32P-Postlabeling Analysis of DNA Adducts of Styrene 7,8-Oxide at the

George Cruzan , Gary P. Carlson , Keith A. Johnson , Larry S. Andrews , Marcy I. Banton , Christopher Bevan , Janette R. Cushman. Regulatory Toxicolog...
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Chem. Res. Toxicol. 1999, 12, 93-99

93

32P-Postlabeling

Analysis of DNA Adducts of Styrene 7,8-Oxide at the O6-Position of Guanine Michael Otteneder, Erwin Eder, and Werner K. Lutz*

Department of Toxicology, University of Wu¨ rzburg, Versbacher Strasse 9, D-97078 Wu¨ rzburg, Germany Received February 17, 1998

A 32P-postlabeling method was established for the quantitative characterization of 2′deoxyguanosyl O6-adducts of styrene 7,8-oxide in DNA. The two regioisomeric adducts, O6-(2hydroxyl-1-phenylethyl)-2′-deoxyguanosine 3′-phosphate (R-isomer) and O6-(2-hydroxyl-2phenylethyl)-2′-deoxyguanosine 3′-phosphate (β-isomer), were synthesized and used for optimizing and quantifying the various analytical steps. The adducts were stable at pH 7 and 10, but not at pH 4. The adducts were sensitive to dephosphorylation during the standard nuclease P1 (NP1) treatment. Within 30 min, 73 and 94% of the R- and β-isomers were digested. Adducts could not be extracted into butanol, and micropreparative chromatography on reversedphase thin layers resulted in a loss of adducts at low levels. Therefore, further methods of enrichment had to be investigated. Micropreparative reversed-phase HPLC chromatography on a C18 column resulted in a many thousand-fold purification from the normal nucleotides. Further enrichment was achieved with a mild NP1 treatment. The phosphorylation efficiency with polynucleotide kinase was 5 and 15% for the R- and β-isomers, respectively. Adduct analysis was performed with reversed-phase TLC followed by contact transfer of the origin to a polyethyleneimine-cellulose sheet and two-dimensional development. Addition of various amounts of adduct standard to the hydrolysate of 30 µg of DNA isolated from a control rat liver showed limits of detection of three and two adducts per 107 nucleotides for the R- and β-isomers, respectively. The applicability of the newly developed method was demonstrated by the DNA analysis of styrene-exposed rats.

Introduction Styrene is an important industrial chemical used in the production of plastics, resins, and polymers. In animals and humans, it is metabolized mainly via styrene 7,8-oxide which has a weak electrophilic reactivity (1). Reaction with guanine in DNA has been shown to occur at the 7(N)-, N2-, and O6-positions, and the 1- or 2-position of the 2-carbon side chain of styrene 7,8-oxide can be involved. This results in the formation of regioisomeric alkylation products (called the R- and β-isomers; see Figure 1). The proportion of 7(N)-, N2-, and O6-alkylated guanine nucleotides formed in double-stranded DNA was reported to be 74:22:4 after in vitro incubation with styrene 7,8-oxide (2). The 32P-postlabeling method for DNA adduct detection has been used in a number of studies published by two groups. Hemminki and co-workers reported labeling efficiencies of 20% for the 2′-deoxyguanosyl N2-adducts and 10 and 5% for the R- and β-isomers of 2′-deoxyguanosyl-O6-styrene 7,8-oxide adducts, respectively. The predominantly formed deoxyguanosyl 7(N)-adducts were barely substrates for the T4 polynucleotide kinase (PNK) (3). Deoxyguanosyl-N2- and O6-styrene adducts were reported to be resistant toward nuclease P1 (NP1)1 treatment (4), and this enrichment procedure was also used for biomonitoring studies in humans. In the DNA * To whom correspondence should be addressed. Telephone: +49-931-201-5402. Fax: +49-931-201-3446. E-mail: [email protected].

