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1 醚菊酯对日本囊对虾肝胰腺细胞 DNA 损伤效应的研究 刘 波, 陈宇锋, 郑惠东, 许贻斌, 郑盛华, 钟硕良 (, ) 摘要 : 为检测中国近海海洋环境中菊酯类农药等污染物对日本囊对虾 (Marsupenaeus japonicas) 的基因毒性及其危害程度, 作者首次使用醚菊酯对日本囊对虾进行毒理实验, 将日本囊对虾分为 6 组进行不同剂量的处理, 采用彗星实验分析技术对处理培育 25 d 的日本囊对虾 DNA 损伤情况进行检测, 并通过 Comet Score 1.5 分析软件对实验图谱进行拖尾率 尾长 彗尾 DNA 相对含量 尾距 Olive 距等 DNA 损伤指标的统计分析, 在对各指标分别建立一元回归方程的基础上, 又通过 SPSS 软件进行判定分析, 筛选影响作用更大的检测指标, 建立了多参数的多元回归方程 实验结果发现, 与空白对照组相比, 日本囊对虾各实验处理组肝胰腺细胞 DNA 均有不同程度的损伤且各检测指标均有显著性差异 (P<0.01), 各检测指标与处理浓度表现出良好的剂量效应关系, 随着处理浓度递增, 各检测指标都呈规律性的增长趋势 回归分析和相关系数表明 : 各检测指标与处理浓度具有高度的相关性, 多项式回归方程 (P<0.05) 和多元回归方程 (P<0.005) 均有显著性的统计学意义, 通过多元回归方程可以有效地推断醚菊酯处理浓度和处理时间 关键词 : 日本囊对虾 (Marsupenaeus japonicas); 醚菊酯 ; DNA 损伤 ; 彗星实验中图分类号 : R994.6 文献标识码 : A 文章编号 : (2014) doi: /hykx (Single Cell Eletrophoresis, SCGE), Ostling [1] 1984, DNA DNA, SCGE, DNA, DNA [2-4], SCGE DNA [5-7],, SCGE (Pagrosomus major) DNA DNA [8-9],, 20 70, [10],,,, [11-12],,, (Marsupenaeus japonicas) DNA, DNA,, 1 材料与方法 1.1 试剂与仪器 ; DNA EDTA (0.5 mol/l) 10 (0.1 mol/l Tris, 10%, 10% Triton X-100) CELL BIOLABS ; NaCl NaOH Na 2 HPO 4 NaH 2 PO 4 : ; : : ( ) : (1980-),,,,,. : , liubo8090@aliyun.com Marine Sciences / Vol. 38, No. 11 /

2 , Cell Biolabs, SW-CJ-1D, DK-S26, Thermo EC250-90, JY-SP-E, Leica DM5500 B 1.2 实验动物, (11.36±0.93)cm, (10.72±2.70)g,, 1.3 处理方法, (P<0.01), - -, h (LC 50 ) mg/l LC 50 (Tumbel) (SC=48 h LC /(24h LC 50 48h LC 50 ) 2 ),,, 6, 1, 5 ( , , , , mg/l), 30 1 m 2, (5.62±0.18)mg/L ph(8.06±0.08) (18.36±0.26) (30.00±0.02),, 25 d 3, 1.4 细胞悬液制备 0.2~0.5 g 1.5 ml, 3, mol/l PBS ( 0.02 mol/l EDTA)1 ml,, 5 min, 3000 r/min 2 min, mol/l PBS, /ml, 1.5 彗星实验方法 Cell Biolabs [13] ~95 20 min, min , 75μL, 4 15 min (2.5 mol/l NaCl, 0.1 mol/l EDTA, 0.01 mol/l Tris, 1%, 1% Triton X-100, ph10.0), 4 30~ 60 min, (0.3 mol/l NaOH, 1 m mol/l EDTA, ph>13), 4 30 min 1.5.3, (0.3 mol/l NaOH, 1 mmol/l EDTA, ph>13), 1 V/cm, 300 ma 15~30 min, 2 min,, 70% 5 min, 100 μl DNA, 15 min 1.6 图像分析和数据处理,, 60, Comet Score 1.5, DNA Olive DNA = / 100% DNA = DNA / DNA 100% = DNA 100% Olive = DNA 100% Excel SPSS16.0, 2 结果 2.1 醚菊酯处理对日本囊对虾肝胰腺细胞 DNA 的影响 24h 48h 30 / 2014 / 38 / 11

3 (LC 50 ) mg/l mg/l, LC 50 (Tumbel) (SC=48 h LC /(24h LC 50 48h LC 50 ) 2 ) mg/l,, 180, DNA,, ; DNA DNA,, DNA,,, 1 DNA 1 DNA,, DNA Olive (P<0.01),, 5.06%±0.87%, DNA Olive 0, DNA ; ~ mg/l 25 d, mg/l,, 1 DNA Fig.1 The comet assay images of DNA damage in hepatopancreas cells of Marsupenaeus japonicas exposed to different concentrations of ethofenprox 1-1. ; mg/l ; mg/l ; mg/l ; mg/l ; mg/l 1-1. control group; mg/l exposure concentration; mg/l exposure concentration; mg/l exposure concentration; mg/l exposure concentration; mg/l exposure concentration 表 1 醚菊酯不同处理浓度对日本囊对虾肝胰腺细胞 DNA 损伤的彗星实验指标数据 Tab.1 The parameters of DNA damage in hepatopancreas cells of Marsupenaeus japonicas exposed to different concentrations of ethofenprox using comet assay method (mg/l) (%) (μm) DNA (%) (% μm) Olive (% μm) ± ±0.76 <0.001 <0.001 < ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.28 :. (P<0.01); mg/l (P<0.01) Marine Sciences / Vol. 38, No. 11 /

