autotrophic Organisms www.butbn.cas.cz/ccala.
PCC: The Pasteur Culture Collection of Cyanobacteria www.pasteur.fr/ip/easysite/go/03b-00000r-0g3/research/collections.
SAG: Culture Collection of Algae (Sammlung von Algenkulturen der Universität Göttingen) epsag.uni-goettingen.de.
UTEX: The Culture Collection of Algae at University of Texas at Austin web.biosci.utexas.edu/utex/.
RCX: RECETOX Culture Collection of Cyanobacteria and Algae.
a
Collections of cyanobacteria and algae.
b
Measured concentrations of microcystins (MC) and cylindrospermopsin (CYN) in biomass, concentrations are rounded.
c
n.d. – no microcystins (MC) and/or cylindrospermopsin (CYN) detected.
d
n.s. – no significant estrogenity at any of the tested concentrations.
e
This species originates from CCALA (strain 008), but has been long-term cultivated at RECETOX.
f
n.i. - no induction at concentration 1x.
extracted by sonication and centrifuged. Solid phase extraction (SPE)
was used to concentrate CYN from the extract using tandem columns
of C18 and ENVI-Carb Supelclean SPE cartridges (Supelco, Bellefonte,
PA, USA). Cylindrospermopsin was eluted with 10 mL of 100% methanol acidified with 0.1% v/v trifluoroacetic acid and the solvent was
evaporated. The extracts were dissolved in milliQ® water and analyzed by an Agilent 1200 HPLC coupled to 6410 Triple-Quad MS (Agilent, USA) with an electrospray (ESI) interface. Separation was
achieved on C18 Supelcosil ABZ + Plus column at 35 °C,
150 × 4.6 mm I.D., 5 μm (Supelco, Bellefonte, PA, USA) with a flow
rate 0.4 mL/min and gradient elution with mobile phases containing
methanol and water acidified with 5 mM ammonium acetate. The
mass spectrometer was operated in multiple reaction monitoring
mode (MRM) with collision energy 40 eV. The capillary voltage and
fragmentation energy were 4000 V and 140 V, respectively. The cylindrospermopsin transition ions m/z 416.2 (M+ H +) to 194.2 and m/z
416.2 to 176.1 were monitored for 250 ms dwell time. Quantification
of CYN was based upon the primary and transition ions of 194.2 and
176.1, respectively at a retention time of 8.25 min and based on external calibration of CYN standard (Sigma-Aldrich, Prague, Czech Republic). Method limit of detection was 10 ng/g dw.
2.2. In vitro bioassays
Estrogen receptor-mediated effects were assessed by use of the
human breast carcinoma cell line MVLN transfected with the ERlinked luciferase gene under control of estrogen responsive element
(ERE) (Willemsen et al., 2004). MVLN cells were cultivated in
DMEM/F12 medium (Sigma-Aldrich, Prague, Czech Republic) supplemented with 10% foetal calf serum Mycoplex (PAA, Pasching, Austria)
at 5% CO2 and 37 °C. Cell culture bioassays were performed in 96 well
microplates with final volume of 200 μL exposure medium per well.
MVLN cells were seeded at densities of 15,000 cells/well. MVLN
cells were exposed in DMEM/F12 supplemented with 5% dialyzed
foetal calf serum (PAA, Austria), which was treated with dextran/
charcoal to further decrease background concentrations of estradiol.
Before measurement of receptor-mediated potency, cytotoxicity
of samples was assessed by use of the Neutral Red assay (NR) (Babich,
1990). Only concentrations of extracts that were not cytotoxic were
further tested for anti/estrogenicity. The exposure was conducted
the same way as for the assessment of estrogenicity. Five milligrams
of neutral red (NR) was dissolved and filtered (0.22 μm) in 10 mL
DMEM/F12 medium without foetal calf serum. At the end of the exposure, 50 μL of this solution was added to cells in culture medium and
incubated for 30 min after which the NR-containing medium was removed. Cells were suspended in 150 μL of lysis solution containing
water, ethanol and acetic acid, and shaken for 30 min (Orbital Shaker
OS-20, BIOSAN, at 120 rpm). The absorbance was measured using the
spectrophotometer (Tecan-Genios®, BIOTEK, USA, program KC4,
λ = 570 nm).
