The Korean Journal of Pesticide Science (농약과학회지)

The Korean Society of Pesticide Science (한국농약과학회)
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Application of Neutral Red Uptake Assay Using EPC Cells as an Alternative to the Fish Acute Toxicity Test for Pesticide

어류급성독성시험 대체법으로서 잉어표피세포를 이용한 Neutral Red Uptake 분석법 적용

  • Seo, Ji-Hyun (Future Environmental Research Center, Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology) ;
  • Park, June-Woo (Future Environmental Research Center, Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology) ;
  • Lee, Sung-Kyu (Future Environmental Research Center, Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology) ;
  • Kim, Woo-Keun (Future Environmental Research Center, Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology)
  • 서지현 (안전성평가연구소 경남환경독성본부 미래환경연구센터) ;
  • 박준우 (안전성평가연구소 경남환경독성본부 미래환경연구센터) ;
  • 이성규 (안전성평가연구소 경남환경독성본부 미래환경연구센터) ;
  • 김우근 (안전성평가연구소 경남환경독성본부 미래환경연구센터)
  • Received : 2013.11.07
  • Accepted : 2014.01.16
  • Published : 2014.03.31
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Abstract

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.

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.

Keywords

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