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32 P‐postlabeling analysis of DNA adducts formed by leukotriene A 4 (LTA 4 )
Author(s) -
Funk Dorothee,
Sorg Bernd L.,
Lindner Sabine C.,
Schmeiser Heinz H.
Publication year - 2010
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.20547
Subject(s) - adduct , chemistry , dna , nucleotide , nuclease , deoxyguanosine , dna adduct , nucleoside , biochemistry , carcinogen , stereochemistry , organic chemistry , gene
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 μM) 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. Environ. Mol. Mutagen. 2010. © 2010 Wiley‐Liss, Inc.