32P‐postlabeling analysis of DNA adducts formed by leukotriene A4 (LTA4)

Abstract

Leukotriene A(4) (LTA(4)), a reactive electrophilic intermediate formed during the biosynthesis of inflammation-related lipid mediators, has been found to bind covalently to DNA. The major DNA adducts formed by LTA(4) in vitro and human cells have been identified by mass spectrometry on the nucleoside level. Here we investigated whether the thin-layer chromatography (TLC) (32)P-postlabeling method is suitable for the detection of LTA(4)-DNA adducts. The reaction of individual deoxynucleoside 3'-monophosphates with LTA(4) in aqueous basic solution yielded numerous adduct spots when analyzed by the two enrichment procedures of the (32)P-postlabeling method-nuclease P1 digestion and butanol extraction. Highest LTA(4)-adduct levels were found with deoxyguanosine 3'-phosphate (around one adduct per 10(4) normal nucleotides). Under similar reaction conditions LTA(4) (25-320 microM) was incubated with calf thymus DNA, then DNA adduct patterns and levels were determined with the TLC (32)P-postlabeling method using both enrichment versions. The same DNA adduct pattern consisting of up to seven spots was observed with both enrichment versions. DNA adduct formation by LTA(4) was concentration-dependent with major adducts being derived from deoxyguanosine. When a human monocytic cell line (Mono Mac 6) was stimulated with arachidonic acid and calcium ionophore LTA(4)-DNA adducts were detected by (32)P-postlabeling. However, the level of these endogenously formed DNA adducts was close to the detection limit (3 +/- 2 adducts per 10(8) normal nucleotides). In summary, the TLC (32)P-postlabeling method is suitable for studying DNA adduct formation by LTA(4) and can be used for further investigations on the link between inflammation and cancer.