어류급성독성시험 대체법으로서 잉어표피세포를 이용한 Neutral Red Uptake 분석법 적용 Application of Neutral Red Uptake Assay Using EPC Cells as an Alternative to the Fish Acute Toxicity Test for Pesticide원문보기
본 연구는 5가지 제품농약을 이용하여 어류 급성독성시험 결과 (반수치사농도)와 잉어의 표피에서 유래된 EPC 세포를 이용한 neutral red uptake 결과 (반수저해농도)를 비교함으로써 동물 실험의 대체 가능성을 평가하기 위하여 수행되었다. 어류 급성 독성시험은 왜몰개 (Aphyocypris chinensis)를 포함하여 OECD와 농촌진흥청의 농약에 대한 독성시험기준에서 추천하는 어종인 송사리 (Oryzias latipes)와 잉어 (Cyprinus carpio)를 이용하여 수행하였다. 5가지 제품 농약에 대한 민감도는 어류에 비하여 세포에서 약 10배 더 낮게 확인되었지만, 독성을 서열화 하였을 때 나타나는 순서는 두 가지 방식에서 모두 비슷하게 나타났다. 5가지 제품 농약에 대한 세포와 어류 독성값의 상관성을 분석한 결과는 A. chinensis, O. latipes와 C. carpio에서 각각 $r^2=0.38$ (p = 0.26), $r^2=0.76$ (p = 0.05), $r^2=0.90$ (p = 0.01)을 나타내었다. 본 시험의 결과, EPC 세포를 이용한 NRU 시험은 O. latipes와C. carpio에 대한 어류 독성시험 결과와 상관성이 높으므로 향후 더 많은 약제시험을 통해 어류 급성독성시험의 대체시험법으로서의 가능성이 기대된다.
본 연구는 5가지 제품농약을 이용하여 어류 급성독성시험 결과 (반수치사농도)와 잉어의 표피에서 유래된 EPC 세포를 이용한 neutral red uptake 결과 (반수저해농도)를 비교함으로써 동물 실험의 대체 가능성을 평가하기 위하여 수행되었다. 어류 급성 독성시험은 왜몰개 (Aphyocypris chinensis)를 포함하여 OECD와 농촌진흥청의 농약에 대한 독성시험기준에서 추천하는 어종인 송사리 (Oryzias latipes)와 잉어 (Cyprinus carpio)를 이용하여 수행하였다. 5가지 제품 농약에 대한 민감도는 어류에 비하여 세포에서 약 10배 더 낮게 확인되었지만, 독성을 서열화 하였을 때 나타나는 순서는 두 가지 방식에서 모두 비슷하게 나타났다. 5가지 제품 농약에 대한 세포와 어류 독성값의 상관성을 분석한 결과는 A. chinensis, O. latipes와 C. carpio에서 각각 $r^2=0.38$ (p = 0.26), $r^2=0.76$ (p = 0.05), $r^2=0.90$ (p = 0.01)을 나타내었다. 본 시험의 결과, EPC 세포를 이용한 NRU 시험은 O. latipes와C. carpio에 대한 어류 독성시험 결과와 상관성이 높으므로 향후 더 많은 약제시험을 통해 어류 급성독성시험의 대체시험법으로서의 가능성이 기대된다.
This study evaluated in vitro cytotoxicity of 5 pesticides, including 2 herbicides, 2 germicides, and an insecticide, as an alternative to the fish acute toxicity test. The in vitro cytotoxicity was tested using a neutral red uptake (NRU) assay with epithelioma papulosum cyprini (EPC) cells that ori...
This study evaluated in vitro cytotoxicity of 5 pesticides, including 2 herbicides, 2 germicides, and an insecticide, as an alternative to the fish acute toxicity test. The in vitro cytotoxicity was tested using a neutral red uptake (NRU) assay with epithelioma papulosum cyprini (EPC) cells that originated from the epidermal tissue of Cyprinus carpio (common carp). An in vivo fish acute toxicity test was conducted according to OECD Test Guideline No. 203 using Aphyocypris chinensis (Chinese bleak), Oryzias latipes (Japanese medaka), and C. carpio. The results showed that the sensitivity of the cell viability assay for the pesticides was similar to the fish acute test in ranking order despite having approximately 10 times less absolute sensitivity. The $r^2$ correlation values were calculated as 0.38 (p = 0.26), 0.76 (p = 0.05) and 0.90 (p = 0.01) for A. chinensis, O. latipes, and C. carpio, respectively. These results suggested that the potential of EPC cell viability assay as an alternative to the fish acute toxicity test due to their good correlation and NRU assay is expected to serve as a useful tool for predicting acute fish lethality for pesticides if further studies with a large set of pesticides are conducted.
