To determine whether polyamines could facilitate the formation of Cr-PdG, dG was incubated with AA, polyamine, or both in the presence of neutral phosphate buffer. Treatment of dG with AA alone () resulted in a single peak with a retention time of 19.7 min. This peak is most likely a carbinolamine intermediate (with a Schiff base component) in the reaction of AA with dG since it was unstable upon isolation and reinjection, and could be converted to the stable adduct N2
-ethyl dG by reducing agents (data not shown) consistent with previous observations (21
). Reaction of dG with polyamine alone resulted in no detectable adduct formation (). However, when the AA reaction was carried out in the presence of spermidine, neither the intermediate nor N2
-ethyl dG was formed. Instead, two closely eluting product peaks (retention times: 1st peak, 23.5 min; 2nd peak, 24.5 min) were observed (). The same two peaks were observed when spermine was used instead of spermidine (data not shown). These peaks were consistent with the properties of the diastereomers of Cr-PdG adducts formed from the reaction of AA in the presence of histone (33
) and from the reaction of dG with CrA (25
To confirm the identity of the peaks resulting from the reaction of AA, dG and spermidine as Cr-PdG adducts, we analyzed the Cr-PdG adducts prepared by directly reacting dG with CrA. The synthetic Cr-PdG adducts derived from the CrA reaction showed the same elution properties as those from the AA and spermidine reaction (). The areas under both diastereomer peaks were essentially the same (1st peak/2nd peak ratio: 1.2/1). Preparation of Cr-PdG adducts by incubation of dG with arginine and AA () also resulted in the same two peaks, although the ratio of the peak areas (1st peak/2nd peak ratio: 1.77/1) was clearly different from that observed with dG and CrA or with dG, AA and spermine.
To rigorously confirm their identities, the two adducts resulting from the reaction of AA and polyamine with dG were purified by preparative HPLC and analyzed by GC-MS analysis after trimethylsilylation. Both diastereomers of the Cr-PdG gave identical mass spectra (), which are expected for Cr-PdG with four trimethylsilyl groups. Characteristic ions were observed at m/z 625, 610, 365 and 350. The ion at m/z 625 is the molecular ion (M+•) and the m/z 610 ion is the typical (M-15)+ ion, which results from the loss of methyl radical (•CH3) from M+•. The ion at m/z 365 represents the base moiety after cleavage of the sugar moiety. The most intense peak at m/z 350 results from the loss of •CH3 from the base moiety, which is common in trimethylsilyl derivatives. In the GC column, the second diastereomer of Cr-PdG (in HPLC) elutes earlier than the first diastereomer.
(a) Total-ion chromatogram obtained during GC-MS analysis of the synthesized Cr-PdG after trimethylsilylation; (b) mass spectrum of the trimethylsilylated Cr-PdG; and (c) mass spectrum of the trimethylsilylated Cr-PdG-13C10,15N5.
For quantification purposes, we synthesized the isotopically labeled Cr-PdG adducts using dG-13C10,15N5 and AA in the presence of arginine. The mass spectrum of the trimethylsilylated Cr-PdG-13C10,15N5 gave the expected corresponding mass spectrum of Cr-PdG with 15 additional mass units for M+• and (M-15)+ ions and 10 additional mass units for ions representing the base moiety ().
Initial attempts to detect the Cr-PdG adduct formation from dG or DNA incubated with lower concentration of AA and spermidine using GC-MS were unsuccessful, due to the partial instability of the Cr-PdG adduct under GC-MS conditions. While others have used either post labeling (25
) or LC-MS analysis of free Cr-PdG base (33
), we chose to develop an LC-MS assay for the Cr-PdG adducts. Analysis of synthetic Cr-PdG adducts by LC-MS yielded typical ions at m
222, 338 and 360. The ion at m
222 represents the base moiety with two additional H atoms (BH2+
ion). The ion at m
338 corresponds to the protonated molecular ion (MH+
) and the ion at m
360 is the sodium adduct ion (MNa+
). These types of ions are typical of nucleosides in LC-MS analysis. The corresponding ions from the isotopically labeled standard were at m
232, 353 and 375, respectively. No ions corresponding to free dG were observed from the purified adduct preparations, indicating that in contrast to GC-MS, the Cr-PdG adducts are stable under LC-MS conditions.
Using LC-MS with isotope dilution, we next assessed the dependence of Cr-PdG lesion formation with dG in response to different, biologically relevant concentrations of AA or spermidine. As shown in , Cr-PdG levels increase with increasing AA concentrations in the range from 25 to 500 μM (using 5.0 mM spermidine). Similarly, in the presence of 200 μM AA, Cr-PdG levels increased with increasing spermidine concentrations in the range 500 μM–2 mM (). In both cases, the concentration curve data could be best fit by a third order polynomial function.
We next investigated the effect of varying concentrations of AA on the formation of Cr-PdG adducts in DNA in the presence or absence of 5 mM spermidine. This value was chosen as the high end of the physiological polyamine concentration range (34
). The identification and quantification of Cr-PdG adducts were performed using the SIM mode, monitoring the ions m
222, 338 and 360. illustrates a typical SIM analysis of the Cr-PdG adducts with ion-current profiles of the ions at m
222 and 338 (Cr-PdG), and m
232 and 353 (Cr-PdG-13
). The dependence of the Cr-PdG adduct formation in DNA on AA concentration is shown in . In the presence of 5 mM spermidine, Cr-PdG adducts were observed with AA concentrations as low as 100 μM, and increased with increasing AA concentrations. The ratio of the two diastereomers was 1.1:1. As observed in reactions with AA, spermidine and dG (), the data were best fit by a third order polynomial function. Under these same conditions, we could not detect any formation of N2
-EtdG (data not shown).
Spermidine reacts directly with AA to form CrA
The fact that we observed the same ratio of Cr-PdG diastereomers from combined AA and spermidine treatment as we did from CrA alone () suggested that spermidine could directly react with AA to form CrA. To test this hypothesis, we incubated AA in phosphate buffer with or without spermidine and monitored the formation of CrA by GC-MS. AA elutes at 0.62 min and CrA elutes at 0.82 min under these experimental conditions. CrA was measured using the SIM mode by monitoring the ions at m/z 39, 41, 69 and 70 (molecular ion). As shown in , CrA was produced by incubating AA and spermidine together in the presence of phosphate buffer, but not by incubating either compound alone. CrA formation from AA and spermidine was time dependent (), and CrA formation could be detected from 100 μM AA incubated in the presence of 5 mM spermidine (). Since no DNA or dG was present in these reactions, the results indicate that spermidine directly reacts with AA to generate CrA.
Figure 5 (a) Time dependence of CrA formation. Ion-current profiles of the ion at m/z 70 obtained during GC-MS-SIM analysis of the samples from the reaction of spermidine (5 mM) and AA (different concentrations) in the presence of 0.1 M phosphate buffer at pH (more ...)