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ToxSci Advance Access originally published online on December 7, 2005
Toxicological Sciences 2006 90(1):133-141; doi:10.1093/toxsci/kfj067
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© The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Establishment of an In Vitro High-Throughput Screening Assay for Detecting Phospholipidosis-Inducing Potential

Toshihiko Kasahara*,1, Kazuo Tomita*, Hiroyuki Murano{dagger}, Tsuyoshi Harada{ddagger}, Keisuke Tsubakimoto*, Takuo Ogihara*, Syuhei Ohnishi* and Chihaya Kakinuma*

* Pharmaceutical Research Center, {dagger} Research Planning and Management, and {ddagger} Development Research, Mochida Pharmaceutical Company Limited, Shizuoka, Japan

Received August 29, 2005; accepted November 16, 2005

Excessive accumulation of phospholipids results in phospholipidosis (PL), which may interfere with cellular functions, leading to acute or chronic disease or even death. Electron-microscopic detection of cytoplasmic lamellar bodies is often used as a diagnostic criterion of PL, but a faster, more convenient procedure is required for high-throughput assay of the PL-inducing potential of candidate drugs. We have developed a 96-well microplate cell-culture method for detecting PL, using a phosphatidylcholine-conjugated dye (NBD-PC) and a fluoro-microplate reader. The fluorescence intensity due to NBD-PC was normalized to that of Hoechst33342, used as an indicator of cell number, to obtain the amount of NBD-PC taken up per living cell. To select a suitable cell type, we examined the PL-detection sensitivity of five cell lines, as well as human and rat primary hepatocyte cultures, with five cationic amphiphilic drugs (CAD) as PL inducers and a negative control compound. The cell lines CHO-K1 and CHL/IU gave the best results. The NBD-PC uptake per CHO-K1 cell showed a high correlation with the pathological score of PL for 24 compounds, including PL-positive and negative compounds. This high-throughput screening assay for PL-inducing potential (HTS-PL assay) offers high sensitivity and accuracy, and it allows simultaneous determination of cytotoxicity.

Key Words: phospholipidosis; cationic amphiphilic drug; NBD-PC; Hoechst33342; HTS-PL assay.


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