Potassium bromate as positive assay control for the Fpg-modified comet assay

Area of Science:

• Biomonitoring
• Genotoxicity assessment
• Oxidative stress research

Background:

• The comet assay is widely used in biomonitoring to detect DNA strand breaks and lesions.
• Accurate assessment of oxidative DNA damage requires specific controls, but a suitable positive control for the Fpg-modified comet assay has been lacking.
• Ethical considerations preclude using human subjects as positive controls for DNA-damaging agents.

Purpose of the Study:

• To evaluate potassium bromate (KBrO3) as a reliable positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay.
• To establish a standardized protocol for using KBrO3 as a positive control in inter-laboratory comet assay studies.

Main Methods:

• Ten laboratories participated in a study using monocytic THP-1 cells treated with varying concentrations of KBrO3 (0.5, 1.5, and 4.5 mM) for 1 hour at 37°C.
• Cells were subsequently cryopreserved and analyzed using the Fpg-modified comet assay.
• Statistical analysis was performed on the data, excluding one laboratory due to technical issues.

Main Results:

• A concentration-response relationship was observed across all participating laboratories for cryopreserved samples treated with KBrO3.
• Regression coefficients ranged from 0.80 to 0.98, indicating a strong correlation between KBrO3 concentration and Fpg-sensitive sites.
• The number of Fpg-sensitive sites per 1 mM KBrO3 varied between laboratories (1.25 to 11.9 %DNA in tail), highlighting the need for standardized protocols.

Conclusions:

• Potassium bromate (KBrO3) is demonstrated to be a suitable positive control for the Fpg-modified comet assay.
• The use of KBrO3 as a positive control can enhance the reliability and standardization of biomonitoring studies assessing oxidative DNA damage.