Anti/estrogenic potencies of extracts and exudates were determined either individually or in combination with competing endogenous ligand, 17β-estradiol (E2). The anti-estrogenicity was assessed
by simultaneous exposure of cells to the sample and E2 (33.3 pM).
This competitive concentration corresponds to the EC50 of the E2 calibration curve. Dose–response relationships were developed in triplicate. Final concentrations of exudates ranged from 0.5 to 10-fold
concentrated compared to the original concentration of the exudates.
The aqueous extracts were tested at final concentrations corresponding
to 0.001–0.25 g dw/L. Solvent control and calibration with 1–500 pM
17β-estradiol (E2) were tested along with the samples. The final concentration of organic solvent did not exceed 0.5% of final volume in
E. Sychrová et al. / Environment International 39 (2012) 134–140
150
2.3. Data analysis
100
3. Results
The model cultures obtained from several international collections of cyanobacterial and algal species originate from various water bodies in Europe and North America
(Table 1). Cyanobacterial toxins microcystin or cylindrospermopsin were detected in
four of the tested biomasses, while the other samples did not contain measurable
levels of these toxins (Table 1). The greatest concentration of microcystin, 2500 μg/g
dw, was found in cultures of M. aeruginosa PCC, while concentrations of approximately
200 μg/g dw were found in pure cultures of Planktothrix agardhii from both collections.
Cylindrospermopsin was found only in Aph. flos-aquae PCC at a concentration of
3100 μg/g dw, while there was no cylindrospermopsin detected in the same species
from the SAG collection.
There was no cytotoxicity observed for exudates of cyanobacteria up to the greatest tested concentration (10-fold concentrated original media) on MVLN cells. But, extracellular products of the algae caused some cytotoxicity at tested concentrations.
Toxicity of exudates to MVLN cells was greatest for the alga Scenedesmus quadricauda.
Its greatest tested concentration (10-fold concentrated) caused a 50% decrease in cell
viability compared to control. The same 10-fold concentration of exudate of Chlorella
kessleri caused 30% decrease in viability of MVLN cells (Fig. 1). However, the 1-fold
concentration of exudates corresponding to the original culture did not cause any significant toxicity to MVLN cells. There were no significant cytotoxic effects observed
after exposure to aqueous extracts except for the greatest concentration of extract
from S. quadricauda (0.25 g dw/L), which caused approximately a 30% decrease in viability of MVLN cells.
Except for Aph. flos-aquae from both the PCC and SAG collections, exudates from all
tested pure strains of cyanobacteria and from both algal species were estrogenic
(Table 1). The greatest induction of the luciferase reporter gene (140% E2max) was
caused by extracts of M. aeruginosa. The greatest tested concentrations of exudates
from Aph. gracile SAG induced maximum response of luciferase expression, while the
other exudates did not cause a maximum response (Figs. 2, 3). The estrogen equivalents (EEQ) based on the EC20 or EC50 as well as on the 1x concentration were in
good agreement and documented similar rank of estrogenic potency among the exudates (Table 1). The EEQ50 values of the exudates with detectable estrogenic potency
ranged from 0.2 to 7.2 ng/L. The greatest EEQ was observed for exudates of M. aeruginosa PCC. The exudate from S. quadricauda, with an EEQ50 of 2.7 ng/L, was the more potent of the algae. The effects of exudates from the same species but different laboratory
strains were similar. Aph. flos-aquae from both collections exhibited no measurable estrogenic potency. Comparable EEQ values were obtained for the two exudates of Aph.
Aphanizomenon gracile CCALA
Aphanizomenon flos-aquae PCC
Anabaena flos-aquae
Chlorella kessleri
Scenedesmus quadricauda
50
0
control
1x
5x
10x
concentration factor
Fig. 1. Cytotoxicity of cyanobacterial and algal exudates after 24 h exposure in MVLN cell
line determined by the neutral red (NR) assay. Values represent the mean± standard
error (n= 3).
gracile as well as for both exudates of P. agardhii, which also caused a similar maximal
response (around 70% E2max) (Fig. 3, Table 1).
