Improved toxicogenomic screening for drug-induced phospholipidosis using a multiplexed quantitative gene expression...

Area of Science:

• Toxicogenomics
• Drug safety assessment
• Molecular biology

Background:

• Previous toxicogenomics analysis identified 12 gene markers for drug-induced phospholipidosis.
• An in vitro real-time PCR assay was established for screening phospholipidosis potential.

Purpose of the Study:

• To adapt the existing PCR-based assay into a higher-throughput 96-well microplate ArrayPlate assay.
• To increase the throughput for assessing the phospholipidosis-inducing potential of compounds.

Main Methods:

• Human hepatoma HepG2 cells were treated with amiodarone and 80 proprietary compounds for 24 hours.
• Gene expression of 12 marker genes was determined using both real-time PCR and the ArrayPlate assay.
• Performance criteria included sensitivity, repeatability, and correlation between the two assay methods.

Main Results:

• The ArrayPlate assay detected mRNA expression for all target genes at quantifiable levels.
• Signal intensities and fold change values for each marker gene demonstrated high repeatability.
• Fold change values from the ArrayPlate assay showed good correlation with those from the real-time PCR assay.

Conclusions:

• The ArrayPlate-based method is a sensitive and repeatable assay for assessing drug-induced phospholipidosis.
• This higher-throughput assay is transferable from the PCR-based method, enabling more efficient screening.
• The ArrayPlate assay provides a reliable tool for evaluating the phospholipidosis potential of chemical compounds.