GENE EXPRESSION PROFILES OF MEDAKA EMBRYOS
EXPOSED TO PCB11 OR PCB126
Katsumasa Hanno1, Takeshi Nakano2, Shoji Oda3, Hiroshi Mitani3
1Chiba
Prefectural Environmental Research Center (1-8-8 Iwasaki-nishi, Ichihara,Chiba 290-0046, Japan, e-mail : k.hnn@pref.chiba.lg.jp )
University, Research Center for Environ. Preservation; Osaka 565-0871, Japan,
3 Department of Integrated Bioscience, Graduate School of Frontier Sciences, The University of Tokyo; Chiba 277-8562, Japan
2 Osaka
BACKGROUND: Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals or endocrine-disrupting compounds. The
non-Aroclor congener 3,3’-dichlorobiphenyl (PCB11) has been detected ubiquitously in air, water, biota, sediment, and suspended
sediment since at least the 1970s. However, this congener was produced inadvertently during the production of current commercial
yellow paint pigments, the detailed effects of this congener on medaka and medaka embryo have not yet been elucidated. In this
study, we exposed medaka embryos in early developmental stage to PCB11 or PCB126 respectively for 48 hours, and after
exposure, the 20 selected candidate genes expression profiles were analyzed using real-time quantitative PCR analysis.
PCB11; 3,3’-dichlorobiphenyl
PCB126; 3,3’,4,4’,5-pentachlorobiphenyl
Ortho-Position
3'
PCB11
2'
2
3
4'
Cl
PCB126
4
5'
6'
6
5
Cl
PCB ｓ
Figure 1: Structure of the PCB. Hydrogen of the biphenyl is
substituted for 1-10 chlorine.
MATERIALS AND METHODS
Medaka embryos and exposure to PCB11 or PCB126:
Fertilized eggs and embryos were obtained from natural mating of
adult orange-red strain medaka fish originally derived from the
colony kept in The University of Tokyo and maintained at 26±0.5
oC under a 14 h-light and 10 h-dark cycle. Newly fertilized eggs
(200-300 embryos) were placed in a 100 ml beaker with distilled
water and incubated for 24 hours at 26±0.5 oC to develop. On the
next day, live embryos (stage17) were settled into wells of
microtiter plates filled with 200l of waterborne vehicle (DMSO)
with 10, 50, 100g/L of PCB11 or 1g/L of PCb126.
Total RNA isolation and real-time PCR experiments :.
Total RNA was isolated from the exposed embryos by ISOGEN
(Nippon Gene Co., Tokyo, Japan) and then purified with RNeasy
Mini Kit (QIAGEN). cDNAs were synthesized from 1 g of total
RNA using Rever TraAce-TM (Toyobo, Osaka, Japan) and
oligo(dT)20 primers. The relative amounts of the transcripts were
quantified using real-time PCR which was performed using
SYBR® Premix Ex TaqTM in a Smart Cycler®II System (Takara,
Shiga, Japan) and specific primers (Hanno et al., in preparation),
according to the manufacturer’s instructions. For each sample,
gene expression was analyzed in triplicate with the following
protocol: 95 oC for 10 s, followed by 45 cycles of 95 oC for 5 s and
60 oC for 20 s. At the completion of each PCR run, the obtained
PCR products were subjected to melting curve analysis to ensure
that only a single product was amplified. At least three technical
replicates of each RNA sample were conducted. Expression data
were quantified based on threshold cycle (Ct) values. For each
gene, the Ct values for each sample were averaged and
normalized to the mean Ct of EF-1which is expressed
ubiquitously in tissues and widely used as gene expression
control..
Medaka (Oryzias latipes )
Small teleosts such as Medaka or Zebrafish are established
as model organisms in risk evaluation of environmental
contaminants, however, little is known about molecular
machineries of dioxin reception and toxicity expression in nonmammalian species and we do not know whether we can
apply the TEQ concept to fish system as well as mammalian
system. Recently, a draft genome of Medaka (Oryzias latipes)
was released and 4 AhR genes were found in Medaka
genome, while 3 AhRs are known for the other nonmammalian genomes and only 1 AhR in mammals. .
