한국산 무당개구리 배아에서 중금속에 의한 발생 및 등쪽 중배엽 형성 저해

Abstract

OECD에 따르면 수은 (mercury; Hg), 구리 (copper; Cu), 카드뮴 (cadmium; Cd), 철 (iron; Fe), 아연 (zinc; Zn) 등은 산업공정에 사용되는 잠재적 독성을 갖는 금속에 속한다. 본 연구에서는 standard protocol for frog embryo teratogenesis assay (FETAX)를 참고하여 염화수은 (mercuric chloride; HgCl2),염화구리 (copper chloride; CuCl2),염화카드뮴 (cadmium chloride; CdCl2),염화아연 (zinc chloride; ZnCl2),염화철 (iron chloride; FeCl3)이 무당개구리 (Bombina orientalis) 배아의 발생, 기형, 성장에 미치는 효과를 분석하였다. 또한 세포사멸과 중배엽형성에 미치는 영향을 분석하였다. 연구결과 염화수은, 염화구리, 염화카드뮴, 염화철의 LC50 (Lethal concentration 50)은 각각 1.92 μM, 6.50 μM, 9.41 μM, 97.5 μM로 확인되었고, 염화아연은 실험농도구간에서 독성효과가 없었다. 기형유발 EC50 (effective concentration)은 각각 0.91 μM, 2.13 μM, 4.95 μM, 135.7 μM, 246.1 μM로 확인되었다. 기형율에 근거한 No observable effective concentration (NOEC)는 각각 0.1 μM, 1 μM, 1 μM, 100 μM, 100 μM 이었다. 기형율에 근거한 Lowest observed effective concentration (LOEC)은 염화수은은 1 μM, 염화구리는 3 μM, 염화카드뮴은 5μM이었고, 염화철과 염화아연은 실험농도구간에서 LOEC를 산출할 수 없었다. 기형유형은 염화수은, 염화구리, 염화카드뮴 처리 168시간에 두부기형 (Head malformation) 비율이 가장 높았다. 처리 168시간 후 유생의 9개의 신체부위에 대한 계측결과, 염화수은 1 μM 처리군의 maximum tail height (MTH)와 2 μM 처리군의 eye diameter (ED), tail length (TL), total length (TAL)가 유의적으로 감소하였다. 염화구리 3 μM 처리군의 MTH, body length (BL), inter orbital distance (IOD), eye to nostril distance (END)와 5 μM 처리군의 TL, TAL, eye to mouth distance (EMD)와 7 μM 처리군의 TMH가 유의적으로 감소하였다. 염화카드뮴은 10 μM 처리군의 TL, BL, TAL이 유의적으로 감소하였다. 염화아연과 염화철의 경우 유의적인 성장저해가 확인되지 않았다. 이를 통하여, 염화수은, 염화구리, 염화카드뮴이 무당개구리 배아에 기형 발생과 성장 저해 효과를 갖는 것으로 확인되었다. 트리판블루 (trypan-blue)염색 결과, 염화수은 처리 12시간에 3 μM 이상 처리군에서, 염화구리는 처리 12시간에 10 μM 이상 처리군에서 염색이 유의적으로 증가하였다. 염화카드뮴은 처리 12시간에 염색이 되지 않았고 24시간에 50 μM 이상 처리군에서 분산된 할구세포가 염색되었다. 동물극 분리체 (Animal cap explant)에서 MTT 분석결과, 염화수은과 염화구리는 10 μM 이상 처리군에서, 염화카드뮴은 50 μM 이상 처리군에서 세포생존율이 유의적으로 감소하였다. 이를 통해 염화수은, 염화구리, 염화카드뮴은 배아세포에 독성이 있음을 알 수 있다. Activin-A를 동물극 분리체에 처리하여 중배엽을 유도한 실험결과, 염화수은은 3 μM 이상의 농도에서 길이 (length)와 길이/폭 값이, 염화구리는 1 μM 이상의 농도에서 길이와 길이/폭 값이, 염화카드뮴은 10 μM 이상의 농도에서 길이와 길이/폭 값이 유의적으로 감소하였다. 중배엽으로 유도된 동물극 분리체에서 중배엽 형성에 관련한 유전자들의 발현 양상 분석결과 염화수은은 1 μM 이상 처리군에서 goosecoid, chordin mRNA가 유의적으로 감소하였고, 10 μM 이상 처리군에서 epidermal keratin mRNA가 유의적으로 증가하였다. 염화구리는 1 μM 이상 처리군에서 goosecoid, noggin, chordin, follistatin mRNA가 유의적으로 감소하였고, 10 μM 이상 처리군에서 epidermal keratin mRNA가 유의적으로 증가하였다. 염화카드뮴은 5 μM 이상 처리군에서 goosecoid, noggin, chordin, follistatin mRNA가 유의적으로 감소하였고, 50 μM 이상 처리군에서 epidermal keratin mRNA가 유의적으로 증가하였다. 중배엽으로 유도된 동물극 분리체에서 세포자살 (apoptosis) 관련 유전자들의 발현 양상 분석결과 염화수은은 10 μM 이상 처리군에서 BAK mRNA가 유의적으로 증가하였다. 염화구리는 10 μM 이상 처리군에서 BAK mRNA가 유의적으로 증가하였고, BAX mRNA는 유의적으로 감소하였다. 염화카드뮴은 50 μM 이상 처리군에서 Bcl-2 mRNA가 유의적으로 증가하였고, 5 μM 처리군에서 BAX mRNA가 유의적으로 감소하였다. 이를 통해 염화수은, 염화구리, 염화카드뮴에서 일부 독성 효과와 중배엽 형성 저해는 세포자살에 의한 것으로 사료되며, 중배엽형성 저해효과는 중배엽 형성 유전자 발현을 저해하는 것으로 사료된다. 본 연구에서는 FETAX 분석법을 이용하여 중금속이 국내 토종 양서류 배아의 발생에 미치는 독성을 최초로 분석하였다. 실험에 사용된 중금속들은 무당개구리 배아에 기형을 유발하며, 세포독성을 갖고, 중배엽 형성을 저해하였다. 무당개구리 배아에서 확인된 독성데이터베이스는 한국의 담수생태계에서 중금속 관리에 대한 가이드라인을 구축하는데 유용하게 사용될 것이다. 독성 시험 모델로서의 무당개구리 배아는 환경시료의 생태 독성 시험뿐만 아니라 개별 화학물질 독성시험에도 적용할 수 있을 것이다.|According to OECDs, mercury (Hg), copper (Cu), cadmium (Cd), iron (Fe), zinc (Zn) are defined as heavy metals. They are widely used for industry and annual usage has been constantly increasing. In this study, we examined developmental toxicities of Hg, Cu, Cd, Fe and Zn according to the standard protocol for frog embryo teratogenesis assay in Bombina orientalis, one of the most common amphibia in Korea. Cell death, cell apoptosis, and inhibition of mesoderm formation by Hg, Cu, and Cd were also examined. In result, 168 h LC50 of HgCl2, CuCl2, CdCl2, and FeCl3 were 1.92 μM, 6.50 μM, 9.41, and 97.5 μM, respectively. Also, 168 h EC50 for malformation were 0.91 μM, 2.13 μM, 4.95 μM, 135.7 μM, and 246.1 μM, respectively. No observable effective concentration (NOEC) for malformation of