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

Leukotriene A₄ (LTA₄), 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 LTA₄ in vitro and human cells have been identified by mass spectrometry on the nucleoside level. Here we investigated whether the thin‐layer chromatography (TLC) ³²P‐postlabeling method is suitable for the detection of LTA₄‐DNA adducts. The reaction of individual deoxynucleoside 3′‐monophosphates with LTA₄ in aqueous basic solution yielded numerous adduct spots when analyzed by the two enrichment procedures of the ³²P‐postlabeling method—nuclease P1 digestion and butanol extraction. Highest LTA₄‐adduct levels were found with deoxyguanosine 3′‐phosphate (around one adduct per 10⁴ normal nucleotides). Under similar reaction conditions LTA₄ (25–320 μM) was incubated with calf thymus DNA, then DNA adduct patterns and levels were determined with the TLC ³²P‐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 LTA₄ 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 LTA₄‐DNA adducts were detected by ³²P‐postlabeling. However, the level of these endogenously formed DNA adducts was close to the detection limit (3 ± 2 adducts per 10⁸ normal nucleotides). In summary, the TLC ³²P‐postlabeling method is suitable for studying DNA adduct formation by LTA₄ and can be used for further investigations on the link between inflammation and cancer. Environ. Mol. Mutagen. 2010. © 2010 Wiley‐Liss, Inc.