Quantitative analysis of gene expression changes in response to genotoxic compounds

Area of Science:

• Toxicology
• Molecular Biology
• Biochemistry

Background:

• High-throughput in vitro screening requires sensitive molecular quantification techniques to identify toxic compounds.
• Gene expression profiling is a key method for assessing cellular responses to chemical exposure.

Purpose of the Study:

• To develop and validate an acridinium-ester chemiluminescent assay for quantifying the expression of three genes (RAD51C, TP53, and cystatin A) in HEPG2 cells.
• To compare the performance of the chemiluminescent assay with quantitative PCR (Q-PCR) for measuring gene expression changes induced by various chemical compounds.

Main Methods:

• HEPG2 cells were exposed to 18 different compounds with varying genotoxicity profiles.
• Gene expression levels of RAD51C, TP53, and cystatin A (CSTA) were measured using both Q-PCR and a newly developed acridinium-ester chemiluminescent reporter assay.
• Analytical sensitivity and linear amplification were optimized for the chemiluminescent assay.

Main Results:

• Q-PCR detected dysregulated expression of at least one of the three genes in most tested compounds, with CSTA showing significant changes in ten compounds.
• The acridinium-ester chemiluminescent assay demonstrated high analytical sensitivity, with limits of detection in the attomole range.
• The chemiluminescent method detected statistically significant increases in RAD51C and CSTA expression, showing good agreement with Q-PCR results.

Conclusions:

• The developed acridinium-ester chemiluminescent assay is a sensitive and reliable method for quantifying gene expression.
• This technology shows potential for high-throughput in vitro screening of chemical toxicity by accurately measuring molecular endpoints.
• The broad agreement between the chemiluminescent assay and Q-PCR validates its wider applicability in gene expression analysis.