Evaluation of a CRISPR/Cas9-engineered Zebrafish GSTP1 Reporter Cell Line for In Vitro Toxicity Assessment

Sammanfattning

Environmental micropollutants are present in aquatic environments at low concentrations and often occur as complex chemical mixtures, making their effects difficult to detect. There is also an increasing need for new alternative methods that reduce the use of animals in toxicity testing, while still providing biologically reliable results. Oxidative stress is a common cellular response to micropollutants in aquatic organisms and represents a suitable mechanism for toxicity assessment. The aim of this study was to evaluate a CRISPR/Cas9-engineered zebrafish GSTP1 reporter cell line as an in vitro model for detecting oxidative stress responses mediated via the Nrf2/ARE pathway. The ZF4-gstp1 zebrafish cell line was exposed to two known inducers of the Nrf2/ARE pathway, sulforaphane (SFN) and tert-Butylhydroquinone (tBHQ), for 6 h and 24 h. Reporter activation was assessed using Firefly luciferase normalised to the constitutively expressed Renilla luciferase, and cytotoxicity was evaluated using an AB/CFDA assay. The results showed that SFN induces GSTP1 reporter activity at low, non-cytotoxic concentrations after both exposure durations, indicating pathway-specific activation. In contrast, tBHQ did not induce GSTP1 reporter activity at any tested concentrations or exposure durations. Cytotoxicity increased at higher concentrations for both compounds; however, SFN-induced reporter activation occurred independently of cytotoxic effects. These results indicate that the GSTP1 reporter cell line can detect pathway-specific oxidative stress responses at low, non-cytotoxic concentrations and show potential as an in vitro approach that can contribute to the development of alternative toxicity testing methods.