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Supplementary Material
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Measuring cytochrome P450 activity in aquatic invertebrates: A critical
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evaluation of in vitro and in vivo methods
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Journal: Ecotoxicology
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Authors: Michele Gottardi,* Andreas Kretschmann, Nina Cedergreen
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*corresponding author, Department of Plant and Environmental Sciences, University of
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Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark, email: migo@plen.ku.dk
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18 pages, 13 figures and 1 table
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Contents
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Section 1. In vivo cytochrome P450 activity assay - Optimization ..................................................... 2
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Section 2. In vitro cytochrome P450 activity assay - Optimization..................................................... 6
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Section 3. Microsome preparation - Optimization ............................................................................. 12
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Section 4. 7-hydroxycoumarin standard curves ................................................................................. 13
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Section 5. The effect of microsomes on 7-hydroxycoumarin fluorescence ....................................... 15
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REFERENCES................................................................................................................................... 18
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S1
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Section 1. In vivo cytochrome P450 activity assay - Optimization
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In vivo measurements of ECOD activity were based and adapted from the existing method
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described in Gagnaire et al. (2010). In order to optimize the number of organisms needed, one, two
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and three Chironomus riparius larvae (n = 2 biological replicates) or 10 and 20 Daphnia magna (n
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= 2 biological replicates) were incubated for 6 hours in 2 mL M7 medium containing the substrate
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7-ethoxycoumarin (0.006 mM). The incubation was conducted in 4 mL amber glass vials. The
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organisms were transferred together with their own growth medium, subsequently the medium was
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removed and the incubation medium was added. A first blank was made with 7-ethoxycoumarin
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(0.006 mM) dissolved in M7 medium to correct for the substrate own fluorescence, a second blank
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was made with organisms in M7 medium to detect possible fluorescence due to molecules excreted
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by the organisms. Moreover, in order to verify whether product formation was cytochrome P450
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dependent, D. magna were continuously pre-exposed to prochloraz (500 µg L-1) for 18 hours prior
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and during incubation. This was done in agreement with one of our previous investigations, which
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showed that prochloraz was found to be the strongest inhibitor of in vitro ECOD activity of rat liver
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microsomes as compared to other azoles fungicides (tebuconazole, epoxiconazole, propiconazole)
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and the cytochrome P450 inhibitor piperonyl butoxide (Fig. S1). The experiments were conducted
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at 22 ˚C. During incubation, 100 µL aliquots of medium were removed from the incubation vial and
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transferred directly into a 96-well plate every 30 min. The samples were stored at -20˚C until
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fluorescence measurement. Fluorescence due to the product formation (7-hydroxycoumarin;
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excitation: 380 nm, emission: 480 nm) was measured with a 96-well plate spectrofluorometer at
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room temperature (25 ˚C).
S2
CN
A
PB
O
on
uc
Te
b
Pr
op
ico
na
az
ol
e
zo
le
zo
le
na
xic
o
Ep
o
Pr
oc
hl
or
az
ECOD Activity (1/min)
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10
9
8
7
6
5
4
3
2
1
0
40
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Fig. S1 ECOD activity of rat liver microsomes (0.01 mg protein mL-1) in the presence of azole
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fungicides and piperonyl butoxide, PBO (0.060 mM). All groups were statistically significantly
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different from acetonitrile (ACN, control) (One-way ANOVA, Tukey Test: p < 0.05). Prochloraz,
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epoxiconazole and propiconazole were not statistically significantly different from one another
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(One-way ANOVA, Tukey Test: p > 0.05). Data are means ± S.E. (3 analytical replicates)
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As it is shown in Fig. S2 and Fig. S3, detectable fluorescence increased within 6 hours for both
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organisms at all densities. Linearity was observed within the first three hours for D. magna and for
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approximately five hours for C. riparius, depending on density. For C. riparius there was a lag-
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phase of approximately half an hour before the fluorescent product was released to the water. For D.
