The measurement of oxidized bases and nucleosides in DNA may be used to gain insights into the nature and importance of chemical reactions generated in DNA by oxidizing agents. Significant improvement for the measurement of oxidatively generated damage to DNA has been obtained by the use of HPLC coupled to MS and MS/MS (3
). This method has been gaining prominence and has overcome many of the limitations that previously hindered the measurement of oxidatively generated DNA damage (3
). It now represents the usual method of choice for identifying modified DNA bases (1
IST, which can be combined with MS and MS/MS, is a relatively new method for studying DNA radical damage (18–21
). Our recent investigations with EPR and with IST with MS and MS/MS have allowed the characterization of nitrone adducts formed in Fenton systems by the hydroxyl radical (26
). The objective of the present work was to determine the time course of formation and repair of free radical-derived DNA nitrone adduct, and to use confocal microscopy to visualize the damage to the DNA in the nucleus. Separation of cytosolic and nuclear fractions and subsequent DNA purification demonstrated the presence of DNA–DMPO adduct, presumably of the known DMPO/N6-centered adenosine nitrone adduct in the nucleus (26
). MS and MS/MS confirmed the DMPO/N6-centered adenosine in the nuclear fraction (data not shown).
Using ESR, MS and MS/MS, we determined that the radical trapped by DMPO was located at N6 of the adenosine (26
). Hydroxyl radical can add to the double bond at C4, C5 and C8 of the adenosine (33–37
), of which C4 constitutes the most abundant addition site (81%) (11
). This adduct undergoes proton or hydrogen transfer, followed by dehydration, to form an N6-dehydrogenated radical. N6 has also been shown elsewhere to be the most likely site of hydrogen abstraction by hydroxyl radical (38
). However, the absence of detection of other DNA radicals is not proof of their absence. It should be noted that this type of site-specific hydroxyl radical generation should not form as many DNA-derived radicals as ionizing radiation where freely diffusing hydroxyl radical is formed.
When the RAW 267.4 cells were treated with CuCl2
, the radical generated was trapped by DMPO immediately upon formation to form a DMPO nitrone adduct. The DMPO radical adduct formed is initially EPR active but subsequently decays to the more stable nitrone adduct, which can be very conveniently detected by the IST method (20
) and confirmed and identified by MS and MS/MS. The radical is trapped spontaneously by DMPO, and artifactual generation of DMPO adducts during the extraction and digestion steps is minimized, if not eliminated. Furthermore, as it is a DMPO adduct that is formed, its identity can be confirmed by MS/MS from its fragmentation to DMPO and the parent radical ion, thereby eliminating the requirement of a pure product for identification.
It should be recognized that the oxidation event does not have to take place inside the cell nucleus, but may occur either within the cytosol or even in the extracellular compartment. However, the damage inflicted by CuCl2/H2O2 Fenton chemistry to the RAW 267.4 macrophages was indeed found to be largely localized to the nuclear DNA. This localization was evident from the confocal data as well as from the immunochemical detection of the cytosolic and nuclear fractions.
We also examined the persistence of the DMPO–DNA adducts formed. When the adducts were monitored over a period of time, they formed detectable amounts within 30
min, then increased slowly over the next several hours before reaching a maximum at ~12
h. Thereafter, repair of the DMPO–DNA nitrone adduct apparently dominates, accompanied by a drop in the adduct level to a value of ~50% of the maximum. However, because this effect could also be caused by turnover due to DNA synthesis, we also studied the repair of the preformed DNA–DMPO adduct added to cell lysate. Preformed DMPO–DNA nitrone repair by treatment with cell lysate was largely complete within an hour. Clearly, the DMPO–DNA nitrone adduct concentrations detected were the result of continuous hydroxyl radical attack on DNA balanced against DNA repair processes. DNA repair enzymes modify DNA damage including removal of DNA adducts, and there are multiple and overlapping DNA repair pathways (40
). Inhibition of the repair pathways will block this mechanism. We used two known inhibitors, namely methoxyamine (TRC 102) of the BER pathway (41
) and NiCl2
of the NER pathway (42
). Methoxyamine is a small molecule repair inhibitor (41
) and inhibited the repair of the single-base lesions in preformed DMPO–DNA nitrones.
In summary, with our method of combining IST with ESR, we were able to confirm that DMPO traps a radical adduct from DNA in accordance with published ELISA studies (18–21
). This methodology provides an unequivocal assignment of the radical formed in DNA damage induced by the hydroxyl radical in a copper/hydrogen peroxide Fenton system (26
). The radical produced when cellular DNA was treated with a Fenton-like system was found to be at the adenosine moiety, which may be an intermediate in a sequence of events leading to the formation of 8-oxo-dGuo (43
) and other products such as 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxodAdo). Although speculative for us, it has been predicted by hole-trapping researchers that the adenine radical cation contributes to the hole-transfer process through A/T sequences and exists as a real chemical intermediate (44–48
). One-electron oxidation of DNA results in migration and localization of the electron loss center to guanine (43
The damage was found to be localized in the nuclear DNA, but was repaired over a period of time, probably through the BER pathway. The repair study is preliminary in nature and makes clear the interfering effect of DNA repair in a biological system. It is necessary that in vivo experiments studying DMPO–DNA nitrone adducts be conducted over a range of times as repair of the DMPO–DNA nitrone adduct does occur.