Assessments of the effects of nicotine and ketamine using tyrosine hydroxylase-green fluorescent protein transgenic zebrafish as biosensors.

Transgenic zebrafish are a common vertebrate model system for the study of addictive behavior. In the present study, plasmid constructs containing green fluorescent protein (GFP) and the promoter of tyrosine hydroxylase (TH), a key synthetic enzyme for catecholamines, were produced. The TH-GFP constructs were microinjected into zebrafish embryonic cells. Three days post-fertilization, GFP began expressing in distinct catecholaminergic areas. The TH-GFP transgenic zebrafish were employed as live biosensors to test the effects of the commonly abused drugs nicotine and ketamine. First, locomotion assays were used to study the general excitatory effects of the drugs. Maximal locomotor activity was obtained after treatment with a high concentration of nicotine (10 µM), but with a much lower concentration of ketamine (0.1 µM). Second, TH protein levels in zebrafish brains were assessed by Western blot. TH protein levels were significantly increased, with maximal protein levels found after treatment with the same drug concentrations that gave maximal locomotor activity. Importantly, analysis of GFP in the zebrafish catecholaminergic areas revealed the same expression patterns as was obtained by Western blot. The present results indicate that increased locomotor activity can be correlated to TH protein expression, as indicated by Western blot and expression of TH-GFP. We have shown that TH-GFP expression is a reliable method to show the effects of drugs on TH expression that may be employed as a novel high-throughput live biosensor for screening drugs of abuse.

Detection of water toxicity using cytochrome P450 transgenic zebrafish as live biosensor: for polychlorinated biphenyls toxicity.

Cytochrome P450 (CYPs) is significant in degradation of endogenous substrates and detoxification of carcinogens, therefore it is a biomarker for assessment of polycyclic aromatic hydrocarbons (PAHs) level in aquatic environment. In the present study, a transgenic line of zebrafish had been generated using a CYP-green fluorescence protein (CYP-GFP) construct, driven by CYP1A1 promoter. Polychlorinated biphenyls (PCBs) were used as toxicant, in concentrations of 0.02 µg/ml, 0.04 µg/ml, 0.08 µg/ml, 0.4 µg/ml, and 0.8 µg/ml. The transgenic control fish showed low intensity of fluorescence in the liver. After exposed to PCBs, zebrafish had morphological changes such as expansion of yolk, contortion of tails and inflation of pericardial area. Green fluorescence signals were found to express according to concentrations and time. The green fluorescence signal was most intense after treatment with 0.08 µg/ml PCBs. However, the maximum area of green fluorescent signal was found at 0.04 µg/ml PCBs. GFP started to express at 3h exposure to PCBs, increasing its intensity until 6 h exposure, and then level off. Since the GFP expression is fast responding and is sensitive to low PAHs concentrations, transgenic fish is a good tool for live imaging and monitoring of aquatic contamination.