Method of egg collection and observation
② Medaka pair （♂1－♀1or2）
① Water tanks breeding Medaka
⑥ Observe the embryos under
stereo-microscope.
⑦RNA extraction
③ Collect fertilized eggs
every morning.
⑤ Settle the eggs into each
well of a microtiter plate wit
h 200m l of dioxin solution.
⑧Quantitative RT- PCR
④ Incubate the eggs in an incubator
for 24 hours at 26±0.5℃.
On the next day , remove dead eggs.
Table1: The 20 genes for biomarker on the evaluation of environmental water.
(bio-marker genes)
Group
1
2
No.
Biomaker
symbol
1
CYP1A1
2
UDPGT
3
AhR1b-1
4
AhR2a
5
AhRR
6
ER-β
7
CACHD1
8
RAR-α
9
ER-α
10
VEGF-R
11
3
12
13
14
Gene ID
(Ensemble Gene ID)
cp1a1
(ENSORLG00000014421)
ENSORLG00000013131
ahr
(ENSORLG00000000137)
ENSORLG00000000135
AHRR
(ENSORLG00000005309)
Q8UW75_ORYLA
(ENSORLG00000017721)
CACHD1
(ENSORLG00000010145)
RARA
(ENSORLG00000004347)
ESR1
(ENSORLG00000014514)
ENSORLG00000001940
AGXT(2 of 2)
(ENSORLG00000016389)
MTF1
MTF1
(ENSORLG00000014411)
Tropomyosin ENSORLG00000007803
HSP90AA1(2 of 2)
HSP90
(ENSORLG00000017525)
AGXT
Gene title
Intercellular reaction
Cytochrome P450 1A
detoxication metabolism
UDP glucuronosyltransferase
xenobiotic metabolism
Aryl Hydrocarbon Receptor 1
arylhydrocarbon receptor
Aryl Hydrocarbon Receptor 2
arylhydrocarbon receptor
AhR Repressor
arylhydrocarbon receptor repressor
Estrogen receptor beta
cell proliferation
VEGFR
angiogenesis
cell proliferation
angiogenesis
bone differentiation
reproduction function
cell proliferation
vascular endothelial cell function
Alanin-glyoxylate aminotransferase
hepatocellular proliferation
Cache domain-containing protein 1
Retinoic acid receptor alpha
Estrogen receptor
Tropomyosin
metal-response
homeostasis
muscle-contraction regulation
Heat shock protein hsp90
transcriptional regulation
transcriptional regulation
Metal-response transcription factor1
15
TBP
TBP
(ENSORLG00000010757)
TATA-box binding protein
16
TNF-R
ENSORLG00000016219
TNF receptor
17
CDC37
18
HSP70
19
MT
20
Ependymin1
4
CDC37(1 of 2)
(ENSORLG00000005543)
HSP71_ORYLA
(ENSORLG00000000233)
MT_ORYLA
(ENSORLG00000015580)
EPDR1
(ENSORLG00000006266)
Hsp90 co-chaperone Cdc37
Heat shock 70 kDa protein 1 (HSP70-1)
metallothionein
Ependymin related protein 1
inflammatory supression
immunological control
protein stabilization
folding
anti-cell death
anti-inflammatory
homeostasis
nervous system heavy metal poisoning
neuroplasticity
angiogenesis
We classified the biomarker
genes into four groups, using
a medaka customized
microarray :
Group1 (CYP1A1, UDPGT,
AhR1b-1, AhR2a, AhRR, ER) ; detoxication metabolism
genes,
Group2 (CACHD1, RAR-,
ER-, VEGF-R) ; endocrine /
reproduction genes,
Group3 (AGXT, MTF1,
Tropomyosin, HSP90) ; cell
proliferation genes,
Group4 (TBP, TNF-R, CDC37,
HSP70, MT, Ependymin) ;
immunity / nervous system
genes,
considering the physiological
functions and induction by
chemicals (Hanno et al., in
preparation).
In particular, Group1 genes
were highly inducible by
dioxins and dioxin-like
compounds exposure.