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magna no detectable lag-phase was observed. Based on these observations the number of organisms
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were set to ten D. magna and three C. riparious using an incubation time of three hours. Since the
S3
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organisms in M7 medium did not show any increase in fluorescence over time (Fig. S2 and Fig.
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S3), this particular control was omitted in the subsequent studies. D. magna exposed to prochloraz
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prior and during incubation did not show any detectable product formation, hence, pre-exposure to
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prochloraz (500 µg L-1) was therefore used as a control for cytochrome P450 dependent activity.
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Fluorescence (flu)
200
150
100
50
0
0
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1
2
3
4
5
6
7
Time (h)
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Fig. S2 Daphnia magna in vivo ECOD activity. Raw data showing the fluorescence increase over
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time measured in samples of 0.100 mL M7 medium. The background fluorescence of the substrate
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(7-ethoxycoumarin) is shown with black circles, the background fluorescence of ten and 20
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organisms incubated without substrate is shown with white circles and black triangles, respectively.
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The change in fluorescence in beakers with substrate and ten or 20 organisms are given with white
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triangles and black squares, respectively, while change in fluorescence in beakers with substrate and
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ten or 20 organisms pre-exposed to prochloraz (500 ug L-1) for 18 h prior to incubation and during
S4
incubation are given with white squares and black diamonds, respectively. Data are means ± S.E. (n
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= 2)
Fluorescence (flu)
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95
90
85
80
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70
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60
55
50
45
40
35
30
0
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1
2
3
4
5
6
7
Time (h)
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Fig. S3 Chironomus riparius larvae in vivo ECOD activity. Raw data showing the fluorescence
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increase over time measured in samples of 0.100 mL M7 medium. The background fluorescence of
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the substrate (7-ethoxycoumarin) is shown with black circles, background fluorescence of one, two
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or three organisms incubated without substrate are given with white circles, black triangles and
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white triangles, respectively, while changes in fluorescence in beakers incubated with substrate and
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one, two or three organisms are given with black squares, white squares and black diamonds,
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respectively. Data are means ± S.E. (n = 2)
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Section 2. In vitro cytochrome P450 activity assay - Optimization
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The starting point for measuring ECOD activity was the method adopted by Bach and Snegaroff
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(1989) for the liver, intestines and other organs of the rainbow trout (Salmo gairdneri) using
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phosphate buffer (KH2PO4/K2HPO4) (50 mM, pH = 7.5), MgCl2 (1.5 mM), NADP (0.25 mM),
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glucose 6-phosphate (2.5 mM), glucose 6-phosphate dehydrogenase (3 units) and 7-ethoxycoumarin
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(0.1 mM). The optimal concentrations of microsomes, substrate and of the two cofactors, NADPH
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and MgCl2 were then investigated using rat liver microsomes (Product nr: M9066, Sigma Aldrich),
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which provided a system known to possess high P450 activity. The aim was to get a very sensitive
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test with a low detection limit for ECOD activity that would be able to capture the low activities
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known to occur in aquatic invertebrates. The five parameters were varied one at a time to obtain the
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optimal concentration as shown in Table S1. All tests were run in phosphate buffer (50 mM, pH =
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7.5).
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Table S1. Summary of chemicals and their concentrations used during in vitro ECOD activity assay
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optimization. Underlined values represent the range tested during optimization, while the other
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factors were kept constant.
Microsomes
MgCl2 NADPH Substrate Solvent
Microsomes (mg protein mL-1) 0.0006 – 0.08 0.03
0.03
0.03
0. 03
MgCl2 (mM)
1. 5
0 - 26
-
-
-
NADPH (mM)
0.25
0. 25
0 – 0.5
0.0156
0.0156
7-ethoxycoumarin (mM)
0.1
0.1
0.1
0.04 – 2.5
0.1
ACN (%)
0.12
0.12
0.12
3
0.12 – 10.12
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S6
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Geometrically distributed dilutions of microsomes were made and fluorescence was monitored
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over time (Fig. S4). A concentration of microsomes in the range of detectable product formation
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within 30 min equal to 0.03 mg protein mL-1 was used for further investigations.
