High-Performance Liquid

of M1G) with a known modification level as an internal standard. This improved ... it is relevant to determine whether this M1G adduct, and possibly t...
0 downloads 0 Views 121KB Size
Download PDF
1032

Chem. Res. Toxicol. 1998, 11, 1032-1041

An Improved 32P-Postlabeling/High-Performance Liquid Chromatography Method for the Analysis of the Malondialdehye-Derived 1,N2-Propanodeoxyguanosine DNA Adduct in Animal and Human Tissues Ping Yi, Xin Sun, Daniel R. Doerge, and Peter P. Fu* National Center for Toxicological Research, Jefferson, Arkansas 72079 Received March 12, 1998

Malondialdehyde (MDA) is a major lipid peroxidation product that is mutagenic and tumorigenic. The MDA-modified DNA adduct, 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-R]purin-10(3H)-one (M1G), has been detected in human tissues and may be a marker of human cancer risk. In this paper, we describe an improved 32P-postlabeling/HPLC method for sensitive detection and quantitation of this MDA-modified 2′-deoxyribonucleotide adduct. Specific improvements include (i) unequivocal structural identification of the postlabeling products, both the 3′,5′-bisphosphate of M1G (MDA-3′,5′-dGDP) and the 5′-monophosphate of M1G (MDA-5′-dGMP); (ii) efficient separation of the 32P-postlabeling products by HPLC; and (iii) the incorporation of a synthetically prepared MDA-modified DNA (or the 3′-monophosphate of M1G) with a known modification level as an internal standard. This improved quantitative methodology provides high intra- and inter-assay reproducibility and has been applied to the analysis of this adduct in rodent and human samples.

Introduction Lipid peroxidation can be induced endogenously or exogenously (1-11). Peroxidation of endogenous polyunsaturated fatty acids is a chain reaction that initiates the formation of carbon-centered radicals, which react with molecular oxygen to produce peroxy radicals. Peroxy radicals can in turn propagate the same sequence by abstraction of the hydrogen from another lipid molecule contained in polyunsaturated fatty acids, and the subsequent reactions result in the formation of primary lipid radicals, hydroperoxides, and alkoxy radicals (7, 8). Upon disproportionation, the alkoxy radicals generate nonvolatile and volatile end products (low-molecular weight end products) (7). A number of these end products, such as malondialdehyde, formaldehyde, acetaldehyde, acrolein, crotonaldehyde, and 4-hydroxy-2-nonenal (HNE),1 have been found to be highly toxic to cells (4, 6, 7, 12, 13), cause DNA strand breaks (13, 14) and DNAprotein cross-linking (15), form DNA adducts (11, 1534), and be mutagenic in bacterial systems and tumorigenic in experimental animals (7, 19, 35-46). Malondialdehyde (MDA), also named malonaldehyde or propanedial, and HNE are the most studied end * To whom correspondence should be addressed. Telephone: (870) 543-7207. Fax: (870) 543-7136. E-mail: [email protected]. 1 Abbreviations: MDA, malondialdehyde; HNE, 4-hydroxy-2-nonenal; M1G adduct, 3-(2-deoxy-β-D-erythro-pentafuranosyl)pyrimido[1, 2-R]purin-10(3H)-one; MDA-3′-dGMP, 3′-monophosphate of M1G; 3′dGMP, 2′-deoxyguanosine 3′-monophosphate (ammonium salt); 5′dGMP, 2′-deoxyguanosine 5′-monophosphate; 3′-dCMP, 2′-deoxycytidine 3′-monophosphate; 3′-dTMP, 2′-deoxythymidine 3′-monophosphate; 3′-dAMP, 2′-deoxyadenosine 3′-monophosphate; MDA-3′,5′-dGDP, 3′,5′-bisphosphate of M1G; MDA-5′-dGMP, 5′-monophosphate of M1G; ATP, adenosine 5′-triphosphate (disodium salt); MN, micrococcal nuclease; SPD, spleen phosphodiesterase; PNK, cloned T4 polynucleotide kinase; NCTR, National Center for Toxicological Research.

S0893-228x(98)00049-6

Figure 1. Structure and abbreviation of 3-(2-deoxy-β-D-erythropentafuranosyl)pyrimido[1,2-R]purin-10(3H)-one.

products (7, 8). MDA, a difunctional and highly reactive electrophile, has been shown to participate in a variety of chemical and biological electrophilic reactions, including covalent binding to protein, RNA, and DNA. The MDA-modified DNA adduct is one of the most studied endogenous DNA adducts (19, 24, 33, 47-50). A major MDA-modified DNA adduct, identified as 3-(2-deoxy-βD-erythro-pentofuranosyl)pyrimido[1,2-R]purin-10(3H)one (M1G) (Figure 1), has been detected in human liver, breast, white blood cells, and pancreas (19, 24, 33, 51). It has also been reported that this adduct is a mutagenic lesion in Escherichia coli (52). Thus, it is probable that this adduct may induce cancer in rodents treated with exogenous chemicals that induce lipid peroxidation and may also cause cancer initiation in humans. Therefore, it is relevant to determine whether this M1G adduct, and possibly the other endogenous DNA adducts, such as those derived from acetaldehyde (32, 34), crotonaldehyde

This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society Published on Web 08/05/1998

32P-Postlabeling/HPLC

Analysis of MDA-DNA Adducts

(29), and HNE (17, 20, 25, 53), can be used as biomarkers of cancer risk. Consequently, development of sensitive and reliable analytical methodology for detection and quantification of the MDA-modified DNA adduct contained in DNA of animal and human samples is highly useful for risk assessment and for molecular epidemiological studies. Marnett and co-workers have reported the development of a sensitive mass spectrometric method for detection and quantification of this M1G adduct in animal and human samples (19, 51). 32P-Postlabeling/TLC and 32 P-postlabeling/HPLC techniques have also been reported for detection and quantification of this adduct contained in animal and human tissues (24, 33). In this paper, we report an improved 32P-postlabeling/HPLC method that we can use to efficiently separate and accurately quantify this adduct. To validate this improved 32P postlabeling method, separation and quantification of the M1G adduct contained in DNA of rat and mouse liver samples and human liver, pancreas, and lung samples are also presented.

Materials and Methods Caution: MDA has been determined to be highly cytotoxic, mutagenic, and carcinogenic in animal bioassays. Therefore, appropriate safety procedures should be followed when working with this compound. Materials. MDA-bisdiethyl acetal (1,1,3,3-tetraethoxypropane, TEP) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Proteinase K, ribonuclease A type II A, calf thymus DNA (sodium salt, type I), 2′-deoxyguanosine 3′-monophosphate (ammonium salt) (3′-dGMP), 2′-deoxycytidine 3′-monophosphate (3′-dCMP), 2′-deoxyadenosine 3′-monophosphate (3′-dAMP), 2′deoxythymidine 3′-monophosphate (3′-dTMP), 2′-deoxyguanosine 5′-monophosphate (5′-dGMP), adenosine 5′-triphosphate (disodium salt) (ATP), nuclease P1, micrococcal nuclease (MN), spleen phosphodiesterase (SPD), bicine, spermidine, and dithiothreitol were purchased from Sigma Chemical Co. (St. Louis, MO). Cloned T4 polynucleotide kinase (PNK) was from U.S. Biochemical Corp. (Cleveland, OH). Commercial calf thymus DNA was purified as previously described (54). Triethylammonium acetate (0.1 M) was prepared by titrating a triethylamine solution with acetic acid to pH 5.0. All solvents used were HPLC grade. Male B6C3F1 mice (16-day-old) and F344 rats (27-month-old) were obtained from the National Center for Toxicological Research (NCTR) breeding colony. DNA of mouse and rat livers was extracted following the procedures previously reported by Beland et al. (55). Human liver, pancreas, and lung DNA samples were provided by F. Kadlubar (NCTR). The isolated DNA was dissolved in distilled water, and the DNA concentration and purity were analyzed spectrophotometrically. The concentration of DNA was determined by the peak height at 260 nm. The ratios of A260/A280 and A230/A260 were 1.7-1.8 and ∼0.4, respectively. After enzymatic hydrolysis of DNA by MN/SPD to 3′-monophosphate 2′-deoxynucleotides, RNA contamination was analyzed by reversed-phase HPLC. Under experimental conditions, no 3′-monophosphate ribonucleotide contamination was detected. Synthesis and Quantification of the MDA-5′-dGMP Adduct. The 5′-monophosphate deoxyguanosinyl adduct of MDA (MDA-5′-dGMP) was synthesized following the procedures described previously for the synthesis of the 3′-monophosphate of M1G (MDA-3′-dGMP) adduct (47, 49, 56) with modification. The MDA, obtained from acid-catalyzed hydrolysis of MDA-bisdiethyl acetal, was reacted with 400 mg (1.2 mmol) of 5′-dGMP in 40 mL of 100 mM KH2PO4 (pH 4.5) at 37 °C for 4 days. The reaction mixture was purified by passage through an ion-exchange column, SAX trimethylaminopropyl Bond Elut (Analytichem International, 60 cm3/10 g), eluted with