RESULTS AND DISCUSSION
Relative Quantity Chart (CYP1A1)
1000
1. Group1 (CYP1A1, AhRR)
Cytochrome P450 1A1 (CYP1A1) and aryl hydrocarbon receptor
repressor (AhRR) genes are well known to be highly inducible by
dioxin and dioxin-like compounds exposure. Both genes are
regulated through the aryl hydrocarbon receptor (AhR)-mediated
pathway, which is ubiquitously functional in both mammalian
tissues and medaka (Oryzia latipes) embryos.
CYP1A1
100
Relative Quantity (Crossing Point)
Induced expression of biomarker genes mRNA in medaka
embryos exposed to PCB11, PCB126 and 2,3,7,8-T4CDD
10
1
0.1
BL (DMSO 0.1%)
PCB11_10ug/L
(1) CYP1A1
(2) AhRR
The relative expression of AhRR was 3.0 fold induction over
vehicle-control by PCB126 or 2,3,7,8-T4CDD exposure, but
PCB11 induced little.
These results suggested that the ecotoxcities of PCB11 might
not be strong as those of PCB126 and 2,3,7,8-T4CDD with high
TEF (each TEF value was 0.1 and 1), and PCB11 might have
another unknown toxic mechanisms.
Further assessment was necessary to make clear the potential
ecotoxicities of PCB11.
PCB11_100ug/L
PCB126_1ug/L
2378T4CDD_1ug/L
Target-Sample
Figure 1. CYP1A1 mRNA expression in medaka embryos exposed to PCB11 and PCB126.
The amount of CYP1A1 mRNA was measured by quantitative real-time PCR and normalized to
the solvent control (0.1% DMSO) for 48 h exposure, the error bars represents SD (n=3).
Relative Quantity Chart (AhRR)
4.0
AHRR
3.0
Relative Quantity (Crossing Point)
The relative expression of CYP1A1 in medaka embryos was
highly related to the WHO-TEF (the toxic equivalent factor) for
mammals (Hanno, et al., 2010).
PCB126 (TEF=0.1) and 2,3,7,8-T4CDD (TEF=1) induced
CYP1A1 expression 35 or 330 fold over respectively, but PCB11
(10g/L , 50g/L, 100g/L) induced a little .
PCB11_50ug/L
2.0
1.0
0.0
BL (DMSO 0.1%)
PCB11_10ug/L
PCB11_50ug/L
PCB11_100ug/L
PCB126_1ug/L
2378T4CDD_1ug/L
Target-Sample
Figure2. AhRR mRNA expression in medaka embryos exposed to PCB11 and PCB126.
The amount of AhRR mRNA was measured by quantitative real-time PCR and normalized to
the solvent control (0.1% DMSO) for 48 h exposure, the error bars represents SD (n=3).
RESULTS AND DISCUSSION
Relative Quantity Chart (CACHD1)
2.5
CACHD1 contains two cache domains and a VWFA (von
Willebrand factor A) domain which is known to be involved in
hemostasis (Anantharaman, et al., 2000). This gene was
induced by the exposure to heavy-metals or PCBs with low TEF,
but was suppressed by PCDDs with high TEF (e.g. 2,3,7,8T4CDD).
Retinoic acid (RA), the active derivative of vitamin A. The lack
or excess of RA result in developmental malformation on
vascular, blood vessel and bone formation (Sharon et al., 2000)
(1) CACHD1
The relative expression of CACHD1 was induced by PCB11
exposure. The induced expression level of CACHD1 exposed to
PCB11 was 1.5 - 2.0 fold induction over vehicle-control, while
PCB126 or 2,3,7,8-T4CDD didn’t.
(2) RAR-
The relative expression of RAR- was induced by PCB11 and
PCB126 exposure, while 2,3,7,8-T4CDD didn’t.
Relative Quantity (Crossing Point)
2. Group2 (CACHD1, RAR-)
CACHD1
2.0
1.5
1.0
0.5
0.0
BL (DMSO 0.1%)
PCB11_10ug/L
PCB11_50ug/L
PCB11_100ug/L
PCB126_1ug/L
2378T4CDD_1ug/L
Target-Sample
Figure3. CACHD1 mRNA expression in medaka embryos exposed to PCB11 and PCB126.
