Establishing a qPCR method for analyzing biomarkers of compound-induced liver toxicity in Zebrafish larvae

Liver diseases represent a significant universal health challenge. Large groups of chemicals have already been screened in cell culture to see how they influence particular cellular features; however, many biological processes cannot be replicated in 2D cultured cells and subsequently need the 3D environment of cells to be studied. Moreover, the metabolism of substances in entire organisms might differ significantly from the procedures carried out in vitro. Therefore, it is preferable to screen the biological activity of small compounds in entire organisms. Zebrafish has emerged as a trustful model organism for observing different aspects of liver disease. The zebrafish liver has many similarities to the human liver including structure, function, and regenerative capacity. These animals have been acknowledged as a useful tool for developmental biologists and researchers in many other domains because of their translucent embryos and quick organogenesis compared to other laboratory animals. In this study, we have successfully induced liver damage in zebrafish using PFOS, an emergent hepatotoxic compound. We tested three different concentrations on whole Zebrafish larvae and then we performed gene expression analysis. In recent years, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has appeared as the classical technique to measure gene expression due to its accuracy, sensitivity, specificity, reproducibility, and robustness. However, to provide accurate data, normalization with stably expressed reference genes is mandatory. Thus, the first goal of our study was to identify a combination of reference genes for an optimal normalization of qPCR expression data in these experimental conditions. Then, the second objective was to optimize a qPCR protocol for the analysis of some biomarkers of xenobiotic-induced liver toxicity in Zebrafish larvae after exposure to PFOS. Primers for both internal control and target genes were designed on unique gene sequences of Zebrafish and were optimized for specificity and sensitivity. Gel electrophoresis was used to ensure the accuracy of the qPCR results by ruling out the possibility of non-specific amplification. The stability of different candidate internal control genes (i.e., ACTB1, ACTB2, EEF1A1, GAPDH, RP113A, TUBA1) and several liver toxicity target genes including CERULOPLASMIN, GC, FABP10, TFA, CYP3A65 were evaluated through a qualitative approach due to the lack of a sufficient number of samples.