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1033 50 mM ammonium formate (pH 3.5). Further elution with 3.5 M ammonium formate (pH 3.5) gave a fraction containing the yellow fluorescent MDA-5′-dGMP adduct. The crude MDA5′-dGMP product was purified twice by ion-exchange HPLC on a Hypersil 5 µm SAX column (Phenomenex, 10 mm × 250 mm) eluted with 50 mM KH2PO4 (pH 4.5). The collected MDA-5′dGMP adduct was further purified by reversed-phase HPLC using a Prodigy ODS column (Phenomenex, 4.6 mm × 250 mm, diameter of 5 µm) eluted with a gradient solvent system (gradient program I): 0-10 min, 20 mM NaH2PO4 (pH 4.5) isocratically; 10-40 min, 0 to 20% of methanol in 20 mM NaH2PO4 (pH 4.5); and 40-50 min, 20 to 30% methanol in 20 mM NaH2PO4 (pH 4.5) at a flow rate of 1 mL/min. To remove the salt present in the adduct, the MDA-5′-dGMP was further purified by reversed-phase HPLC on a Prodigy ODS column (Phenomenex, 4.6 mm × 250 mm, diameter of 5 µm) eluted with water for 30 min followed by methanol at a flow rate of 1 mL/ min. The structure of the purified MDA-5′-dGMP (purity of >99%) was verified using electrospray LC/MS analysis. Proton NMR was used to quantify this adduct (88 µg), on the basis of the response of a known amount of internal standard. On the basis of the determined quantity and the absorbance of the UV/visible absorption spectrum measured in methanol, the 254nm (UV molar extinction coefficient) of MDA-5′-dGMP was determined to be 7.81 × 104 M-1 cm-1. Synthesis and Quantification of the MDA-3′-dGMP Adduct. The 3′-monophosphate adduct of M1G (MDA-3′dGMP), previously synthesized in our laboratory (47), was prepared again on a scale (50 mg of 3′-dGMP) needed for developing the method. Under the assumption that the 254nm UV molar extinction coefficients of the MDA-3′-dGMP and MDA-5′-dGMP adducts are practically identical, the quantity of the purified MDA-3′-dGMP adduct was 10 µg and the reaction yield was determined accordingly. 5′-Phosphorylation of MDA-3′-dGMP and Identification of the 3′,5′-Bisphosphate of M1G (MDA-3′,5′-dGDP). Phosphorylation of MDA-3′-dGMP with nonradioactive ATP and PNK in a quantity more than 106-fold higher than that of the regular 32P-postlabeling was performed. Thus, about 6.5 nmol of MDA-3′-dGMP dissolved in 20 µL of distilled water was reacted with 20 µL of a PNK mixture containing 0.4 µmol of ATP, 150 units of PNK, 40 mM bicine-NaOH (pH 9.6), 20 mM MgCl2, 2 mM spermidine, and 20 mM dithiothreitol at 37 °C for 40 min. The reaction mixture was injected onto a Prodigy 5 µm ODS column (Phenomenex, 4.6 mm × 250 mm) and eluted with a gradient solvent system (gradient program I) described above. The fraction containing MDA-3′,5′-dGDP was collected and the structure identified by UV/visible absorption and electrospray mass spectral analysis. 3′-Dephosphorylation of MDA-3′,5′-dGDP. The MDA3′,5′-dGDP adduct (about 4 nmol) was 3′-dephosphorylated by incubation with 125 units of nuclease P1 in 50 µL of nuclease P1 buffer (1 mM ZnCl2 and 120 mM sodium acetate at pH 5.0) at 37 °C for 5 h. The reaction mixture was separated by reversed-phase HPLC, using the same conditions (gradient program I) described above. Chemical Reaction of MDA with Calf Thymus DNA. To obtain different levels of MDA-modified DNA, 15 mg of purified calf thymus DNA in 10 mL of distilled water was reacted with different amounts of MDA from hydrolysis of TEP at 37 °C for 4-24 h. After incubation, the reaction mixture was extracted twice with 10 mL of chloroform/isoamyl alcohol (v/v, 24/1). The DNA was precipitated by adding 20 mL of cold ethanol and 1 mL of 3 M NaCl and the mixture washed with 70% ethanol and redissolved in distilled water. The DNA adduct was then analyzed by 32P-postlabeling/HPLC. 32P-Postlabeling/HPLC Analysis of MDA-3′-dGMP. Ten micrograms of DNA (0.5-1 µg in 1 µL of distilled water) was enzymatically hydrolyzed to the corresponding 2′-deoxyribonucleoside 3′-monophosphates at 37 °C for 4 h with 20 µL of a mixture containing 1.25 units of MN, 62 milliunits of SPD, 20 mM sodium succinate, and 10 mM calcium chloride (pH 6). Two

