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Applications of the SR4G Transgenic Zebrafish Line for Biomonitoring of Stress-Disrupting Compounds: A Proof-of-Concept Study.

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.

SUBMITTER: Nozari A

PROVIDER: S-EPMC8635770 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The neutrophil enzyme myeloperoxidase (MPO) is a major enzyme made by neutrophils to generate antimicrobial and immunomodulatory compounds, notably hypochlorous acid (HOCl), amplifying their capacity for destroying pathogens and regulating inflammation. Despite its roles in innate immunity, the importance of MPO in preventing infection is unclear, as individuals with MPO deficiency are asymptomatic with the exception of an increased risk of candidiasis. Dysregulation of MPO activity is also linked with inflammatory conditions such as atherosclerosis, emphasising a need to understand the roles of the enzyme in greater detail. Consequently, new tools for investigating granular dynamics in vivo can provide useful insights into how MPO localises within neutrophils, aiding understanding of its role in preventing and exacerbating disease. The zebrafish is a powerful model for investigating the immune system in vivo, as it is genetically tractable, and optically transparent. To visualise MPO activity within zebrafish neutrophils, we created a genetic construct that expresses human MPO as a fusion protein with a C-terminal fluorescent tag, driven by the neutrophil-specific promoter lyz. After introducing the construct into the zebrafish genome by Tol2 transgenesis, we established the Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496 line, and confirmed transgene expression in zebrafish neutrophils. We observed localisation of MPO-mEmerald within a subcellular location resembling neutrophil granules, mirroring MPO in human neutrophils. In Spotless (mpxNL144) larvae-which express a non-functional zebrafish myeloperoxidase-the MPO-mEmerald transgene does not disrupt neutrophil migration to sites of infection or inflammation, suggesting that it is a suitable line for the study of neutrophil granule function. We present a new transgenic line that can be used to investigate neutrophil granule dynamics in vivo without disrupting neutrophil behaviour, with potential applications in studying processing and maturation of MPO during development.

Dataset Information

Applications of the SR4G Transgenic Zebrafish Line for Biomonitoring of Stress-Disrupting Compounds: A Proof-of-Concept Study.

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