Chem. Res. Toxicol. 2000, 13, 1259-1264
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Quantification of Etheno-DNA Adducts Using Liquid Chromatography, On-Line Sample Processing, and Electrospray Tandem Mass Spectrometry Daniel R. Doerge,* Mona I. Churchwell, Jia-Long Fang, and Frederick A. Beland Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079 Received July 19, 2000
Etheno-DNA adducts are promutagenic lesions present in normal animal and human tissues that are believed to be important in the etiology of cancer related to diet and lifestyle. A method has been developed for the quantification of trace levels of etheno-DNA adducts using on-line sample preparation coupled with liquid chromatography and electrospray tandem mass spectrometry. The use of automated solid-phase extraction and stable labeled internal standards permitted the robust determination of ethenodeoxyadenosine contained in crude DNA hydrolysates from untreated rodent and human tissues at levels on the order of one adduct in 108 normal nucleotides from 100 µg of DNA. Inherent analyte response and matrix interference made sensitivity for simultaneous determination of ethenodeoxycytidine approximately 5-fold lower. The method was applied to the analysis of liver DNA from untreated and urethanetreated B6C3F1 mice, untreated rat liver, human placenta, and several commercial DNA preparations. Some sources of potential artifactual formation of etheno-DNA adducts were investigated.
Introduction The etheno derivatives of guanine (1,N2- and N2,3etheno-2-deoxyguanosine), adenine [1,N6--2′-deoxyadenosine (-dA)1], and cytosine [3,N4-etheno-2′-deoxycytidine (-dC)], shown in Figure 1, are promutagenic lesions in DNA (1). These adducts are produced by either endogenous metabolic processes [e.g., lipid peroxidation (2)] or by metabolic activation of exogenous chemicals [e.g., vinyl chloride (3) or urethane (4)]. Etheno-DNA adducts are persistent in target tissues of experimental animals (3, 5), and it is likely that these adducts play an important role in human carcinogenesis caused by diet and lifestyle factors (2). The 32P-postlabeling technique, in conjunction with immunoaffinity purification, has been the principal analytical method used for assessment of etheno adducts with dA and dC (6, 7). Using this method, etheno-DNA adducts have been reported at levels of fewer than one adduct in 109 normal nucleotides using 10-50 µg of DNA. More recently, LC with electrospray/MS detection has been used to quantify -dA and -adenine (8) and -guanine (9). However, the lower sensitivity of MS detection relative to 32P-postlabeling, albeit with higher specificity, required the use of milligram quantities of human DNA for measuring adduct levels in the range of one in 107106 normal nucleotides in these studies. * To whom correspondence should be addressed. Telephone: (870) 543-7943. Fax: (870) 543-7720. E-mail:
[email protected]. 1 Abbreviations: BW, body weight; dA, 2′-deoxyadenosine; dC, 2′deoxycytidine; dG, 2′-deoxyguanosine; -dA, etheno-2′-deoxyadenosine; -dC, etheno-2′-deoxycytidine; IAC, immunoaffinity chromatography; LOD, limit of detection (S/N ) 3); LOQ, limit of quantitation (S/N ) 10); M1dG, pyrimido[1,2-R]purine-10(3H)-one-2′-deoxyribose; MRM, multiple reaction monitoring; RSD, relative standard deviation (SD/ mean × 100).
10.1021/tx0001575
Figure 1. Structures of etheno-DNA adducts. Asterisks indicate the labeled sites in the internal standards.
