International Journal of Marine Science 2016, Vol.6, No.4, 1-12
7
Time (hpf)
48
72
96
Heart rate (BPM)
100
120
140
160
180
Control
0.5 M 4-t-OP
1.0 M 4-t-OP
a
ab
b
a
ab
b
a
ab
b
Figure 2
The effect of 4-t-OP on Tg (fil-1: EGFP) zebrafish
heart rate following the 96 hours exposure. Data are presented
as mean±SD of three independent experiments. The asterisk (*)
denotes statistically significant differences (
p <0.05
) between
control and 4-t-OP treated groups as measured by Student’s t
test.
3.4 Effects of 4-t-OP on the expression of
cardiovascular system-related genes in zebrafish
The significant harmful effect on cardiovascular
development and function in zebrafish motivated us to
investigate the effect of 4-t-OP on the change of
molecular level in cardiovascular system. First, we
analyzed the effect of 4-t-OP on the expression of
estrogen receptor. Expression levels of ERα and ER 2
genes are significantly increased in zebrafish
following exposure to 4-t-OP at 48 and 72 hpf. The
result showed that 1
M of 4-t-OP treated zebrafish
produced 3.15- and 8.9-fold significantly higher
mRNA expression level of ER and ER 2 at 72 hpf
compared to control, respectively; however although 1
M of 4-t-OP also induced 1.5- and 2.0-fold
significantly increasing in mRNA expression level of
ER 1 at 48 and 72 hpf, the increasing level was not
strong compared to that in ER and ER 2 (Fig. 3).
Moreover the expression level of genes, which was
associated with cardiovascular development and
function including Nkx2.5, Nkx2.7, Hand2, Tbx2a,
Tbx2b, Tbx5a, FGF1a and GATAs families, in 4-t-OP
exposed zebrafish were analyzed by real-time PCR.
Compare to the expression of Nkx2.5, the expression
of Nkx2.7 were significantly declined in 4-t-OP
exposed zebrafish embryos at 48 and 72 hpf. The
mRNA expression level of Hand2, Tbx5a, FGF1a and
GATAs families including GATA-4, -5 and -6 were
significantly suppressed in 4-t-OP exposed zebrafish
at 48 and 72 hpf. The expression level of Tbx2a and
Tbx2b are significantly suppressed only in the
presence of 1
M 4-t-OP at 48 hpf, and significantly
suppressed in the presence of 0.5
M and 1
M
4-t-OP at 72 hpf (Fig. 4). These results suggested that
4-t-OP suppresses the expression level of
cardiovascular development-related genes during
zebrafish embryogenesis.
Figure 3
Quantitative PCR analyses of gene expression in
4-t-OP exposed Tg (fil-1:EGFP) zebrafish embryos at 48 and
72 hpf. The expression of ER, ER1 and ER2 were quantified.
The data of the control group at 48 hpf was set as 1. The results
represented triplicated experiments, and the different letters
were considered significantly at p < 0.05.