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Fluorescence (flu)
600
400
200
0
0
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5
10
15
20
25
30
Time (min)
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Fig. S4 ECOD activity of rat liver microsomes – Fluorescence increase over time of geometrical
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dilutions of rat liver microsomes. Five of the eight concentrations are shown: 0.08 mg protein mL-1
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by black circles, 0.04 mg protein mL-1 by white circles, 0.02 mg protein mL-1 by black triangles,
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0.01 mg protein mL-1 by white triangles and 0.005 mg protein mL-1 by black squares. Data are
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means ± S.E. (3 analytical replicates)
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Subsequently, ECOD activity was monitored over 30 min using different geometrically
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distributed MgCl2 concentrations (Fig. S5). MgCl2 did not seem to greatly enhance nor inhibit
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ECOD activity and was therefore excluded in all following experiments.
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ECOD activity (flu min )
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10
9
8
0.2
102
103
104
0.4
0.8
1.6
3.3
6.6 13.1 26.2
[MgCl2 ] (mM)
Fig. S5 ECOD activity of rat liver microsomes – Optimization of MgCl2 concentration. Data are
means ± S.E. (3 analytical replicates)
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Fluorescence increase over time was monitored in relation to geometrically distributed dilutions
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of NADPH (Fig. S6). In order to minimize background noise (cause by NADPH fluorescence at the
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working wavelengths) (Aitio 1978) the lowest concentration that showed no depletion (linear
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product formation) within 30 minutes was considered as optimal. For the chosen microsome
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concentration this was 0.0156 mM NADPH.
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Fluorescence (flu)
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200
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100
50
0
0
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5
10
15
20
25
30
Time (min)
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Fig. S6 ECOD activity of rat liver microsomes – Optimization of NADPH concentration.
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Fluorescence increase over time with geometric dilutions of NADPH. Four of the eight
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concentrations are shown: 0.0312 mM by black circles, 0.0156 mM by white circles, 0.0078 mM by
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black triangles and 0 mM by white triangles. Data are means ± S.E. (3 analytical replicates)
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In order to find the optimal substrate concentration, ECOD activity was investigated in relation to
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ten geometrical dilutions of substrate and by estimating kinetic parameters such as Vmax and KM
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(Michaelis Menten kinetics: rate = Vmax [S]/([S]+KM). Curve fitted using SigmaPlot 12.5) (Fig. S7).
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Moreover, since the substrate was solubilized in a solvent: 30 % v/v acetonitrile (ACN) in MilliQ
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water, the possible inhibition of activity due to the solvent (Li et al. 2010) was tested by addition of
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known amounts of solvent to the test system (Fig. S8). A concentration of substrate within the
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range of Vmax that did not significantly decrease activity due to the solvent was considered optimal.
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We chose the concentration of 0.6 mM 7-ethoxycoumarin, giving a total acetonitrile concentration
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in the sample of 0.72 % v/v.
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-1
ECOD activity (flu min )
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20
15
10
5
0
0.0
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0.5
1.0
1.5
2.0
2.5
[7-ethoxycoumarin] (mM)
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Fig. S7 ECOD activity of rat liver microsomes – Optimization of substrate (7-ethoxycoumarin)
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concentration. ECOD activity as a function of substrate concentration. Kinetic parameters: Vmax:
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29.4 ± 0.6 flu min-1, KM: 0.19 ± 0.01 mM 7-ethoxycoumarin estimated with Michealis-Menten
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equation fit (black line). Data are means ± S.E. (3 analytical replicates)
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-1
ECOD activity (flu min )
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8
6
4
2
0
0
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2
4
6
8
10
[Solvent] (%)
Fig. S8 ECOD activity of rat liver microsomes – Optimization of solvent (Acetonitrile)
concentration. Data are means ± S.E. (3 analytical replicates)
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The lowest detectable activity of the method was investigated by measuring the activity of
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geometrical dilutions of rat liver microsomes (0.008 to 0.0001 mg protein mL-1) and Chironomus
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riparius larvae microsomes (0.04 to 0.002 mg protein mL-1) with the optimized amounts of
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reagents. The lowest fluorescence increase over time that was found to be statistically significant
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from the blank (no substrate) (ANCOVA, p < 0.01, run with the program "R") was considered to be
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the lowest detectable activity of the method developed in the present study. For rat liver
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microsomes, the lowest detectable activity was 0.21 ± 0.08 flu min-1 (± S.D.) corresponding to ~34
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fmol min-1 and a concentration of microsomes of 0.001 mg protein mL-1. For Chironomus riparius
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larvae microsomes, the lowest detectable activity was 0.25 ± 0.07 flu min-1 (± S.D.) corresponding
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to ~37 fmol min-1 and a concentration of microsomes of 0.036 mg protein mL-1.
