Supporting Information
Developmental Toxicity of Diclofenac and Elucidation of Gene
Regulation in zebrafish (Danio rerio)
Jia-Bin Chen1, Hong-Wen Gao1, Ya-Lei Zhang1,*, Yong Zhang2, Xue-Fei Zhou1, Chun-Qi Li2, and
Hai-Ping Gao1
1
State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science
and Engineering, Tongji University, Shanghai, 200092, China
2
Hunter Biotechnology, Inc., Hangzhou, 311231, China
*
To whom correspondence should be addressed. Tel.:+86 021 65980624; fax: +86 021 65989961;
e-mail:
zhangyalei2003@163.com
Tests
Test S1. Determination of diclofenac by HPLC-UV.
Test S2. Determination of diclofenac in the cytoplasm and membrane by HPLC-MS/MS.
Test S1. Determination of diclofenac by HPLC-UV.
Diclofenac was determined on a high performance liquid chromatography (HPLC) with a UV
detector. The Kromasil ODS C18 (250×4.6 mm, 5 μm) was used as the LC column for separation.
The mobile phase was composed of 60% acetonitrile and 40% water with 0.4% acetic acid. The
flow rate was 1 ml·min-1, and the detection wavelength was set at 280 nm. The calibration curve
and chromatogram were shown in Fig. S1. The limit of detection (LOD) for diclofenac was 0.13
μM, which was determined as the concentration of diclofenac giving a signal to noise ratio greater
than 3:1.
Test S2. Determination of diclofenac in the cytoplasm and membrane by HPLC-MS/MS.
The concentrations of diclofenac in the cytoplasm and membrane were determined by
UPLC-MS/MS1. Briefly, LC analysis was conducted on Thermo Accela UPLC equipped with a
zorbax extend-C18 column (100×2.1 mm, 1.8 μm). The mobile phase consisted of a binary
mixture of solvents A (0.4% acetic acid in water) and B (acetonitrile) with the flow rate of 0.2
ml·min-1. The gradient was performed by increasing the content of B from 38% at 1 min, then
increased to 60% at 12 min, finally back to 38% at 17-20 min. MS analysis was performed on a
TSQ Quantum Access triple quadrupole mass spectrometer in the negative ion electro spray mode.
The sheath gas pressure and AUX gas pressure were 40 and 10 Arb, respectively. The spray
voltage and skimmer offset were set at 3500 and 10 V, respectively. The collision voltage was 20
eV. The daughter ion of m/z 214 was used for detection and quantification and the other one of
m/z 250 for confirmation. Series of diclofenac standard solutions (0.016, 0.063, 0.157. 0.314,
0.943, and 3.145 μM) were prepared for the determination of calibration curve. Good linearity of
the calibration curve was observed with R2 being 0.9996. LOD (S/N=3) for diclofenac was
determined to be 0.0007 μM.
Tables
Table S1. Concentration and purity of total RNA.
Table S2. Parameters of gene primers.
Table S3. Mortality of embryos at different exposed time.
Table S4. Percentage of zebrafish with developmental abnormality during the exposure at 4
dpt.
Table S1. Concentration and purity of total RNA.
time
Concentration
(μM)
A260
0
3.38
10.13
15.2
0
3.38
10.13
15.2
0.124
0.100
0.102
0.097
0.126
0.146
0.111
0.108
1 dpt
2 dpt
A280
RNA concentration
（μg/ml）
A260/A280
0.074
0.058
0.062
0.059
0.076
0.09
0.068
0.065
1.49
1.20
1.22
1.16
1.51
1.75
1.33
1.30
1.68
1.72
1.65
1.64
1.66
1.62
1.63
1.66
Table S2. Parameters of gene primers.
Gene
β-actin
Gata4
Nkx2.5
Wnt3a
Wnt8a
Function
Reference
gene
Development
of heart and
intestine
Development
of heart
Development
of heart and
body axis
Development
of body axis
and tail
Tm
Best
cycle
Product
length
(℃)
number
(bp)
60
30
389
57.5
38
286
51
38
281
56
38
198
59
40
334
Primers
Forward
5’-CATCAGCATGGCTTCTGCTCTGTATGG-3’
Reverse
5’-GACTTGTCAGTGTACAGAGACACCCT-3’
Forward
5'-TCCAGGCGGGTGGGTTTATC-3’
Reverse
5'-TGTCTGGTTCAGTCTTGATGGGTC-3’
Forward
5'-GTCCAGGCAACTCGAACTACTC-3’
Reverse
5'-AACATCCCAGCCAAACCATA-3
Forward
5'-TACGCCTTCTTCAAGCATCC-3’
Reverse
5'-CTCTTTGCGCTTTTCTGTCC-3’
Forward
5'-CAAGCAAGGAAGTTGGAGATGG-3’
Reverse
5'-CGCATTTGACTGTGCAGCAC-3’
Table S3. Mortality of embryos at different exposed time.
fish water
1.01 μM
3.38 μM
10.13 μM
15.2 μM
1 dpt
0
1
0
0
0
2 dpt
0
0
0
0
0
3 dpt
0
0
0
4
13
4 dpt
0
0
8
26
17
Total
0
1 (3.3%)
8 (26.7%)
30 (100%)
30 (100%)
Table S4. Percentage of zebrafish with developmental abnormality during the exposure at
4 dpt.