There was a significant dose-dependent estrogenic response caused by aqueous
extracts of two strains of Aph. gracile from both the RCX and SAG collection (Fig. 4). Extracts of Aph. gracile from RCX consistently caused 300% of the maximum response to
E2, while the SAG strain only caused about 90% of the maximal E2 response. Extracts
of the other cyanobacterial strains did not exhibit significant estrogenic potency. Alternatively, there was some estrogenic potential observed in aqueous extracts from both
algal species (Fig. 4). The EEQ50 values for the four potent aqueous extracts ranged
from 15 to 280 ng/g dw with EEQ20 to EEQ80 values in the range of 10 to 677
(Table 2). Thus, both algal species and Aph. gracile were the only species where estrogenic potency was observed for both the extracellular and intracellular compounds.
Antiestrogenic (antagonistic) effects compared to the response to 33 pM E2 were
observed only for the exudate of S. quadricauda. None of the cyanobacterial exudates
were anti-estrogenic (data not shown). The IC50 value for the exudate of S. quadricauda
was a 0.78 dilution, while a 0.5 × dilution caused 40% inhibition of the E2 effect.
Among the aqueous extracts, only that of Aph. flos-aquae PCC suppressed the effect
induced by estradiol (33 pM) in a dose-dependent manner. The greatest tested concentration
(0.25 g/L) decreased the response to 30% of that caused by E2 alone (Fig. 5). The extract of
Aph. flos-aquae SAG in the presence or absence of E2 induced strong estrogenic responses
at lesser concentrations, but at concentrations above 0.003 g/L, the responses were significantly less than the control values (Fig. 6), indicating an antiestrogenic effect.
4. Discussion
There are both natural and synthetic hormonally active compounds in
the environment, including those arising from metabolization and breakdown of other compounds. Effects of endocrine disrupting chemicals
(EDCs) on animals in the aquatic environment have been documented
(Burkhardt-Holm, 2010; Sumpter and Johnson, 2005). The results of
150
% E2max induction
The mean solvent control response was subtracted from both
sample and standard dose responses and the detected luminescence
induced by sample dilutions was related to the maximal response of
standard ligand 17β-estradiol (E2max) and converted into percentages. The estrogen equivalent (EEQ) values, expressed in ng E2/L for
exudates and ng E2/g dw for biomass, were determined by relating
the amount of unknown sample required to give the response equal
to 20, 50 and 80% E2max (EC20, EC50, EC80) respectively, to the equivalent amount of E2 required to cause the same magnitude of response.
To account for violation of assumptions of the data analysis, such as
the slopes of the standard and unknown dose–response relationships
not being parallel, the multiple point estimate approach over the
range of responses from EC20 to EC80 was applied for biomass extracts
where the maximal induction caused by the samples was greater than
the EC80 induction level of the standard estradiol (Villeneuve et al.,
2000). Since the maximal induction reached with exudate samples
was generally less than the EC80 level of response to estradiol, the
point estimates based on the EC20 and EC50 levels of response are
reported. For exudates, a point estimate of EEQ at 1× concentration
(equal to the original concentration in the cultivation) is reported as
well. EC values were calculated by nonlinear logarithmic regression of
dose–response curve of calibration standard and samples (Graph Pad
Prism, GraphPad® Software, San Diego, California, USA). The IC50 was
defined as the concentration that resulted in 50% inhibition relative to
the response to the competing ligand E2. This was determined from
the logarithmic regression of the inhibition dose–response curve
obtained by simultaneous exposure of the sample with added competing ligand E2 (33 pM).
% control
the bioassays. The exposure lasted 24 h. After the exposure, the intensity of luciferase luminescence was measured using the Promega Steady
Glo Kit® (Promega, Madison, WI, USA).
137
Anabaena flos-aquae
Microcystis aeruginosa
Cylindrospermopsis raciborskii
Chlorella kessleri
100
50
0
1.23 3.7 11.1 33.3 100 500
calibration curve E2 (pM)
0.5x
1x
5x
10x
concentration factor
Fig. 2. Examples of the dose–response curves of the estrogenic activity (expressed as %
of maximal estrogen receptor-mediated induction caused by standard estradiol —
E2max) in MVLN cell line after 24 h exposure to exudates from tested cyanobacteria
and algal species. Values represent the mean ± standard error (n = 3).
E. Sychrová et al. / Environment International 39 (2012) 134–140
Aphanizomenon gracile CCALA
Aphanizomenon gracile SAG
Planktothrix agardhii CCALA
Planktothrix agardhii SAG
Table 2
Determined EEQ values for samples of aqueous extracts, which elicited significant
estrogenicity.