This study evaluated in vitro cytotoxicity of 5 pesticides, including 2 herbicides, 2 germicides, and an insecticide, as an alternative to the fish acute toxicity test. The in vitro cytotoxicity was tested using a neutral red uptake (NRU) assay with epithelioma papulosum cyprini (EPC) cells that originated from the epidermal tissue of Cyprinus carpio (common carp). An in vivo fish acute toxicity test was conducted according to OECD Test Guideline No. 203 using Aphyocypris chinensis (Chinese bleak), Oryzias latipes (Japanese medaka), and C. carpio. The results showed that the sensitivity of the cell viability assay for the pesticides was similar to the fish acute test in ranking order despite having approximately 10 times less absolute sensitivity. The $r^2$ correlation values were calculated as 0.38 (p = 0.26), 0.76 (p = 0.05) and 0.90 (p = 0.01) for A. chinensis, O. latipes, and C. carpio, respectively. These results suggested that the potential of EPC cell viability assay as an alternative to the fish acute toxicity test due to their good correlation and NRU assay is expected to serve as a useful tool for predicting acute fish lethality for pesticides if further studies with a large set of pesticides are conducted.
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제안 방법
Therefore, this study was conducted to evaluate the sensitivity of the correlation between the NRU assay and fish acute toxicity tests. To accomplish this, the NRU assay used an EPC cell line that is commonly used and has a high sensitivity.
대상 데이터
To accomplish this, the NRU assay used an EPC cell line that is commonly used and has a high sensitivity. A total of 3 kinds of fish, Aphyocypris chinensis (A. chinensis), Oryzias latipes (O. latipes), and Cyprinus carpio (C. carpio), were tested with 5 pesticides.
The tests lasted for 96 hrs in static systems without medium changes. Each test unit contained 7 fish, both male and female, in 5 liters of test media, and the test was performed with no replicates.
Epithelioma papulosum cyprini (EPC) cells from carp (C. carpio) epithelium, which is an established monolayer-type cultured fish cell line, were used. The EPC cells were distributed by Chonnam National University (Department of Aqualife Medicine).
The A. chinensis, O. latipes, and C. carpio used in this study were obtained from the Korea Institute of Toxicology (Daejeon, Korea). The fish were maintained in dechlorinated tap water at 22 ± 1℃ with a photoperiod of 16:8 hrs (light:dark).
carpio) epithelium, which is an established monolayer-type cultured fish cell line, were used. The EPC cells were distributed by Chonnam National University (Department of Aqualife Medicine). The cells were maintained at 20℃ in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
데이터처리
All of the statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS) 20 (IBM, United States), and all of the data were plotted using Sigma Plot 10.0 (Systat Software Inc., Germany). Probit analysis was used for calculation the IC50 and LC50 values.
성능/효과
In conclusion, the NRU assay using EPC cell was well correlated with acute toxicity of C. carpio and O. latipes. Therefore, it will serve a potential tool as an alternative to the fish acute toxicity test.
The in vivo LC50 values for the 3 species of fish had a similar rank order as the IC50 values in A. chinensis and O. latipes: Insecticide B (0.01 and 0.09 mg/L, respectively) > Germicide B (0.07 and 0.13 mg/ L, respectively) > Insecticide A (0.16 and 1.05 mg/L, respectively) > Herbicide (3.54 and 1.27 mg/L, respectively) > Germicide A (3.81 and 2.43 mg/L, respectively).
value were also determined. The results revealed that a linear correlation between the in vitro IC50 for the EPC cells and the in vivo LC50 for C. carpio were the most highly significant (p = 0.01 with r2= 0.90) followed by O. latipes (p = 0.05 with r2= 0.76). For A.
carpio are shown in Table 1. The results showed that the IC50 values were closely correlated with the whole fish LC50 values and those highly significant linear correlations between the in vitro and in vivo values were found for C. carpio and O. latipes. However, A.
후속연구
Therefore, it will serve a potential tool as an alternative to the fish acute toxicity test. However, further studies with a larger set of pesticides are needed to strengthen the reliability of the assays and to validate the correlation with in vivo data.
참고문헌 (23)
Babich, H., S. H. Goldstein and E. Borenfreund (1990) In vitro cyto- and genotoxicty of organomercurials to cells in culture. Toxicology Letters. 50:143-149.
Brandao, J. C., H. H. L. Bohets, I. E. van de Vyver and P. J. Dierickx (1992) Correlation between the in vitro cytotoxicity to cultured fathead minnow fish cells and fish lethality data for 50 chemicals. Chemosphere. 25:553-562.
