Oklahoma State University, Stillwater, OK 74078, USA.
Oklahoma State University, Stillwater, OK 74078, USA.
Comp Biochem Physiol C Toxicol Pharmacol. 2023 Aug;270:109638. doi: 10.1016/j.cbpc.2023.109638. Epub 2023 May 4.
RTgill-W1 cells cannot be directly exposed to freshwater (FW) or seawater (SW) due to osmotic stress. Adjustments of exposure solutions are needed, but these might reduce the bioavailability and toxicity of pollutants. To facilitate cell polarization and allow direct exposure of water samples, cells were cultured on transwell inserts. Monolayer formation was measured by trans-epithelial electrical resistance (TEER) and an apparent permeability (P) assay. At 14 days both TEER and P indicated the lowest permeability. Cell viability showed that cells can tolerate apical FW with complete medium (L-15/FBS) in the basolateral compartment but SW reduced cell viability. However, when reference toxicants, silver nitrate and sodium dodecyl benzene sulfonate, were added no toxicity was detected. Increased osmolality in the apical side and presence of proteins indicated diffusion from the basolateral to the apical side. Thus, reduced toxicity was likely caused by complexation with media salts and amino acids. A protein and amino acid free exposure medium (L-15/ex) was applied in the basolateral compartment. However, FW exposures with basolateral L-15/ex resulted in reduced cell viability. To reduce osmotic stress, mannitol was added to apical FW maintaining basolateral L-15/ex which improved cell viability and allowed detection of silver toxicity. Finally, RTgill-W1 cells did not show normal tight junction protein (ZO-1) immunocytochemical staining, which fits with the formation of a leaky epithelium. Overall, culturing of RTgill-W1 cells on transwell inserts allowed direct exposure to mannitol FW medium but showed a reduced sensitivity to toxicants. Thus, exposure on flat bottom wells is recommended for routine toxicity testing.
由于渗透压的影响，RTgill-W1 细胞不能直接暴露于淡水（FW）或海水（SW）中。需要调整暴露溶液，但这可能会降低污染物的生物利用度和毒性。为了促进细胞极化并允许直接暴露于水样，将细胞培养在 Transwell 插入物上。通过跨上皮电阻（TEER）和表观渗透率（P）测定来测量单层形成。在第 14 天，TEER 和 P 均表明渗透率最低。细胞活力表明，细胞可以在基底外侧腔中用完全培养基（L-15/FBS）耐受顶侧 FW，但 SW 降低了细胞活力。然而，当添加参考毒物硝酸银和十二烷基苯磺酸钠时，未检测到毒性。顶侧渗透压增加和蛋白质的存在表明蛋白质从基底外侧扩散到顶侧。因此，毒性降低可能是由于与培养基盐和氨基酸的络合。在基底外侧腔中应用了不含蛋白质和氨基酸的暴露培养基（L-15/ex）。然而，用基底外侧 L-15/ex 进行 FW 暴露会导致细胞活力降低。为了减轻渗透压的影响，在顶侧 FW 中添加甘露醇以维持基底外侧 L-15/ex，这改善了细胞活力并允许检测银的毒性。最后，RTgill-W1 细胞没有显示正常的紧密连接蛋白（ZO-1）免疫细胞化学染色，这与形成渗漏的上皮细胞相符。总体而言，将 RTgill-W1 细胞培养在 Transwell 插入物上可以直接暴露于甘露醇 FW 培养基，但对毒物的敏感性降低。因此，建议在平底孔中进行常规毒性测试。
