Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation...

Area of Science:

• Biotechnology
• Toxicology
• Cellular Biology

Background:

• The p21 gene is a crucial p53 target, activated by cellular DNA damage.
• High-throughput genotoxicity testing is essential for new drug development.
• Existing in vitro assays have limitations in detecting certain genotoxic agents.

Purpose of the Study:

• To develop and validate a fish cell biosensor for high-throughput genotoxicity detection.
• To assess the sensitivity and specificity of the biosensor for various genotoxic and non-genotoxic agents.
• To evaluate the biosensor's utility in detecting the genotoxicity of Di(2-ethylhexyl) phthalate (DEHP).

Main Methods:

• Development of a stable fish cell line (p21FGLuc) by integrating two reporter plasmids (pGL3-p21-luc and pRL-CMV-luc) into flounder gill (FG) cells.
• Validation of the biosensor's p53 signaling pathway and response to known genotoxic agents (bleomycin, mitomycin C, cyclophosphamide with/without metabolic activation).
• Testing the biosensor's response to non-genotoxic agents (ethanol, D-mannitol) and DEHP.

Main Results:

• The p21FGLuc biosensor demonstrated a wild-type p53 signaling pathway.
• The system showed high sensitivity and specificity, correctly identifying genotoxic agents and distinguishing them from non-genotoxic ones.
• The biosensor provided a strong indication of DEHP genotoxicity, overcoming limitations of other assays.

Conclusions:

• The p21FGLuc fish cell biosensor is a validated, sensitive, and specific tool for high-throughput genotoxicity testing.
• This system offers a reliable method for assessing the safety of new drugs and identifying genotoxic compounds like DEHP.
• A potential limitation includes false positives from agents that activate the p21 gene independently of p53.