The amount of CACHD1 mRNA was measured by quantitative real-time PCR and normalized
to the solvent control (0.1% DMSO) for 48 h exposure, the error bars represents SD (n=3).
Relative Quantity Chart (RAR- )
2.0
RAR-
1.5
Relative Quantity (Crossing Point)
Induced expression of biomarker genes mRNA in medaka
embryos exposed to PCB11, PCB126 and 2,3,7,8-T4CDD
1.0
0.5
0.0
BL (DMSO 0.1%)
PCB11_10ug/L
PCB11_50ug/L
PCB11_100ug/L
PCB126_1ug/L
2378T4CDD_1ug/L
Target-Sample
Figure4. RAR- mRNA expression in medaka embryos exposed to PCB11 and PCB126.
The amount of RAR- mRNA was measured by quantitative real-time PCR and normalized to
the solvent control (0.1% DMSO) for 48 h exposure, the error bars represents SD (n=3).
RESULTS AND DISCUSSION
Relative Quantity Chart (HSP90)
1.5
Induced expression of biomarker genes mRNA in medaka
embryos exposed to PCB11, PCB126 and 2,3,7,8-T4CDD
Relative Quantity (Crossing Point)
HSP90
1.0
HSPs (heat shock proteins) are stress-defenssive proteins and
are highly induced in response to stresses caused by changes
in environmental factors such as high temperature, heavy metal
administrations, reactive oxygen production, salinity, etc
(Beckmann, et al.,1990) and protect the structure and function
of proteins and cells from damages, maintaining the cellular
homoeostasis.
Tropomyosin is an actin-binding protein that regulates actin
mechanics. It is important, among other things, for muscle
contraction.
(1) HSP90
The relative expression of HSP90 was suppressed by PCB126
or 2,3,7,8-T4CDD, and PCB11 didn’t induce.
(2) Tropomyosin
The relative expression of Tropomyosin was induced by PCB11
at fifty g/L concentration, and was not induced by PCB126 or
2,3,7,8-T4CDD.
0.5
0.0
BL (DMSO 0.1%)
PCB11_10ug/L
PCB11_50ug/L
PCB11_100ug/L
PCB126_1ug/L
2378T4CDD_1ug/L
Target-Sample
Figure5. HSP90 mRNA expression in medaka embryos exposed to PCB11 and PCB126.
The amount of HSP90 mRNA was measured by quantitative real-time PCR and normalized to
the solvent control (0.1% DMSO) for 48 h exposure, the error bars represents SD (n=3).
Relative Quantity Chart (Tropomyosin)
3.0
2.5
Tropomyosin
Relative Quantity (Crossing Point)
3. Group3 (HSP90, Tropomyosin)
2.0
1.5
1.0
0.5
0.0
BL (DMSO 0.1%)
PCB11_10ug/L
PCB11_50ug/L
PCB11_100ug/L
PCB126_1ug/L
2378T4CDD_1ug/L
Target-Sample
Figure6. Tropomyosin mRNA expression in medaka embryos exposed to PCB11 and PCB126.
The amount of Tropomyosin mRNA was measured by quantitative real-time PCR and
normalized to the solvent control (0.1% DMSO) for 48 h exposure, the error bars represents
SD (n=3).
RESULTS AND DISCUSSION
MT
Relative Quantity (Crossing Point)
Induced expression of biomarker genes mRNA in medaka
embryos exposed to PCB11, PCB126 and 2,3,7,8-T4CDD
Relative Quantity Chart (Metallothionein, MT)
1.5
1.0
Metallothionein (MT) is a protein that has a high cysteine
content, a low molecular mass about 7 kDa, and a high affinity
for metals. It binds metals and regulates the homeostasis of
essential trace metals such as copper and zinc, also taking a
part in counteracting the toxic effects of heavy metals such as
cadmium, mercury, and silver (Choi, et al., 2007).
Ependymin is a glycoprotein associated with the consolidation
of long-term memory, claims involving protection from strokes,
and neuronal regeneration.
(1) Metallothionein (MT)
The relative expression of Metallothionein was suppressed by
PCB126, 2,3,7,8-T4CDD and PCB11(100ug/L). PCB11 (50ug/L)
induced a little.
