Concern about genetic alterations in fish populations arising from anthropogenic activities has led to the adaptation and/or development of new tests and techniques that shed light on these alterations. The high number and the reduced size of chromosomes and the long cell cycle associated with most fish species preclude the use of most accepted genotoxicity assays. The purpose of this work was to study the capability of the randomly amplified polymorphic DNA technique to show genotoxic effects induced by chemicals in fish cells. To do that we studied the effect of 0.5 microg/ml mitomycin C (MMC) on an established rainbow trout cell line (RTG-2). To increase the sensitivity of detecting altered copies of DNA and to avoid the presence of false positives and a lack of reproducibility, the amounts of DNA template and primer present in amplification reactions were studied and optimized after comparison between the control and exposed fingerprints for 4, 6 and 8 h. Results show that 5 ng of DNA template and 4 pM chosen primer were optimum to show differences between control and exposed cells and to obtain reproducible results. The results obtained, after optimum conditions were established, show that this system could be useful for the assessment of DNA alterations in in vitro genotoxicity studies.
The technique of random amplified polymorphic DNA (RAPD) permits the study of genetic ecotoxicology without having to face the problem posed by the high number of chromosomes generally presented by the fish species used in traditional methods. Additionally, the use of in vitro systems allows the study of a large number of samples in environmental-risk assessment. Good standardization of the parameters involved in the RAPD reaction, such as primer concentration and the DNA template used and its integrity, is fundamental for obtaining reliable and repeatable results. This is especially important when the differences in DNA fingerprints between control cells and cells exposed to genotoxic agents are interpreted as toxic-dependent alterations. The use of more than one primer increases sensitivity in the detection of such differences, provided the amplification is carried out under optimum conditions. This article demonstrates that the conditions established for certain primers in previous studies can be acceptable for others, independent of the complexity of band patterns generated. Furthermore, the integrity of the DNA is shown to be stable for several months in the genomic extracts stored at 4 degrees C, which to a large extent facilitates the application of this methodology.
The detection of genotoxic effects using in vitro cell systems can be extremely useful in risk assessment procedures. However, care should be taken in the extrapolation of in vitro results since, amongst other factors, established cell lines may deviate from the genetic characteristics of their species. In this work, the genetic similarities between the RTG-2 cell line and rainbow trout individuals (Oncorhynchus mykiss) from several fish farms have been studied by the RAPD technique. Results show a significant analogy in the band patterns obtained for both systems, up to 73% of the bands composing the fingerprint of the RTG-2 cell line were found in all the individuals analysed. The inter-population similarity index (Lynch, 1990), considering the RTG-2 cell line as a population, gives a value of 0.931 between both systems. The dendrogram constructed from all the individuals, considering the RTG-2 cell line as just another individual of a single population, showed that the genetic structure of the cell line was not different from those of the other individuals tested. The strong genetic similarity of both systems, together with the previously proven capability of the RAPD technique to detect genetic alterations caused in vitro by genotoxic agents, can be very useful in genetic ecotoxicological studies.
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