1034 Chem. Res. Toxicol., Vol. 11, No. 9, 1998 enrichment methods, nuclease P1 and HPLC enrichment, were compared. For the nuclease P1 method, the MN/SPD digested DNA solutions were incubated at 37 °C for 30 min with 4 µL of nuclease P1 solution (2 µg of nuclease P1 in 1 µL of buffer containing 0.24 M sodium acetate and 2 mM ZnCl2 at pH 5) to remove normal 2′-deoxynucleotides. The resulting mixture was then evaporated to dryness under reduced pressure and redissolved in 10 µL of distilled water for 32P-postlabeling. For the method of HPLC enrichment, the MN/SPD digested DNA solutions were injected onto a Prodigy 5 µm ODS column (Phenomenex, 4.6 mm × 250 mm) eluted isocratically with 10% methanol in 0.1 M triethylammonium acetate (pH 5) at a flow rate of 1.5 mL/min. The fraction containing MDA-3′-dGMP was collected for the 32P-postlabeling reaction. (1) Analysis of [32P]MDA-3′,5′-dGDP Adducts by HPLC. Each of the MDA-3′-dGMP adduct samples, obtained either from enzymatic digestion of DNA of mouse liver, rat liver, human liver, human pancreas, and human lung or from the synthetic MDA-3′-dGMP standard, was dissolved in 10 µL of distilled water and 32P-phosphorylated by incubating with 10 µL of a PNK mix containing 100 µCi of [γ-32P]ATP (specific activity of 3000-4000 Ci/mmol), 12 units of PNK, and 2 µL of 10× PNK buffer [200 mM bicine-NaOH (pH 9.6), 100 mM dithiothreitol, 100 mM MgCl2, and 10 mM spermidine in distilled water] at 37 °C for 40 min. The labeled mixture was injected onto two Prodigy 5 µm ODS columns in series (Phenomenex, 4.6 mm × 250 mm) and eluted using a linear gradient between methanol (A) and 20 mM NaH2PO4 (pH 4.5) (B) (gradient program II) as follows: 0-30 min, 0 to 10% A in B; 30-55 min, 10 to 20% A in B; 55-65 min, 20% A in B isocratically; 65-66 min, 20 to 100% A in B; and 66-75 min, 100% A isocratically. The HPLC flow rate was 0.8 mL/min, and the scintillation fluid flow rate was 2.4 mL/min. To avoid interference by the high radioactivity of the free 32P and the unreacted [γ-32P]ATP, the on-line FLO-ONE radioactivity detector (Radiomatic Instruments, Tampa, FL) was equipped with a diverter. Thus, the eluent from the first 20 min was diverted away from the radioactivity detector. Similarly, for analysis of the [32P]MDA-5′-dGMP adducts described below, the eluent for the first 30-40 min was diverted. The columns were equilibrated for 15 min with 20 mM NaH2PO4 (pH 4.5) before each run. (2) Analysis of [32P]MDA-5′-dGMP Adducts by HPLC. The labeled mixture containing [32P]MDA-3′,5′-dGDP adducts obtained above was first adjusted to approximately pH 5 by adding 4 µL of 0.4 M acetic acid. The resulting solutions were then 3′-dephosphorylated with 17.5 µg of nuclease P1 (5 µg/µL in 0.42 M sodium acetate and 2 mM ZnCl2) at 37 °C for 5 h to yield the corresponding [32P]MDA-5′-dGMP, which was then subjected to HPLC analysis. To verify that the radioactive chromatographic peak (retention time) contained the [32P]MDA-5′-dGMP adduct, about 2 nmol of the nonradioactive synthetic standard MDA-5′-dGMP was added to each dephosphorylated sample as a UV marker. 32P-Postlabeling/HPLC Analysis of MDA-3′-dGMP Using 1 µg of DNA. 32P-Postlabeling/HPLC analysis was also conducted on 1 µg of DNA using conditions similar to those described above, with the exception that the ratio of [γ-32P]ATP and PNK employed was 5-fold higher. 32P-Postlabeling/TLC Analysis of the MDA-3′-dGMP Adduct. An aliquot containing 5 µg of DNA was digested by MN/SPD, enriched by nuclease P1, and 32P-postlabeled as described previously (47, 49). As a control experiment, about 3 fmol of the synthetic MDA-3′-dGMP was also 32P-postlabeled in parallel under the same conditions. To analyze the [32P]MDA-3′,5′-dGDP adduct, the labeled mixture was applied onto 10 cm × 10 cm PEI cellulose plates (Macherey-Nagel) and a two-dimensional development as previously described was carried out (47, 49). Autoradiography was performed on Dupont Cronex films, and the radioactivity on the TLC spots was quantitated by Cerenkov counting. The relative adduct levels (RALs) were calculated following the procedure of Reddy and Randerath (57).

Yi et al. Instrumentation. HPLC analysis was performed with a Waters Associates, Inc., instrument consisting of two model 510 pumps, a model 680 solvent programmer, a model U6K injector, and a Hewlett-Packard 1040A UV detection system. For radiochromatography analysis, the HPLC system, with on-line detections of both radioactivity and UV absorbance, consisted of a solvent gradient programer (Waters model 680), two HPLC pumps (Waters model 510), a UV detector (Waters model 440), and a radiochromatography detector (Hewlett-Packard FLOONE/Beta A-500) equipped with a diverter and an autosampler (Waters model 717). UV/visible spectra were determined with a Beckman model DU-65 spectrophotometer. 1H NMR spectra were recorded at 500 MHz on a Bruker AM500 spectrometer (Bruker Instruments, Billerica, MA). The samples were dissolved in methanol-d4. Electrospray (ES) mass spectrometry was performed using a Platform II single quadrupole instrument (Micromass, Inc., Altrincham, U.K.), and ES tandem mass spectrometry was performed using a Q-Q-TOF hybrid instrument (Q-TOF, Micromass). Nucleotides were analyzed in negative ion mode using either LC/MS introduction or MS/MS product ion scans from sample introduction by nanoflow electrospray using a gold-plated borosilicate nanoprobe (total volume introduced of ca. 1 µL). Separate MS functions were used to acquire full scan data at a low and high cone voltage in a single chromatographic run (e.g., 20 and 40 V, respectively, for m/z 100-500). Product ion scans were obtained from CID of selected ions using a constant cone voltage of 30 V and collision energies between 25 and 40 eV. The collision gas was Ar at a pressure of 4 × 10-3 mbar. LC/MS samples (5 µL injection volume) were introduced into the ES probe following separation using a Beckman Ultrasphere ODS column (5 µm particle size) eluted with 20% methanol in water at a flow rate of 1.0 mL/min split to approximately 0.2 mL/min entering the probe.

Results Synthesis of MDA-3′-dGMP, MDA-5′-dGMP, and MDA-3′,5′-dGDP. To characterize unequivocally the 32 P-postlabeling reaction products, the MDA-3′-dGMP, MDA-5′-dGMP, and MDA-3′,5′-dGDP adducts were synthesized and used as standards (markers) and/or reaction substrates. (1) MDA-5′-dGMP Adduct. Following the synthetic and purification procedures described in Materials and Methods, the resulting pure MDA-5′-dGMP was obtained as a sharp HPLC chromatographic peak (data not shown). The UV/visible absorption spectrum was very similar to that of MDA-3′-dGMP. Figure 2B shows the mass spectrum obtained from negative ion LC/MS analysis of this MDA-5′-dGMP adduct. The spectra exhibited three characteristic ions, a prominent [M - H]- ion at m/z 382, a fragment ion at m/z 186 corresponding to MDA-modified guanine, and a fragment ion at m/z 195 corresponding to deoxyribose monophosphate. On the basis of these results, together with the structural assignment for the MDA-3′-dGMP analogue determined previously by Seto et al. (56) and Vaca et al. (49), the structure of this MDA-5′-dGMP adduct was determined to be 5-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2R]purin-10(3H)-one. On the basis of the analysis by proton NMR spectral measurements using an internal standard (1,4-dioxane) of known quantity, only 88 µg of this adduct was obtained after repeating purification procedures. On the basis of this quantity and the UV/ visible absorbance measured in methanol, the molar extinction coefficient of MDA-5′-dGMP at 254 nm was determined to be 7.81 × 104 M-1 cm-1. Analysis of the

32P-Postlabeling/HPLC

Analysis of MDA-DNA Adducts

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1035

Figure 2. Negative ion electrospray mass spectra of synthetic standards of (A) MDA-3′-dGMP, (B) MDA-5′-dGMP, and (C) MDA3′,5′-dGDP. Mass spectra A and B were obtained using a single quadrupole instrument with in-source CID, and spectrum C shows the product ions from m/z 462 obtained using a hybrid tandem MS instrument.

