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The zebrafish (Danio rerio)
- model species for ecotoxicology research
Summerschool, University of Bern, FIWI, Lisa Baumann
Ecotoxicology is the study of the effects of toxic chemicals on biological organisms, especially at
the population, community, ecosystem level. Ecotoxicology is a multidisciplinary field, which integrates toxicology, physiology and ecology.
What is ecotoxicology?
- What kind of chemicals can be found in the environment?
- Where do they come from?
- How do they reach wildlife and humans?
- What can be done to investigate the resulting problems?
- What can be done to solve them?
Some general questions...
How do toxicants reach us?
adapted from: Wise et al. 2011
Are Oral Contraceptives a Significant Contributor to theEstrogenicity of Drinking Water?†
A M B E R W I S E , ‡ , § K A C I E O ’ B R I E N , ‡ A N DT R A C E Y W O O D R U F F * , ‡
Program on Reproductive Health and the Environment, University of California,San Francisco, 1330 Broadway Street, Suite 1100, Oakland, California 94612, andDepartment of Environmental Studies and Biological Sciences, Asian Universityfor Women, 20G M.M. Ali Road, Chittagong-4000 Bangladesh
Received April 30, 2010. Revised manuscript received August 31, 2010. AcceptedSeptember 14, 2010.
Recent observed feminization of aquatic animals has raisedconcerns about estrogenic compounds in water supplies andthe potential for these chemicals to reach drinking water.Public perception frequently attributes this feminization to oralcontraceptives (OCs) in wastewater and raises concernsthat exposure to OCs in drinking water may contribute to therecent rise in human reproductive problems. This paper reviewsthe literature regarding various sources of estrogens, insurface, source and drinking water, with an emphasis on theactive molecule that comes from OCs. It includes discussion ofthe various agricultural, industrial, and municipal sourcesand outlines the contributions of estrogenic chemicals to theestrogenicity of waterways and estimates that the risk of exposureto synthetic estrogens in drinking water on human health isnegligible.Thispaperalsoprovidesrecommendationsforstrategiesto better understand all the potential sources of estrogeniccompounds in the environment and possibilities to reduce thelevels of estrogenic chemicals in the water supply.
Introduction
The recent increase in examples of intersex fish and organismsfound in global waterways has led people to be concernedabout estrogenic compounds in the environment (1-13).Often, oral contraceptives (OCs) are blamed, as they are aneasily identifiable source of estrogen, with 11.6 million womenof reproductive age using OCs in the U.S. (14). Use of OCsallows women a significant level of reproductive freedomand additionally has societal and global ramifications onpopulation levels. However, after wastewater treatment, lowlevels of the main estrogenic ingredient in OCs, 17 alpha-ethinylestradiol (EE2), have been detected in some surfacewaters (15-18), and this has caused some concern aboutdrinking water contamination. The Endocrine Society re-cently published a position statement expressing concernthat low level, chronic exposure to such environmentalendocrine disruptors cause or contribute to adverse humanhealth effects (19). Intersex fish have been observed nearsewage treatment plants in the U.S., across Europe, and inJapan (5, 11, 20). There is growing concern that a connection
exists between estrogenic surface water, the occurrence ofintersex fish in these rivers, lakes, and streams, and the risein human reproductive problems (7). The peer-reviewedliterature and popular media have pointed to EE2 from OCsas a major estrogenic endocrine disrupting chemical con-tributing to these phenomena (6, 21-23). We review thescientific literature to qualitatively assess the contributionof other estrogenic chemicals to the estrogenicity of water-ways, to evaluate the pathway of EE2 from ingestion todrinking water, and to explore what is known about the effectsof EE2 exposure in drinking water on human health. Studiesfrom western Europe and the U.S. are highlighted becausethey have similar industrial practices and contraceptive use.We conclude with possible solutions to reducing the presenceof estrogenic compounds, including EE2, in water.
