A toxicogenomic approach to drug-induced phospholipidosis: analysis of its induction mechanism and establishment of a novel in vitro screening system.
Phospholipidosis is a lipid storage disorder in which excess phospholipids accumulate within cells. Some cationic amphiphilic compounds are known to have the potential to induce phospholipidosis. This study was undertaken to examine the molecular mechanisms that contribute to the development of phospholipidosis and to identify specific markers that might form the basis of an in vitro screening test. Specifically, we performed a large-scale gene expression analysis using DNA microarrays on human hepatoma HepG2 cells after they were treated with each of 12 compounds known to induce phospholipidosis. In electron microscopy, HepG2 cells developed lamellar myelin-like bodies in their lysosomes, the characteristic change of phospholipidosis, after treatment with these compounds for 72 h. DNA microarray analysis performed 6 and 24 h after treatment showed alterations in gene expression reflecting the inhibition of lysosomal phospholipase activity and lysosomal enzyme transport, and the induction of phospholipid and cholesterol biosynthesis. Seventeen genes that showed a similar expression profile following treatment were selected as candidate markers. Real-time PCR analysis confirmed that 12 gene markers showed significant concordance with lamellar myelin-like body formation. Furthermore, the average fold change values of these markers correlated well with the magnitude of this pathological change. In conclusion, microarray analysis revealed that factors such as alterations in lysosomal function and cholesterol metabolism were involved in the induction of phospholipidosis. Furthermore, comprehensive gene expression analysis enabled us to identify biomarkers of this condition that we then used to develop a rapid and sensitive in vitro screening test for drug-induced phospholipidosis.
Sawada
Hiroshi
H
Biomedical Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Osaka 532-8686, Japan. Sawada_Hiroshi@takeda.co.jp
Takami
Kenji
K
Asahi
Satoru
S
D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D045744:Cell Line, Tumor; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling / Q000379:methods*; D005819:Genetic Markers; D006801:Humans; D008064:Lipidoses / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D020411:Oligonucleotide Array Sequence Analysis; D010740:Phospholipases / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018675:Toxicity Tests / Q000379:methods; D043922:Toxicogenetics; D015262:Xenobiotics / Q000633:toxicity
D005819:Genetic Markers; D010743:Phospholipids; D012333:RNA, Messenger; D015262:Xenobiotics; D010740:Phospholipases