N-Methyl- N-nitrosourea Induced 3'-Glutathionylated DNA-Cleavage Products in Mammalian Cells

Abstract

Apurinic/apyrimidinic (AP) sites, that is, abasic sites, are among the most frequently induced DNA lesions. Spontaneous or DNA glycosylase-mediated β-elimination of the 3'-phosphoryl group can lead to strand cleavages at AP sites to yield a highly reactive, electrophilic 3'-phospho-α,β-unsaturated aldehyde (3'-PUA) remnant. The latter can react with amine or thiol groups of biological small molecules, DNA, and proteins to yield various damaged 3'-end products. Considering its high intracellular concentration, glutathione (GSH) may conjugate with 3'-PUA to yield 3-glutathionyl-2,3-dideoxyribose (GS-ddR), which may constitute a significant, yet previously unrecognized endogenous lesion. Here, we developed a liquid chromatography tandem mass spectroscopy method, in combination with the use of a stable isotope-labeled internal standard, to quantify GS-ddR in genomic DNA of cultured human cells. Our results revealed the presence of GS-ddR in the DNA of untreated cells, and its level was augmented in cells upon exposure to an alkylating agent, N-methyl-N-nitrosourea (MNU). In addition, inhibition of AP endonuclease (APE1) led to an elevated level of GS-ddR in the DNA of MNU-treated cells. Together, we reported here, for the first time, the presence of appreciable levels of GS-ddR in cellular DNA, the induction of GS-ddR by a DNA alkylating agent, and the role of APE1 in modulating its level in human cells.

Figure 1.

Generation of GS-ddR-MX-containing ODNs. (A) Scheme illustrating the reaction between the aldehyde terminus of GS-ddR and methoxyamine (MX). (B) Schematic diagram depicting the procedures for the generation of a GS-ddR-MX-containing ODN from a single-stranded uracil-containing ODN. (C) Chemical structures of GS-ddR-MX and [¹³C₂, ¹⁵N]-GS-ddR-MX.

Figure 2.

LC–MS/MS characterizations of GS-ddR-MX monophosphate in the negative-ion mode. (A) Representative SICs for monitoring the m/z 531 → 306 and 534 → 309 transitions for the [M – H]⁻ ions of GS-ddR-MX and [¹³C₂, ¹⁵N]-GS-ddR-MX monophosphates, respectively, in the nuclease P1 digestion mixtures of the corresponding ODNs. (B) Negative-ion ESI-MS/MS of GS-ddR-MX (left) and [¹³C₂, ¹⁵N]-GS-ddR-MX (right) monophosphates. (C) Proposed fragmentation patterns of product ions observed in the MS/MS of the [M – H]⁻ ions of GS-ddR-MX (left) and [¹³C₂, ¹⁵N]-GS-ddR-MX (right) monophosphates.

Figure 3.

Recovery of GS-ddR-MX from the lesion-containing ODNs. (A) Workflow of sample preparation for the LC–MS/MS analysis of GS-ddR-MX in DNA samples. “X” in the “Enzymatic Digestion” part represents modified nucleobases. (B) Representative HPLC trace for the enrichment of GS-ddR-MX and O⁶-MedG from the nucleoside digestion mixture of DNA samples isolated from HEK293T cells treated with MNU. (C) Recovery calibration curve of GS-ddR-MX obtained from LC–MS/MS analyses of the enzymatic digestion mixtures of calf thymus DNA spiked with different amounts of a GS-ddR-MX-containing ODN and a fixed amount (20 fmol) of [¹³C₂, ¹⁵N]-GS-ddR-MX-containing ODN, which was used as the internal standard. Plotted are the detected versus spiked-in levels of GS-ddR in calf thymus DNA.

Figure 4.

Time- and dose-dependent induction of GS-ddR in HEK293T cells treated with MNU. (A,B) Quantification results for the levels of GS-ddR (A) and O⁶-MedG (B) in HEK293T cells treated with 0, 50, 100, 200, 500 μM, and 1 mM of MNU for 1 h. p-values were calculated by using Kruskal–Wallis one-way ANOVA and Conover’s multiple comparisons test further adjusted by the Benjamini–Hochberg FDR method for the comparison between control and MNU treatments with different concentrations and the comparison between different concentration treatments. (C,D) Quantification results for the levels of GS-ddR (C) and O⁶-MedG(D) in HEK293T cells treated with 100 μM or 1 mM of MNU for 0, 1, 8, and 24 h. The data in (A–D) represent the mean ± S.D. (n = 3) of results from three independent experiments. The results for GS-ddR and O⁶-MedG were acquired by LC–MS/MS analysis on a QE Plus quadrupole-Orbitrap and an LTQ XL linear ion trap mass spectrometer, respectively. The p values were calculated by using the unpaired two-tailed t test for the comparison between 0 h and different treatment times and the comparison between different treatment times. #, p > 0.05; *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001.

Figure 5.

Effects of APE1 inhibition on the generation of GS-ddR. (A,B) Quantification results for the levels of GS-ddR (A) and O⁶-MedG (B) in the HEK293T cells treated with MNU, APE1 inhibitor (CRT0044867), alone or in combination. The data represent mean ± S.D. (n = 3) of results from three independent experiments. The p values were calculated by using the unpaired two-tailed t test (#, p > 0.05; *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; ***, p < 0.001).

Scheme 1.

GS-ddR Arising from the Strand Cleavage at an AP Site in the Presence of Glutathione

Scheme 2.

MNU-Induced Generation of N7-Methyl-2′-deoxyguanosine and O⁶-Methyl-2′-deoxyguanosine and the Subsequent Formation of GS-ddR