Sources of Estrogens and Estrogenic Compounds. Manychemicals found in our waterways, both natural and syn-thetic, have the ability to mimic or disrupt the naturalestrogens found in humans and animals (11, 12, 18, 24-47).Estrogenic chemicals of varying potency and persistenceoriginate from agriculture, industry, humans, householdproducts, and other pharmaceuticals. Figure 1 diagrams the
various points of entry into waterways for estrogenicchemicals. The following sections will outline in more detailsome of the different sources and their contribution toestrogenic contamination.
† This manuscript is part of the Environmental Policy: Past,Present, and Future Special Issue.
* Corresponding author phone: (510)986-8924; e-mail:[email protected].
‡ University of California.§ Asian University for Women.
FIGURE 1. Simplified diagram outlining points of entry ofestrogenic chemicals into the water supply, adapted fromVelicu et al., 2009 (12).
Environ. Sci. Technol. 2011, 45, 51–60
10.1021/es1014482 © 2011 American Chemical Society VOL. 45, NO. 1, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 51Published on Web 10/26/2010
FOOD
oberhalb unterhalb
KlÑranlagenauslauf
0.00
0.20
0.40
0.60
0.80
Êg Vite
llogenin/ml Plasm
a
The pill problem....
before after!!
outflow of STP !
• The observations of reproductive disturbances in wildlife and man gave rise to the „endocrine disruption hypothesis“
• It suggests that the alterations of the reproductive system observed in wildlife are caused by environmental chemical substances that modulate or disrupt the endogenous hormone system of the exposed organisms, in particularly the sexual/reproductive system:
„endocrine-disrupting compounds (EDCs)“
The „endocrine disruptor“-Hypothesis (1992)
Observations in man: increasing incidence of diseases related to the endocrine system
Sharpe and Irvine 2004
Observa(ons in wildlife:
reproduc(ve and developmental diseases
Gonad morphological changes: • feminized/intersex gonads in fish and
amphibia, • reduced phallus size in alligators • imposex in molluscs Physiological changes • altered sex hormone levels in fish and
alligators • elevated vitellogenin levels in male fish • reduced T3/T4 levels in gulls, seals, and
polar bears • altered parental care behaviour in fish
and birds
A diversity of hormone systems and signals can be impacted by EDCs
estrogen receptor signaling/E2
mimics
sex steroid system
cortisol system
thyroid system
neuroendo-crine system
GH/IGF system
steroid synthesis/ aromatase
androgen receptor signaling
• A range of industrial chemicals including alkylphenols, bisphenol A, PCBs, etc.
• Pesticides, for instance, o,p-DDT, imidazoles
• Plant-derived substances such as sitosterol from wood processing in paper mills
• Pharmaceuticals such as ethinylestradiol (EE2) from contraceptive pills
• Natural hormones from animals and humans (e.g., daily urinary excretion of estradiol per female: 10 µg) released via wastewater effluents
⇒ Broad variety of substances
⇒ Many non-classical pollutants
Which environmental pollutants can cause reproductive disturbances ?
Estrogens
ogie Endocrine disruptionEndocrine disruption
xiko
l
a major mode of damage a major mode of damage to reproductionto reproduction
koto
x to reproductionto reproduction
ng Ö
kle
sun
Vorl
1
Ökotoxikologie XVÖkotoxikologie XV
Natural and synthetic estrogens
ogie
17ß-estradiol estrone 17D-ethinylestradiol diethylstilbestrol
Endocrine Endocrine disrupting disrupting
chemicalschemicalsxi
kol estrone 17D ethinylestradiol diethylstilbestrol
Industrial chemicals
chemicals chemicals
...en...en
koto
x
bisphenol A benzylbutylphthalate 2,2´,5-trichlorobiphenylp-octylphenol
ndocrindocri
ng Ö
k
Pesticides
ne disne dis
lesu
n
o,p-DDTdieldrin atrazine kepon / chlordecon
sruptorsruptor
VorlPhytoestrogens
rsrs
2coumestrol enterolacton E-sitosterol
Basically through two mechanisms:
• binding as agonists or antagonists to endogenous hormone receptors (hormone mimics) thereby modu-lating receptor-controlled pathways and processes
• altering endogenous hormone metabolism, i.e. altering hormone synthesis, transport, catabolism and/ or excretion
How can these diverse groups of compounds disrupt the hormone system of exposed organisms ?