crude reaction product indicated the yield of the MDA5′-dGMP adduct was about 2-3%. (2) MDA-3′-dGMP Adduct. Reaction of MDA with 3′-dGMP in KH2PO4 (pH 4.5) was carried out at 37 °C for 4 days. The reaction mixture was purified by sequential ion-exchange and reversed-phase HPLC. The resulting adduct had an UV/visible absorption spectrum identical to that previously reported (47). The negative ion mass spectrum of the adduct was also measured. Parts A and B of Figure 2 show the spectra obtained from LC/ MS analysis of synthetic MDA-3′-dGMP and MDA-5′dGMP derivatives, respectively, at a cone voltage that yielded molecular and fragment ions. Both spectra are similar and are characterized by a prominent [M - H]ion (m/z 382) and fragmentation to deprotonated ions corresponding to MDA-modified guanine (m/z 186) and deoxyribose monophosphate (m/z 195). Analysis of the crude reaction product indicated the yield of this adduct was about 3%. (3) Phosphorylation of MDA-3′-dGMP and Identification of MDA-3′,5′-dGDP. To prepare MDA3′,5′-dGDP in a quantity sufficient for structural identification by UV/visible absorption and mass spectral analysis, the MDA-3′-dGMP synthesized above was phosphorylated with nonradioactive ATP catalyzed by PNK. The reaction mixture was separated by reversedphase HPLC (Figure 3). The materials eluted at 26 min had an UV/visible absorption spectrum similar to those of MDA-3′-dGMP and MDA-5′-dGMP. Negative ESMS analysis of this product indicated that it had a molecular ion at m/z 462 (Figure 2C), consistent with the proposed structure of MDA-3′,5′-dGDP. The product ion spectrum was obtained using tandem MS from collisioninduced dissociation of the mass corresponding to the deprotonated molecule of MDA-3′,5′-dGDP (m/z 462).

Figure 3. Reversed-phase HPLC profiles of (A) the reaction mixture from phosphorylation of the synthetic MDA-3′-dGMP standard with cold ATP and PNK, (B) co-injection of the reaction mixture with the MDA-3′-dGMP standard, and (C) co-injection of the reaction mixture with the MDA-5′-dGMP standard. For the experimental conditions, see Materials and Methods.

The spectrum contained deprotonated ions corresponding to deoxyribose bisphosphate (m/z 275) and two successive water losses (m/z 257 and 239), MDA-modified guanine

1036 Chem. Res. Toxicol., Vol. 11, No. 9, 1998

Figure 4. Structure and proposed mass spectral fragmentation of MDA-2′-deoxyribose 3′,5′-bisphosphate (MDA-3′,5′-dGDP).

(m/z 186), pyrophosphate (m/z 177) and a water loss (m/z 159), and HPO4 (m/z 97) and a water loss (m/z 79) (Figure 4). The material eluted at 41 min (Figure 3A) had an UV/ visible absorption spectrum and ES-mass spectrum (data not shown) identical to those of the authentic MDA-5′dGMP (Figure 2), respectively. When the synthetic MDA-3′-dGMP standard was mixed with the reaction mixture for HPLC under identical conditions, the MDA3′-dGMP eluted earlier than this adduct (Figure 3B). When the synthetic MDA-5′-dGMP was mixed with the reaction mixture for HPLC, this MDA-5′-dGMP and the adduct cochromatographed. Thus, all these results unequivocally indicate that this adduct was MDA-5′dGMP. (4) 3′-Dephosphorylation of the MDA-3′,5′-dGDP adduct. To confirm further the MDA-3′,5′-dGDP adduct and subsequent MDA-5′-dGMP formation, the nonradioactive synthetic MDA-3′,5′-dGDP adduct (about 4 nmol) was 3′-dephosphorylated by incubation with nuclease P1. The product had a HPLC retention time, UV/visible absorption spectrum, and mass spectrum identical to those of the authentic MDA-5′-dGMP. Thus, the formation of MDA-5′-dGMP adduct was verified. 32 P-Postlabeling/HPLC Analysis of MDA-modified Adducts. (1) Nuclease P1 Enrichment followed by [32P]MDA-3′,5′-dGDP Detection. After the synthetic MDA-3′-dGMP adduct was 5′-32P-phosphorylated with [γ-32P]ATP, the resulting reaction mixture was analyzed by reversed-phase HPLC (Figure 5A). Comparison of the HPLC retention times of the radioactive chromatographic peaks with those of the synthetic MDA-3′,5′-dGDP, MDA-3′-dGMP, and MDA-5′-dGMP standards indicated that the [32P]MDA-3′,5′-dGDP adduct was formed (marked with an arrow in Figure 5A), without the formation of MDA-5′-dGMP. This result is different from that obtained from phosphorylation of MDA-3′dGMP using the nonradioactive ATP described above (Figure 3A). 32P-Postlabeling of MDA-3′-dGMP derived from MDAmodified calf thymus DNA produced similar results.

Yi et al.

Figure 5. Reversed-phase HPLC profiles of (A) [32P]MDA3′,5′-dGDP adducts formed by 32P-postlabeling the MDA-3′dGMP synthetic standard, (B) [32P]MDA-3′,5′-dGDP adducts formed by 32P-postlabeling the synthetically prepared MDAmodified calf thymus DNA, (C) the [32P]MDA-5′-dGMP adduct from 3′-dephosphorylation of [32P]MDA-3′,5′-dGDP described in part A, (D) the [32P]MDA-5′-dGMP adduct from 3′-dephosphorylation of [32P]MDA-3′,5′-dGDP described in part B, (E) the product from 32P-postlabeling of blank calf thymus DNA (10 µg) followed by 3′-dephosphorylation, and (F) the MDA-5′dGMP cold standard cochromatographed with the 32P-postlabeling product described in part C, to serve as a UV marker detected at 254 nm. The HPLC analysis was performed on a combination of two Prodigy 5 µm ODS columns with on-line detections of radioactivity and UV absorbance using a gradient solvent system (gradient program II). HPLC elution was diverted away from the radioactivity detector from 0 to 20 min in parts A and B and from 0 to 35 min in parts C-E. Arrows indicate the positions of the adducts. For details on the experiments and HPLC conditions, see Materials and Methods. The radioactivity scale is the same for chromatograms A-E and is 100 000 cpm full scale.

Since the chromatographic peak containing the [32P]MDA-3′,5′-dGDP adduct was hidden in multiple unidentified peaks, the [32P]MDA-3′,5′-dGDP adduct was unable to be quantified (Figure 5B). Therefore, 3′dephosphorylation of the [32P]MDA-3′,5′-dGDP adduct by nuclease P1 to the [32P]MDA-5′-dGMP adduct was used for identification and quantification. (2) Nuclease P1 Enrichment Followed by [32P]MDA-5′-dGMP Detection. After the 32P-postlabeling reaction, the labeled mixture containing [32P]MDA-3′,5′dGDP adduct was 3′-dephosphorylated with nuclease P1. For verification of the HPLC retention time, nonradioactive MDA-5′-dGMP was added to the 3′-dephosphorylation product as a UV marker. The reversed-phase HPLC profiles of the dephosphorylated mixtures derived from the MDA-3′-dGMP synthetic standard, the MDAmodified calf thymus DNA, and the unmodified calf thymus DNA are shown in parts C-E of Figure 5, respectively. The HPLC profile of the nonradioactive MDA-5′-dGMP adduct, detected by a UV detector at 254 nm, is shown in Figure 5F. Comparison of HPLC retention times indicates that the chromatographic peaks eluted at 61 min in parts C and D of Figure 5 match that of the cold MDA-5′-dGMP standard (Figure 5F).