Figure 1. Structures of O6-(2-hydroxy-1-phenylethyl)-2′-deoxyguanosine 3′-phosphate (R-isomer) and O6-(2-hydroxy-2-phenylethyl)-2′-deoxyguanosine 3′-phosphate (β-isomer), two regioisomeric adducts formed by the reaction of styrene 7,8-oxide with DNA.

of lymphocytes of lamination workers, the R-isomer of 2′deoxyguanosyl-O6-styrene 7,8-oxide adducts was detected at a level of five adducts per 108 nucleotides (5, 6). Postlabeling analysis of adducts in DNA of styrenetreated animals was only recently reported. The adduct level of O6-adducts in the lungs of mice, 3 h after a single 1 Abbreviations: BG, background; [γ-32P]ATP, adenosine [γ-32P]triphosphate; MN, micrococcal nuclease; NP1, nuclease P1; PEI, polyethyleneimine; RAL, relative adduct labeling; SPDE, spleen phophodiesterase.

10.1021/tx980028c CCC: $18.00 © 1999 American Chemical Society Published on Web 12/15/1998

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ip dose of 0.5 mmol of styrene/kg of body weight, was about 15 O6-adducts per 108 nucleotides (7). A modification of the postlabeling technique later enabled the detection also of 2′-deoxyguanosyl 7(N)adducts (8, 9). Up to 18 adducts per 108 nucleotides were detected in the liver of mice after a single ip dose of 0.5 mmol/kg (7). Bodell and co-workers also described a postlabeling method using nuclease P1 enrichment (10, 11). After incubation of calf thymus DNA with styrene 7,8-oxide, six adduct spots were detected. Three of them were identified by cochromatography with a chemically synthesized standard as the O6-R-, O6-β-, and N2-styrene adducts (10, 11). In human lymphocytes of styreneexposed workers, only the N2-adduct was detected. The reported mean adduct level was 16 adducts per 108 nucleotides (12). There was no evidence of the presence of the 2′-deoxyguanosyl-O6-styrene 7,8-oxide adducts in these studies. It was not possible to explain the divergent results obtained by the two groups with respect to the styrene adduct patterns in similar samples. A comparison of the results was rendered difficult in view of the different chromatographic methods used. Furthermore, information on recoveries, quantification, and detection limits was not available. In view of the importance of quantitative data in human biomonitoring studies, it was considered necessary to investigate the postlabeling procedure in a more quantitative manner. For this purpose, adduct standards were synthesized in sufficient amounts, and labeling efficiency, enrichment procedures, and chromatographic properties were thoroughly investigated. The limit of detection was determined by a standard addition procedure. The applicability of the newly developed method was demonstrated by the DNA analysis of styrene-exposed rats.

Materials and Methods Caution: Styrene oxide is mutagenic and/or carcinogenic and should be handled with care. Chemicals and Equipment. Analytical separation and identification of the mixture of regioisomers were performed with a Hewlett-Packard HPLC system (model 1050) equipped with a diode array detector, using a C18 reversed-phase column (LiChrospher, Merck). Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were recorded with Bruker WM 250 and Bruker WM 600 instruments. UV spectra were obtained with a Kontron Uvikon 860 apparatus. Mass spectra were recorded with a HPLC-coupled mass spectrometer with an electrospray interface (Fisons Trio 2000). Detection and quantification of radioactive spots on TLC plates were performed using a PhosphorImager (Molecular Dynamics) or an InstantImager (Canberra Packard). [γ-32P]ATP (specific activity of 5000 Ci/mmol, 10 µCi/µL) was purchased from Amersham and cloned T4 polynucleotide kinase from U.S. Biochemicals. Micrococcal nuclease (MN), potato apyrase, and nuclease P1 (NP1) were supplied by Sigma Chemical Co. (St. Louis, MO). Spleen phosphodiesterase (SPDE) was purchased from Boehringer-Mannheim Corp. Polyethyleneimine-cellulose thin-layer chromatography (TLC) plates were from Macherey and Nagel (Du¨ren, Germany). C18 reversedphase TLC plates were from Merck. All other chemicals and biochemicals were of analytical grade and were used without further purification. Animals. Liver samples of styrene-exposed rats could be obtained from the Styrene Information and Research Center (SIRC). Treatment was by inhalation of 1000 ppm styrene, 6 h per day, 5 days per week, for 104 weeks.

