background: polychlorinated biphenyls (pcbs) are known as neurotoxic chemicals or...

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.

GENE EXPRESSION PROFILES OF MEDAKA EMBRYOSEXPOSED 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 : [email protected] ) 2 Osaka 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

Figure 1: Structure of the PCB. Hydrogen of the biphenyl is substituted for 1-10 chlorine.

C l C lC l C l

PCB ｓ

2'3'

4'

6'5'

2 3

4

56

Ortho-Position

PCB11; 3,3’-dichlorobiphenyl

PCB126; 3,3’,4,4’,5-pentachlorobiphenyl

PCB11 PCB126

mailto:[email protected]

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 non-mammalian 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 non-

mammalian genomes and only 1 AhR in mammals..

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 200l of waterborne vehicle (DMSO) with 10, 50, 100mg/L of PCB11 or 1mg/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-1which is expressed ubiquitously in tissues and widely used as gene expression control..

Medaka (Oryzias latipes )

① Water tanks breeding Medaka

② Medaka pair （♂ 1 －♀ 1or2 ） ③ Collect fertilized eggs

every morning.

⑥ 　 Observe the embryos under stereo-microscope.

④ 　 Incubate the eggs in an incuba-tor for 24 hours at 26±0.5℃. On the next day , remove dead eggs.

Method of egg collection and observation

⑤ 　 Settle the eggs into each well of a microtiter plate with 200m l of dioxin solution.

⑦RNA extraction ⑧Quantitative RT- PCR

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.

Table1: The 20 genes for biomarker on the evaluation of environmental water.

(bio-marker genes)

Group No.Biomaker

symbol

Gene ID

(Ensemble Gene ID)Gene title Intercellular reaction

1 CYP1A1cp1a1

(ENSORLG00000014421)Cytochrome P450 1A detoxication metabolism

2 UDPGT ENSORLG00000013131 UDP glucuronosyltransferase xenobiotic metabolism

3 AhR1b-1ahr

(ENSORLG00000000137)Aryl Hydrocarbon Receptor 1 arylhydrocarbon receptor

4 AhR2a ENSORLG00000000135 Aryl Hydrocarbon Receptor 2 arylhydrocarbon receptor

5 AhRRAHRR

(ENSORLG00000005309)AhR Repressor arylhydrocarbon receptor repressor

6 ER-βQ8UW75_ORYLA

(ENSORLG00000017721)Estrogen receptor beta cell proliferation

7 CACHD1CACHD1

(ENSORLG00000010145)Cache domain-containing protein 1

angiogenesis

cell proliferation

8 RAR-αRARA

(ENSORLG00000004347)Retinoic acid receptor alpha

angiogenesis

bone differentiation

9 ER-αESR1

(ENSORLG00000014514)Estrogen receptor

reproduction function

cell proliferation

10 VEGF-R ENSORLG00000001940 VEGFR vascular endothelial cell function

11 AGXTAGXT(2 of 2)

(ENSORLG00000016389)Alanin-glyoxylate aminotransferase hepatocellular proliferation

12 MTF1MTF1

(ENSORLG00000014411)Metal-response transcription factor1

metal-response

homeostasis

13 Tropomyosin ENSORLG00000007803 Tropomyosin muscle-contraction regulation

14 HSP90HSP90AA1(2 of 2)

(ENSORLG00000017525) Heat shock protein hsp90 transcriptional regulation

15 TBPTBP

(ENSORLG00000010757)TATA-box binding protein transcriptional regulation

16 TNF-R ENSORLG00000016219 TNF receptorinflammatory supression

immunological control

17 CDC37CDC37(1 of 2)

(ENSORLG00000005543) Hsp90 co-chaperone Cdc37

protein stabilization

folding

18 HSP70HSP71_ORYLA

(ENSORLG00000000233)Heat shock 70 kDa protein 1 (HSP70-1)

anti-cell death

anti-inflammatory

19 MTMT_ORYLA

(ENSORLG00000015580)metallothionein

homeostasis

nervous system heavy metal poisoning

20 Ependymin1EPDR1

(ENSORLG00000006266)Ependymin related protein 1

neuroplasticity

angiogenesis

1

2

3

4

RESULTS AND DISCUSSION

Induced expression of biomarker genes mRNA in medaka embryos exposed to PCB11, PCB126 and 2,3,7,8-T4CDD

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.

(1) CYP1A1The 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 (10mg/L , 50mg/L, 100mg/L) induced a little .

(2) AhRRThe 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.

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).

