Fluorescent transgenic zebrafish as a biosensor for growth-related effects of methyl parathion.

Transgenic fish models are potential alternative subjects in toxicological studies, since they can provide in vivo information on the deleterious effects of different substances. Here, we used a transgenic zebrafish (Danio rerio) lineage, which expresses a destabilized fluorescent protein (DsRED) driven by the myosin light chain promoter (Mylz2), in order to propose a new research tool for environmental biomonitoring. For validating the MYO-RED lineage, we exposed fish to the organophosphorated pesticide methyl parathion (MP). The effect of MP on fish growth was assessed by evaluating weight, length, condition factor and muscle fiber diameter. All factors suffered reduction at both tested concentrations (0.13µM and 13µM of MP). Similarly, fluorescence intensity decreased in a concentration-dependent manner, suggesting muscle protein catabolism. However, DsRED gene expression lowered only at the higher MP concentration. Results indicate that the MYO-RED transgenic zebrafish is an interesting model for detecting the growth-related effects of pollutants. Destabilized proteins such as reporter genes are apparently sensitive biomarkers, since effects were observed even at the lower, environmentally acceptable concentration. Therefore, this transgenic fish is a promising candidate model for sensitive, fast, and easy environmental monitoring.

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio).

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na(+), K(+)-ATPase, H(+)-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na(+), K(+)-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na(+). Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na(+) import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.

The effect of GH overexpression on GHR and IGF-I gene regulation in different genotypes of GH-transgenic zebrafish.

Most biological actions of growth hormone (GH) are mediated by the insulin-like growth factor I (IGF-I) that is produced after the interaction of the hormone with a specific cell surface receptor, the GH receptor (GHR). Even though the GH excess on fish metabolism is poorly known, several species have been genetically engineered for this hormone in order to improve growth for aquaculture. In some GH-transgenic fish growth has been dramatically increased, while in others high levels of transgene expression have shown inhibition of the growth response. In this study, we used for the first time different genotypes (hemizygous and homozygous) of a GH-transgenic zebrafish (Danio rerio) lineage as a model for studying the GH resistance induced by different GH transgene expression levels. The results obtained here demonstrated that homozygous fish did not grow as expected and have a lower condition factor, which implies a catabolic state. These findings are explained by a decreased IGF-I and GHR gene expression as a consequence of GH resistance. Together, our results demonstrated that homozygous GH-transgenic fish showed similar characteristics to the starvation-induced fish and could be an interesting model for studying the regulation of the GH/GHR/IGF-I axis in fish.