Otteneder et al. Synthesis of O6-(2-Hydroxyl-1-phenylethyl)-2′-deoxyguanosine 3′-Phosphate (r-Isomer) and O6-(2-Hydroxyl2-phenylethyl)-2′-deoxyguanosine 3′-Phosphate (β-Isomer). The 2′-deoxyguanosyl-O6-styrene 7,8-oxide adducts were synthesized according to the published procedure by Pongracz et al. (10). The regioisomeric mixture was separated with a semipreparative RP-18 reversed-phase column (Lichrospher, 8 mm i.d., 250 mm length, 10 µm particle size) by a linear gradient of 10 mM ammonium acetate (pH 5.1) (A) and methanol (B) from 0 to 70% B over the course of 30 min (3 mL/min flow rate). The fraction showing O6-substitution (according to UV spectra) was collected (R at 20.0 min and β at 22.0 min) and lyophilized (yield of 2.9 mg, 3%). For R and β: UVmax 247, 281 nm; UVmin 260 nm; HPLC/ electrospray MS (m/z) 468 (MH+), 272 (BH+); 1H NMR (600 MHz, D2O) δ 8.00 (s, 1H, C-8), 7.45-7.30 (m, 5H, aromatic protons), 6.29 (t, 1H, H-1′, J1′2′ ) 7 Hz), 6.22 (m, 1H, R-CH), 5.13 (m, 1H, β-CH), 4.87 (m, 1H, H-3′), 4.60 (m, 2H, β-CH2), 4.24 (m, 1H, H-4′), 3.92 (m, 2H, R-CH2), 3.76 (m, 2H, H-5′), 2.812.58 (m, 2H, H-2′); 13C NMR (150 MHz, D2O) δ 165.5 (C-6), 162.5 (C-2), 155.6 (C-4), 142.1 (C-8), 131.4-129.1 (aromatic ring), 117.3 (C-5), 88.1 (C-1′), 86.1 (C-4′), 80.3 (R-CH), 76.9 (C-3′), 73.1 (β-CH2), 72.1 (R-CH), 67.4 (R-CH2), 63.2 (C-5′), 39.4 (C-2′). pH Stability of Adducts and Analysis. Two hundred fifty microliters of an aqueous solution of a mixture of the R- and β-isomers was added to 750 µL of buffer. The following buffers were used: 100 mM ammonium formate/formic acid (pH 4.0), 200 mM NaH2PO4/NaOH (pH 7.0), and 200 mM ammonium carbonate/NaOH (pH 10.0). At different times, the incubation mixture was analyzed by HPLC (see above) with a linear gradient of 25 mM ammonium formate/formic acid (pH 5.1) (A) and methanol (B) from 0 to 70% B over the course of 30 min (1 mL/min flow rate; analytical RP18 reversed-phase column, Lichrospher, 4 mm i.d., 250 mm length, 5 µm particle size). Preparation of Styrene 7,8-Oxide-Modified DNA. One milligram of calf thymus DNA was dissolved in 1 mL of 10 mM Tris-HCl (pH 7.5). To this mixture was added 20 µL of styrene 7,8-oxide, and the mixtute was incubated at 37 °C for 12 h. The mixture was extracted twice with 1 mL of diethyl ether to remove traces of unreacted styrene 7,8-oxide. DNA was further treated with RNAse and isolated as described below. DNA Isolation and Hydrolysis. DNA was isolated from rat liver according to the method of Beach and Gupta (13) with the following modifications. The final concentrations of RNAse A and RNAse T1 were 150 µg/mL and 1 unit/µL, respectively. Sodium dodecyl sulfate (10%) was added prior to proteinase K (200 µg) treatment. DNA (100 µg) was digested to nucleoside 3′-phosphates with 25 µL of 10× MN-SPDE buffer [100 mM sodium succinate and 50 mM CaCl2 (pH 6.0)] and 25 µL of MN-SPDE (1 µg/µL of each) at 37 °C for 4 h in a total volume of 250 µL. Enrichment Procedures. (1) Nuclease P1 Treatment. The nuclease P1-enhanced 32P-postlabeling procedure as described by Reddy and Randerath (14) was used. The stability of the adducts toward 3′-dephosphorylation was assayed by incubating 200 fmol of each isomer of the 2′deoxyguanosyl-O6-styrene 7,8-oxide adduct standard with 5 µg of NP1. The enzymatic reaction was stopped by adding 2.4 µL of 500 mM Tris base after incubation for 10, 20, and 30 min at 37 °C. The control value (t ) 0 min) was obtained by adding Tris base before incubating with NP1. The mixture was 32Plabeled as described later. NP1 (5 µg) was added to the DNA digest and the mixture incubated for 30 min at 37 °C. The digest was radiolabeled as described below. (2) Butanol Extraction. Adducts were enriched by butanol extraction as described by Gupta (15). The labeling reaction was performed as described below. (3) Micropreparative TLC. The DNA digest was applied to the C18 plate (four samples per plate, 10 cm × 10 cm). The plate was developed in 0.4 M ammonium formate to 6 cm onto