Collectiona Maximal
EEQ50
EEQ20–EEQ80
induction (ng E2/g dw) (ng E2/g dw)
(% E2max)
4
RCX
319
280
113–667
SAG
92
15
10–24
CCALA
CCALA
86
66
62
58
54–72
113–l.i.b
Aphanizomenon
gracile
Aphanizomenon
gracile
Chlorella kessleri
Scenedesmus
quadricauda
5
11
12
1x
5x
10x
a
Fig. 3. Estrogenic response (expressed as % of maximal estrogen receptor-mediated induction caused by standard estradiol — E2max) in MVLN cell line after 24 h exposure to
exudates from two cyanobacteria species from different collections. Values represent
the mean ± standard error (n = 3).
our study demonstrate estrogenic potential of compounds produced by
cyanobacteria and algae, which was generally greater for extracellular
compounds than intracellular compounds. The observed estrogenic
potencies are important namely in relation to the possible mass
expansion of cyanobacteria under favorable conditions. There can
be significant amounts of extracellular products of cyanobacteria
released to the surrounding water during the life time of cyanobacteria.
The greatest estrogenicity was detected for exudates of M. aerigunosa, a
common species in environmental water blooms, with EEQ approximately 10-fold greater than those of most other species. Concentrations
of EEQ in the range of ng/L found in the extracellular products could be
considered relatively large since equivalent potencies of estrogens have
been shown to cause reproductive toxicity to aquatic animals. Even a
complete collapse of a fish population was documented during a
seven year whole-lake experiment with ethinylestradiol (EE2) at concentrations of 5–6 ng/L (Kidd et al., 2007). EE2 concentration as little
as 0.2 ng/L completely inhibited reproduction of the F-1 offspring in a
multigeneration study of Chinese rare minnows (Gobiocypris rarus)
(Zha et al., 2008b). In the same species, 4 ng/L ethinylestradiol significantly affected fecundity, fertility, and laying interval and lead to male
feminization, ova-testis and increased plasma VTG in both males
and females already after 21 day exposure (Zha et al., 2008a).
% E2max induction
350
Aphanizomenon gracile CCALA
Aphanizomenon gracile SAG
Chlorella kessleri
Scenedesmus quadricauda
300
250
200
150
100
50
E2calibration(pM)
0.25
0.083
0.028
0.0093
0.0031
500
100
33.3
11.1
3.7
1.23
0
concentration(g/L)
Fig. 4. Dose–response curves of the estrogenic action of the active aqueous extracts
samples prepared from cyanobacteria and algae in MVLN cell line after 24 h exposure
(expressed as % of maximal estrogen receptor-mediated induction caused by standard
estradiol — E2max). Concentrations (sample dilutions) on X-axis are expressed as
equivalent g dry weight of biomass from which the extract was prepared per L in the
bioassay. Values represent the mean ± standard error (n = 3).
b
Abbreviations of Collections as in Table 1.
l.i. — low induction — not sufficient to determine EEQ80.
Aqueous extracts caused significant estrogenicity in case of both
algal species, but only one cyanobacterium (Aph. gracile). The effect
was caused by intracellular compounds, which can be released especially during degradation of cells at the end of the growing season. In
a previous study, estrogenic potency of aqueous extracts from complex cyanobacterial bloom biomasses collected from the environment
had EEQ20 values ranging from 730 to 970 ng/g dw (Stepankova et al.,
2011). Some of those environmental bloom biomasses, aqueous extracts of which were estrogenic, were dominated by several species,
including P. agardhii or M. aeruginosa, the extract of which was not estrogenic in the present study. The differences could be partly caused
by the fact that in the other study, the complex bloom biomasses
were collected by use of a plankton net and the exudates were not
completely separated by centrifugation, which could influence the results. However, our study also showed differences in estrogenic potency of samples prepared from different strains of the same species
in some cases. Extracellular products collected from different strains
of the same species caused no effect (Aph. flos-aquae) or their EEQs
were comparable (Aph. gracile, P. agardhii). Alternatively, even
though the aqueous extracts from the two Aph. gracile cultures from
different collections elicited (as the only cyanobacteria) clear dose
dependent estrogen-like response, there was a 20-fold difference in
their EEQs, and one of them caused much greater maximal induction
than the other. The difference in the responses to extracts from Aph.
