Do cyanobacteria produce
estrogenic compounds?
Klára Hilscherová
K. Nováková, E. Sychrová, T. Štěpánková, J.P.Giesy, L. Bláha
Research Centre for Toxic Compounds in the Environment (RECETOX),
Faculty of Science, Masaryk University, Brno, Czech Republic
Biomedical Sciences and Toxicology Centre, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada
Bioactive compounds in cyanobacteria
Extracts from aquatic water blooms – greater effects than according to the
cyanotoxin content (fish, amphibians, invertebrates, mammals)
- cyanotoxins are produced in mixture with other cyanobacterial compounds
- cyanobacterial blooms can accumulate various anthropogenic chemicals
Effects of mixtures from cyanobacterial water bloom = result of interactions of
many endogenous and accumulated compounds
COOH
CH3 O
N
NH
OCH3
H3C
O
NH
C H3 CH3
CH3
NH
O
NH
O H2C
H3C
O
NH
NH
COOH O
O
C H3
CH3
NH
HN
NH2
Known cyanotoxins
(MCs, CYN, ATX, SXT,..)
O3SO
H3C
H
OH
H
H
N
NH NH
NH
O
???
Additivity
Other bioactive
compounds
produced by
cyanobacteria
Antagonism
Synergism
NH
O
Anthropogenic contaminants
In vivo studies: effects on fecundity, reproduction,
development, sperm quality in mammals, male
reproductive system in mammals and birds,
survival, reproduction in invertebrates
(Ding et al., 2006; Li et al., 2008, Kozlowsky-Suzuki et al., 2009; Trubetskova
and Haney, 2006)
? Specific mechanisms of effects – ? Endocrine disruption
Possible consequences: disruption of homeostasis, reproduction,
development, behavior, defective sexual development, low
fecundity/fertility/immunity, malformations
The aim of study:
Find out if cyanobacteria can produce compounds that
could act through important specific mechanisms and
thus disrupt functioning of endocrine system
Disruption of hormonal
regulation
Synthesis
Transport
Inhibition
Interaction with receptors
Metabolization
Stimulation
NUCLEAR RECEPTOR MEDIATED EFFECTS
– important mechanisms involved in chronic toxicity
Dioxin-like activity
Aryl hydrocarbon receptor (AhR)
• induction of detoxification systems
• hepatotoxicity, immunotoxicity
• affects reproduction, endocrine and
nervous system, carcinogenesis,
embryotoxicity
• „cross-talk“ with signaling pathways of
hormones
Anti/estrogenicity
Estrogen receptor (ER)
• sexual development, control of
reproduction, carcinogenesis, affects
cellular proliferation and differentiation,
development and homeostasis
Anti/androgenicity
Androgen receptor (AR)
• sexual development, namely male
sexual characteristics, control of
reproduction, carcinogenesis, affects
growth, spermatogenesis
Glucocorticoid activity
Glucocorticoid receptor (GR)
• affects growth, metabolisms, immune
response, reactions to stress
Anti/retinoid activity
Retinoid acid receptor (RAR)
• regulates growth, morphogenesis,
apoptosis and differentiation, affects
nervous and immune system, vision
and embryonal development
In vitro reporter gene trans-activation assays
= cell lines with reporter gene luciferase under control:
AhR – dioxin-like toxicity
ER – anti/estrogenicity
AR – anti/androgenicity
GR – glucocorticoid activity
RAR – anti/retinoid activity
Complex cyanobacterial blooms from the environment
Sample Species composition
Date of sampling
Locality
A
Microcystis aeruginosa
(90%), Microcystis
wesenbergii (10%)
Microcystis aeruginosa
(30%), Microcystis
wesenbergii (20%),
Microcystis
ichthyoblabe (25%),
Aphanizomenon sp. (25%)
Anabaena spiroides
8 October 2004
Brno - reservoir
Total
microcystin
content
(µg/g dw)
n.d.
1 October 2007
Brno - reservoir
410
19 August 1998
n.d.
Aphanizomenon flosaquae (70%), Microcystis
viridis (30%)
Planktothrix agardhii
14 August 1997
Jedovnice - pond
Olšovec
Skalka - dam
reservoir
Dubice - flooded pit
2290
B
C
D
E
8 September 2004
715
Complex cyanobacterial blooms from the environment
Sample Species composition
Samples
A
B
C
D
E
Date of sampling
Locality
Total
microcystin
content
(µg/g dw)
n.d.
Brno - reservoir
Microcystis aeruginosa
8 October 2004
(90%), Microcystis
Aqueous
extract from the biomass
wesenbergii1(10%)
Fraction
(SPE C18 permeate) – polar
Microcystis aeruginosa
1 October 2007
Brno - reservoir
410
Fraction
2
(SPE
eluate)
–
less
polar
containing
microcystins
(30%), Microcystis
wesenbergii (20%),
Microcystis
Results
ichthyoblabe (25%),
Aphanizomenon sp. (25%)
No
significant
concentrations:
Anabaena
spiroideseffects at
19noncytotoxic
August 1998
Jedovnice - pond
n.d.
