Detection of phospholipidosis induction: a cell-based assay in high-throughput and high-content format

Abstract

Drug-induced phospholipidosis is characterized by the accumulation of intracellular phospholipids in cells exposed to cationic amphiphilic drugs. The appearance of unicentric or multicentric multilamellar bodies viewed under an electron microscope (EM) is the morphological hallmark of phospholipidosis. Although the EM method is the gold standard for detecting cellular phospholipidosis, this method has its drawbacks, including low throughput, high cost, and unsuitability for screening a large chemical library. In this study, a cell-based phospholipidosis assay has been developed using the LipidTOX Red reagent in HepG2 cells and miniaturized into a 1536-well plate format. To validate this assay for high-throughput screening (HTS), the LOPAC library of 1280 compounds was screened using a quantitative HTS platform. A group of known phospholipidosis inducers, such as amiodarone, propranolol, chlorpromazine, desipramine, promazine, clomipramine, and amitriptyline, was identified by the screen, consistent with previous reports. Several novel phospholipidosis inducers, including NAN-190, ebastine, GR127935, and cis-(Z)-flupentixol, were identified in this study and confirmed using the EM method. These results demonstrate that this assay can be used to evaluate and profile large numbers of chemicals for drug-induced phospholipidosis.

Keywords: LipidTOX; phospholipidosis; qHTS.

Figure 1

Effect of amiodarone on PLD induction in HepG2 cells. (A) Concentration response curve of amiodarone after 24 h treatment. Each value represents the mean ± SD of eight independent experiments. (B) Representative images of intracellular staining with LipidTOX red dye and nuclear staining with Hoechst dye in the absence or in the presence of amiodarone. Images acquired in ImageXpress microsystem using a 20× objective. Nuclei were stained blue and PLD were stained red.

Figure 1

Effect of amiodarone on PLD induction in HepG2 cells. (A) Concentration response curve of amiodarone after 24 h treatment. Each value represents the mean ± SD of eight independent experiments. (B) Representative images of intracellular staining with LipidTOX red dye and nuclear staining with Hoechst dye in the absence or in the presence of amiodarone. Images acquired in ImageXpress microsystem using a 20× objective. Nuclei were stained blue and PLD were stained red.

Figure 2

Induction of intracellular phospholipidosis (PLD) accumulation labeled with LipidTOX dye in HepG2 cells after 24 h of compound treatment. Representative images of fixed cells stained with LipidTOX (red color) and Hoechst (blue color) are from 24 compounds with the indicated concentration around EC₅₀ values; butaclamol, 4.8 µM; propranalol, 19.16 µM; 5-(N,N-hexamethylene) amiloride, 19.16 µM; amiodarone, 4.8 µM; BIX 01294, 2.39 µM; BW 723C86, 19.16 µM; CGS-12066A, 9.58 µM; cis-(Z)-flupenthixol, 4.79 µM; ebastine, 0.3 µM; eliprodil, 9.58 µM; ellipticine, 9.58 µM; GR 127935, 1.2 µM; GW405833,19.16 µM; ifenprodil, 9.58 µM; L765,314, 4.8 µM; metergoline, 4.79 µM; methiothepin, 4.79 µM; mibefradil, 9.58 µM; NAN-190, 2.4 µM; NNC 55-0396, 9.58 µM; octoclothepin, 9.58 µM; ouabain, 2.39 µM; spiperone, 2.4 µM; vinblastine, 19.16 µM.

Figure 3

Transmission electron micrographs of PLD induction in HepG2 cells treated with drugs for 24 h. (A) amiodarone (6 µM), (B) NAN-190 (3 µM), (C) ebastine (1 µM), (D) GR127935 (3 µM), (E) cis-(Z)-flupenthixol (6 µM), and (F) DMSO only. Insets from drug treatment show large multicentric myeloid bodies in the cytoplasm. Bar = 2 µm for main panel and bar = 500 nm for inset.

Figure 4

Phospholipidosis induction in HepG2 cells using LipidTOX dye. After treatment with (A) amiodarone (6 µM), (B) NAN-190 (3 µM), (C) ebastine (1 µM), (D) GR127935 (3 µM), (E) cis-(Z)-flupenthixol (6 µM), and (F) DMSO only for 24 h, the cells were fixed and stained with LipidTOX and Hoechst. Images were acquired in ImageXpress microsystem using a 20× objective.