32P-Postlabeling/HPLC

Analysis of MDA-DNA Adducts

Figure 6. Radioactivity HPLC response curve of 32P-postlabeling of the MDA-3′-dGMP adduct. The abscissa is the amount of standard added to the reaction, and the ordinate is the radioactivity calculated from the peak area of [32P]MDA-5′dGMP. For experimental details, see Materials and Methods. 32 P-Postlabeling of the unmodified calf thymus DNA resulted in only background radioactivity at 61 min (Figure 5E), which corresponds to the retention time of [32P]MDA-5′-dGMP. Thus, these results indicate the product obtained from 32P-postlabeling followed by 3′dephosphorylation is unequivocally determined to be the [32P]MDA-5′-dGMP adduct and that, under the HPLC conditions, separation and quantification of the [32P]MDA-5′-dGMP adduct are highly reliable. The HPLC analysis was performed using two Prodigy 5 µm ODS columns in series. Use of only one column resulted in less satisfactory separation and made quantification less reliable. The use of several other HPLC columns, including a Vydac 219 diphenyl column (250 mm × 4.6 mm i.d.), also resulted in less satisfactory separations (data not shown). (3) HPLC Enrichment Followed by [32P]MDA-5′dGMP Detection. Using the developed HPLC conditions that can be used to separate MDA-3′-dGMP from the unmodified monophosphate 2′-deoxynucleotides, the MDA-3′-dGMP from DNA digestion was enriched by HPLC. After 32P-postlabeling and subsequent 3′-dephosphorylation of the resulting [32P]MDA-3′,5′-dGDP, HPLC analysis of the [32P]MDA-5′-dGMP was performed. HPLC analysis showed that the chromatographic peak corresponding to the [32P]MDA-5′-dGMP adduct was hidden in multiple chromatographic peaks, and consequently, identification and quantification of this adduct are not feasible (data not shown). Therefore, in this paper, unless specifically indicated, the nuclease P1 method was used for enrichment of the MDA-3′-dGMP adduct formed from enzymatic digestion of DNA samples. Radioactivity HPLC Response Curve. The substrate concentration-dependent 32P-postlabeling curve of MDA-3′-dGMP was obtained by 32P-postlabeling of 1.5, 3, 15, and 75 fmol of the MDA-3′-dGMP synthetic standard followed by 3′-dephosphorylation (catalyzed by nuclease P1). The resulting [32P]MDA-5′-dGMP adduct was then analyzed by HPLC. As shown in Figure 6,

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1037

there is a good linear correlation (r ) 0.999) between substrate (quantity of MDA-3′-dGMP) and response (radioactivity of [32P]MDA-5′-dGMP), within the range studied. Quantification of the MDA-Modified DNA Adduct Using 32P-Postlabeling/HPLC. Method 1, Based on Recovery. Recovery of MDA-3′-dGMP with the 32Ppostlabeling/HPLC procedure was determined as follows: (i) enzymatic digestion of 10 µg of unmodified calf thymus DNA in triplicate by MN/SPD to an unmodified 3′-dNMP (3′-dGMP, 3′-dCMP, 3′-dAMP, and 3′-dTMP) mixture; (ii) adding 3, 15, or 150 fmol of the synthetic MDA-3′-dGMP to the 3′-dNMP mixture; (iii) following the 32P-postlabeling procedure described above, each of the reaction mixtures subjected to nuclease P1 enrichment, 32P-postlabeling, 3′-dephosphorylation of the [32P]MDA-3′,5′-dGDP adduct with nuclease P1, and HPLC analysis. The radioactivity contained in the resulting [32P]MDA-5′-dGMP adduct was quantified. Based on the radioactivity, the average recovery for these three concentrations was found to be 74, 75, and 75%, respectively. On the basis of calculations, 10 µg of DNA with a modification level of 1 MDA-3′-dGMP adduct/107 nucleotides contains 3 fmol of the MDA-3′-dGMP adduct. To match the level of MDA-3′-dGMP adduct contained in biological (animal and human) DNA samples, from 3 (for mouse liver or human DNA samples) to 30 fmol (for 27month-old rat liver DNA samples) of the synthetic MDA3′-dGMP standard was used as an external standard for 32P-postlabeling in parallel. The MDA-3′-dGMP adduct contained in the DNA samples was quantified on the basis of the ratio of the radioactivity of the [32P]MDA5′-dGMP peak of the sample and the standard and then normalized on the basis of recovery described above. Subtraction of background radioactivity was done automatically for each sample by the peak integration function of the Packard FLO-ONE program. Method 2, Based on 32P-Postlabeling in Parallel with an MDA-3′-dGMP External Standard. As described above for method 1, 10 µg of unmodified calf thymus DNA was digested by MN/SPD to the unmodified 3′-dNMP, 3-30 fmol of the synthetic MDA-3′-dGMP was added to the digested DNA, and then the resulting mixture was used as an external standard subjected to nuclease P1 enrichment and 32P-postlabeling in parallel with the biological samples. After 32P-postlabeling, 3′dephosphorylation of the [32P]MDA-5′-dGMP adduct with nuclease P1, and HPLC analysis, the radioactivity contained in the resulting [32P]MDA-5′-dGMP adduct was determined. The MDA-3′-dGMP adduct contained in the biological samples was quantified based on the ratio of the radioactivity of [32P]MDA-5′-dGMP peak of the sample and the standard. Method 3, Based on 32P-Postlabeling in Parallel with an MDA-Modified DNA External Standard. Two MDA-modified DNA standards were synthesized. As described previously, the quantity of the MDA-3′-dGMP adduct was determined on the basis of the 1H NMR measurements with a known quantity of an internal standard (1,4-dioxane). Thus, by parallel 32P-postlabeling with the MDA-3′-dGMP adduct, the levels of modification of these two DNA samples were determined to be 3.2 adducts/107 nucleotides and 1.4 adducts/106 nucleotides, respectively. Accordingly, when 32P-postlabeling of the biological samples was carried out, these DNA

1038 Chem. Res. Toxicol., Vol. 11, No. 9, 1998

Yi et al.

Table 1. Level of M1G Adduct Detected in DNA of Rodent and Human Tissues

species

sex

B6C3F1 male mouse Fischer female 344 rat human male and female human male and female human male and female

age

tissue

M1G level (adducts/107 nucleotides, X h ( SD)

liver

4.83 ( 1.51 (n ) 14)

27 months liver

16.8 ( 11.0 (n ) 15)

16 days

adult

pancreas 3.58 ( 1.46 (n ) 10)

adult

liver

1.40 ( 0.28 (n ) 4)

adult

lung

1.00 ( 0.40 (n ) 3)

standards were employed as an external standard for 32Ppostlabeling in parallel. The MDA-3′-dGMP adduct contained in DNA (to use either 10 or 1 µg) from biological samples was then quantified on the basis of the ratio of the radioactivity of the [32P]MDA-5′-dGMP peak of the sample and the DNA standard. Evaluation of Intra- and Inter-Experimental Reproducibility of 32P-Postlabeling. The synthetic MDAmodified calf thymus DNA standard was used for determination of intra- and inter-experimental reproducibility. This standard in triplicate was then subjected to 32Ppostlabeling at the same time for the intraexperimental trial. The resulting values were 2.02, 3.04, and 4.43 h ( SD ) 3.16 ( 1.21), respecadducts/107 nucleotides (X tively. For the interexperimental trial, this same standard was 32P-postlabeled at four different times. The values were 4.86, 2.43, 3.04, and 4.34 adducts/107 nucleotides (X h ( SD ) 3.37 ( 1.17), respectively. 32P-Postlabeling/HPLC Analysis of the MDA-3′dGMP Adduct in DNA Samples of Animal and Human Tissues. To validate the established 32P-postlabeling/HPLC method, the MDA-3′-dGMP adduct in MDA-modified calf thymus DNA, mouse and rat liver DNA, and human pancreas, liver, and lung DNA was assayed. In each experiment, an external standard was also analyzed in parallel with the biological samples (Table 1). The standard is either MDA-modified DNA containing a known level of adduct (1.4 adducts/106 nucleotides or 3.2 adducts/107 nucleotides) or a known quantity of MDA-3′-dGMP standard that closely matched the modification level expected in each DNA sample. To each sample was added a cold standard of MDA-5′dGMP as an UV marker to verify the chromatographic peak of [32P]MDA-5′-dGMP. The HPLC profiles obtained from 32P-postlabeling of the DNA extracted from mouse liver, rat liver, human pancreas, and human liver are shown in Figure 7A-D. These results indicate that a clear chromatographic peak of [32P]MDA-5′-dGMP was obtained and the peak was well separated from the interfering peaks. Thus, it is this efficient chromatographic separation that makes it possible to accurately quantify the MDA-3′-dGMP adduct level in animal and human tissues.