Carvan et al. 2005
EDC
Binding to endogenous hormone receptors: activation of the estrogen receptors (ER) by EDCs
Tyler and Jobling 2008
estrogen receptors
ER agonist mode of action: what are targets and toxicological consequences ?
Reproductive system of fish
• Mammals: homogamy XX implicates a female phenotype, heterogamy XY a male phenotype
• Birds: heterogamy (ZW) implicates a female phenotype, homogamy (ZZ) a male phenotype
• Fish: „everything“
• Heterogamy: XY/XX as well as ZZ/ZW
• In many fish species, morphologically recognizable sex chromosomes do not exist
• Polygenetic sex determination: several genes located on various chromosomes determine sex
• Environmental sex determination of particular importance
Genetic determination of sex in vertebrates
Which model organism is suitable
for investigations on EDCs?
Why zebrafish (Danio rerio)?
- standard laboratory organism, used in 930 labs and 175 companies worldwide (zfin.org, 2014)
- high availability of information and material
- good knowledge about genomics, physiology, morphology and development
- easy and cheap maintenance without need of much space
- transparent eggs, observation of development possible
- fast development, sexually mature after 3 months
- high fecundity, females give 100-500 eggs per week
- special sexual differentiation: „juvenile hermaphroditism“
10 20 30 40 50 60 days
Adult Baby Child Teenager
undifferentiated sexual organs
NON-FUNCTIONAL female sexual organs
female AND male sexual organs
female OR male sexual organs
Sexual development of the zebrafish (Danio rerio)
Egg
primordial gonad (100 %)
immature
ovary (100 %)
50 %
(50 %)
embryo-larval juvenile adult
high aromatase, high estrogen
ovary
low aromatase, low estrogen
testis
exogenous estrogen supply arrests testis
differentiation
immature ovary = arrested testis
Hormonal regulation of sexual
differentiation in zebrafish
Exposure of developing zebrafish to estrogens
results in 100 % phenotypic females at the end of the juvenile period
Example: zebrafish to study mechanism(s) of disruption of sexual differentiation by estrogen-active compounds
Exposure of mature zebrafish to estrogens does not change the
sex ratio
0 0.05 0.3 1.7 3 10
ng ethynylestradiol/L
0
20
40
60
80
100
sex
ratio
(%)
fish with ovaries fish with testes 0 0.05 0.3 1.7 3 10
ng ethynylestradiol/L
0
20
40
60
80
100
sex
ratio
(%)
fish with ovaries fish with testes
male female0
20
40
60
80
100
% m
ale/
fem
ale
Sex ratio of brown trout exposed during sensitive window at end of
exposure
male female0
20
40
60
80
100
% m
ale/
fem
ale
Sex ratio of brown trout exposed during sensitive window at 1-year-
age
male female0
20
40
60
80
100
% m
ale/
fem
ale
Sex ratio of zebrafish exposed during sensitive window at end of
exposure
Sex ratio of zebrafish exposed during sensitive window at adult
stage
male female0
20
40
60
80
100
% m
ale/
fem
ale
Gonochorist: irreversible effect
Juvenile hermaphrodite: reversible effect
Is the environmentally induced gonad feminization reversible or irreversible ? It depends on the species
Skewed sex ratios of zebrafish after exposure to EDCs from 0-100 dph
Feminization with Ethinylestradiol (estrogen):
Masculinization with Trenbolone (androgen):
Baumann et al. 2014, dph: days post hatch, IS/UD: intersex/undifferentiated, C: continous exposure, R: recovery of 40 days
Histopathology of zebrafish gonads
Female
Stage 0 Stage 2 Stage 4
Male
Stage 0 Stage 2 Stage 4
Intersex
Grade 1 Grade 2 Grade 4
Gonad maturity as sensitive marker for EDC effects
38 L. Baumann et al. / Aquatic Toxicology 128– 129 (2013) 34– 42
Females
Contro l 32 10 0 32 00
50000
100000
150000
200000
0
1
2
3
4-Tert-Pentylphenol( µg/L)
VTG
(ng/
mlh
omog
enat
e)
***
******
**
Vitellogenin
MaturityIndex
Males
Contro l 32 10 0 32 00
5000
10000
15000
1
2
3
4-Tert-Pentylphenol( µg/L)
VTG
(ng/
mlh
omog
enat
e)
Maturtiy
IndexM
aturtiyIndex
***
***
Fig. 2. Induction of vitellogenin and maturity index in female and male zebrafish(Danio rerio) after exposure to 4-tert-pentylphenol at 60 dph (**p < 0.05, ***p < 0.01;Dunnett’s test).