HgCl2, CuCl2, and CdCl2 were 1 μM, 5 μM and 5 μM, respectively. The lowest observed effective concentration (LOEC) of HgCl2, CuCl2, and CdCl2 were 2 μM, 7 μM, and 10 μM, respectively. NOEC of FeCl3 and ZnCl2 for malformation was 100 μM. Embryonic exposure to HgCl2, CuCl2, and CdCl2 produced several types of abnormalities such as head malformation, bent tail, gut abnormal coiling, bent trunk, and ventral blister. Head malformation was the most frequent abnormal type. In tadpoles exposed HgCl2 for 168 h, maximum tail height (MTH) were significantly decreased at 1 μM. Eye diameter (ED), tail length (TL), total length (TAL) were significantly decreased at 2 μM. In tadpoles exposed CuCl2 for 168 h, MTH, BL, interorbital distance (IOD), and eye to nostril distance (END) were significantly decreased at 3 μM. TL, TAL, and EMD were significantly decreased at 5 μM. TMH were significantly decreased in 5 μM. In tadpoles exposed CdCl2 for 168 h, TL, BL, and TAL were significantly decreased at 10 μM. Even at 100 uM FeCl3 and ZnCl2 did not alter the growth parameters. Together, heavy metals were teratogenic as well as growth inhibitory in B. orinetalis embryos. In cell death assay using trypan blue staining, cell death was significantly increased at 3 μM HgCl2 (12h), 10 μM CuCl2 (12h), and 50 μM CdCl2 (24h). Activin A-induced convergent-extension of animal cap explants were significantly inhibited by 3 μM HgCl2, 1 μM CuCl2, and 10 μM CdCl2. In MTT assay for activin A-induced convergent-extension of animal cap explants cellular viability were significantly inhibited by 10 μM HgCl2, 10 uM CuCl2, and 50 μM CdCl2. In the analysis of expression of genes associated with dorsal mesoderm formation goosecoid and chordin mRNA were significantly decreased at 1 μM HgCl2 together with significantly increase in epidermal keratin mRNA at 10 μM HgCl2. Noggin and follistatin mRNA levels were not changed. In CuCl2 treated animal cap explants goosecoid, noggin, chordin and follistatin mRNA were significantly decreased at 1 μM together with significantly increased in epidermal keratin mRNA at 10 μM. In CdCl2 treated animal cap explants, goosecoid, noggin, chordin, and follistatin mRNA were significantly decreased at 5 μM together with significantly increased in epidermal keratin mRNA at 50 μM. Together, heavy metals may inhibit mesoderm development in B. orinetalis embryos. In animal cap explants, apoptosis related gene BAK mRNA was significantly increased at 10 μM In HgCl2. In CuCl2 treated animal cap explants, BAK mRNA was significantly increased at 10 μM together with significant decrease in BAX mRNA at 10 μM. In CdCl2 treated animal cap explants, bcl-2 mRNA were significantly decreased at 50 μM together with significant decrease in BAX mRNA at 5 μM. Together, toxic effects and mesoderm development inhibition might be partially mediated by apoptosis. This is the first report on the developmental toxicity of heavy metals in Korean amphibian species based on the FETAX protocol. Tested heavy metals were teratogenic, cytotoxic and inhibitory for mesoderm development in Bombina orientalis embryos. Toxic database obtained from Bombina orientalis embryos will be helpful for building the guide line for management of heavy metals in the freshwater system in Korea. Furthermore, Bombina orientalis embryos as a toxicity test model will be applicable for ecotoxicity test of environmental media as well as individual chemicals.; According to OECDs, mercury (Hg), copper (Cu), cadmium (Cd), iron (Fe), zinc (Zn) are defined as heavy metals. They are widely used for industry and annual usage has been constantly increasing. In this study, we examined developmental toxicities of Hg, Cu, Cd, Fe and Zn according to the standard protocol for frog embryo teratogenesis assay in Bombina orientalis, one of the most common amphibia