Urethane (ethyl carbamate) is carcinogenic in many organs (e.g., liver and lung) in numerous species of experimental animals (e.g., rodents and non-human primates) and has been classified as a possible/probable human carcinogen by the International Agency for Research on Cancer and the U.S. Environmental Protection Agency (10). Urethane carcinogenicity is mediated by conversion to vinyl carbamate (11) which is catalyzed by P450 2E1 (12). Because ethanol both induces and acts as a substrate for P450 2E1, the possibility exists for ethanol to affect urethane carcinogenicity. Human exposure to urethane results principally from consumption
This article not subject to U.S. Copyright. Published 2000 by the American Chemical Society Published on Web 11/15/2000
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Chem. Res. Toxicol., Vol. 13, No. 12, 2000
of bread and alcoholic beverages (13, 14). For these reasons, the U.S. Food and Drug Administration has nominated urethane for carcinogenicity testing in a chronic mouse bioassay through the National Toxicology Program, with an added evaluation of the effects of ethanol co-administration. A critical requirement for this study of urethane carcinogenesis was an analytical method capable of conveniently measuring, with high specificity, small amounts of urethane-derived etheno-DNA adducts in target tissues. This paper describes the development and validation of a method for on-line, automated processing of hydrolyzed DNA from in vitro modification using chloroacetaldehyde, tissues of experimental animals, and human placenta coupled with an LC separation and tandem mass spectrometric detection using multiplereaction monitoring (MRM).
Experimental Section Reagents. The synthesis of -dA and -dC was achieved by the method of Green and Hathway (15) and purified by HPLC. Synthesis of -dG was attempted, but no stable product was isolated. Normal nucleosides, synthetic oligonucleotides, purified DNA samples, and all other biochemical reagents were obtained from Sigma Chemical Co. (St. Louis, MO) and used as provided. DNA Modified in Vitro. Calf thymus DNA in 100 mM sodium cacodylate buffer (pH 7.5) reacted overnight with several chloroacetaldehyde to DNA nucleotide concentration ratios (1.85 × 10-5, 1.85 × 10-4, and 1.85 × 10-3) according to the method of Young and Santella (16). The DNA was precipitated with ethanol and sodium chloride, washed with 70% ethanol, and dissolved in 5 mM Bis-Tris, 0.1 mM EDTA buffer (pH 7.1). The DNA concentration and purity were measured spectrophotometrically using 260 nm absorbance and the 260 nm/280 nm ratio, respectively. DNA Modified in Vivo. Male and female 8-week-old B6C3F1 mice (four per sex per group) from the NCTR colony and maintained on NIH-31 diet were administered 0 or 90 ppm urethane in their drinking water for 28 days and then sacrificed by exposure to carbon dioxide. The livers and lungs were removed and nuclei isolated (17), and DNA was prepared (18) and dissolved in 5 mM Bis-Tris, 0.1 mM EDTA buffer (pH 7.1) for analysis. Human placenta DNA samples were a gift from P. Chiarelli (University of Loyola, Chicago, IL). Rat liver was removed from a 13-week-old female Sprague-Dawley rat from the NCTR colony. The human placental and rat liver DNA samples were prepared in a manner similar to that described for the mouse samples. Enzymatic Hydrolysis of DNA. The in vitro- and in vivomodified DNA samples (∼100 µg in a total volume of 100-200 µL) were quantified spectrophotometrically and then spiked with the internal standards (10-20 pg). Initially, the DNA was hydrolyzed enzymatically to nucleotides with DNase I, followed by snake venom phosphodiesterase and alkaline phosphatase (19). Because unsatisfactory results were obtained (see the Results), subsequent hydrolyses were conducted with micrococcal nuclease, spleen phosphodiesterase, and nuclease P1. Specifically, the samples were incubated with 2 units of micrococcal nuclease (Sigma) and 0.2 unit of spleen phosphodiesterase (Sigma) for 4 h at 37 °C in 20 mM sodium succinate, 10 mM calcium chloride (pH 6) buffer. Before use, these enzymes were dialyzed against water as described by Randerath et al. (20). Complete hydrolysis of the DNA to nucleotides was verified using HPLC/UV analysis. Nuclease P1 (5 µg, Sigma) was then added, and the hydrolysis was continued for an additional 2 h. The dA present in each hydrolysate was converted to deoxyinosine by incubation with adenosine deaminase (Sigma). It was determined that 0.01 unit per sample was sufficient to hydrolyze >99% dA in 0.5 h (not shown) and that -dA and -dC were