Females
Contro l DMSO 0.1 1 3 10100
102
104
106
1
2
3
17 -Ethinylestradiol(ng/L)
VTG
(ng/
mlh
omog
enat
e)
***
**
Vitellogenin
MaturityIndex
Males
Contro l DMSO 0.1 1 3 10100
102
104
106
0
1
2
3
4
5
17 -Ethinylestradiol(ng/L)
VTG
(ng/
mlh
omog
enat
e)
Maturtiy Index
Maturtiy Index
***
***
***
***
Fig. 3. Induction of vitellogenin and maturity index in female and male zebrafish(Danio rerio) after exposure to 17!-ethinylestradiol at 60 dph (**p < 0.05, ***p < 0.01;Dunnett’s test).
Females
Contro l DMSO 1 3 100
200000
400000
600000
800000
1000000
0
1
2
3
4
17 -Trenbolone(ng/L)
VTG
(ng/
mlh
omog
enat
e)
Maturtiy Index
Maturtiy Index
Vitellogenin
MaturityIndex
Males
Contro l DMSO 1 3 10 300
500
1000
1500
1.5
2.0
2.5
3.0
17 -Trenbolone(ng/L)
VTG
(ng/
mlh
omog
enat
e)
**
**
*
*
Fig. 4. Induction of vitellogenin and maturity index in female and male zebrafish(Danio rerio) after exposure to 17"-trenbolone at 60 dph (*p < 0.05, **p < 0.01; Dun-nett’s test). No female individuals developed in treatment groups >10 ng/L.
Females
Contro l 10 00
500000
1000000
1500000
2000000
3.2
3.3
3.4
3.5
3.6
3.7
Dihydrotestosterone(ng/L)
VTG
(ng/
mlh
omog
enat
e)
Maturtiy Index
Maturtiy Index
Vitellogenin
MaturityIndex
Males
Contro l 10 0 32 0 100 00
100
200
300
400
500
3.2
3.4
3.6
3.8
4.0
4.2
Dihydrotestosterone(ng/L)VT
G(n
g/m
lhom
ogen
ate)
*********
*********
Fig. 5. Induction of vitellogenin and maturity index in female and male zebrafish(Danio rerio) after exposure to dihydrotestosterone at 60 dph (***p < 0.01; Dunnett’stest). No female individuals developed in treatment groups >100 ng/L.
Baumann et al. 2013!
Adult estrogen exposure: impaired reproduction relates to impaired germatogenesis (gonad dysfunction)
females: oocyte atresia
0 0.05 0.3 1.7 3 10ng EE2/L
0
300
600
900
1200
1500
1800
sper
mat
id c
yst s
ize
(µm
2 )
males: spermatid cyst size
21-day-exposure to ethynylestradiol EE2
Estrogen exposure of adults to (xeno)estrogens results in reduced fecundity and fertility
0 0.05 0.3 1.7 3 10ng EE2/L
0
24
48
72
96
120
fert
ility
(%
)0 0.05 0.3 1.7 3 10
ng EE2/L
0
24
48
72
96
120
fecu
ndity
(%
)
21-day-exposure to ethinylestradiol EE2
