Artificial sweeteners are sugar substitutes that, due to their widespread use and environmental persistence, have been detected in rivers, lakes, groundwater, and sediments worldwide, raising concerns about their potential impacts on aquatic organisms. In this master's thesis, we investigated the effect of five artificial sweeteners (acesulfame K, aspartame, sodium cyclamate, saccharin, and sucralose) and the natural sweetener stevioside on the zebrafish (Danio rerio) liver cell line (ZFL). During a 72-hour exposure period, we tested a wide range of concentrations (0,01 µM, 0,1 µM, 1 µM, 10 µM, 100 µM, 1000 µM in 10000 µM), which also included concentrations in the range of environmentally relevant values. Using MTS and MTT assays, we determined the effect of the sweeteners on cell viability and evaluated the occurrence of oxidative stress immediately after sweetener exposure using the fluorescent probe H2DCFDA. Flow cytometry was used to monitor the expression of a mitotic activity marker (pH3) and a DNA damage marker (γH2AX). Cytotoxic effects were detected only for aspartame and sucralose at the highest tested concentration of 10,000 µM. Acesulfame K, sodium cyclamate, and saccharin significantly increased oxidative stress in ZFL cells at the highest tested concentrations (5,000 µM or 10,000 µM). None of the studied sweeteners induced changes in the expression of pH3 and γH2AX markers. Stevioside, as a natural sweetener, did not affect cell viability, oxidative activity, or the expression of the studied markers. The results indicate that the studied sweeteners at environmentally relevant concentrations do not cause measurable cytotoxic effects, increased oxidative stress, or changes in expression of γH2AX and pH3 markers in the ZFL model. Nevertheless, findings from numerous in vivo studies suggest that long-term exposure to artificial sweeteners can affect aquatic organisms, making their environmental impacts an important area for further research.
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