Development of a 32P-Postlabeling Method for the Detection of 1,N2-Propanodeoxyguanosine Adducts of trans-4-Hydroxy-2-nonenal in Vivo

Abstract

A (32)P-postlabeling method was developed for the sensitive detection of 1,N(2)-propanodeoxyguanosine adducts of the lipid peroxidation product trans-4-hydroxy-2-nonenal in vivo. The method development was based on the chemically synthesized HNE-1, N(2)-propanodeoxyguanosine adduct standard, which was characterized by NMR and mass spectra. The adducts were enriched by nuclease P1. They were subsequently reacted with [gamma-(32)P]ATP to give the respective 3'-5'-bisphosphates, which were two-directionally separated on PEI-cellulose TLC and quantitated by autoradiography. The medium labeling efficiency for the mixture of the two pairs of diastereomers was 27%, and the recovery of spiked amounts of adduct standard in the enzymatical procedure was about 80%. The method is applicable for the separation and quantitation of HNE-dGp-propano adducts in vivo. It was applied to DNA from colon and brain tissue of untreated Fischer 344 rats and humans. The determination of the limit of quantitation in DNA from rat colon by spiking of adduct standard revealed a sensitivity of <21 adducts/10(9) nucleotides. The analytical quantitation of 4-HNE-dGp-propano adducts resulted in adduct-levels per 10(9) normal nucleotides +/- the standard deviation of 223.32 +/- 79.84 in rat colon tissue, 90.37 +/- 11.94 in rat brain tissue, 378.44 +/- 52.42 in human colon tissue, and 185.15 +/- 6.48 in human brain tissue. The results clearly demonstrate the applicability of this method for the sensitive detection of endogenously formed 1,N(2)-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal, a specific marker for the lipid peroxidation process.