0 μM
1.01 μM
3.38 μM
10.13 μM
15.2 μM
0
0
100
-
-
abnormal jaw
0
0
100
-
-
smaller eye
0
0
100
-
-
lack of liver
0
0
100
-
-
lack of intestine
0
0
100
-
-
0
0
100
-
-
0
0
100
-
-
0
0
80
-
-
0
0
60
-
-
0
0
60
-
-
pericardial
edema
shorter body
length
abnormal
muscle and
somite
abnormal
pigmentation
lack of
circulation
body edema
-: no data, because all zebrafish were dead in the 10.13 and 15.20 μM treatment groups at 4 dpt.
Figures
Figure S1. The calibration curve and chromatogram of diclofenac (162 μM).
Figure S2. Agarose gel electrophoresis of total RNA extract of zebrafish.
Figure S3. Effect of pH (A), ionic strength (B), temperature (C) on the binding number (γ) of
diclofenac (81μM) to embryos after 8 h incubation.
Figure S4. Distribution of diclofenac (5-162 μM) in different parts (ectracellular, membrane,
cytoplasm) of zebrafish embryos.
Figure S5. Toxic characteristics of zebrafish related to the eye, muscle, liver, intestine and
circulation during the exposure at 4 dpt. (A) Control group; (B) 1.01 μM exposure group; (C)
3.38 μM exposure group
Figure S6. Toxic characteristics of zebrafish related to pericardial and body edema during
the exposure at 4 dpt. (A) Control group; (B) 1.01 μM exposure group; (C) 3.38 μM exposure
group
Figure S7. Toxic characteristics of zebrafish related to abnormal pigmentation during the
exposure at 4 dpt. (A) Control group; (B) 1.01 μM exposure group; (C) 3.38 μM exposure group
Figure S8. Agarose gel electrophoresis of β-actin (a), Wnt3a (b), Wnt8a (c), Gata4 (d) and
Nkx2.5 (e) at 1 and 2 dpt. (A) The treatment groups of 0, 3.38,10.13, 15.2 μM; (B) The
treatment groups of 0 and 1.01 μM and blank control (supplemental experiments).
Figure S9. Full-length blots/gels of Wnt8a
5000
4000
y=3.277x+0.8714,
2
R =0.9999
Aera
3000
2000
1000
0
0
200
400
600
800 1000 1200 1400 1600
Cdiclofenac (μM)
Figure S1. The calibration curve and chromatogram of diclofenac (162 μM).
Figure S2. Agarose gel electrophoresis of the total RNA extract of zebrafish.
0.60
B
A
C
0.55
0.50
 (nmol/embryo)
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
5 6 7 8 9 10
0.0
0.1
pH
0.2
20 25 30 35 40
o
I (M)
T ( C)
Figure S3. Effect of pH (A), ionic strength (B), temperature (C) on the binding number (γ) of
diclofenac (81μM) to embryos after 8 h incubation.
16
Membrane
Cytoplasm
Extracellular
14
 (nmol/embryo)
12
10
8
6
4
2
0
5.0
10.1
20.2
40.5
81.0
162.0
C0 (M)
Figure S4. Distribution of diclofenac (5-162 μM) in different parts (extracellular, membrane,
cytoplasm) of zebrafish embryos.
Figure S5. Toxic characteristics of zebrafish related to the eye, muscle, liver, intestine and
circulation during the exposure at 4 dpt. (A) Control group; (B) 1.01 μM exposure group; (C)
3.38 μM exposure group.
Figure S6. Toxic characteristics of zebrafish related to pericardial and body edema
during the exposure at 4 dpt. (A) Control group; (B) 1.01 μM exposure group; (C) 3.38 μM
exposure group
Figure S7. Toxic characteristics of zebrafish related to abnormal pigmentation during
the exposure at 4 dpt. (A) Control group; (B) 1.01 μM exposure group; (C) 3.38 μM
exposure group
Figure S8. Agarose gel electrophoresis of β-actin (a), Wnt3a (b), Wnt8a (c), Gata4 (d) and
Nkx2.5 (e) at 1 and 2 dpt. (A) The treatment groups of 0, 3.38,10.13, 15.2 μM; (B) The
treatment groups of 0, 1.01 μM and blank control (supplemental experiments).
Figure S9. Full-length blots/gels of Wnt8a
Reference
1.
Yuan H, Zhou X, Zhang YL. Degradation of Acid Pharmaceuticals in the UV/H2O2 Process:
Effects of Humic Acid and Inorganic Salts. CLEAN – Soil, Air, Water 2013, 41(1): 43-50.