flos-aquae from two different collections also indicates that different
strains can have different potencies (Figs. 5,6). Thus, there are
150
100
Aphanizomenon flos-aquae PCC
50
Aphanizomenon klebahnii CCALA
0
1.23
concentration
factor
% competing E2 induction
calibration curve E2 (pM)
E2 concentration (pM)
0.25
0.5x
0.083
1.23 3.7 11.1 33.3 100 500
0.028
0
0.0093
50
Sample Species
0.0031
100
100
% E2max induction
150
33.3
138
concentration (g/L)
Fig. 5. Anti/estrogenic activity (% of induction compared to the effect of competing
33 pM E2) of cyanobacterial extracts determined in MVLN cell line after 24 h exposure.
Sample dilutions on X-axis as in Fig. 4. Values represent the mean ± standard error
(n = 3).
2.5
2
no E2 added
added 33 pM E2
1.5
1
0.5
control
DMSO
E2 calibration
(pM)
0.25
0.083
0.028
0.0093
0.0031
0.001
0.00034
100
33.3
11.1
0
0
Relative luminiscence units
E. Sychrová et al. / Environment International 39 (2012) 134–140
Aphanizomenon flos-aquae
SAG (g/L)
Fig. 6. Biphasic effect observed after 24 h exposure of MVLN cells to aqueous extract
from Aphanizomenon flos-aquae SAG. The dark columns show the responses without
addition of any competing ligand, the white columns after addition of the 33 pM E2.
Sample dilutions on X-axis as in Fig. 4. Values represent the mean ± standard error
(n = 3).
probably other unknown parameters influencing production of compounds able to interact with ER signaling pathways by cyanobacteria
and algae.
There are few reports on the endocrine disruptive influence or reproductive effects of cyanobacterial substances, but they indicate the
potential to modulate hormonally regulated processes. Intraperitoneal exposure of male mice to extracts of M. aeruginosa cells
or MC-LR for 2–4 weeks lead to decreased body weight, smaller and
damaged testes, decreased quality of mature sperm, reduced sperm
motility and viability as well as lesser sperm concentration and concentrations of testosterone, FSH and LH in blood serum (Ding et al.,
2006; Li et al., 2008). The results of an in vitro study documented lesser testosterone production along with greater cytotoxicity, production of reactive oxygen species and lipid peroxidation after exposure
of Leydig cells to 500 nM of MC-LR (Li et al., 2008). Some of the observed effects could be associated with potential estrogenic potency
of cyanobacterial products. Exposures of laboratory animals and wildlife to estrogenic chemicals have been shown to significantly affect
the reproductive system and in some cases to result in abnormalities
including reduced gonad size, feminization of genetic males, and low
sperm count and quality (Akingbemi, 2005; Carreau et al., 2007; Min
and Lee, 2010). However, administration of extracts of a bloom of cyanobacterium M. aeruginosa as a source of drinking water to mice for one
year had no effect on reproduction (Falconer et al., 1988). There was no
difference in number, gender, viability and body weight of young of exposed and non exposed parents.
A recent study of the estrogenic potency of the cyanotoxins
microcystin-LR and nodularin-R by use of a transactivation assay
with a cell line stably transfected with an estrogen-regulated luciferase gene (Oziol and Bouaďcha, 2010), such as used in our study,
have also reported estrogenic effects. Both nodularin-R and
microcystin-LR exhibited weak estrogenic potency, which was inhibited when pure estrogen receptor antagonist was added to the cells.
This result is consistent with ER-dependent effects. The authors of
that paper suggested that the estrogenic mechanism of nodularin-R
and microcystin-LR is probably mediated through phosphorylation
of signaling pathways similar to ocadaic acid (phosphatases inhibitor)
that at comparable concentrations (50 nM) activates estrogen receptors without the presence of their ligands. Another possible mechanism of the estrogenic potential of these cyanotoxins might be
oxidative stress through production of reactive oxygen species (ROS)
that can significantly affect signaling proteins including transcription
factors (Morel and Barouki, 1998).