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Section 3. Microsomes preparation - Optimization
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As a proper extraction of microsomes from whole organisms with an exoskeleton requires severe
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homogenization to ensure cell breakage, we chose to use an ultrasonic stick (Digital Sonifier cell
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disruptor Model 450, Branson Ultrasonics, U.S.). Homogenization with ultrasonic stick can,
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however, potentially heat and thereby denature proteins resulting in a substantial decrease in
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detectable ECOD activity. Therefore two types of homogenization trials were conducted: First,
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studies using rat liver microsomes were conducted to identify optimal mode and processing times
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that would not halter P450 activity. Three settings: 3x3 sec, 6x3 sec and 12x3 sec with 10 sec pause
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in between on ice and the power set to 20 % were tested on rat liver microsomes. Foaming was
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observed for 6x3 sec and 12x3 sec, and subsequent measurements of ECOD activity showed that
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12x3 sec decreased the activity of rat liver microsomes by 27 % as compared to the control.
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Secondly, homogenization trials were made with D. magna using either 20 or 40 five day old
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organisms in 0.9 mL of phosphate buffer (50 mM, pH = 7.5) and glycerol (10 % v/v). Two different
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power outputs, 10 % and 20 %, and various processing times starting with 1x3 sec to 14x3 sec with
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10 sec pause on ice in between were tested. Results showed that the total number of organisms was
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not fully homogenized when low power output or processing time shorter than 12 sec were used.
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On the other hand, when high power or long processing time were used, the homogenate was
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foaming, which was previously shown to decrease P450 activity in rat-liver microsomes. To
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minimise the homogenization time using the ultrasonic stick while still ensuring full cell disruption,
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a preliminary cell disruption carried out by hand with a tissue grinder (Econo Grind, Radnoti, U.S.)
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was therefore adopted. After that, ultrasonic homogenization was carried out with the optimal
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settings found for rat liver microsomes: power output equal to 20 % and a time sequence of 3x3 sec
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with 10 sec pause in between.
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Section 4. 7-hydroxycoumarin standard curves
4000
3500
Fluorescence (flu)
3000
2500
2000
1500
1000
500
0
0
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100
200
300
400
500
600
7-hydroxycoumarin (pmol)
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Fig. S9 Standard curve of 7-hydroxycoumarin in MilliQ water and rat liver microsomes (0.03 mg
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protein mL-1) (Total volume 200 µL). Fit: y = 51.1 + 6.266x. Data are means ± S.E. (2 analytical
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replicates)
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1400
1200
Fluorescence (flu)
1000
800
600
400
200
0
0
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172
173
20
40
60
80
100
120
140
160
7-hydroxycoumarin (pmol)
Fig. S10 Standard curve of 7-hydroxycoumarin in MilliQ water (Total volume 100 µL). Fit: y =
27.1 + 8.218x. Data are means ± S.E. (3 analytical replicates)
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700
600
Fluorescence (flu)
500
400
300
200
100
0
0
174
175
176
10
20
30
40
50
60
70
80
90 100
7-hydroxycoumarin (pmol)
Fig. S11 Standard curve of 7-hydroxycoumarin in phosphate buffer (0.13 M) (Total volume 200
µL). Fit: y = 46.1 + 6.803x. Data are means ± S.E. (3 analytical replicates)
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Section 5. The effect of microsomes on 7-hydroxycoumarin fluorescence
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Different amounts of microsomes (final concentration in the well ranging from 0 to 0.05 mg
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protein mL-1) obtained from C. riparius larvae and D. magna were incubated with the ECOD
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product 7-hydroxycoumarin (60 pmol) in order to investigate possible decrease of fluorescence due
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to the conversion of the product to non-fluorescence molecules (Fig. S12) and possible scattering of
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fluorescence signal due to turbidity of the samples (Fig. S13).