Discussion 32

Although P-postlabeling has been established as one of the most sensitive analytical methodologies for detection and quantification of carcinogen-modified DNA adducts, this methodology often suffers from critical pitfalls. Because of low synthetic yields, high polarity, and low mass spectral ionization efficiency, the 32P-

Figure 7. Reversed-phase HPLC profiles of 32P-postlabeled DNA adducts of MDA determined as [32P]MDA-5′-dGMP resulting from (A) 16-day-old mouse liver DNA (10 µg), (B) 27month-old rat liver DNA (10 µg), (C) human pancreas DNA (10 µg), and (D) human liver DNA (10 µg). Enrichment of the adducts was achieved by nuclease P1 hydrolysis. The HPLC analysis of the adducts was performed under the same conditions shown for Figure 5. The adduct peaks are indicated by the arrows. The HPLC eluate was diverted away from the detector from 0 to 35 min in parts A-C and from 0 to 40 min in part D. The radioactivity scale is the same for chromatograms A-D and is 100 000 cpm full scale.

postlabeling products, [32P]-3′,5′-bisphosphates of carcinogen-modified 2′-deoxyribonucleotides, are generally not fully characterized. Separation of the 32P-postlabeling products, by either HPLC or TLC, is frequently not satisfactory for unequivocal identification and/or quantification. Even though determination of recovery between different trials performed at different times can be normalized on the basis of the observed substrate concentration-response relationship (curve), possible variation in enzyme activity and experimental conditions can result in quantitative deviations between different trials. Thus, without 32P-postlabeling of a known quantity of cold authentic standard in parallel with the tested samples, quantitative comparison of 32P-postlabeling products obtained from different trials is not reliable. As a consequence, to establish an improved 32P-postlabeling methodology, we have performed the following steps. (1) The postlabeling products, MDA-3′,5′-dGDP and MDA-5′-dGMP, were characterized by comparison of HPLC retention times, UV/visible absorption, and ESMS spectra with those of synthetic standards. (2) An efficient HPLC separation of the 32P-postlabeling products was carried out. (3) In each experiment, an external standard, a DNA synthetic standard containing a known level of adduct that closely matches the range of the modification level of the biological DNA samples, was also analyzed in parallel with the biological samples so that the quantity of the 32P-postlabeling products obtained from different trials can be reliably compared. By using these improved features, we eliminated critical defects, and the improved 32P-postlabeling/HPLC methodology was used for the analysis of MDA-modified 1,N2-propanodeoxyguanosine adduct M1G contained in mouse liver, rat liver, human liver, human pancreas, and human lung. We previously reported the use of 32P-postlabeling/TLC for identification and quantification of the MDA-3′-

32P-Postlabeling/HPLC

Analysis of MDA-DNA Adducts

dGMP adduct formed from metabolism of chloral hydrate by rat and mouse liver microsomes in the presence of calf thymus DNA (47). Although this methodology was useful in our initial studies, re-examination of this method indicated that the resultant TLC spot was frequently contaminated by unidentified radioactive materials (data not shown). Therefore, it was desirable to investigate 32 P-postlabeling/HPLC as an alternative with the potential for enhanced chromatographic resolution and sensitivity for detection of the MDA-3′-dGMP adduct. In this paper, we describe the experimental procedures and conditions for an improved 32P-postlabeling/HPLC method and provide unequivocal structural identification of the 32P-postlabeling/HPLC products, both MDA-3′,5′dGDP and MDA-5′-dGMP. To optimize the experimental conditions, several different quantities of the MN/SPD used for DNA digestion, the nuclease P1 used for adduct enrichment and 3′-dephosphorylation of the [32P]MDA3′,5′-dGDP adduct, and the PNK and [γ-32P]ATP used for postlabeling were employed. The quantities of each enzyme and [γ-32P]ATP that provided the maximum yield have been employed for all the experiments described above in each of the reactions. The improved procedure was used to analyze DNA from rodent and human tissues for the presence of M1G adducts (see Table 1). These results shown are similar to those reported previously. Marnett and co-workers showed that M1G adduct levels in human leukocytes and liver are 0.6 and 9.0 adducts in 107 normal nucleotides, respectively (19), and Vaca et al. found that M1G adduct levels in leukocytes and breast tissue are 2.6 and 3.0 adducts in 107, respectively (24, 33). This experimental procedure is superior to other reported procedures in terms of overall selectivity and sensitivity. The results reported in this paper are the first to provide HPLC conditions that can be used to effectively separate the final 32P-postlabeling product, the [32P]MDA-5′-dGMP adduct, for reliable quantification. The intra- and inter-experimental results indicate high reproducibility and accuracy. The response for the [32P]MDA-5′-dGMP adduct equivalent to 3.16 ( 1.21 adducts per 107 nucleotides in 10 µg of synthetic MDA-modified calf thymus DNA gave a signal/noise ratio of about 80130; therefore, it is estimated that the detection limit (s/n ) 3) for the [32P]MDA-5′-dGMP adduct contained in 10 µg of MDA-modified calf thymus DNA is 8.8 ( 0.14 adducts/109 nucleotides. Similarly it is estimated that the detection limits (s/n ) 3) for 10 µg of mouse liver (n ) 5), rat liver (n ) 5), human pancreas (n ) 6), human liver (n ) 3), and human lung (n ) 4) DNA are around 1.0, 4.4, 1.9, 1.8, and 1.5 adducts per 108 nucleotides, respectively. These levels of sensitivity indicate that this methodology is highly suitable for detection of this adduct in biological samples, including humans. When the quantity of biological samples is limited, we have demonstrated that our improved 32P-postlabeling/HPLC method can be used even with as little as 1 µg of DNA. To further improve performance of the method, an external standard was used in parallel for 32P-postlabeling so that the level of DNA adduct can be quantified accurately. As described in the Results, using quantification of MDA-3′-dGMP as an example, there are three possible approaches: (i) use of MDA-3′-dGMP as an external standard for 32P-postlabeling, followed by normalization based on recovery; (ii) use of unmodified calf thymus DNA for enzymatic digestion, followed by adding

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1039

MDA-3′-dGMP as an external standard for 32P-postlabeling; and (iii) use of DNA that contains the level of MDA-3′-dGMP that is similar to that of the tested DNA sample. Although all these methods have been employed for quantification, the first two methods have the potential for confounding results, neglecting the possible different digestion efficiencies of the DNA samples by the micrococcal nuclease (MN) and spleen phosphodiesterase (SPD) enzymes among different trials. As a consequence, if enzyme activities vary between trials, quantification thus cannot be compared between different trials. Even in the first method, if the nuclease P1 activity varies between different trials, comparison of quantification between different trials also would be unreliable. Therefore, to obtain accurate quantification, we highly recommended the use of the MDA-modified DNA standard with a known level of modification. This method should be the choice for interlaboratory trials. 32 P-Postlabeling/HPLC has been employed to detect other lipid peroxidation-derived endogenous DNA adducts, such as HNE (53), crotonaldehyde (29), and acetaldehyde (32, 34). Therefore, our 32P-postlabeling/ HPLC method could be expanded to accommodate the analysis of several lipid peroxidation-dependent endogenous DNA adducts. Since we have reported the separation of the HNE-derived DNA adduct by 32P-postlabeling/ HPLC (53), the use of this technique to simultaneously detect both the MDA- and HNE-modified DNA adducts is under development. Previously, Marnett and co-workers have developed a sensitive mass spectroscopic method for the detection and quantification of the M1G adduct in various human tissues (19, 51, 58). Recently, this group reported the development of monoclonal antibodies to the M1G adduct and its uses in the quantitation and purification of the M1G adduct (58, 59). Our improved 32P-postlabeling/ HPLC method described in this paper has also been demonstrated to be highly reliable. Therefore, this 32Ppostlabeling/HPLC method together with the mass spectrometric method and the immunoassay can be employed in a complementary manner to quantify the MDA-3′dGMP adduct contained in biological samples.