(2) Ependymin
The relative expression of Ependymin was induced by PCB11
exposure. The induced expression level of Ependymin exposed
to PCB11 was about 2.0 fold induction over vehicle-control,
while PCB126 or 2,3,7,8-T4CDD induced a little.
0.5
0.0
BL (DMSO 0.1%)
PCB11_10ug/L
PCB11_50ug/L
PCB11_100ug/L
PCB126_1ug/L 2378T4CDD_1ug/L
Target-Sample
Figure7. MT mRNA expression in medaka embryos exposed to PCB11 and PCB126.
The amount of MT mRNA was measured by quantitative real-time PCR and normalized to the
solvent control (0.1% DMSO) for 48 h exposure, the error bars represents SD (n=3).
Relative Quantity Chart (Ependymin)
2.5
Ependymin
2.0
Relative Quantity (Crossing Point)
4. Group4 (Metallothionein, Ependymin)
1.5
1.0
0.5
0.0
BL (DMSO 0.1%)
PCB11_10ug/L
PCB11_50ug/L
PCB11_100ug/L
PCB126_1ug/L
2378T4CDD_1ug/L
Target-Sample
Figure8. Ependymin mRNA expression in medaka embryos exposed to PCB11 and PCB126.
The amount of Ependymin mRNA was measured by quantitative real-time PCR and
normalized to the solvent control (0.1% DMSO) for 48 h exposure, the error bars represents
SD (n=3).
RESULTS AND DISCUSSION
PCB126 (1ug/L) or 2,3,7,8-T4CDD (1ug/L) induced CYP1A1 in proportion to its
TEF values, AhR2a and AhRR (Group1), and suppressed the genes of Group3
and Group4. On the other hand, PCB11 (10, 50, 100 ug/L) induced CYP1A1
(Group1), CACHD1, RAR- (Group2), and Tropomyosin (Group3).
The relative mRNA expressions of these genes were presented in radar figure of
20 square. (Figure9)
CYP1A1 /5
Ependymin1 5
UDPGT
MT
AhR1b-1
4
HSP70
CDC37
TNF-R
3
2
1
0
AhR2a
AhRR
ER-β
TBP
CACHD1
HSP90a
RAR-α
Tropomyosin
MTF1
ER-α
VEGF-R
CYP1A1
Ependymin1 5
MT
4
HSP70
CDC37
TNF-R
3
2
1
0
AhR2a
AhRR
ER-β
TBP
CACHD1
HSP90a
RAR-α
Tropomyosin
MTF1
ER-α
VEGF-R
AGXT
AGXT
PCB126_1ug/L
UDPGT
AhR1b-1
2378T4CDD_1ug/L
PCB11_10ug/L
PCB11_100ug/L
PCB11_50ug/L
Figure 9. The biomarker genes mRNA expressions in medaka embryos exposed
to PCB11, PCB126 and 2,3,7,8-T4CDD, presented in radar figure of 20 square.
The amount of those mRNA were measured by quantitative real-time PCR and
normalized to the solvent control (0.1% DMSO) for 48 h exposure. (n=3)
CONCLUSIONS
We have evaluated the ecotoxicities of wastewaters by investigating the biomarker genes expression profiles in
the early developmental stage of medaka embryos exposed to wastewaters containing dioxins and dioxin-like
compounds.
In this study, we evaluated the toxicities of PCB11, obtaining the following conclusions :
-1. PCB11 (10, 50, 100 ug/L) induced CYP1A1 (Group1), CACHD1, RAR- (Group2), and Tropomyosin (Group3).
-2. PCB126 (1ug/L) and 2,3,7,8-T4CDD (1ug/L) induced CYP1A1 in proportion to its TEF values, AhR2a, AhRR
(Group1), and suppressed the genes of Group3 and Group4.
-3. The ecotoxcities of PCB11 might not be strong as those of PCB126 and 2,3,7,8-T4CDD with high TEF (each
TEF value was 0.1 and 1).
-4. PCB11 might have another unknown toxic mechanisms.
-5. Further assessment was necessary to make clear the potential ecotoxicities of PCB11.
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E-mail is as follows;
k.hnn@pref.chiba.lg.jp ( Katsumasa HANNO )