32P-Postlabeling

of Styrene-DNA Adducts

a Whatman #17 paper wick. The plates were dried with an airdryer for 10 min with warm air. To extract adducts, the C18 material from the origins was scraped off, transferred to 1.5 mL polypropylene microcentrifuge tubes, and extracted with 600 µL of acetonitrile/water (70:30). After centrifugation at 10000g for 5 min, the supernatant was transferred to a new tube and the pellet was extracted again with 600 µL of acetonitrile/water. The combined supernatant fractions were centrifuged again for 10 min at 10000g to remove fine particles and transferred to a new tube before evaporation in a vacuum. The residue was dissolved in 10 µL of water and radiolabeled. (4) Micropreparative HPLC. Fifty micrograms of the DNA digest was separated by HPLC on a RP-18 reversed-phase column (Lichrospher, 4 mm i.d., 125 mm length, 5 µm particle size) with a linear gradient of 10 mM ammonium formate/formic acid (pH 5.1) (A) and methanol (B) from 0 to 70% B over the course of 30 min (1 mL/min flow rate). One-milliliter fractions (tR ) 15-25 min) of the column eluate were collected, lyophylized, and redissolved in 10 µL of water. (5) Micropreparative HPLC, followed by Mild Nuclease P1 Treatment. Separation was performed as described above except that three-milliliter fractions were analyzed. The 10 µL samples were further hydrolyzed by adding 5.8 µL of nuclease P1 solution [1.0 µL of NP1 (0.1 µg/µL), 3.0 µL of 250 mM NaOAc, and 1.8 µL of 0.3 mM ZnCl2] and incubated at 37 °C for 15 min to reduce unmodified nucleotides. At the end of the incubation, 2.4 µL of 500 mM Tris was added. The samples were lyophylized and redissolved in 10 µL of water. 32P-Postlabeling with [γ-32P]ATP. The sample was converted into 32P-labeled deoxyguanosine 3′,5′-bisphosphate by adding 7.4 µL of “hot mix” prepared by mixing 5 µL of [γ-32P]ATP (50 µCi), 2.2 µL of labeling buffer [200 mM bicine/NaOH (pH 9.6), 100 mM MgCl2, 100 mM dithiothreitol, and 10 mM spermidine], 0.1 µL of 100 µM ATP, and 0.3 µL of T4 polynucleotide kinase (8 units) and incubating for 45 min at room temperature. To the labeled sample was added 4 µL of potato apyrase (40 milliunits), and the mixture was incubated for 20 min. The labeling mixture (1 µL) was diluted with water to 100 µL prior to adding apyrase. Aliquots of 5 µL were chromatographed on PEI-cellulose TLC plates using 0.4 M sodium phosphate (pH 6.0). The amount of [γ-32P]ATP consumed during the labeling reaction was determined by comparing the amount of radioactivity of the ATP spot with the amount of radioactivity of the normal nucleotide spots. Adduct Analysis by Reversed-Phase TLC and Contact Transfer to PEI-Cellulose. For mapping the adducts, the undiluted samples of the 32P-labeling mixture were applied to a 10 cm × 10 cm reversed-phase thin-layer plate. The plate was developed in 0.4 M ammonium formate/formic acid (pH 6.2) to 6 cm onto a Whatman #17 paper wick which had been attached to the top of the plate. Square chips (1.5 cm × 1.5 cm) containing the origin areas were excised and attached with a clothes peg at 2 cm both from the bottom and from the left edge of the PEIcellulose plate (10 cm × 13 cm). The adducts were transferred to the cellulose plate by developing in n-propanol/H2O (1:1) containing 1% Nonidet P40 for 90 min at 45 °C in preheated tanks. The plates were washed for 10 min with H2O and airdried. Paper wicks (Whatman #1, 3 cm) were attached, and the plates were developed in 1.5 M formic acid/lithium hydroxide and 3.8 M urea (pH 3.9) from the bottom to the top of the plate. The plates were washed in H2O, air-dried, and redeveloped at a right angle to the previous direction in 1.5 M NaH2PO4/NaOH (pH 6.0) onto a #1 paper wick of 3.5 cm. The DNA adduct spots on the TLC plates were visualized and quantified using a counting gas-based autoradiography system. Standard Addition Curve and Limit of Detection. Different amounts of synthesized standard were added to the combined 16-22 min fractions of the HPLC eluate of a 30 µg DNA digest from a rat liver. The samples were subjected to mild NP1 treatment, radiolabeled, and chromatographed as described above. Measured counts per minute values at the position of