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400
380
380
Fluorescence (flu)
Fluorescence (flu)
400
360
340
320
300
340
320
A) C. riparius larvae
10
183
360
12
14
16
300
18
20
Time (min)
B) D. magna
10
12
14
16
18
20
Time (min)
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Fig. S12 Fluorescence variation of 7-hydroxycoumarin over time in the presence of C. riparius
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larvae and D. magna microsomes in different concentrations: 0.05 mg protein mL-1 by black circles,
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0.025 mg protein mL-1 by white circles, 0.00125 mg protein mL-1 by black triangles, 0.0063 mg
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protein mL-1 by white triangles, 0.0031 mg protein mL-1 by black squares, 0.0016 mg protein mL-1
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by white squares and 0 mg protein mL-1 by black diamonds. The time frame shown corresponds to
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the time frame used for activity measurements
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Average Fluorescence (% relative to control)
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10
8
6
4
2
0
-2
-4
-6
-8
-10
0.00 0.01 0.02 0.03 0.04 0.05 0.06
Microsomes (mgprotein mL-1)
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Fig. S13 The effect of different microsomes concentrations on the average fluorescence of 7-
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hydroxycoumarin. Microsomes of C. riparius larvae and D. magna are represented by black and
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white circles, respectively
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The fluorescence of 7-hydroxycoumarin over time did not decrease due to the addition of C.
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riparius larvae or D. magna microsomes (Fig. S12). Differently from what has been previously
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found for EROD activity of fish liver S9 fraction (Vehniainen et al. 2012), it can be excluded that
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the present microsomes contained enzymes that were able to convert the product into other non–
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fluorescent molecules. The addition of increasing concentrations of C. riparius larvae and D. magna
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microsomes did not have any clear effect on fluorescence detection of the product 7-
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hydroxycoumarin (Fig. S13, averages obtained from Fig. S12). Both the second lowest (0.0063 mg
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protein mL-1) and the highest amount of C. riparius larvae microsomes (0.05 mg protein mL-1) were
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able to decrease fluorescence intensity by approx. 6% and the highest amount of D. magna
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microsomes resulted in increase of fluorescence detection. Therefore, it can be assumed that
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microsomes concentrations within the tested range do not have a substantial scattering effect on the
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measured fluorescence.
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REFERENCES
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Aitio A (1978) A simple and sensitive assay of 7-ethoxycoumarin in deethylation. In: Leonard BJ
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(ed) Toxicological Aspects of Food Safety, vol 1. Archives of Toxicology. Springer Berlin
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Heidelberg, pp 275-275. doi:10.1007/978-3-642-66896-8_53
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Bach J, Snegaroff J (1989) Effects of the fungicide prochloraz on xenobiotic metabolism in
Rainbow trout - in vivo induction Xenobiotica 19:1-9
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Gagnaire B, Geffard O, Noury P, Garric J (2010) In vivo indirect measurement of cytochrome
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P450-associated activities in freshwater gastropod molluscs Environmental Toxicology
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25:545-553 doi:10.1002/tox.20515
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Li D et al. (2010) Effect of regular organic solvents on cytochrome P450-mediated metabolic
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activities in rat liver microsomes Drug Metabolism and Disposition 38:1922-1925
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doi:10.1124/dmd.110.033894
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Vehniainen ER, Schultz E, Lehtivuori H, Ihalainen JA, Oikari AO (2012) More accuracy to the
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EROD measurements--the resorufin fluorescence differs between species and individuals
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Aquatic Toxicology 116-117:102-108 doi:10.1016/j.aquatox.2012.03.007
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