Development and validation of the in vivo alkaline comet assay for detecting genomic damage in marine flatfish

被引:106
作者
Belpaeme, K
Cooreman, K
Kirsch-Volders, M
机构
[1] Free Univ Brussels, Lab Cellular Genet, B-1050 Brussels, Belgium
[2] Sea Fisheries Dept, Agr Res Ctr, B-8400 Oostende, Belgium
关键词
flatfish; comet assay; micronucleus test; mutagenicity;
D O I
10.1016/S1383-5718(98)00062-X
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Biomonitoring is an important subject within environmental sciences. Biomonitoring tests are required to be quick, relatively inexpensive, accurate, and reproducible. No genetic test currently fulfils all of these requirements. The chromosome aberration and sister chromatid exchange tests are very time consuming, the DNA adduct technique is rather expensive, and the micronucleus test has not inconclusively proven its use as a reliable monitoring tool. This work is focused on the validation of the comet assay as a candidate for monitoring marine ecosystems. For the comet assay, this work deals with the effectiveness of tissue dissociation, storage of cells in lysing buffer and in liquid nitrogen, different electrophoretic conditions, neutralisation and fixation of slides, interindividual variation between samples, and responsiveness of four tissue types to ethyl methanesulphonate (EMS). The main conclusions are: (i) dissociation of solid tissues in a phosphate buffer supplemented with 200 mM N-t-butyl-alpha-phenylnitrone provides cells with an acceptable background DNA damage; (ii) freezing of cells or tissues in liquid nitrogen generally leads to an increase in DNA breakage, especially for liver, gill and kidney tissue; (iii) storage of slides in the lysing solution for up to one week gives minor changes in comet tails; (iv) differences in protocols for neutralisation and fixation may influence the results; (v) high intra- and interindividual variations in comets (length and DNA content) may obscure the interpretation of comet results; (vi) blood, gill, liver and kidney all showed a statistically significant increase of DNA damage after exposure to 50 mg EMS/l; (vii) electrophoresis at low voltage for longer periods is to be preferred to high voltage and short electrophoresis times. The simplicity and sensitivity of the comet assay make it an adequate test system for biomonitoring of chronic low level exposure. However, protocols and experimental conditions have to be chosen carefully. (C) 1998 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:167 / 184
页数:18
相关论文
共 24 条
[1]   FISH MICRONUCLEI FOR ASSESSING GENOTOXICITY IN WATER [J].
ALSABTI, K ;
METCALFE, CD .
MUTATION RESEARCH-GENETIC TOXICOLOGY, 1995, 343 (2-3) :121-135
[2]   Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay [J].
Belpaeme, K ;
Delbeke, K ;
Zhu, L ;
KirschVolders, M .
MUTAGENESIS, 1996, 11 (05) :485-492
[3]   The DNA 'comet assay' as a rapid screening technique to control irradiated food [J].
Cerda, H ;
Delincee, H ;
Haine, H ;
Rupp, H .
MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS, 1997, 375 (02) :167-181
[4]   The comet assay: What can it really tell us? [J].
Collins, AR ;
Dobson, VL ;
Dusinska, M ;
Kennedy, G ;
Stetina, R .
MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS, 1997, 375 (02) :183-193
[5]  
DEBOECK M, UNPUB CARCINOGENESIS
[6]  
DEVAUX M, 1997, IN VITRO, V11, P71
[7]   Detection of genotoxic effects on cells of liver and gills of B-rerio by means of single cell gel electrophoresis [J].
Deventer, K .
BULLETIN OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY, 1996, 56 (06) :911-918
[8]   The advantages and disadvantages of the cytokinesis-block micronucleus method [J].
Fenech, M .
MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS, 1997, 392 (1-2) :11-18
[9]  
*ICES, 1997, IN PRESS REP ICES AD
[10]  
*ICES, 1996, ICES CM 1996