Doerge et al. stable under these conditions. Complete hydrolysis of the DNA to nucleosides was verified using HPLC/UV analysis. The etheno-DNA adducts were quantified by direct injection of incubations into the LC system. Liquid Chromatography. The liquid handling system consisted of an autosampler (AS3500, Dionex, Sunnyvale, CA), two automated switching valves (TPMV, Rheodyne, Cotati, CA), and two HPLC pumps (a Dionex GP40 quaternary gradient pump and a Hewlett-Packard 1050 pump, Palo Alto, CA). Valve 1 allowed the quaternary pump eluent to either load a sample onto the trap column and then wash it or to bypass the trap column and clean the analytical column (see ref 21 for details). Valve 2 was used to divert the trap column effluent to either waste or the analytical column. The quaternary pump was used for sample injection, cleanup, and regeneration of the trap and analytical columns; the isocratic pump, containing a 90% H2O/ 10% acetonitrile mixture, was used to backflush the trap column to the analytical column during analysis and to keep a constant flow of mobile phase going into the mass spectrometer during sample loading and preparation periods. The sample was loaded and washed for 4.5 min at a rate of 1 mL/min with 100% H2O onto a reverse phase trap column [Luna C18(2), 2 mm × 30 mm, 3 µm, Phenomenex, Torrance, CA], and then the trap column was washed with a 95% H2O/ 5% acetonitrile mixture for 1.5 min at a rate of 1 mL/min to waste. After valve 2 was switched, the concentrated sample zone was backflushed from the trap column onto the analytical column [Luna C18(2), 2 mm × 150 mm, 3 µm, Phenomenex] at a rate of 0.2 mL/min with a 90% H2O/10% acetonitrile mixture, and the sample components were eluted into the mass spectrometer. When the 12 min run was finished, valve 2 was switched and the trap column was cleaned with a 95% acetonitrile/5% H2O mixture for 2 min at a rate of 1 mL/min to waste. Next, valve 1 was switched, and the analytical column was cleaned with a 95% acetonitrile/5% H2O mixture for 2 min at a rate of 0.2 mL/min. Then both valves were switched to their initial positions to equilibrate both the trap and analytical columns at the starting mobile phase compositions, and the process was repeated. Mass Spectrometry. A Quattro LC triple-quadrupole mass spectrometer (Micromass, Manchester, U.K.) equipped with an ES interface was used with a source block temperature of 150 °C and a desolvation temperature of 450 °C. Nitrogen gas was used as the desolvation gas (750 L/h) and nebulizing gas (90 L/h). Argon was used as the collision gas, at a collision cell pressure of 1.5 × 10-3 mbar. Positive ions were acquired in MRM mode (dwell time of 0.3 s, span of 0.02 Da, and interchannel delay of 0.03 s) for the (M + H)+ to BH2+ transitions for both -dA (m/z 276 to 160) and -dC (m/z 252 to 136) and the internal standards [15N5]--dA (m/z 281 to 165) and [15N3]-dC (m/z 255 to 139). The cone voltage was 25 V for the -dA transitions and 20 V for the -dC transitions. The collision energy was 15 eV for all four transitions. Preparation and Characterization of [15N]-E-dA and [15N]-E-dC Internal Standards. Uniformly labeled [15N5]dA and [15N3]dC were obtained from Cambridge Isotope Laboratories (Andover, MA). Etheno derivatives were prepared by reaction with chloroacetaldehyde and purification using preparative HPLC. The chemical concentrations were determined by using LC with UV detection (260 nm) in comparison with authentic unlabeled analogues. The isotopic distribution was determined using full scan LC-ES/MS (m/z 100-600). The -dA was 93.8% 15N5, 5.9% 15N4, and 0.3% 15N3; no unlabeled -dA was detected (
