Determination of phospholipidosis potential based on gene expression analysis in HepG2 cells.
Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.
Atienzar
Franck
F
UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l'Alleud, Belgium. franck.atienzar@ucb-group.com
Gerets
Helga
H
Dufrane
Simon
S
Tilmant
Karen
K
Cornet
Miranda
M
Dhalluin
Stephane
S
Ruty
Bernard
B
Rose
Geoffrey
G
Canning
Michael
M
D015415:Biomarkers / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D064420:Drug-Related Side Effects and Adverse Reactions; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008064:Lipidoses / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D004364:Pharmaceutical Preparations / Q000145:classification; D010743:Phospholipids / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D018675:Toxicity Tests / Q000379:methods; D015854:Up-Regulation / Q000187:drug effects
D015415:Biomarkers; D004364:Pharmaceutical Preparations; D010743:Phospholipids; D012333:RNA, Messenger