Olšovec
• AhR (dioxin-like toxicity)
Aphanizomenon flos14 August 1997
Skalka - dam
715
•aquae
GR
(glucocorticoid
activity)
(70%), Microcystis
reservoir
•viridis
RAR
(anti/retinoid activity)
(30%)
Planktothrix agardhii
8 September 2004 Dubice - flooded pit
2290
E
E1
0.00078
D
D1
250
150
100
50
0
equivalent biomass concentration (g dw/L)
E2
D2
0.0625
0.03125
B1
0.0078
0.125
B
0.0625
0.03125
0.0125
0.0078
0.0039
0.00078
0.0625
A2
0.000078
C2
0.0625
0.03125
0.0078
C1
0.00625
0.000625
0.78
A1
0.00625
0.00078
0.000625
C
0.078
0.0078
0.0078
0.00078
A
0.000078
0.003125
0.00156
0.00078
0.000078
% of E2max induction
% of E2max induction
0.025
0.0625
0.25
2.5
0.0015
0.015
0.0625
0.15
0.0015
0.015
0.0625
0.15
0.000625
0.00625
0.0078
0.03125
0.0625
0.000625
0.00625
0.0078
0.03125
0.0625
0.000078
0.00078
0.0078
PRO-ANDROGENIC ACTIVITY
0.000078
200
In both polar (without MC) and less
polar fraction (with MC)
% of E2max induction
ESTROGENIC ACTIVITY
200
150
100
50
0
B2
150
100
50
0
Model species cultured in laboratory
Sample
Species
Collection
Place of origin
Country
Water body
CYANOBACTERIA
1
Anabaena flos-aquae
UTEX
USA
Mississippi
2
Aphanizomenon flos-aquae
PCC
Netherland
lake Brielse Meer
3
Aphanizomenon flos-aquae
SAG
Canada
unspecified
4
Aphanizomenon gracile
RCX
Ireland
lake LoughNeagh
5
Aphanizomenon gracile
SAG
Germany
lake Plussee
6
Aphanizomenon klebahnii
Great Britain
reservoir Queen Elizabeth
7
Cylindrospermopsis raciborskii
SAG
Hungary
lake Balaton
8
Microcystis aeruginosa
PCC
Netherland
reservoir Braakman
9
Planktothrix agardhii
CCALA
Germany
lake Plussee
10
Planktothrix agardhii
SAG
France
Rendeau
CCALA
ALGAE
11
Chlorella kessleri
CCALA
USA
12
Scenedesmus quadricauda
CCALA
Germany
Greifswald
Model species cultured in laboratory
Sample
Species
Collection
Place of origin
Country
Water body
CYANOBACTERIA
1
Anabaena flos-aquae
UTEX
USA
Mississippi
2
Aphanizomenon flos-aquae
PCC
Netherland
lake Brielse Meer
3
Aphanizomenon flos-aquae
SAG
Canada
unspecified
4
Aphanizomenon gracile
RCX
Ireland
lake LoughNeagh
5
Aphanizomenon gracile
SAG
Germany
lake Plussee
6
Aphanizomenon klebahnii
Great Britain
reservoir Queen Elizabeth
7
Cylindrospermopsis raciborskii
SAG
Hungary
lake Balaton
8
Microcystis aeruginosa
PCC
Netherland
reservoir Braakman
9
Planktothrix agardhii
ExtracellularCCALA
spent media
Germany (Exudates)
lake Plussee
10
Planktothrix agardhii
Preconcentration of organic compounds on tandem SPE
SAG
France
Rendeau
(C18 OASIS HLB + ALLTECH CARBOGRAFF)
SamplesCCALA
Aqueous extracts from biomass
ALGAE
11
Chlorella kessleri
CCALA
USA
12
Scenedesmus quadricauda
CCALA
Germany
Greifswald
Estrogenity in model species cultured in laboratory
Significant EEQs - in exudates of most species
- in extracts of both algal species and in cyanobacteria
Aphanizomenon gracile from two different collections
160
E2 calibration
120
80
40
0
%E2 max induction
% E2 max induction
160
Anabaena flosaquae
120
80
Microcystis
aeruginosa
40
Cylindrospermopsis
raciborskii
0
Chlorella kessleri
-40
-40
0.1
1
10
1
10
100
1000
Effects not
related
to
the
concentration
of
studied
cyanotoxins
(MC, CYN)
concentration of exudate
E concentration (pM)
0.1
1
10
concentration of exudate
E2 calibration (pM)
0.25
0.083
-40
0.028
Planktothrix
agardhii SAG
0.0093
0
0.0031
Planktothrix
agardhii UTEX
500
40
100
Aphanizomenon
gracile SAG
33.3
80
11.1
Aphanizomenon
gracile CCALA
Aphanizomenon gracile CCALA
Aphanizomenon gracile SAG
Chlorella k essleri
Scenedesmus quadricauda
3.7
120
350
300
250
200
150
100
50
0
-50
1.23
%E2 max induction
160
% E2max induction .