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 95 Table 1. Analysis of the Two Regioisomeric 2′-Deoxyguanosyl-O6-styrene 7,8-Oxide Adducts r and β in Liver DNA of Female CD Rats, after Exposure by Inhalation to 1000 ppm Styrene for 2 Yearsa (6 h/day and 5 days/week) BG animal R-spot β-spot area BG area net R net β no. (cpm) (cpm) (cpm) (cpm/mm2) (cpm/mm2) (cpm/mm2) 397 675 397b

3703 7301 8343

2241 11 127 3430 20 431 9350 26 911

10.74 20.04 19.05

9.31 19.42 26.05

1.40 -1.50 31.50

a Animal 397 was an untreated control, and animal 675 was styrene-exposed. b Control sample spiked with 66 fmol of adduct standard of both isomers.

the R- and β-isomers were normalized to a specified time to account for the radioactive decay and plotted against the amount of standard added. A linear regression and a 95% confidence band were calculated using the StatView Student v1.0 software package. The confidence limits were approximated by the two straight lines parallel to the linear regression. The range is equivalent to the 95% confidence interval at the background (x ) 0 fmol added). The limit of detection was calculated as described previously (16). A horizontal line was drawn from the point of intersection between the ordinate and the upper limit of the confidence band to the lower limit of the confidence band. The respective value on the abscissa represents the limit of detection for single values. Quantification of Adducts. The amount of radioactivity in the areas of the two adduct spots was counted. To account for the individual TLC background, a larger area combining both adduct spots and extending to the left and right was also counted and the difference was used for background subtraction. The amount of net radioactivity in the adduct spots (counts per minute per square millimeter) was converted to absolute amounts of adducts (femtomoles) on the basis of the measurements obtained with the added standard, analyzed in parallel. Adduct concentrations (adducts per 107 nucleotides) were calculated from the amounts of adduct and the amount of DNA, determined on the basis of the amount of normal nucleotides eluting via HPLC. An example for the quantification of the R-adduct isomer is given below (data from Table 1). Addition of 66 fmol of the R-isomer adduct to the control sample resulted in an increase of the net count of 16.74 (26.05 - 9.31) cpm/mm2. Therefore, 0.25 (16.74/66) cpm/mm2 represent 1 fmol of adduct. In the styrene-exposed rat, a treatment-related increase of 10.11 (19.42 - 9.31) cpm/mm2 was noted. This corresponds to 40 fmol of adduct. The DNA equivalent loaded on the TLC was 19 µg, i.e., 61 nmol of nucleotides. Therefore, the adduct concentration of the R-isomer in the treated animal was seven per 107 nucleotides.

