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BACKGROUND: Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals or endocrine-disrupting compounds. The non-Aroclor congener 3,3’-dichlorobiphenyl.

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Presentation on theme: "BACKGROUND: Polychlorinated biphenyls (PCBs) are known as neurotoxic chemicals or endocrine-disrupting compounds. The non-Aroclor congener 3,3’-dichlorobiphenyl."— Presentation transcript:

1 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 EMBRYOS EXPOSED TO PCB11 OR PCB126 Katsumasa Hanno 1, Takeshi Nakano 2, Shoji Oda 3, Hiroshi Mitani 3 1 Chiba Prefectural Environmental Research Center (1-8-8 Iwasaki-nishi, Ichihara,Chiba , Japan, e - mail : 2 Osaka University, Research Center for Environ. Preservation; Osaka , Japan, 3 Department of Integrated Bioscience, Graduate School of Frontier Sciences, The University of Tokyo; Chiba , Japan Figure 1: Structure of the PCB. Hydrogen of the biphenyl is substituted for 1-10 chlorine. PCB11; 3,3’-dichlorobiphenyl PCB126; 3,3’,4,4’,5-pentachlorobiphenyl PCB11PCB126

2 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 ( 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 o C for 10 s, followed by 45 cycles of 95 o C for 5 s and 60 o C 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 )

3 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 incubato r 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 wit h 200 m l of dioxin solution. ⑦ RNA extraction ⑧ Quantitative RT- PCR

4 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)

5 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) CYP1A1 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. (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. CYP1A1 AHRR

6 RESULTS AND DISCUSSION Induced expression of biomarker genes mRNA in medaka embryos exposed to PCB11, PCB126 and 2,3,7,8-T4CDD 2. Group2 (CACHD1, RAR-  ) 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) CACHD1 The relative expression of CACHD1 was induced by PCB11 exposure. The induced expression level of CACHD1 exposed to PCB11 was 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. CACHD1 RAR- 

7 RESULTS AND DISCUSSION Induced expression of biomarker genes mRNA in medaka embryos exposed to PCB11, PCB126 and 2,3,7,8-T4CDD 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) 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. Tropomyosin HSP90

8 RESULTS AND DISCUSSION Induced expression of biomarker genes mRNA in medaka embryos exposed to PCB11, PCB126 and 2,3,7,8-T4CDD 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) 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. Ependymin MT

9 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)

10 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 Group 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. REFERENCES Besselin Anantharaman V, Aravind L. (2000) Trends Biochem Sci. 25(11), Beckmann R.P., Mizzen L.E., Welch W.J. (1990) Science. 248, Choi C.Y., An K.W., Nelson E.R., Habibi H.R. (2007) Comp. Biochem. Physiol. C145, Hahn ME., Allan LL., Sherr DH. (2009) Biochem, Pharmacol. 77, Hanno K, Oda S, and Mitani H. (2010) Chemosphere 78, Sharon A., Peter J., Luigi M.D.L. (2000) Physiol. Rev. 80, Weber R, Hagenmaier H (1999); Chemosphere 38 (3): is as follows; ( Katsumasa HANNO )


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