Ah-receptor-agonist activity of sediment extracts

Ah-receptor-agonist activity of sediment extracts from
Hamburg Harbour and the Rhine River in Danio rerio
J. Bräunig1, S. Peddinghaus1, T. Bui 1, T. Hoen1, K. Winkens1, H. Hollert1 & S. Keiter1
1
Institute
Institutefor
for
Environmental
EnvironmentalResearch
Research
Department of Ecosystem Analysis, Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
Introduction
The European Water Framework Directive (EWFD) aims to achieve a good ecological and chemical status in the surface water of European rivers until 2015.
However, there is still need for basic research in order to fulfill this legal obligation. Sediment toxicology plays a major role in this intention as sediments can act
as a secondary source of pollution. It is important to detect the bioavailable fraction of sediment-bound contamination and thus, the risk of sediment bound
substances.
The collaborative project DanTox will develop a fish embryo-based test concept to assess contaminated sediments with regard to selected mechanism-specific
biological endpoints (teratogenicity, genotoxicity, mutagenicity, Ah-receptor-mediated toxicity, neurotoxicity). As part of the DanTox project the present study
investigates dioxin-like effects of acetonic sediment extracts from the Rhine River and Hamburg Harbour in zebrafish (Danio rerio) embryos.
Discussion and Outlook
Materials and Methods
To investigate the activity of AhR-agonists in Danio rerio embryos the fish
embryo toxicity test (FET) and the 7-ethoxyresorufin-O-deethylase (EROD)
assay were combined: Embryos were exposed to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) or acetonic sediment extracts for 48 h according to the FET
(DIN 38415-6). Subsequently, embryos were homogenized to isolate cell
proteins, including CYP1A, and used in the EROD assay. The induction of
EROD indicates dioxin‐like activity of the samples on the test species. Over
the course of the study the procedure was further developed and optimized.
Results
B
1,0
0,5
0,0
Ehrenbreitstein (48 h)
 The present results of the positive control with TCDD (Fig. 2) indicate that
the natural barrier function of the chorion prevents TCDD from entering and
harming the embryo. This assumption is reinforced by a) the highly
increased acute toxicity of TCDD after hatching as reported by Otte et. al
(2010) and b) the comparison of the maximal induction in RTL-W1 cell
lines and Danio rerio (Fig. 3) which shows a 14-fold increased EROD
induction.
Log [EROD activity [pmol/(mg*min)]]
1,5
EROD activity pmol/(mg*min)
1,0
0,5
100
10
1
0.1
0.01
0,0
1,17
2,34
4,67
9,37
18,75
NK
0,625 1,25
Concentration [mg SEQ/mL]
C
EROD activity pmol/(mg*min)
2,5
5
10
20
-W
TL
R
NK
Concentration [mg SEQ/mL]
Hamburg harbor (48 h)
before optimization
after optimization
1
D
1,0
 A comparison of the induction of EROD activity in the negative controls
(Fig. 4) in Danio rerio embryos and RTL-W1 cell lines, representing basal
activity, shows that the method is an applicable tool to investigate AhR
0.4
mediated toxicity.
0,5
0,2
0,39
0,78
1,56
3,125
NK
Concentration [mg SEQ/mL]
Fig. 1: Comparison of EROD activity induced in Danio rerio embryos after 48 h exposure to acetonic
sediment extracts (Altrip (A), Ehrenbreitstein (B), and Hamburg Harbour (C)). Bars represent
measurment before and after test optimization. The negative controls were conducted with artificial
water only.
0.3
0.2
0.1
0.0
-W
TL
R
1
D
io
an
ri o
re
Fig. 4: Comparison of the induction of EROD activity in the negative controls of RTL-W1 cell
lines (n = 61) and Danio rerio embryos (n = 42). Data for RTL-W1 cell lines taken from Keiter
et al. 2009
TCDD (48 h)
EROD activity pmol/(mg*min)
rio
re
1,5
0,0
1,5
before optimization
after optimization
1,0
 Further improvements of the testing system are necessary to obtain
stable results and a higher induction of EROD activity. These include:
0,5
0,0
6
60
100
500
900 1500 3000 NK
Concentration [pM]
Fig. 2: Comparison of EROD activity induced in Danio rerio embryos after 48 h
exposure to TCDD. The concentration of 3000 pM was only tested after
optimization of the procedure. The negative control was conducted with artificial
water only.
The DanTox Project is
funded by:
io
an
Fig. 3: Comparison of the maximal induction of RTL-W1 cell lines (n = 64) and Danio rerio
embryos (n = 7) exposed to TCDD in the EROD assay. Data for RTL-W1 cell lines taken
from Keiter et al. 2009
EROD activity [pmol/(mg*min)]
EROD activity pmol/(mg*min)
Altrip (48 h)
A
1,5
The optimized testing method, using an electric dispersing device and
continuously cooling the sample, led to higher inductions of EROD activity
(Fig. 1) 2)The fish egg EROD assay has shown to be an applicable test to
investigate dioxin-like activity in Danio rerio embryos; however, the results
 Finding a suitable positive control
 lengthen exposure time
 testing further chemicals
References
Keiter, S., Braunbeck, T., Heise, S., Pudenz, S., Manz, W. and Hollert, H. (2009) A fuzzy logic-classification of
sediments
based on data from in vitro biotests. J. Soils Sediments 9: 168-179.
Otte, J.C., Schmidt, A.D., Hollert, H. and Braunbeck, T. (2010) Spatio-temporal development of CYP1 activity in early
life-stages of zebrafish (Danio rerio). Aquatic Toxicology 100: 38-50.
The RWTH Aachen University Undergraduate Funds, as part of the German Excellence Initiative provided funding for
participating the ATW meeting by a personal travel grant to the first author.
Undergraduate Funds

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