Quantification of the 2-deoxyribonolactone and nucleoside 5'-aldehyde products of 2-deoxyribose oxidation in DNA and cells by isotope-dilution gas chromatography mass spectrometry: differential effects of gamma-radiation and Fe2+-EDTA.

The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

GC/MS methods to quantify the 2-deoxypentos-4-ulose and 3'-phosphoglycolate pathways of 4' oxidation of 2-deoxyribose in DNA: application to DNA damage produced by gamma radiation and bleomycin.

DNA oxidation plays a substantive role in the pathophysiology of human diseases, such as cancer. While the chemistry of nucleobase lesions has dominated studies of DNA damage, there is growing evidence that the oxidation of 2-deoxyribose in DNA plays a critical role in the genetic toxicology of oxidative stress. As part of an effort to define the spectrum of 2-deoxyribose oxidation products arising in vitro and in vivo, we now describe methods for quantifying products arising from 4' oxidation of 2-deoxyribose in DNA. The chemistry of 4' oxidation partitions between either of two pathways to form either a 2-deoxypentos-4-ulose abasic site (oxAB) or a strand break comprised of a 3'-phosphoglycolate (3PG) residue and a 5'-phosphate, with the release of either malondialdehyde and free base or a base propenal. Highly sensitive gas chromatography/mass spectrometry (GC/MS) methods were developed to quantify both lesions. The abasic site was converted to a 3'-phosphoro-3-pyridazinylmethylate derivative by treatment of the damaged DNA with hydrazine, which was released from DNA as 3-hydroxymethylpyridazine (HMP) by enzymatic hydrolysis. Similarly, 3PG was released as 2-phosphoglycolic acid (PG) by enzymatic hydrolysis. Following HPLC prepurification, both PG and HMP were silylated and quantified by GC/MS, with limits of detection of 100 and 200 fmol and sensitivities of 2 and 4 lesions per 10(6) nucleotides (nt) in 250 microg of DNA, respectively. Following validation of the methods with oligodeoxynucleotides containing the two lesions, the methods were applied to DNA damage produced by bleomycin and gamma radiation. As expected for an agent known to produce only 4' oxidation of DNA, the quantities of 3PG and oxAB accounted for all 2-deoxyribose oxidation events, as indicated by slopes of 0.8 and 0.3, respectively, in plots of the lesion frequency against total 2-deoxyribose oxidation events, with the latter determined by a plasmid-nicking assay. 3PG residues and oxAB were produced at the rate of 32 and 12 lesions per 10(6) nt per microM, respectively. For gamma radiation, on the other hand, 4' oxidation was found to comprise only 13% of 2-deoxyribose oxidation chemistry, with 3% oxAB (4 per 10(6) nt per Gy) and 10% 3PG (13 per 10(6) nt per Gy).

Formation of 1,4-dioxo-2-butene-derived adducts of 2'-deoxyadenosine and 2'-deoxycytidine in oxidized DNA.

Oxidation of deoxyribose in DNA produces a variety of electrophilic residues that are capable of reacting with nucleobases to form adducts such as M(1)dG, the pyrimidopurinone adduct of dG. We now report that deoxyribose oxidation in DNA leads to the formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA. We previously demonstrated that these adducts arise in reactions of nucleosides and DNA with trans-1,4-dioxo-2-butene, the beta-elimination product of the 2-phosphoryl-1,4-dioxobutane residue arising from 5'-oxidation of deoxyribose in DNA, and with cis-1,4-dioxo-2-butene, a metabolite of furan. Treatment of DNA with enediyne antibiotics capable of oxidizing the 5'-position of deoxyribose (calicheamicin and neocarzinostatin) led to a concentration-dependent formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA, while the antibiotic bleomycin, which is capable of performing only 4-oxidation of deoxyribose, did not give rise to the adducts. The nonspecific DNA oxidant, gamma-radiation, also produced the adducts that represented approximately 0.1% of the 2-phosphoryl-1,4-dioxobutane residues formed during the irradiation. These results suggest that the oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA could represent endogenous DNA lesions arising from oxidative stresses that also give rise to other DNA adducts.

5'-(2-phosphoryl-1,4-dioxobutane) as a product of 5'-oxidation of deoxyribose in DNA: elimination as trans-1,4-dioxo-2-butene and approaches to analysis.

Oxidation of deoxyribose in DNA leads to the formation of a spectrum of electrophilic products unique to each position in the sugar. For example, chemical reactions following abstraction of the C5'-hydrogen atom partition to form either a nucleoside 5'-aldehyde residue attached to the 5'-end of the DNA strand or a 5'-formyl phosphate residue attached to the 3'-end of the DNA strand that is accompanied by a four-carbon fragment on the 5'-end. We now present two approaches that both identify the latter fragment as 5'-(2-phosphoryl-1,4-dioxobutane) and provide a means to quantify the formation of this residue by different oxidizing agents. The first approach involves oxidation of DNA followed by reaction with O-benzylhydroxylamine to form stable dioxime derivatives of the putative 5'-(2-phosphoryl-1,4-dioxobutane) residues. The beta-elimination product of this dioxime proved to be the expected trans-1,4-dioxo-2-butene, as judged by gas chromatographic and mass spectrometric (GC/MS) comparison to authentic dioximes of cis- and trans-1,4-dioxo-2-butene, which revealed a unique pattern of three signals for each isomer, and by X-ray crystallography. Using a benzylhydroxylamine dioxime derivative of [2H4]-labeled cis-1,4-dioxo-2-butene as an internal standard, the dose-response for the formation of 5'-(2-phosphoryl-1,4-dioxobutane) was determined to be linear for gamma-radiation, with approximately 6 lesions per 10(6) nt per Gy, and nonlinear for Fe2+-EDTA. A comparison of 5'-(2-phosphoryl-1,4-dioxobutane) formation to total deoxyribose oxidation suggests that gamma-radiation produces approximately 0.04 lesions per deoxyribose oxidation event. As a positive control for 5'-oxidation of deoxyribose, the enediyne calicheamicin was observed to produce 5'-(2-phosphoryl-1,4-dioxobutane) at the rate of approximately 9 lesions per 10(6) nt per microM. A second approach to identifying and quantifying the sugar residue involved derivatization with hydrazine and beta-elimination to form pyridazine followed by quantification of the pyridazine by GC/MS. Using this approach, it was observed that the enediyne, neocarzinostatin, produced a linear dose-response for pyridazine formation, as expected given the ability of this oxidant to cause 1'-, 4'-, and 5'-oxidation of deoxyribose in DNA. The antitumor antibiotic, bleomycin, on the other hand, produced pyridazine at a 10-fold lower rate, which is consistent with 4'-chemistry as the predominant mode of deoxyribose oxidation by this agent. These results provide novel insights into the chemistry of deoxyribose oxidation in DNA and two approaches to quantifying the 5'-(2-phosphoryl-1,4-dioxobutane) precursor of trans-1,4-dioxo-2-butene, an electrophile known to react with nucleobases to form novel DNA adducts.

A general synthesis of specifically deuterated nucleotides for studies of DNA and RNA.

An efficient procedure is described for the preparation of ribonucleotides and deoxyribonucleotides with deuterium incorporated at the 1', 4', or 5' position. Three intermediates-[1-2H]-D-ribose, [4-2H]-D-ribose, and [5-2H(2)]-D-ribose-were prepared by chemical synthesis and subsequently converted to ribonucleotides and deoxyribonucleotides via enzymatic reactions. Milligram quantities of the desired products were obtained with an average deuterium content of 96+/-1%.