Oxidative DNA adducts have been associated with the pathway of multistage carcinogenesis, as indicators of increased reactive oxygen species generation, and as products of lipid peroxidation. The chemical structures of this class of adducts vary considerably, from “simple” adducts obtained by the hydroxylation of a normal nucleic acid base, to the formation of an additional ring on guanine as seen in the cases of certain aldehydes (e.g. 4-hydroxy-2-nonenal [HNE]). Different analytical techniques and methods have been used in order to separate (e.g. TLC, HPLC, GC) and quantify (e.g. 32P-postlabeling, MS, electrochemical detection) individual oxidative DNA adducts. In our continued effort to develop methodology for analyzing many oxidative adducts collectively, we explored the possibility of using HPLC in conjunction with multichannel electrochemical detection (CoulArray). DNA was hydrolyzed using micrococcal endonuclease and spleen phosphodiesterase, after which the enzymes were removed by either acetonitrile precipitation or use of centrifugal concentrators. The digest is lyophilized, and the residue reconstituted in a mobile phase consisting of a citric acid/sodium acetate buffer. The deoxynucleotide 3’-monophosphates (dNp) were then separated by a reversed-phase C18 HPLC using a citric acid/sodium acetate/methanol buffer as the isocratic mobile phase. Synthetic 8-oxodGp was resolved from dNp, and was also distinguished from dGp electrochemically by the difference in the potential ranges that each compound was detected (250 to 550 mV vs 650 to >1000 mV, respectively). With the exception of dGp which was only weakly electrically active, the other normal nucleotides (dAp, dCp and Tp) were transparent to electrochemical detection under the conditions run. dGp adducts from crotonaldehyde (Crot), hexenal (Hex), or HNE were tested in this separation system, and were found to elute with retention times well beyond those of the normal nucleotides (range: 5.2-7.2 min). The elution order and times for these adducts were Crot-dGp (26.2 min) < Hex-dGp (31.8 min) < HNE-dGp (51.5 min). When 100 μg DNA digest, spiked with 2 pmoles of the HNE-dGp standard, was purified by precipitation with acetonitrile, sieved through a centrifugal filter and analyzed by HPLC-CoulArray, the HNE-dGp adduct was clearly detected at 50.2 min within the same potential range as the standard alone. The limit of detection, however, remains to be determined. Using this methodology, it is possible to detect these aldehyde-derived cyclic adducts by electrochemical detection in a runtime of <60 minutes.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]