Results Synthesis of 2′-Deoxyguanosyl-O6-styrene 7,8Oxide Adduct Standards. O6-(2-Hydroxy-1-phenylethyl)-2′-deoxyguanosine 3′-phosphate and O6-(2-hydroxy2-phenylethyl)-2′-deoxyguanosine 3′-phosphate (Figure 1) were characterized by UV and electrospray MS and 1H NMR and 13C NMR. The data are given in Materials and Methods. pH Stability and 32P-Labeling Efficiency. The stability of the adducts at various pH values was investigated by incubating aliquots of the regioisomeric derivatives at 37 °C for different amounts of time. At pH 7 and 10, no degradation was observed for up to 92 h. At pH 4, the adducts were not stable; the half-lives of the R- and β-isomers were about 12 and 30 h, respectively (data not shown). The time-dependent formation of 2′deoxyguanosine 3′-phosphate detected by HPLC indi-

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Figure 2. Stability of 2′-deoxyguanosyl-O6-styrene 7,8-oxide adduct isomers incubated for various periods of time with nuclease P1, determined by 32P-postlabeling. Means of two independent experiments ( 1 SD (shown if larger than the size of the symbol).

cated a cleavage of the styrene moiety from the nucleotide by hydrolysis of the O6-aralkyl ether bond. The different half-lives of the R- and β-isomers show that the linkage to the primary benzylic R-carbon is more acid-labile than the linkage to the secondary benzylic β-carbon. The adduct decay may not operate at physiological pH but should be taken into account during DNA analysis. The labeling efficiencies of the adducts were determined by measuring the amount of labeled deoxyguanosine bisphosphate formed in relation to the amount of standard incubated (femtomole amounts). Labeling efficiencies of 5 and 15% were calculated for the R- and β-isomers, respectively. It was independent of small contamination with normal nucleotides, as long as an excess of ATP was available. Two-Step TLC of Adducts. For the separation of adducts from [32P]ATP and other contaminants after the 32P-postlabeling step, the multidirectional TLC using 1 M sodium phosphate as the D1 solvent (a standard procedure for highly lipophilic adducts) was not suitable, because the styrene oxide DNA adducts did not bind strongly enough to the PEI-cellulose material. Therefore, C18 reversed-phase TLC and PEI-cellulose TLC were combined for adduct analysis (17). This was performed by in situ contact transfer of adducts from the C18 layer to the PEI-cellulose layer. The recovery of the contact transfer was 50%. The amount of background radioactivity was dramatically reduced by this additional step. The subsequent adduct separations were achieved by chromatography with more dilute electrolyte/urea solvents than those needed for lipophilic aromatic DNA adducts. Comparison of Different Enrichment Procedures. (1) Nuclease P1 Treatment. The 2′-deoxyguanosyl-O6-styrene 7,8-oxide adducts were not stable toward 3′-dephosphorylation by NP1 in our hands. Seventythree percent and 94% of the applied R- and β-isomers were digested by NP1 within 30 min, as determined by 32P-postlabeling (Figure 2). To confirm our results in the presence of the DNA digest, a styrene-adducted calf thymus DNA was also examined. Adduct levels determined after NP1 treatment (R- and β-isomer values of 200 and 100 per 105 nucleotides, respectively) were

Figure 3. 32P-Postlabeling analysis of calf thymus DNA treated with styrene 7,8-oxide: (A) nuclease P1 enrichment, (B) butanol extraction, and (C) TLC enrichment. Different exposure times for autoradiography in panels A-C.

considerably lower than with the C18 TLC enrichment procedure (430 and 1200 per 105 nucleotides, respectively; Figure 3, top chart). (2) Butanol Extraction. The recovery of both isomer standards was lower (R- and β-isomer values of 10 and 40 per 105 nucleotides, respectively) compared to those obtained with NP1 and chromatographic enrichment procedures (Figure 3, center chart). This procedure was therefore not appropriate for styrene adducts. (3) Micropreparative TLC. Separation of adducts from normal nucleotides by reversed-phase TLC (18) resulted in the highest recoveries on average (R- and β-isomer values of 430 and 1200 per 105 nucleotides, respectively; Figure 3, bottom chart). However, this method produced largely variable results. Small amounts

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of Styrene-DNA Adducts

Figure 4. HPLC elution profile of digested DNA with UV detection at 250 nm. Retention times (tR) for the chemically synthesized R- and β-isomers were 16 and 19 min, respectively.