in Korea. Cell death, cell apoptosis, and inhibition of mesoderm formation by Hg, Cu, and Cd were also examined. In result, 168 h LC50 of HgCl2, CuCl2, CdCl2, and FeCl3 were 1.92 μM, 6.50 μM, 9.41, and 97.5 μM, respectively. Also, 168 h EC50 for malformation were 0.91 μM, 2.13 μM, 4.95 μM, 135.7 μM, and 246.1 μM, respectively. No observable effective concentration (NOEC) for malformation of HgCl2, CuCl2, and CdCl2 were 1 μM, 5 μM and 5 μM, respectively. The lowest observed effective concentration (LOEC) of HgCl2, CuCl2, and CdCl2 were 2 μM, 7 μM, and 10 μM, respectively. NOEC of FeCl3 and ZnCl2 for malformation was 100 μM. Embryonic exposure to HgCl2, CuCl2, and CdCl2 produced several types of abnormalities such as head malformation, bent tail, gut abnormal coiling, bent trunk, and ventral blister. Head malformation was the most frequent abnormal type. In tadpoles exposed HgCl2 for 168 h, maximum tail height (MTH) were significantly decreased at 1 μM. Eye diameter (ED), tail length (TL), total length (TAL) were significantly decreased at 2 μM. In tadpoles exposed CuCl2 for 168 h, MTH, BL, interorbital distance (IOD), and eye to nostril distance (END) were significantly decreased at 3 μM. TL, TAL, and EMD were significantly decreased at 5 μM. TMH were significantly decreased in 5 μM. In tadpoles exposed CdCl2 for 168 h, TL, BL, and TAL were significantly decreased at 10 μM. Even at 100 uM FeCl3 and ZnCl2 did not alter the growth parameters. Together, heavy metals were teratogenic as well as growth inhibitory in B. orinetalis embryos. In cell death assay using trypan blue staining, cell death was significantly increased at 3 μM HgCl2 (12h), 10 μM CuCl2 (12h), and 50 μM CdCl2 (24h). Activin A-induced convergent-extension of animal cap explants were significantly inhibited by 3 μM HgCl2, 1 μM CuCl2, and 10 μM CdCl2. In MTT assay for activin A-induced convergent-extension of animal cap explants cellular viability were significantly inhibited by 10 μM HgCl2, 10 uM CuCl2, and 50 μM CdCl2. In the analysis of expression of genes associated with dorsal mesoderm formation goosecoid and chordin mRNA were significantly decreased at 1 μM HgCl2 together with significantly increase in epidermal keratin mRNA at 10 μM HgCl2. Noggin and follistatin mRNA levels were not changed. In CuCl2 treated animal cap explants goosecoid, noggin, chordin and follistatin mRNA were significantly decreased at 1 μM together with significantly increased in epidermal keratin mRNA at 10 μM. In CdCl2 treated animal cap explants, goosecoid, noggin, chordin, and follistatin mRNA were significantly decreased at 5 μM together with significantly increased in epidermal keratin mRNA at 50 μM. Together, heavy metals may inhibit mesoderm development in B. orinetalis embryos. In animal cap explants, apoptosis related gene BAK mRNA was significantly increased at 10 μM In HgCl2. In CuCl2 treated animal cap explants, BAK mRNA was significantly increased at 10 μM together with significant decrease in BAX mRNA at 10 μM. In CdCl2 treated animal cap explants, bcl-2 mRNA were significantly decreased at 50 μM together with significant decrease in BAX mRNA at 5 μM. Together, toxic effects and mesoderm development inhibition might be partially mediated by apoptosis. This is the first report on the developmental toxicity of heavy metals in Korean amphibian species based on the FETAX protocol. Tested heavy metals were teratogenic, cytotoxic and inhibitory for mesoderm development in Bombina orientalis embryos. Toxic database obtained from Bombina orientalis embryos will be helpful for building the guide line for management of heavy metals in the freshwater system in Korea. Furthermore, Bombina orientalis embryos as a toxicity test model will be applicable for ecotoxicity test of environmental media as well as individual chemicals.