Adverse Outcome Pathway-Informed Integrated Testing to Identify Chemicals Causing Genotoxicity Through Oxidative DNA...

Area of Science:

• Toxicology and Molecular Biology
• Genotoxicity and Risk Assessment
• Adverse Outcome Pathways (AOPs)

Background:

• Adverse Outcome Pathways (AOPs) provide a framework for understanding chemical toxicity and interpreting data from new testing methods.
• AOP #296 links oxidative DNA damage to mutations and chromosomal aberrations, crucial for regulatory decision-making.
• Evaluating chemicals through established AOPs enhances the reliability of risk assessment.

Purpose of the Study:

• To develop and validate an integrated testing strategy informed by AOP #296.
• To assess the genotoxic potential of 4-nitroquinoline 1-oxide (4NQO) using an AOP-based approach.
• To quantitatively characterize the time- and concentration-dependent effects of 4NQO on DNA damage and mutations.

Main Methods:

• Human TK6 cells were exposed to 4NQO in a time-series experiment.
• Oxidative DNA damage and strand breaks were measured using the CometChip assay with and without formamidopyrimidine DNA glycosylase (Fpg).
• Micronucleus (MN) frequency was analyzed by flow cytometry, and mutations were assessed using duplex sequencing (DS).

Main Results:

• 4NQO induced concentration- and time-dependent increases in oxidative DNA damage, strand breaks, MN frequency, and mutation frequency.
• Oxidative DNA damage was detected early, while mutations appeared at later time points (48h).
• Duplex sequencing identified C > A transversions, consistent with oxidative DNA lesions, and revealed repair of oxidative damage with persistent strand breaks.

Conclusions:

• The study provides a validated AOP-informed testing strategy for evaluating chemicals via AOP #296.
• The integrated approach effectively quantifies 4NQO-induced genotoxicity and elucidates the pathway dynamics.
• Duplex sequencing is confirmed as a valuable tool for mutagenicity assessment within AOP frameworks.