139
In the present study, the known cyanotoxins, microcystin and cylindrospermopsin, were detected in the biomass from four cultures
(Table 1). The only extract that caused clear dose-dependent antiestrogenic activity in co-exposure with E2 was Aph. flos-aquae PCC, which
was the only species containing cylindrospermopsin (3100 μg/g dw).
This may indicate interference of cylindrospermopsin with hormonal
signaling and it should be evaluated in further studies. A previous
study documented inhibition of progesterone production in human
granulosa cells after exposure to cylindrospermopsin at a concentration
of 0.0625 μg/mL, without affecting the production of estrogen or cytotoxicity (Young et al., 2008). Alternatively, none of the aqueous extracts
that exhibited significant estrogenic potency, prepared from both Aph.
gracile and both algae cultures, contained detectable cyanotoxins.
Thus, the effects are probably caused by as yet unidentified compounds
and/or their mutual interactions. More pronounced effects of exposure
to cyanobacterial crude extracts than to their known toxins alone have
been documented in previous studies (Oberemm et al., 1999; Palikova
et al., 2007).
The extracts and exudates from both tested algae species exhibited
significant estrogenicity, which indicates the role of compounds not
specific for cyanobacteria, but present also in algae, probably phytoestrogens, in the observed effects. Also, the super-induction of the luciferase activity (>100% E2max), such as observed for the extract of Aph.
gracile RCX, has been previously shown for some phytoestrogens, such
as daidzein, genistein or resveratrol in the MVLN bioassay (Freyberger
and Schmuck, 2005; Li, 2006). However, to our knowledge there is no
information on the presence of these compounds or any other known
phytoestrogens in cyanobacteria or algae, although similar compounds
can be found in other autotrophic organisms (Gross, 2003; Rochester
and Millam, 2009). Exudates of S. quadricauda were agonistic when
tested in media devoid of E2, while there was an antagonistic response
in the presence of competing E2. Phytoestrogens can also act as both estrogens and antiestrogens, and the contradictory effects may be related
to them being weak ER agonists. At low concentration of the endogenous hormone, weak agonists can elicit estrogenic effects, while they
can block the binding sites in case of greater concentration of endogenous
ligand decreasing the response (D'Alessandro et al., 2005).
In the present study, biphasic responses were observed for the extract from Aph. flos-aquae SAG with great responses (super-induction) at lesser concentrations followed by responses that were less
than that of the controls at greater concentrations without any signs
of cytotoxicity. The possible explanation includes inhibition of luciferase activity or its synthesis by some compounds present in the sample
at greater concentrations or a presence of a potent antiestrogen,
which is not effective at lesser concentrations, or compounds with
possible biphasic effects mimicking estrogenic action at lesser concentration and being anti-estrogenic at greater concentrations. Although these effects might be expected in complex mixtures, there
are no experimental studies that have investigated this in detail. Similar biphasic effects have been observed previously for some compounds such as genistein that stimulated growth of breast tumor at
lower concentrations and inhibited at greater ones (Lemos, 2001).
In conclusion, this study provides the first information on the significant presence of compounds able to interfere with estrogen signaling in cyanobacterial and algal exudates and also in biomass of
some species. From the environmental point of view, exudates of cyanobacteria seem to be of greater importance for the aquatic environment as release of bioactive compounds during mass expansion of
blue-green algae may continuously affect other organisms. Since
both exudates and extracts contain mixtures of compounds, the measured responses can be the result of numerous interactions among
compounds and/or their effects. Further studies are needed to better
characterize the parameters influencing the occurrence and levels of
the estrogenic potencies both in model cyanobacterial species and in
the environmental water blooms and their potential environmental
significance.
140
E. Sychrová et al. / Environment International 39 (2012) 134–140
Acknowledgements
We thank Lucie Blahova for technical assistance. This research was
supported by the Czech Science Foundation grant No. GACR 524/08/
0496, the Ministry of Education, Youth and Sports of Czech Republic
Project ENVISCREEN 2B08036 and by the project CETOCOEN (no.
CZ.1.05/2.1.00/01.0001) from the European Regional Development
Fund. Prof. Giesy was supported by the Canada Research Chair program,
and at large Chair Professorship at the Department of Biology and
Chemistry and State Key Laboratory in Marine Pollution, City University
of Hong Kong, the Einstein Professor Program of the Chinese Academy
of Sciences and the Distinguished Visiting Professor program of King
Saud University.
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