Acknowledgment. We thank Joanna Deck for performing the NMR measurements, Linda S. Von Tungeln for DNA extraction, and Dr. James Langridge (Micromass, Inc.) for Q-TOF mass spectral measurements. We also thank Dr. Fred Kadlubar for the human liver, pancreas, and lung samples and his comments on this paper. This research was supported in part by an appointment (Y.P.) to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

References (1) Benedetti, A., Barbieri, L., Ferrali, M., Casini, A. F., Fulceri, R., and Comporti, M. (1981) Inhibition of protein synthesis by carbonyl compounds (4-hydroxyalkenals) originating from the peroxidation of liver microsomal lipids. Chem.-Biol. Interact. 35, 331-340. (2) Benedetti, A., Esterbauer, H., Ferrali, M., Fulceri, R., and Comparti, M. (1982) Evidence for aldehyde bound to liver microsomal protein following CCl4 or BrCCl3 poisoning. Biochim. Biophys. Acta. 711, 346-356.

1040 Chem. Res. Toxicol., Vol. 11, No. 9, 1998 (3) Corongiu, F. P., Lai, M., and Milia, A. (1983) Carbon tetrachloride, bromotrichloromethane and ethanol acute intoxication: new chemical evidence for lipid peroxidation in rat tissue microsomes. Biochem. J. 212, 615-631. (4) Comporti, M. (1985) Biology of disease-lipid peroxidation and cellular damage in toxic injury. Lab. Invest. 53, 599-623. (5) Poli, G., Dianzani, M. U., Chesseman, K. H., Slater, T. F., Lang, J., and Esterbauer, H. (1985) Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions. Biochem. J. 227, 629-638. (6) Esterbauer, H., and Cheeseman, H. (1987) Lipid peroxidation. II: Pathological implications. Chem. Phys. Lipids 45, 103-371. (7) Esterbauer, H., Zollner, H., and Schaur, R. J. (1990) Aldehydes formed by lipid peroxidation: Mechanisms of formation, occurrence, and determination. In Membrane Lipid Oxidation (VigoPelfrey, C., Ed.) pp 239-268, CRC Press, Boca Ration, FL. (8) Esterbauer, H., Schaur, R. J., and Zollner, R. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radical Biol. Med. 11, 81-128. (9) Marnett, L. J., and Burcham P. C. (1993) Endogenous DNA adducts: potential and paradox. Chem. Res. Toxicol. 6, 771-785. (10) Natarajan, A., Scribner, W. M., and Taher, M. M. (1993) 4-Hydroxynonenal, a metabolite of lipid peroxidation, activates phospholipase D in vascular endothelial cells. Free Radical Biol. Med. 15, 365-375. (11) Wang, M.-Y., and Liehr, J. G. (1995) Lipid hydroperoxide-induced endogenous DNA adducts in hamsters: possible mechanism of lipid hydroperoxide-mediated carcinogenesis. Arch. Biochem. Biophys. 316, 38-46. (12) Halliwell, B., and Gutteridge, J. M. C. (1989) Free Radicals in Biology and Medicine, Clarendon Press, Oxford, U.K. (13) Nishikawa, A., Sodum, R., and Chung, F.-L. (1992) Acute toxicity of trans-4-hydroxy-2-nonenal in Fisher 344 rats. Lipids 27, 5458. (14) Eckl, P. M., Ortner, A., and Esterbauer, H. (1993) Genotoxic properties of 4-hydroxyalkenals and analogous aldehydes. Mutat. Res. 290, 183-192. (15) Vaca, C. E., Wilhelm, J., and Harms-Ringdahl, M. (1988) Interaction of lipid peroxidation products with DNA. A review. Mutat. Res. 195, 137-149. (16) Chung, F.-L., Chen, H.-Y. C., and Nath, R. G. (1996) Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts. Carcinogenesis 17, 2105-2111. (17) Sodum, R. S., and Chung, F.-L. (1988) 1,N2-Ethenodeoxyguanosine as a potential marker for DNA adduct formation by trans4-hydroxy-2-nonenal. Cancer Res. 48, 320-323. (18) Sodum, R. S., and Chung, F.-L. (1989) Structural characterization of adducts formed in the reaction of 2,3-epoxy-4-hydroxynonanal with deoxyguanosine. Chem. Res. Toxicol. 2, 23-28. (19) Chaudhary, A. K., Nokubo, M., Reddy, G. R., Yeola, S. N., Morrow, J. D., Blair, L. A., and Marnett, L. J. (1994) Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265, 1580-1582. (20) Douki, T., and Ames, B. N. (1994) An HPLC-EC assay for 1,N2propano adducts of 2′-doexyguanosine with 4-hydroxynonenal and other R,β-unsaturated aldehydes. Chem. Res. Toxicol. 7, 511-518. (21) Nath, R. G., and Chung, F.-L. (1994) Detection of exocyclic 1,N2propanodeoxyguanosine adducts as common DNA lesions in rodents and humans. Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495. (22) Wang, M.-Y., and Liehr, J. G. (1995) Induction by estrogens of lipid peroxidation and lipid peroxide-derived malonaldehyde-DNA adducts in male Syrian hamsters: role of lipid peroxidation in estrogen-induced kidney carcinogenesis. Carcinogenesis 16, 19411945. (23) Li, D., Wang, M., Liehr, J. G., and Randerath, K. (1995) DNA adducts induced by lipid and lipid peroxidation products: possible relationships to I-compounds. Mutat. Res. 344, 117-126. (24) Vaca, C. E., Fang, J.-L., Mutanen, M., and Valsta, L. (1995) 32Ppostlabelling determination of DNA adducts of malonaldehyde in humans: total white blood cells and breast tissue. Carcinogenesis 16, 1847-1851. (25) Winter, C. K., Segall, H. J., and Haddon, W. F. (1986) Formation of cyclic adducts of deoxyguanosine with the aldehydes trans-4hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal in vitro. Cancer Res. 46, 5682-5686. (26) Nath, R. G., Chen, H.-J. C., Nishikawa, A., Young-Sciame, R., and Chung, F.-L. (1984) A 32P-postlabeling method for simultaneous detection and quantification of exocyclic etheno and propano adducts in DNA. Carcinogenesis 15, 979-984. (27) Wilson, V. L., Foiles, P. G., Chung, F.-L., Povey, A. C., Frank, A. A., and Harris, C. C. (1991) Detection of acrolein and crotonal-

Yi et al.