2
concentration (g/L)
Estrogen equivalents in biomass
Environmental samples
Sample
A
B
C
D
E
crude extract
730
970
n.s.
760 *
n.c.
EEQ20 (ng/g dw)
permeate
200
360
n.s.
150
2200
eluate
140
19
n.s.
220
n.c.
Laboratory cultures – model species
Sample
Species
Collection
Maximal
induction
EEQ50
EEQ20 – EEQ80
(ng E2/g dw)
(% E2max)
(ng E2/g
dw)
4
Aphanizomenon gracile
RCX
319
280
113 - 667
5
Aphanizomenon gracile
SAG
92
15
10 - 24
11
Chlorella kessleri
CCALA
86
62
54 - 72
12
Scenedesmus quadricauda
CCALA
66
58
113 – l.i.
Estrogen equivalents in biomass
Environmental samples
Sample
crude extract
A
730
B
970
n.s.
C
D
760 *
E Estimate for
n.c.Brno
EEQ20 (ng/g dw)
permeate
200
360
n.s.
150
lake 2200
eluate
140
19
n.s.
220
n.c.
At the density of 20 000 cells/mL EEQ in biomass about 0.8 ng/L
Laboratory
cultures
model
species
At the density
of 100 000
cels/mL –
EEQ
4.3 ng/L
Sample
Species
Collection
Maximal
induction
EEQ50
EEQ20 – EEQ80
(ng E2/g dw)
(% E2max)
(ng E2/g
dw)
4
Aphanizomenon gracile
RCX
319
280
113 - 667
5
Aphanizomenon gracile
SAG
92
15
10 - 24
11
Chlorella kessleri
CCALA
86
62
54 - 72
12
Scenedesmus quadricauda
CCALA
66
58
113 – l.i.
Exudates of model species cultured in laboratory
Collection
Toxin production
(µg/g dw)
EEQ
(ng E2/L )
Anabaena flos-aquae
UTEX
n.d.
0.19
Aphanizomenon flos-aquae
PCC
CYN 3100
n.s.
Aphanizomenon flos-aquae
SAG
n.s.
Aphanizomenon gracile
RCX
n.d.
n.d.
0.79
Aphanizomenon gracile
SAG
n.d.
0.61
CCALA
n.d.
0.66
Cylindrospermopsis raciborskii
SAG
n.d.
1.7
Microcystis aeruginosa
PCC
MC 2500
7.2
Planktothrix agardhii
CCALA
MC 170
0.54
Planktothrix agardhii
ALGAE
SAG
MC 200
0.81
Species
CYANOBACTERIA
Aphanizomenon klebahnii
Chlorella kessleri
CCALA
0.69
Scenedesmus quadricauda
CCALA
2.7
Environmental relevance
EEQ in ng/L levels can cause reproductive toxicity in aquatic
animals
Zha et al. 2008. Complete inhibition of F-1 generation in multigeneration
study with minnows (0.2 ng/L ethinylestradiol EE2, Gobiocypris rarus).
Toxicol. Appl. Pharmacol. 226: 298-308
Zha et al. 2008. Adverse effects on fecundity and fertility, occurrence of
ova-testes, increased plasma VTG (4 ng/L EE2, Gobiocypris rarus, 21
days). Ecotoxicol. Environ. Safety 71: 390-399
Kidd et al. 2007. Collapse of fish populations after exposure to estrogen
(5 ng/L EE2, 7 years). Proceedings of the National Academy of Sciences
104(21):8897-8901
Controls
+Ethinylestradiol
In search for „cyano“-estrogens
C. raciborskii lab culture spent media
2 major estrogenic fractions
- concentrated (C18/carbon SPE)
- fractionated on HPLC / C18, slow methanol gradient
Sum of fractions > whole sample
- chromatogram UV @205 nm
(masking effects in the mixture)
Summary
• Compounds from cyanobacteria and algae can affect
signaling of important nuclear receptors
– No significant dioxin-like, glucocorticoid or anti/retinoid activity
– Interference with signaling of androgen receptor
– Most important effect: ESTROGENICITY
• Both environmental and laboratory biomasses
• Both cyanobacteria and algae
• Both in extracts and exudates (! Effects for more species!)
• Both in polar and less polar fraction
• Unrelated to known cyanotoxins
• ? cyano-phyto estrogens ?
Acknowledgements
..coworkers and students….
Czech Science Foundation grant No. GACR P503/12/0553
project CETOCOEN (no. CZ.1.05/2.1.00/01.0001) from the
European Regional Development Fund
Thank you for your attention !
http:/www.recetox.muni.cz
E-mail contact: hilscherova@recetox.muni.cz