Ultrasensitive UPLC-MS-MS Method for the Quantitation of Etheno-DNA Adducts in Human Urine

Etheno-DNA adducts are generated from the metabolism of exogenous carcinogens and endogenous lipid peroxidation. We and others have previously reported that 1,N6-ethenodeoxyadenosine (εdA) and 3,N4-ethenodeoxycytidine (εdC) are present in human urine and can be utilized as biomarkers of oxidative stress. In this study, we report a new ultrasensitive UPLC-ESI-MS/MS method for the analysis of εdA and εdC in human urine, capable of detecting 0.5 fmol εdA and 0.3 fmol εdC in 1.0 mL of human urine, respectively. For validation of the method, 20 human urine samples were analyzed, and the results revealed that the mean levels of εdA and εdC (SD) fmol/µmol creatinine are 5.82 ± 2.11 (range 3.0–9.5) for εdA and 791.4 ± 328.8 (range 116.7–1264.9) for εdC in occupational benzene-exposed workers and 2.10 ± 1.32 (range 0.6–4.7) for εdA and 161.8 ± 200.9 (range 1.8–557.5) for εdC in non-benzene-exposed workers, respectively. The ultrasensitive detection method is thus suitable for applications in human biomonitoring and molecular epidemiology studies.


Introduction
Etheno-DNA adducts 1,N 6 -ethenodeoxyadenosine (dA) and 3,N 4 -ethenodeoxycytidine (dC) are formed not only from exogenous carcinogens vinyl chloride and urethane [1][2][3][4], but also peroxidation of arachidonic acid and liver microsomal membranes in the presence of LPO-inducing compounds [5][6][7]. These miscoding lesions are elevated in various diseases, such as Wilson's disease and primary hemochromatosis induced by excess metal storage or chronic inflammation and infections [8,9]. A very high -6 polyunsaturated fatty acid diet strongly increased etheno-DNA adduct levels in white blood cells, particularly in female subjects [10]. Mice injected with RcsX cells, which cause overexpression of inducible nitric oxide synthase (iNOS), exhibited six-fold higher etheno adduct levels in the spleen DNA compared with the controls [11]. Etheno-DNA adducts were also found in the affected tissue of chronic pancreatitis patients [12]. Our recent study found that 4-hydroxy-estradiol metabolite formation and high -6 PUFA intake were both linked to increased formation of LPO-derived adducts in white blood cells of premenopausal women [13]. A recent study first detected the presence of such etheno-modified 5mdC residues (5mdC) in human tissue DNA [14]. These observations suggested that etheno-DNA adducts accumulate in cancer-prone tissues as a result of chronic inflammatory processes causing oxidative stress and LPO.
Non-invasive detection methods, such as urinalysis, will expedite studies in humans aimed to elucidate etiopathological factors that cause oxidative DNA damage. For these reasons, Nair and others have established methods to study the formation and excretion of etheno-DNA base adducts; dA and dC were found to be present in human urine, and these could be biomarkers for DNA damage produced by persistent oxidative stress and lipid peroxidation [15][16][17]. The method, involving immunoprecipitation, high-performance liquid chromatography and fluorescence detection, has been applied to evaluate the effects of some dietary factors on the formation of urinary dA in non-smoking post-menopausal women [18]. Chen et al. developed a gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) method for the detection of 3,N 4 -ethenocytosine (C) and 1,N 6 -ethenoadenine (A) in human urine [19,20]. A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method had been developed for urinalysis compared with a GC/NICI/MS method [21]. We developed a 32 P-postlabeling/TLC method for analysis of dC using a multi-substrate deoxyribonucleoside kinase enzyme in small amounts of human urine [22]. The present method, although highly sensitive, requires the use of a radioisotope or considerable time and effort.
We now report a new ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method for the analysis of dA and dC adducts in human urine. For validation of the method, 20 human urine samples randomly selected from female workers who participated in a cross-sectional epidemiology study of occupational benzene exposure were assayed, and the results indicate that our ultrasensitive method appears to be superior to other methods reported in terms of: (1) high sensitivity and specificity; (2) low amounts of urine sample required; (3) capability to detect background levels of etheno-DNA adducts in human biological samples; and (4) reliable monitoring of the disease-related increase of these substances in patients. We present the details of the method protocol and its application to quantify dA and dC in human urine samples.

Synthesis of Unlabeled 3,N 4 -Etheno-2'-Deoxycytidine (dC)
Synthesis and purification of unlabeled dC were followed by the reported procedure described above. Quantification of dC was performed by UV spectrometry as a standard [26].