4 2.2 日本囊对虾肝胰腺细胞 DNA 损伤各项实验指标与醚菊酯处理浓度的回归分析 1,,, Comet Score 1.5, (R 2 >0.99), 2 Olive DNA Y= X X X X 4 (P<0.005), Y, X 1, X 2 Olive, X 3 DNA, X 4, 表 2 醚菊酯不同处理浓度对日本囊对虾肝胰腺细胞 DNA 损伤各实验指标的回归方程 Tab.2 The regression equation of parameters of DNA damage in hepatopancreas cells of Marsupenaeus japonicas exposed to different concentrations of ethofenprox P y = x x x <0.05 y = x x x <0.05 DNA y = x x x <0.05 y = x x x <0.05 Olive y = 4.194x x x <0.05 : y, x 3 讨论与结论,, [14],,,, DNA [15-17] DNA Olive, Comet Score 1.5, [18],, DNA,, DNA, DNA,,, DNA, Hellman [19] :, DNA Olive, DNA, mg/l, (P<0.01), mg/l, mg/l( Tumbel ), DNA, (P<0.01),,,,,, (P<0.05),, Olive DNA Olive DNA, Olive, Olive (P<0.005),,, Olive,, SPSS 32 / 2014 / 38 / 11

5 ,, DNA,, DNA, [20],,,,,, DNA, DNA, DNA, [21],, DNA,, : [1] Ostling O, Johanson K J. Microelectrophoretic study of radiation induced DNA damages in individual mammalian cells[j]. Biochemical and Biophysical Research Communications, 1984, 123(1): [2] Olive P L, Banath J P, Durand R E. Heterogeneity in radiation induced DNA damage and repair in tumor and normal cells using the comet assay[j]. Radiation Research, 1990, 122(1): [3] Singh N P, McCoy M T, Tice R R, et al. A simple technique for quantification of low levels of DNA damage in individual cells[j]. Experimental Cell Research, 1988, 175(1): [4] Lee R F, Steinert S. Use of the single cell gel electrophoresis comet assay for detecting DNA damage in aquatic (marine and freshwater) animals [J]. Mutation Research, 2003, 544(1): [5],,,. DNA [J]., 2004, 23(1): [6],,,. [J]., 2007, 22(3): [7],,,. [J]., 1999, 25(5): [8],,,. [a] (Pagrosomus major) DNA [J]., 2006, 25(6): [9],,,. DNA [J]., 2006, 36( ): [10],. DNA [J]., 2008, 25(8): [11],,,. γ- [J]., 1999, 19(6): [12],,,. [J]., 2001, 19(4): [13] De Boeck M, Touil N, De Visscher G, et al. Validation and implementation of an internal standard in comet assay analysis[j]. Mutation Research, 2000, 469(2): [14]. [J]., 1989, 28(4): 37. [15] Mitchelmore C L, Chipman J K. DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring [J]. Mutation Research, 1998, 399(2): [16] Mukhopadhyay I, Chowdhuri D K, Bajpayee M, et al. Evaluation of in vivo genotoxicity of cypermethrin in Drosophila melanogaster using the alkaline comet assay [J]. Mutagenesis, 2004, 19(2): [17] Chauhan L K, Agarwal D K, Sundararaman V. In vivo induction of sister chromatid exchange in mouse bone marrow following oral exposure to commercial formulations of alpha-cyano pyrethroids [J]. Toxicology Letters, 1997, 93(3): Marine Sciences / Vol. 38, No. 11 /

6 [18],,,. DNA [J]., 2010, 19(6): [19] Hellman B, Vaghef H, Bostrom B. The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay [J]. Mutation Research, 1995, 336(2): [20],,,. DNA [J]., 2007, 26(4): [21],. [M]. :, The research on DNA damage in hepatopancreas cells of Marsupenaeus japonicas exposed to ethofenprox LIU Bo, CHEN Yu-feng, ZHENG Hui-dong, XU Yi-bin, ZHENG Sheng-hua, ZHONG Shuo-liang (Fisheries Research Institute of Fujian, Xiamen , China) Received: Nov., 22, 2013 Key words: Marsupenaeus japonicas; ethofenprox; DNA damage; comet assay Abstract: In order to analyze the genotoxicity of Marsupenaeus japonicas caused by the pollutant of pyrethroid pesticides in the coastal sea of China, here, we applied ethofenprox in toxicological experiment on M. japonicas for the first time. In our study, M. japonicas were divided into six groups and exposed to different concentrations of ethofenprox for 25 days. DNA damage in hepatopancreas cells of M. japonicas was evaluated with comet assay. The DNA damage parameters such as the comet rate, the tail length, the percentage of tail DNA, the tail moment and the olive moment were statistically analyzed by Comet Score 1.5. On the basis of one-dimensional regression equation, we selected greater influenced parameters after comparing with SPSS 16.0 and constructed the multiple- dimensional regression equation. The results show that DNA damage in hepatopancreas cells was observed in all exposed groups and the parameters of each exposed group were significantly different from the control group (P<0.01). There was a dose-effect relationship between ethofenprox and DNA damage in hepatopancreas cells, which increased regularly with the exposure concentrations. The regression analysis and coefficient indicated that all DNA damage parameters were highly relevant to the exposed concentrations. The one-dimensional regression equation (P<0.05) and multiple-dimensional regression equation (P<0.005) had significant statistic meaning, which might be an effective way to estimate the exposed concentration and time of ethofenprox. ( 本文编辑 : 谭雪静 ) 34 / 2014 / 38 / 11