RESUMO
The monitoring of the impact of chemical pollutants upon marine ecosystems commonly employs a multi-biomarker approach. Functional genomics, using cDNA microarrays, allows for a comprehensive view of how an organism is responding to an exposure, with respect to changes in gene expression. Differentially expressed mRNAs were first isolated from livers of European flounder by means of suppressive, subtractive hybridisation. A clone set containing a total of 284 different potentially differentially expressed mRNAs was produced, of which 84 were tentatively identified. These were combined with previously cloned known stress genes isolated by degenerate PCR to produce a custom 500-clone microarray platform with each clone arrayed to four spots. Subsequent array experiments using cadmium-treated flounder detected up-regulation of 27 transcripts, including Cu/Zn superoxide dismutase, thioredoxin, a peroxiredoxin and a glutathione-S-transferase, reflecting oxidative stress in exposed flounder, while CYP1A expression was down-regulated. These changes were confirmed by real-time PCR. The array experiment highlighted a number of candidate genes for further analysis as potential novel biomarkers of cadmium exposure and demonstrated the applicability of the custom microarray approach in the study of the effects of toxicants.
Assuntos
Cádmio/toxicidade , Linguado/genética , Análise de Sequência com Séries de Oligonucleotídeos , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/química , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Inglaterra , Monitoramento Ambiental , Linguado/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , RNA Mensageiro/análise , Água do MarRESUMO
Commercially farmed European flounder (Platichthys flesus) were treated with benzo[a]pyrene or cadmium under controlled laboratory conditions. In addition, animals were collected from the polluted Tyne and relatively clean Alde estuaries in the UK. Potential differentially expressed genes were isolated from liver tissue by means of suppressive, subtractive hybridisation. A total of 284 different mRNAs were identified as having the potential for being differentially expressed, of which 84 could be tentatively identified by homology with database sequences. These clones will form part of a flounder cDNA array for validation and future applications, with full identification being pursued for those expressed sequence tags for which differential expression can be confirmed by array technology.