Figure 5. 32P-Postlabeling analysis of residual normal nucleotides from DNA digests of 5 and 50 µg of DNA in the retention time range of adduct elution after chromatographic separation with HPLC. Three-milliliter fractions (tR ) 16-25 min) were analyzed.

of added standard were irreversibly bound to the C18 plate, as determined with the respective dose-response curve (data not shown). The adducts could only be detected above approximately 50 fmol added. On the basis of 10 µg of DNA, the limit of detection would at best be three adducts per 106 nucleotides. (4) Micropreparative HPLC. A micropreparative C18 HPLC technique for the separation of 2′-deoxyguanosylO6-styrene 7,8-oxide adducts was developed. The normal nucleoside 3′-phosphates elute earlier than the 2′-deoxyguanosyl-O6-styrene 7,8-oxide adducts on a HPLC reversed-phase column, due to their higher polarity. Under the conditions used, the retention times for the R- and β-styrene adducts were about 16 and 19 min, respectively (see Figure 4 for the optical density profile). Different amounts of DNA digest (5 and 50 µg) were separated by HPLC, and the quantity of residual normal nucleotides in the range of adduct elution was determined by 32Ppostlabeling of 3 mL fractions at elution times between 16 and 25 min (Figure 5). At the retention time of adduct elution, the amount of normal nucleotides was below the optical detectability. Still, up to 120 pmol of normal nucleotides in one 3 mL fraction was measured at the elution time of the adducts. On the basis of the results obtained with 50 µg of DNA, the extent of adduct enrichment was about 1000-fold.

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(5) Micropreparative HPLC and Mild Nuclease P1 Treatment. A modified nuclease P1 treatment (about 250 times less NP1 enzyme compared to the standard protocol) was used after HPLC enrichment to further reduce the amount of normal nucleotides and lower the detection limit. Thirty micrograms of DNA digest (from control rat liver) was spiked with 125 fmol of the 2′-deoxyguanosyl-O6-styrene 7,8-oxide adduct standards, and the 3 mL fractions were analyzed by two-dimensional chromatography (Figure 6). The excess ATP and the amount of normal nucleotides were determined by one-dimensional chromatography. In the fractions containing the adducts, sufficient ATP was available for the phosphorylation reaction. The overall extent of enrichment of adducts over normal nucleotides was more than 10000-fold. Standard Addition Curve and Limit of Detection. A standard addition curve was generated by spiking an HPLC eluate (16-22 min) of 30 µg of DNA digest with different amounts of R- and β-isomer standards. After mild NP1 treatment and two-step TLC, the amount of radioactivity in the R- and β-spots was counted and plotted against the amount of standard added (Figure 7). Each symbol represents an independent analysis. The analyses were performed with the same labeling batch. Regression lines calculated from the duplicate values and their 95% confidence band for the true mean of counts per minute values were used for the determination of the limit of detection (16). The amount of 2′-deoxyguanosyl-O6-styrene 7,8-oxide adduct required to generate a significant increase would be three and two adducts per 107 normal nucleotides for the R- and β-isomers, respectively, for a 30 µg DNA sample and a single determination. Quantification and Recovery. Quantification of the overall recovery was based on a standard addition of DNA digest prior to HPLC. For the R-isomer adduct, recoveries of the HPLC separation, NP1 treatment, phosphorylation, and contact transfer were 80, 100, 5, and 50%, respectively, and the respective recoveries for the β-isomer adduct were 80, 80, 15, and 50%. Any loss of DNA adducts during the DNA isolation and digestion could not be investigated. Therefore, given adduct levels represent minimum values. Detection of Styrene-Derived DNA Adducts in Styrene-Exposed Rats. Liver DNA of female CD rats was analyzed for styrene adducts eluting in the combined fractions (16-22 min). Figure 8 clearly shows a treatment-related formation of the R-adduct isomer. Quantification of the adduct concentration based on the raw data given in Table 1 and as described in Materials and Methods resulted in a value of seven adducts per 107 nucleotides.

