Measurement of the incorporation and repair of exogenous 5-hydroxymethyl-2'-deoxyuridine in human cells in culture using gas chromatography-negative chemical ionization-mass spectrometry

Abstract

The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage. More recently, the corresponding deoxynucleoside, 5-hydroxymethyl-2'-deoxyuridine (HmdU), has proven to be valuable for the introduction of controlled amounts of a single type of damage lesion into the DNA of replicating cells, which is subsequently repaired by the base excision repair pathway. Complicating the study of HmU formation and repair, however, is the known chemical reactivity of the hydroxymethyl group of HmU under conditions used to hydrolyze DNA. In the work reported here, this chemical property has been exploited by creating conditions that convert HmU to the corresponding methoxymethyluracil (MmU) derivative that can be further derivatized to the 3,5-bis-(trifluoromethyl)benzyl analogue. This derivatized compound can be detected by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS) with good sensitivity. Using isotopically enriched exogenous HmdU and human osteosarcoma cells (U2OS) in culture, we demonstrate that this method allows for the measurement of HmU in DNA formed from the incorporation of exogenous HmdU. We further demonstrate that the addition of isotopically enriched uridine to the culture medium allows for the simultaneous measurement of DNA replication and repair kinetics. This sensitive and facile method should prove valuable for studies on DNA oxidation damage and repair in living cells.

Figure 1

GC-MS of derivatized thymine and HmU and identification of major fragments of derivatized HmU, 5-methoxymethyluracil-BTFMBz₂. (A) GC separation of derivatized thymine from HmU. 10 nmol (10⁻⁸ mol) each of thymidine and HmdU were hydrolyzed and derivatized and 2 uL injected into the GC-MS as detailed in Experimental Procedures. (B) Electron ionization mass spectrum of derivatized HmU. (C) Negative chemical ionization of the same compound as in (B). It is unknown which BTFMBz group falls off to produce the two fragments m/z 227 and 381.

Figure 2

GC-MS of a mix of HmdU standards unenriched and enriched with stable-isotopes. HmdU (2.0 pmol), d₂-HmdU (5.0 pmol), and ¹⁵N₂,d₂-HmdU (1.0 pmol) were hydrolyzed and derivatized and 1 uL injected onto GC-MS in NCI mode as detailed in Experimental Procedures. Chromatogram is shown of derivatized unenriched HmU (m/z 381), d₂-HmU (m/z 383), and ¹⁵N₂,d₂-HmU (m/z 385). The retention times of the latter two compounds are slightly earlier than the unenriched compound due to isotope effects from the presence of deuterium atoms.

Figure 3

Toxicity and incorporation of d₂-HmdU in U2OS cells. U2OS cells were treated for 48 h with stable isotope-labeled HmdU (d₂-HmdU). (A) Incorporation of d₂-HmdU into the DNA as compared to thymidine (dThd). DNA was isolated from cells, internal standard was added to DNA (2 μg DNA per sample), and samples were hydrolyzed, derivatized, and analyzed by GC-NCI-MS. Each data point represents three independent plates of cells treated with d₂-HmdU. (B) Toxicity of d₂-HmdU to U2OS cells, given as % viability compared to the untreated control. Toxicity was measured on aliquots of the cells by trypan blue exclusion and counting cells on a hemacytometer. Each data point is an average of one aliquot taken from 3 separate plates of cells treated with d₂-HmdU. Data was fit to a curve using eqn 1 (see Experimental Procedures) for determination of the inhibitory concentration at which 50% of the cells were viable (IC₅₀).

Figure 4

Incorporation and washout of dThd and HmdU by replication and repair. U2OS cells were incubated with 100 μM ¹⁵N-enriched uridine (¹⁵N₂-Urd) for 24 h, then with both 100 μM ¹⁵N₂-Urd and 10μM d₂-HmdU for an additional 24 h. At 48 h, the cells were washed with DPBS and given fresh media containing no isotope labeled compounds. Every 12 h, a plate of cells was harvested and DNA isolated, internal standard was added, and sample was derivatized and analyzed by GC-NCI-MS. (A) Incorporation and washout of ¹⁵N-enriched dThd in DNA. Washout is due to DNA replication only. Exponential growth and decay equations (eqn 2 and 3) were fit to data in SigmaPlot and the half-life of washout was calculated using eqn 6 based on the rate constant obtained from the parameters of the fit. (B) Incorporation and washout of d₂-HmdU. Washout is due to DNA replication plus repair of d₂-HmdU out of the DNA. In addition to data indicating washout both by replication and repair, calculated curves are shown to indicate d₂-HmdU decline by replication alone and d₂-HmdU decline by repair alone. Exponential growth and decay equations (eqn 4 and 5) were fit to data in SigmaPlot and half-lives calculated as mentioned above.

Figure 5

Overall analysis of incorporation, replication, and repair experiment shown in Figure 4. Replication of the DNA is shown as happening once during each 24 h period, with the strands from each round of replication labeled with a different color for clarity. Only one branch from the first round of replication is expanded. At 0 h, 100 μM ¹⁵N₂-Urd is added to the cell media and incorporated into the DNA during the following replication cycle as ¹⁵N₂-Thd (labeled as bold, italic T). At 24 h, 10 μM d₂-HmdU is added in addition to ¹⁵N₂-Urd and both are incorporated into the DNA during the following replication cycle (d₂-HmdU is labeled as bold, italic H). At 48 h, media is replaced with fresh media containing no supplemental ¹⁵N₂-Urd or d₂-HmdU, and the washout of isotope labeled Thd and HmdU is shown over the remaining 48 h. The top branch shows decline of both ¹⁵N₂-Thd and d₂-HmdU from the DNA by replication alone, as only the parental strand retains the isotope labeled compounds, whereas newly replicated strands contain unenriched Thd only. The bottom branch shows decline of ¹⁵N₂-Thd by replication, and d₂-HmdU by replication and repair acting together. DNA replication leads to decline of ¹⁵N₂-Thd and d₂-HmdU by the same amount as seen in the top branch. In addition, one of the two d₂-HmdU residues shown is removed by base excision repair during the first round of replication (from 48 to 72 h – blue H replaced by green T) and the the other is removed during the following round (from 72 to 96 h – blue H replaced by red T).

Figure 6

Base excision of uracil and HmU from oligonucleotides incubated with U2OS cell nuclear extract. Nuclear extracts were prepared and 10 μg of protein were incubated for 60 min at 37°C with 2 pmol ³²P-end labeled 24mer duplex oligonucleotides containing either uracil or HmU in the center position of the labeled strand, both paired opposite adenine (labeled as U:A or H:A, respectively). Cleavage of the oligonucleotide at the position of the modified base is indicated by the appearance of a band at the 12mer position, whereas uncleaved oligonucleotide is shown at the 24mer position. Excision of HmU from the oligonucleotide was observed (lanes 3 and 4), indicating the presence of repair glycosylases in U2OS cells specific for this lesion. Excision of uracil was also observed (lanes 1 and 2), and was included in the figure as a positive control.

Scheme 1

Formic acid hydrolysis of HmU-containing DNA and conversion of both HmU and HmU-formate ester into 5-methoxymethyluracil (MmU).