Preparation of Urine Samples
Preparation of urine samples followed our reported procedure with minor modifications [22]. Briefly, 1.0-mL urine samples were filtered through a 0.22-micron filter and were spiked with 100 pg of [ 15 N5]-dA and [ 15 N3]-dC as internal standards (I.S.). The urinary protein was precipitated by adding 1.5 mL cold ethanol and centrifuged at 4000 rpm for 10 min after standing at -20 °C for 30 min. The supernatant (2.5 mL) was moved to a new tube and concentrated to dryness by vacuum centrifugation after protein precipitation. The dried urine sample was redissolved in 0.5 mL of ddH2O and loaded onto a C18 OH solid-phase silica column (BondElut R , Agilent Technologies, 500 mg, 3 mL, USA). The columns were washed with 12 mL of water followed by 3 mL of 10 % methanol (v/v) and 3 mL of 15% methanol (v/v) in order to remove the bulk of normal nucleosides. The columns were then eluted twice with 2.5 mL of 30% methanol (v/v). Before sample loading, the columns were pre-washed with 15 mL of methanol followed by 15 mL of water. The elute was concentrated to dryness by vacuum centrifugation.

Urinary Analysis of Etheno-DNA Adducts
Purification and enrichment of dA and dC in urine samples were achieved using a solid-phase silica column.

Reproducibility of UPLC-MS-MS Method
The UPLC-MS/MS method was validated with respect to the intra-assay and inter-assay. A control urine sample was prepared by diluting 50:50 (v/v) with a urine sample of non-benzene-exposed workers and water. The control urine sample was spiked with 100 pg of [ 15 N5]-dA, [ 15 N3]-dC as I.S. These single urine samples were prepared following adduct enrichment by loading a C18 OH solid phase silica column after protein precipitation. These single urine samples were injected into the UPLC-MS/MS at the same time for the intra-assay. The recovery level of [

Recovery of UPLC-MS-MS Analysis
The

Etheno-DNA Adducts Detected in Human Urine
To validate this UPLC-MS-MS method, 20 urine samples randomly selected from female workers who participated in a cross-sectional epidemiology study of occupational benzene exposure were assayed. The typical autoradiograms of dA and dC detected in human urine samples are shown in Figure 3. The results revealed a significant difference between levels of εdA (SD) fmol/µmol creatinine, which are 5.82  2.11 (range 3.0-9.5) in female benzene-exposed (BF) workers and 2.10  1.32 (range 0.6-4.7) in female non-benzene-exposed (CF) workers (p = 0.0015). The levels of εdC (SD) fmol/µmol creatinine are 791.4  382.8 (range 116.7-1264.9) in benzene-exposed workers and 161.8  200.9 (range 1.8-557.5) in non-benzene-exposed workers, for which significant differences were also found (p = 0.0013) ( Table 3).  Table 3. Levels of etheno-DNA adducts (dA and dC) in human urine of occupational benzene-exposed and non-exposed workers (fmol/mol creatine) *.

Discussion
Increased oxidative stress and lipid peroxidation are implicated in multistage carcinogenesis. Etheno adducts are formed in DNA bases after reaction with aldehydes, such as trans-4-hydroxy-2-nonenal (HNE), generated during oxidative stress as lipid peroxidation-end products. These DNA adducts are believed to be important in the etiology of cancer [1][2][3][4][5][6]. Existing methods for quantifying DNA adducts use 32 P-postlabeling; although highly sensitive, postlabeling requires the use of an energetic radioisotope and considerable time and effort [15,22,23]. The LC-MS/MS methodology reported everywhere permits automated quantification of trace levels of DNA adducts. Therefore, we now report a new ultrasensitive UPLC-MS/MS method for the analysis of dA and dC adducts in human urine. Our method is based on the effective purification and enrichment of dA and dC in human urine samples; hereby, solidphase silica column chromatography has been developed that allows an efficient separation of dA and dC adducts from the urinary matrix. The absolute sensitivities of our method were found to be 0.5 fmoles of εdA and 0.3 fmoles of dC detectable in 1.0 mL of human urine, thus being a magnitude higher than the currently reported GC/NICI/MS method with a sensitivity of 12 fmoles of dC in 0.1 mL urine [20]. For purification and enrichment of etheno-DNA adducts from normal nucleosides in human urine, Bond Elut LC18-OH (Agilent, 500 mg, 3 mL) and Oasis HLB (Waters, 220 mg, 6 mL) silica-phase columns were used, respectively, to achieve an efficient enrichment of etheno-DNA adducts from human urine. The Bond Elut LC18-OH silica-phase column was shown to be valid for achieving a better recovery of etheno-DNA adducts from urine matrix. For validating the reliability of our method, [ 15 N5]-εdA and [ 15 N3]-εdC were added to each urine sample as internal standards, allowing correction of the recovery. Results on the intra-and inter-assay variability revealed a high reproducibility and accuracy. Therefore, our ultrasensitive method appears to be superior to other methods reported in terms of: (1) high sensitivity and specificity; (2) low amounts of urine sample required; (3) capability to detect background levels of etheno adducts in human urine; and (4) high-throughput and reliable monitoring of the diseaserelated increase of these substances in patients.
A series of studies in animals and humans have demonstrated that etheno-DNA adducts are among the ideal markers for DNA damage produced endogenously as a result of persistent oxidative stress and lipid peroxidation (LPO) [9][10][11][12][13][14]. There is increasing evidence for a role of reactive oxygen species and lipid peroxidation in the etiology of human cancers and the development of other chronic degenerative diseases. The development and application of sensitive and specific detection methods for this class of DNA-adducts should provide valuable tools for investigating their role in human disease pathogenesis and its preventability.