Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna Pot 111, 1000 Ljubljana, Slovenia.
Jozef Stefan International Postgraduate School, 1000 Ljubljana, Slovenia.
Int J Mol Sci. 2023 Feb 15;24(4):3894. doi: 10.3390/ijms24043894.
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.
在过去的 20 年中，已经引入了许多酪氨酸激酶抑制剂（TKI）用于各种类型恶性肿瘤的靶向治疗。由于频繁且不断增加的使用，导致最终随体液排泄，它们的残留已在医院和家庭废水中以及地表水发现。然而，TKI 残留对水生生物的环境影响描述甚少。在本研究中，我们使用体外斑马鱼肝脏细胞（ZFL）模型研究了五种选定的 TKI，即厄洛替尼（ERL）、达沙替尼（DAS）、尼洛替尼（NIL）、regorafenib（REG）和索拉非尼（SOR）的细胞毒性和遗传毒性。通过 MTS 测定和碘化丙啶（PI）死活染色通过流式细胞术测定细胞毒性。DAS、SOR 和 REG 剂量和时间依赖性地降低 ZFL 细胞活力，其中 DAS 是研究的最具细胞毒性的 TKI。在高达其最大溶解度的浓度下，ERL 和 NIL 不影响活力；然而，NIL 是唯一一种显著降低流式细胞术确定的 PI 阴性细胞比例的 TKI。细胞周期进展分析表明，DAS、ERL、REG 和 SOR 导致 ZFL 细胞在 G0/G1 期的细胞周期停滞，同时 S 期细胞分数减少。由于严重的 DNA 片段化，无法获得 NIL 的数据。使用彗星和胞质分裂阻断微核（CBMN）测定评估了所研究 TKI 的遗传毒性活性。NIL（≥2 μM）、DAS（≥0.006 μM）和 REG（≥0.8 μM）诱导 DNA 单链断裂的剂量依赖性诱导，其中 DAS 最有效。所研究的 TKI 均未诱导微核形成。这些结果表明，在与先前报道的人类癌细胞系相似的浓度范围内，正常非靶鱼肝脏细胞对所研究的 TKI 敏感。尽管在暴露的 ZFL 细胞中引起不良反应的 TKI 浓度比目前预期在水生环境中的浓度高几个数量级，但观察到的 DNA 损伤和细胞周期效应表明，环境中 TKI 的残留可能对生活在受 TKI 污染的环境中的非故意暴露的生物体构成危害。