Discussion The 32P-postlabeling procedure was adapted and optimized for the determination of the two regioisomeric 2′deoxyguanosyl O6-adducts formed between styrene 7,8oxide and DNA. Numerous control experiments were performed so adduct levels in DNA samples analyzed from animal experiments and humans could be reliably quantitated. One of the important parts of the 32P-postlabeling assay is the separation of the carcinogen-adducted nucleotides

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Figure 7. Standard addition curves of 2′-deoxyguanosyl-O6styrene 7,8-oxide adducts determined by 32P-postlabeling. Various amounts of R- and β-isomer adduct standards were added after micropreparative reversed-phase HPLC of DNA digests. The uninterrupted line shows the linear regression of the counts per minute values. The interrupted line shows the upper and lower 95% confidence limit. The confidence limits were approximated by two straight lines parallel to the linear regression. The range is equivalent to the 95% confidence interval at the background level (x ) 0 fmol added). The limits of detection were three and two adducts per 107 nucleotides for the R- and β-isomers, respectively.

Figure 6. Autoradiograms of rat liver DNA (30 µg) spiked with 125 fmol (R- and β-isomer each) of the 2′-deoxyguanosyl-O6styrene 7,8-oxide adduct (corresponding to 13 adducts per 107 nucleotides). Three-milliliter fractions with elution times between 16 and 25 min were collected, treated mildly with nuclease P1, 32P-postlabeled, and separated via TLC.

from the normal nucleotides to increase the assay’s sensitivity. The lower the concentration of normal nucleotides, the less 32P-ATP is required and the lower the amount of background radioactivity on the TLC and its variability between experiments and chromatograms.

In contrast to another report (4), the 2′-deoxyguanosylO6-styrene 7,8-oxide adducts were not stable toward NP1 treatment in our hands. The instability was quantified by using both the synthesized standard and styrene 7,8oxide-modified DNA. The adduct levels in styrene 7,8oxide-modified DNA were significantly lower than with other enrichment methods tested. The different intensities of the adduct spots of the R- and β-isomers are in agreement with the different specificity of NP1 for both isomers used as standards. Therefore, despite a higher labeling efficiency for the β-isomer, the major spot corresponded to the R-isomer (Figure 3A). Enrichment by extraction of adducts into butanol or by C18 TLC was not successful, either. The use of C18 TLC resulted in a loss of adducts, particularly when working with small amounts. This is possibly due to some irreversible binding to soluble adsorbent material of the

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the 1H NMR and 13C NMR spectra and Dr. G. Birner from this department for determining the mass spectral data. We thank the summer student M. Welter for his help. This work was supported by the Styrene Steering Committee of CEFIC.

References

Figure 8. 32P-Postlabeling maps of DNA isolated from the liver of an untreated rat and of a rat (control) exposed for 2 years to 1000 ppm styrene (6 h/day and 5 days/week). The autoradiogram of the control rat shows the ellipse chosen for background subtraction (III) and areas I and II of the R- and β-isomers, respectively.

C18 phase and might make this method unreliable for other applications as well. A combination of micropreparative HPLC followed by a mild NP1 treatment and two-step TLC generated the best results. HPLC enrichment should be a generally applicable method for small and polar DNA adducts. It is only limited by a potentially insufficient separation from the normal nucleotides. The additional mild NP1 step helped overcome the restriction for the adducts under investigation. The 2′-deoxyguanosyl-O6-styrene 7,8-oxide adducts were relatively stable toward mild NP1 treatment. The overall recoveries of 2 and 5% for the R- and β-isomers appear to be low. It includes all steps after DNA digestion. To account for the variability between labeling batches, adduct concentrations were always on the basis of a sample spiked with synthetic standard, and analyzed in parallel. For the conversion of the amounts of radioactivity measured in the TLC spots into adduct concentrations, a standard addition procedure was performed. Using criteria generally set by good analytical chemical practice, the limit of detection was two to three adducts per 107 nucleotides. The described method is by a factor of about 10 less sensitive than the method used for levels reported elsewhere (5, 6). The use of more DNA could theoretically lower the detection limit, but it must be considered that the capacity of the enrichment steps is limited. Higher amounts of “contaminating” normal nucleotides would entail the use of larger amounts of radioactive ATP. Higher levels of 32P lead to higher background radioactivity levels on TLC and larger variability. This, in turn, limits the detectability of an adduct-related increase. In view of our extensive attempts to optimize the analysis, reported adduct levels in human lymphocytes (on the order of five adducts per 108 nucleotides) should be viewed with caution.

Acknowledgment. We are indebted to D. Koppler from the Department of Organic Chemistry for recording

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