Abstract
Transgenic zebrafish models have been successfully used in biomonitoring and risk assessment studies of environmental pollutants, including xenoestrogens, pesticides, and heavy metals. We employed zebrafish larva (transgenic SR4G line) with a cortisol-inducible green fluorescence protein reporter (eGFP) as a model to detect stress responses upon exposure to compounds with environmental impact, including bisphenol A (BPA), vinclozolin (VIN), and fluoxetine (FLX). Cortisol, fluorescence signal, and mRNA levels of eGFP and 11 targeted genes were measured in a homogenized pool of zebrafish larvae, with six experimental replicates for each endpoint. Eleven targeted genes were selected according to their association with stress-axis and immediate early response class of genes. Hydrocortisone (CORT)and dexamethasone (DEX) were used as positive and negative controls, respectively. All measurements were done in two unstressed and stressed condition using standardized net handling as the stressor. A significant positive linear correlation between cortisol levels and eGFP mRNA levels was observed (r> 0.9). Based on eGFP mRNA levels in unstressed and stressed larvae two predictive models were trained (Random Forest and Logistic Regression). Both these models could correctly predict the blunted stress response upon exposure to BPA, VIN, FLX and the negative control, DEX. The negative predictive value (NPV) of these models were 100%. Similar NPV was observed when the predictive models trained based on the mRNA levels of the eleven assessed genes. Measurement of whole-body fluorescence intensity signal was not significant to detect blunted stress response. Our findings support the use of SR4G transgenic larvae as an in vivo biomonitoring model to screen chemicals for their stress-disrupting potentials. This is important because there is increasing evidence that brief exposures to environmental pollutants modify the stress response and critical coping behaviors for several generations.
Highlights
The eggs and embryos of aquatic vertebrates such as fish and frogs are directly exposed to the potential adverse effects of environmental contaminants because of their external fertilization and development [1]
Whole-body cortisol and enhanced version of green fluorescent protein (eGFP) mRNA levels were measured to determine the appropriate time for sample collection
The mRNA levels of selected genes in both unstressed and stressed conditions were imported to the model regardless of their level of significance. our findings showed that both predictive models could determine the altered stress response upon exposure to different chemicals (FLX, bisphenol A (BPA), VIN, DEX, and class of genes. Hydrocortisone (CORT)) (Supplementary Table 2)
Summary
The eggs and embryos of aquatic vertebrates such as fish and frogs are directly exposed to the potential adverse effects of environmental contaminants because of their external fertilization and development [1]. Amongst the most studied environmental contaminants endocrine disrupting compounds (EDCs), pharmaceuticals (PCs) and agricultural pesticides have shown to affect many aspects of embryonic development in aquatic vertebrates [4]. The EDCs with their potential to interfere with hormonal systems are of high risk, to a developing embryo [5–8]. These EDCs affect the survivability and normal development of an exposed embryo, but there is an increasing body of evidence that support long-lasting effects of such an exposure to the adulthood and even to the descendant generations [5, 7, 9, 10]. Reduced fertility, altered sexual behaviour, altered locomotion, and reduced stress and anxiety-like responses have been reported in adult teleost exposed to EDCs during early life stages [11, 12]
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