Detection of mitomycin C-induced genetic damage in fish cells by use of RAPD.
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.
Becerril
C
C
Toxicology Department CNA, I.S. Carlos III, E-28220 Majadahonda, Madrid and Division of Environmental Toxicology, CISA-INIA, E-28130, Madrid, Spain. cbeceril@iscii.es
Ferrero
M
M
Sanz
F
F
Castaño
A
A
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000187:drug effects* / Q000235:genetics; D004249:DNA Damage; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005470:Fluorometry; D016685:Mitomycin / Q000633:toxicity*; D009152:Mutagenicity Tests; D017686:Oncorhynchus mykiss; D019105:Random Amplified Polymorphic DNA Technique
D000903:Antibiotics, Antineoplastic; D017931:DNA Primers; D016685:Mitomycin; D004247:DNA