Response of epithelial cell lines from the rainbow trout gut and gill to ammonia

Rainbow trout epithelial cell lines from the gill, RTgill-W1, and gut, RTgutGC, were exposed to NH₄Cl at 18-21 °C in L15 (basal medium) with fetal bovine serum and were found to undergo cytoplasmic vacuolization and cell death, depending on NH₄Cl concentration and exposure time. Vacuolization arose within 24 h of cultures being exposed to 10-100 mM NH₄Cl, and vacuoles disappeared over 24 h after NH₄Cl-exposed cultures were returned to just L15/FBS. RTgill-W1 appeared more sensitive to vacuolization, with one indicator being the maximal proportion of vacuolated cells in a culture, which approached 100% in 50 mM NH₄Cl for 72 h. RTgill-W1 also were more sensitive to NH₄Cl-induced cell killing. For 7-d exposures, the inhibitory concentrations (IC50s) for the 50% loss of cell viability as evaluated with Alamar Blue were 30 mM NH₄Cl for RTgill-W1 and 80 mM for RTgutGC. In a wound-healing assay, RTgutGC cells in 0.1 and 1 mM NH₄Cl were able to migrate and cover a 500-μm gap in 5 d, like the control, but in 50 mM NH₄Cl healing was blocked. In 10 mM NH₄Cl, repair was slowed but by 14 d the gap was covered with cells and many of these were vacuolated. Overall, the results provide a foundation for using these two cell lines to study the physiology and toxicology of ammonia in fish.