Bruschweiler, B. J., F. E. Wurgler and K. Fent (1995) Cytotoxicity in vitro of organotin compounds in fish hepatoma cells PLHC-1 (Poeciliopsis lucida). Aquatic Toxicology. 32:143-160.
Castano, A., M. J. Cantarino, P. Castillo and J. V. Tarazona (1996) Correlations between the RTG-2 cytotoxicity test $EC_{50}$ andin vivo $LC_{50}$ rainbow trout bioassay. Chemosphere. 32:2141-2157.
Castano, A., N. Bols, T. Braunbeck, P. Dierickx, M. Halder, B. Isomaa, K. Kawahara, L. E. J. Lee, C. Mothersill, P. Part, G. Repetto, J. R. Sintes, H. Rufli, R. Smith, C. Wood and H. Segner (2003) The use of fish cells in ecotoxicology. The report and recommendations of ECVAM workshop 47. Alternatives to Laboratory Animals. 31:317-351.
Dierickx, P. J. and I. E. van de Vyver (1991) Correlation of the neutral red uptake inhibition assay of cultured fathead minnow fish cells with fish lethality tests. Bull. Environmental Contamination and Toxicology. 46:649-653.
Dierickx, P. J. (1993) Comparison between fish lethality data and the in vitro cytotoxicity of lipophilic solvents to cultured fish cells in a two-compartment model. Chemosphere. 27:1511-1518.
Kilkenny, C., W. J. Browne, I. C. Cuthill, M. Emerson, D. G. Altman (2010) Improving bioscience research reporting: The ARRIVE Guidelines for reporting animal research. British Journal of Pharmacology. 160(7):1577-1579.
Lammer, E., G. J. Carr, K. Wendler, J. M. Rawlings, S. E. Belanger and Th. Braunbeck (2009) Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comparative Biochemistry and Physiology, Part C. 149: 196-209.
Lange, M., W. Gebauer, J. Markl and R. Nagel (1995) Comparison of testing acute toxicity on embryo of zebrafish, Brachydanio rerio and RTG-2 cytotoxicity as possible alternatives to the acute fish test. Chemosphere. 30:2087-2102.
Lee, H., K. Noh, S. Seok, M. Baek, H. Lee, D. Kim, Y. Na, S. Park, B. Kim, G. Park, J. Lee and J. Park (2008) Establishment of the neutral red uptake assay as alternatives to the Draize test and its validation. Journal of Alternatives to Animal Experiments 2:5-9.
Ni Shuilleabhain, S., C. Mothersill, D. Sheehan, N. M. O'Brien, J. O' Halloran, F. N. A. M. Van Pelt, M/ Davoren (2003) In vitro cytotoxicity testing of three zinc metal salts using established fish cell lines. Toxicology in Vitro. 18:365-376.
Organization of Economic Co-operation and Development (1992) Guideline for the testing of cheminals, Test No. 203; Fish acute toxicity test, Paris, France.
Paluch, E. and C. Heisenberg (2009) Biology and physics of cell shape changes in development. Current Biology. 19: 790-799.
Rasmussen, P. E., K. W. T. Goulding, J. R. Brown, P. R. Grace, H. H. Janzen and M. Korschens (1998) Long-term agroecosystem experiments: Assessing agricultural sustainability and global change. Science. 282:893-896.
Repetto, G., A. Del Peso and J. Zurita (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protocols. 3:1125-1131.
Rural Development Administration (2013) 13-1-1. Freshwater fish acute toxicity test. Public notice no. 2013-21, standards of registration for agricultural chemicals and pesticides. Rural Development Administration, Korea.
Saito, H., T. Koyasu and T. Shigeoka (1993a) Cytotoxicity of anilines and aldehydes to goldfish GFS cells and relationships with 1-octanol/water partition coefficients. Chemosphere. 27:1553-1560.
Saito, H., J. Koyasu, K. Yoshida, T. Shigeoka and S. Koike (1993b) Cytotoxicity of 109 chemicals to goldfish GSF cells and relationships with 1-octanol/water partition coefficients. Chemosphere. 26:1015-1028.
Segner, H. (2004) Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish. Alternatives to Laboratory Animals. 32:375-382.
Taju, G., S. Maheed, K. Nambi, V. Babu, S. Vimal, S. Kamatchiammal and A. Sahul (2012) Comparison of in vitro and in vivo acute toxicity assays in Etroplus suratensis (Bloch, 1790) and its three cell lines in relation to tannery effluent. Chemosphere. 87:55-61.
Van Dartel, D. A. M. and A. H. Piersma (2011) The embryonic stem cell test combined with toxicogenomics as an alternative testing model for the assessment of developmental toxicity. Reproductive Toxicology 32:235-244.
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