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Updated: Aug 9, 2025
Cytotoxicity Assays with Zebrafish Cell Lines
Published on: January 6, 2023
Adverse Toxic Effects of Tyrosine Kinase Inhibitors on Non-Target Zebrafish Liver (ZFL) Cells
Katja Kološa1, Bojana Žegura1,2, Martina Štampar1
1Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna Pot 111, 1000 Ljubljana, Slovenia.
Abstract:
Over the past 20 years, numerous tyrosine kinase inhibitors (TKIs) have been introduced for targeted therapy of various types of malignancies. Due to frequent and increasing use, leading to eventual excretion with body fluids, their residues have been found in hospital and household wastewaters as well as surface water. However, the effects of TKI residues in the environment on aquatic organisms are poorly described. In the present study, we investigated the cytotoxic and genotoxic effects of five selected TKIs, namely erlotinib (ERL), dasatinib (DAS), nilotinib (NIL), regorafenib (REG), and sorafenib (SOR), using the in vitro zebrafish liver cell (ZFL) model. Cytotoxicity was determined using the MTS assay and propidium iodide (PI) live/dead staining by flow cytometry. DAS, SOR, and REG decreased ZFL cell viability dose- and time-dependently, with DAS being the most cytotoxic TKI studied. ERL and NIL did not affect viability at concentrations up to their maximum solubility; however, NIL was the only TKI that significantly decreased the proportion of PI negative cells as determined by the flow cytometry. Cell cycle progression analyses showed that DAS, ERL, REG, and SOR caused the cell cycle arrest of ZFL cells in the G0/G1 phase, with a concomitant decrease of cells in the S-phase fraction. No data could be obtained for NIL due to severe DNA fragmentation. The genotoxic activity of the investigated TKIs was evaluated using comet and cytokinesis block micronucleus (CBMN) assays. The dose-dependent induction of DNA single strand breaks was induced by NIL (≥2 μM), DAS (≥0.006 μM), and REG (≥0.8 μM), with DAS being the most potent. None of the TKIs studied induced micronuclei formation. These results suggest that normal non-target fish liver cells are sensitive to the TKIs studied in a concentration range similar to those previously reported for human cancer cell lines. Although the TKI concentrations that induced adverse effects in exposed ZFL cells are several orders of magnitude higher than those currently expected in the aquatic environment, the observed DNA damage and cell cycle effects suggest that residues of TKIs in the environment may pose a hazard to non-intentionally exposed organisms living in environments contaminated with TKIs.
Insights
Environmental tyrosine kinase inhibitor (TKI) residues impact aquatic life. This study found TKIs cause cytotoxicity and DNA damage in zebrafish liver cells, highlighting potential ecological risks even at low environmental concentrations.
Area of Science:
- Environmental Science
- Toxicology
- Molecular Biology
Background:
- Tyrosine kinase inhibitors (TKIs) are widely used cancer drugs.
- TKI residues are increasingly detected in aquatic environments.
- The ecotoxicological effects of TKIs on aquatic organisms remain largely unknown.
Purpose of the Study:
- To investigate the cytotoxic and genotoxic effects of five TKIs (erlotinib, dasatinib, nilotinib, regorafenib, sorafenib) on zebrafish liver cells (ZFL).
- To assess the potential environmental hazard posed by TKI residues to non-target aquatic organisms.
Main Methods:
- Cytotoxicity assessed via MTS assay and propidium iodide staining.
- Cell cycle progression analyzed by flow cytometry.
- Genotoxicity evaluated using comet and cytokinesis-block micronucleus (CBMN) assays.
Main Results:
- Dasatinib, sorafenib, and regorafenib reduced ZFL cell viability dose- and time-dependently.
- Nilotinib induced DNA fragmentation, while dasatinib, erlotinib, regorafenib, and sorafenib caused G0/G1 cell cycle arrest.
- Dasatinib, nilotinib, and regorafenib induced DNA single-strand breaks, with dasatinib being most potent.
Conclusions:
- Zebrafish liver cells are sensitive to TKIs, exhibiting cytotoxicity and genotoxicity.
- Observed DNA damage and cell cycle effects indicate potential hazards to aquatic organisms from environmental TKI residues.
- Further research is needed to understand the long-term ecological impacts of TKIs in aquatic ecosystems.