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

(46) (47)

(48)

(49)

dehyde DNA adducts in cultured human cells and canine peripheral blood lymphocytes by 32P-postlabeling and nucleotide chromatography. Carcinogenesis 12, 1483-1490. Smith, R. A., Williamson, D. S., Cerny, R. L., and Cohen, S. M. (1990) Detection of 1,N6-propanodeoxyadenosine in acroleinmodified polydeoxyadenylic acid and DNA by 32P-postlabeling. Cancer Res. 50, 3005-3012. Chung, F.-L., Young, R., and Hecht, S. S. (1984) Formation of cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990-995. Nair, J., Barbin, A., Guichard, Y., and Bartsch, H. (1995) 1,N6ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine in liver DNA from humans and untreated rodents detected by immunoaffinity/ 32P-postlabelling. Carcinogenesis 16, 613-617. El Ghissassi, F., Barbin, A., Nair, J., and Bartsch, H. (1995) Formation of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine by lipid peroxidation products and nucleic acid bases. Chem. Res. Toxicol. 8, 278-283. Fang, J. L., and Vaca, C. E. (1995) Development of a 32Ppostlabelling method for the analysis of adducts arising though the reaction of acetaldehyde with 2′-deoxyguanosine-3′-monophosphate and DNA. Carcinogenesis 16, 2177-2185. Fang, J. L., Vaca, C. E., Valsta, L. M., and Mutanen, M. (1996) Determination of DNA adducts of malonaldehyde in humans: effects of dietaty fatty acid composition. Carcinogenesis 17, 10351040. Fang, J. L., and Vaca, C. E. (1997) Detection of DNA adducts of acetaldehyde in peripheral white blood cells of alcohol abusers. Carcinogenesis 18, 627-632. Feron, V. J., Til, H. P., de Vrijer, F., Woutersen, R. A., Cassee, F. R., and van Bladeren, P. J. (1991) Aldehydes: occurrence, carcinogenic potential, mechanism of action and risk assessment. Mutat. Res. 259, 363-385. Cajelli, E., Ferraris, A., and Brambilla, G. (1987) Mutagenicity of 4-hydroxynonenal in V79 Chinese hamster cells. Mutat. Res. 190, 169-171. Marnett, L. J., Hurd, H. K., Hollstein, M. C., Levin, D. E., Esterbauer, H., and Ames, B. N. (1985) Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104. Mutat. Res. 148, 25-34. Chung, F.-L., Chen, H.-J. C., Guttenplan, J. B., Nishikawa, A., and Hard, G. C. (1993) 2,3-Epoxy-4-hydroxynonenal as a potential tumor-initiating agent of lipid peroxidation. Carcinogenesis 14, 2073-2077. Moriya, M., Zhang, W., Johnson, F., and Grollman, A. P. (1994) Mutagenic potency of exocyclic DNA adducts: marked differences between Escherichia coli and simian kidney cells. Proc. Natl. Acad. Sci. U.S.A. 91, 11899-11903. Neudecker, T., Lutz, D., Eder, E., and Henschler, D. (1981) Crotonaldehyde is mutagenic in a modified Salmonella typhimurium mutagenicity testing system. Mutat. Res. 91, 27-31. Curren, R. D., Yang, L. L., Conklin, P. M., Grafstrom, R. C., and Harris, C. C. (1988) Mutagenesis of xeroderma pigmentosum fibroblasts by acrolein. Mutat. Res. 209, 17-22. Lutz, D., Eder, E., Neudecker, T., and Henschler, D. (1982) Structure-mutagenicity relationship in R,β-unsaturated carbonylic compounds and their corresponding allylic alcohols. Mutat. Res. 93, 305-315. Eder, E., Henschler, D., and Neudecker, T. (1982) Mutagenicity properties of allylic and R,β-unsaturated compounds: consideration of alkylating mechanisms. Xenobiotica 12, 831-848. Eder, E., Deininger, C., Neudecker, T., and Deininger, D. (1992) Mutagenicity of β-alkyl substituted acrolein congeners in the Salmonella typhimurium strain TA100 and genotoxicity testing in the SOS chromotest. Environ. Mol. Mutagen. 19, 338-345. Kerns, W. D., Pavkov, K. L., Donofrio, D. J., Gralla, E. J., and Swenberg, J. A. (1983) Carcinogenicity of formaldehyde in rats and mice after long-term inhalation exposure. Cancer Res. 43, 4382-4392. Spalding, J. W. (1988) NTP Technical Report 331, pp 5-13. Ni, Y.-C., Kadlubar, F. F., and Fu, P. P. (1995) Formation of malondialdehyde-modified 2′-deoxyguanosinyl adduct from metabolism of chloral hydrate by mouse liver microsomes. Biochem. Biophys. Res. Commun. 216, 1110-1117. Agarwal, S., and Draper, H. H. (1992) Isolation of a malondialdehyde-deoxyguanosine adduct from rat liver DNA. Free Radical Biol. Med. 13, 695-699. Vaca, C. E., Vodicka, P., and Hemminki, K. (1992) Determination of malonaldehyde-modified 2′-deoxyguanosine-3′-monophosphate and DNA by 32P-postlabelling. Carcinogenesis 13, 593-599.

32P-Postlabeling/HPLC

Analysis of MDA-DNA Adducts

(50) Kautiainen, A., Vaca, C. E., and Granath, F. (1993) Studies on the relationship between hemoglobin and DNA adducts of malonaldehyde and their stability in vivo. Carcinogenesis 14, 705708. (51) Kadlubar, F. F., Anderson, K. E., Lang, N. P., Thompson, P. A., MacLeod, S. L., Chou, M. W., Mikhailova, M., Plastaras, J., Marnett, L. J., Haussermann, S., Nair, J., Velic, I., and Bartsch, H. (1998) Comparison of endogenous DNA adduct levels in human pancreas. Mutat. Res. (in press.) (52) Benamira, M., Johnson, K., Chaudhary, A., Bruner, K., Tibbetts, C., and Marnett, L. J. (1995) Induction of mutations by replication malondialdehyde-modified M13 DNA in Escherichia coli: determination of the extent of DNA modification, genetic requirements for mutagenesis, and types of mutations induced. Carcinogenesis 16, 93-99. (53) Yi, P., Zhan, D.-J., Samokyszyn, V. M., Doerge, D. R., Evans, F. E., and Fu, P. P. (1997) Synthesis and 32P-postlabeling/HPLC separation of diastereomeric 1,N2-(1,3-propano)-2′-deoxyguanosine 3′-phosphate adducts formed from 4-hydroxy-2-nonenal. Chem. Res. Toxicol. 10, 1259-1265. (54) Gupta, R. C. (1993) 32P-Postlabelling analysis of bulky aromatic adducts. In Postlabeling Methods for Detection of DNA Adducts (Phillips, D. P., Castegnaro, M., and Bartsch, H., Eds.) pp 1123, International Agency for Research on Cancer (IARC), Lyon, France.

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1041 (55) Beland, F. A., Fullerton, N. F., and Heflich, R. H. (1984) Rapid isolation, hydrolysis and chromatography of formaldehyde-modified DNA. J. Chromatogr. 308, 121-131. (56) Seto, H., Takesue, T., and Ikemura, T. (1985) Reaction of malonaldehyde with nucleic acid. II. Formation of fluorescent pyrimido[1,2-a]purin-10(3H)-one mononucleotide. Bull. Chem. Soc. Jpn. 58, 3431-3435. (57) Reddy, M. V., and Randerath, K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551. (58) Rouzer, C. A., Chaudhary, A. K., Nokubo, M., Ferguson, D. M., Reddy, G. R., Blair, I. A., and Marnett, L. J. (1997) Analysis of the malondialdehyde-2′-deoxyguanosine adduct pyrimidopurinone in human leukocyte DNA by gas chromatography/electron capture/ negative chemical ionization/mass spectrometry. Chem. Res. Toxicol. 10, 181-188. (59) Sevilla, C. L., Mahle, N. H., Eliezer, N., Uzieblo, A., O’Hara, S. M., Nokubo, M., Miller, R., Rouzer, C. A., and Marnett, L. J. (1997) Development of monoclonal antibodies to the malondialdehydedeoxyguanosine adduct, pyrimidopurinone. Chem. Res. Toxicol. 10, 172-180.

TX9800497