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Validation of an in vitro screen for phospholipidosis using a high-content biology platform

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Abstract

Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to $100 μmol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, nonphospholipidogenic controls were correctly called.

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Abbreviations

CADs:

cationic amphiphilic drugs

DMSO:

dimethyl sulfoxide

EthD-2e:

ethidium homodimer-2 dead cell stain

HCB:

high-content biology

NBD-PE:

N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt

PLD:

phospholipidosis

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Authors and Affiliations

  1. Safety Assessment US, USA

    J. K. Morelli, M. Buehrle, F. Pognan, L. R. Barone & P. J. Ciaccio

  2. Neuroscience, AstraZeneca Pharmaceuticals, Wilmington, Delaware, USA

    W. Fieles

  3. Safety Assessment US, Astrazeneca Pharmaceuticals, 1800 Concord Pike, CRDL 149, P.O. Box 15437, Wilmington, DE, 19850, USA

    J. K. Morelli

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  1. J. K. Morelli
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  2. M. Buehrle
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  3. F. Pognan
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  4. L. R. Barone
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  5. W. Fieles
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  6. P. J. Ciaccio
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Correspondence to J. K. Morelli.

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Morelli, J.K., Buehrle, M., Pognan, F. et al. Validation of an in vitro screen for phospholipidosis using a high-content biology platform. Cell Biol Toxicol 22, 15–27 (2006). https://doi.org/10.1007/s10565-006-0176-z

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  • DOI: https://doi.org/10.1007/s10565-006-0176-z

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