An in Vitro Fluorescence Assay for the Detection of Drug-Induced Cytoplasmic Lamellar Bodies

Abstract

An in vitro fluorescence approach to assessing lamellar body formation was investigated. Human foreskin fibroblasts (HFFs) were incubated with a fluorescent phosphatidylcholine analog, 1-acyl-2-(12[(7-nitro-2–1–3-benzoxadiazol-4-yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), in the presence or absence of gentamicin, trospectomycin, or amiodarone (known to induce phospho-lipidosis in vivo), or amikacin (which does not induce phospholipidosis). Observations were made by fluorescence microscopy, spectrofluorometry, and electron microscopy. When control cells were labeled with NBD-PC at 2°C and then warmed to 37°C, fluorescence was observed diffusely throughout the cell and within occasional punctate inclusions. Incubation for 24 h in the presence or absence of amikacin gave similar results. Incubation with gentamicin routed the fluorescent label to numerous distinct cytoplasmic inclusions within 1.5 h. Similar results were obtained in 24 h incubations with gentamicin, trospectomycin, or amiodarone. The inclusions were interpreted to be lamellar bodies based on transmission electron microscopy. The lamellar bodies were not quickly removed, as fluorescence could still be observed after a 24 h incubation in the absence of drug or additional fluorescent label. Spectrofluorometry did not indicate an unequivocal increase in total fluorescence, indicating fluorescence microscopy to be the more sensitive means of evaluation. Our results suggest that drug-induced phospholipid accumulation may result from altered membrane traffic rather than altered phospholipid catabolism. The methods described here have potential application in drug development and biological mechanism studies.