title: Establishment of an in vitro high-throughput screening assay for detecting phospholipidosis-inducing potential.
abstract: 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.
authors.affiliation: Pharmaceutical Research Center, Mochida Pharmaceutical Company Limited, Shizuoka, Japan. kasat@mochida.co.jp
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Kasahara
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tomita
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Murano
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Harada
authors.forename: Keisuke
authors.initials: K
authors.lastname: Tsubakimoto
authors.forename: Takuo
authors.initials: T
authors.lastname: Ogihara
authors.forename: Syuhei
authors.initials: S
authors.lastname: Ohnishi
authors.forename: Chihaya
authors.initials: C
authors.lastname: Kakinuma
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008064:Lipidoses / Q000139:chemically induced*; D008647:Mesocricetus; D051379:Mice; D010713:Phosphatidylcholines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013501:Surface-Active Agents / Q000145:classification / Q000633:toxicity*
chemical_list: C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D007202:Indicators and Reagents; D010713:Phosphatidylcholines; D010743:Phospholipids; D013501:Surface-Active Agents; D009327:4-Chloro-7-nitrobenzofurazan