0.1

1

10

100

1000

BL (DMSO 0.1%) PCB11_10ug/L PCB11_50ug/L PCB11_100ug/L PCB126_1ug/L 2378T4CDD_1ug/L

Rela

tive

Qua

ntity

(Cro

ssin

g Po

int)

Target-Sample

Relative Quantity Chart (CYP1A1)

0.0

1.0

2.0

3.0

4.0


Rela

tive

Qua

ntity

(Cro

ssin

g Po

int)

Target-Sample

Relative Quantity Chart (AhRR)

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).

CYP1A1

AHRR



2. Group2 (CACHD1, RAR-a)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. 2,3,7,8-T4CDD).

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) CACHD1The 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- aThe relative expression of RAR- was induced by PCB11 and

PCB126 exposure, while 2,3,7,8-T4CDD didn’t.

0.0

0.5

1.0

1.5

2.0

2.5


Rela

tive

Qua

ntity

(Cro

ssin

g Po

int)

Target-Sample

Relative Quantity Chart (CACHD1)

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).

Figure4. RAR-a mRNA expression in medaka embryos exposed to PCB11 and PCB126.The amount of RAR-a 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).

0.0

0.5

1.0

1.5

2.0


Rela

tive

Qua

ntity

(Cro

ssin

g Po

int)

Target-Sample

Relative Quantity Chart (RAR-a)

CACHD1

RAR-a



3. Group3 (HSP90, Tropomyosin)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) HSP90The relative expression of HSP90 was suppressed by PCB126

or 2,3,7,8-T4CDD, and PCB11 didn’t induce.

(2) TropomyosinThe relative expression of Tropomyosin was induced by PCB11

at fifty mg/L concentration, and was not induced by PCB126 or 2,3,7,8-T4CDD.

0.0

0.5

1.0

1.5


Rela

tive

Qua

ntity

(Cro

ssin

g Po

int)

Target-Sample

Relative Quantity Chart (HSP90)

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).

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).

0.0

0.5

1.0

1.5

2.0

2.5

3.0


Rela

tive

Qua

ntity

(Cro

ssin

g Po

int)

Target-Sample

Relative Quantity Chart (Tropomyosin)

Tropomyosin

HSP90



4. Group4 (Metallothionein, Ependymin)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) EpendyminThe 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.

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).

0.0

0.5

1.0

1.5

2.0

2.5


Rela

tive

Qua

ntity

(Cro

ssin

g Po

int)

Target-Sample

Relative Quantity Chart (Ependymin)

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).

0.0

0.5

1.0

1.5


Rela

tive

Qua

ntity

(Cro

ssin

g Po

int)

Target-Sample

Relative Quantity Chart (Metallothionein, MT)

Ependymin

MT

RESULTS AND DISCUSSIONPCB126 (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-a (Group2), and Tropomyosin (Group3). The relative mRNA expressions of these genes were presented in radar figure of 20 square. (Figure9)

012345

CYP1A1 /5UDPGT

AhR1b-1

AhR2a

AhRR

ER-β

CACHD1

RAR-α

ER-αVEGF-R

AGXTMTF1

Tropomyosin

HSP90a

TBP

TNF-R

CDC37

HSP70

MTEpendymin1

PCB126_1ug/L 2378T4CDD_1ug/L

012345CYP1A1

UDPGTAhR1b-1

AhR2a

AhRR

ER-β

CACHD1

RAR-α

ER-αVEGF-R

AGXTMTF1

Tropomyosin

HSP90a

TBP

TNF-R

CDC37

HSP70

MTEpendymin1

PCB11_10ug/L PCB11_50ug/LPCB11_100ug/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)

CONCLUSIONSWe 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-a (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.

REFERENCESBesselin Anantharaman V, Aravind L. (2000) Trends Biochem Sci. 25(11), 535-537 Beckmann R.P., Mizzen L.E., Welch W.J. (1990) Science. 248, 850-854.Choi C.Y., An K.W., Nelson E.R., Habibi H.R. (2007) Comp. Biochem. Physiol. C145, 595-600.Hahn ME., Allan LL., Sherr DH. (2009) Biochem, Pharmacol. 77, 485-497. Hanno K, Oda S, and Mitani H. (2010) Chemosphere 78, 830-839.Sharon A., Peter J., Luigi M.D.L. (2000) Physiol. Rev. 80, 1021-1054. Weber R, Hagenmaier H (1999); Chemosphere 38 (3):529-549.

E-mail is as follows; [email protected] ( Katsumasa HANNO )

background: polychlorinated biphenyls (pcbs) are known as neurotoxic chemicals or...

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