Polyacrylamide Gel Electrophoresis Analysis

Feb 14, 2002 - 32P-Postlabeling analysis is a powerful technique to detect DNA adducts. Polyethylenimine−cellulose TLC plates are generally used to ...
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Chem. Res. Toxicol. 2002, 15, 305-311

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Articles 32P-Postlabeling/Polyacrylamide

Gel Electrophoresis Analysis: Application to the Detection of DNA Adducts Isamu Terashima, Naomi Suzuki, and Shinya Shibutani* Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651 Received April 20, 2001 32P-Postlabeling

analysis is a powerful technique to detect DNA adducts. Polyethyleniminecellulose TLC plates are generally used to separate 32P-labeled adducts using several different buffers. However, separation by TLC is time-consuming and labor-intensive for a large number of DNA samples. To expedite analyses, nondenaturing 30% polyacrylamide gel electrophoresis (PAGE) has been adapted for the 32P-postlabeling analysis. The major advantages of this technique are as follows: (a) many DNA samples can be loaded concomitantly on the PAGE with standard markers; (b) DNA adducts can be resolved in only a few hours; and (c) exposure to 32P during handing can be minimized. To show the usefulness of 32P-postlabeling/PAGE analysis, the formation of a tamoxifen (TAM)-DNA adduct resulting from O-sulfonation of R-hydroxytamoxifen was demonstrated. In addition, to quantify TAM adducts, oligodeoxynucleotides containing diastereoisomers of R-(N2-deoxyguanosinyl)tamoxifen can be used as standards. The detection limit of this assay for 5 µg of DNA was approximately 7 adducts/109 nucleotides. The 32P-postlabeling/PAGE analysis can also be used to detect DNA adducts derived from benzo[a]pyrene diol epoxide, 2-acetylaminofluorene, and 4-hydroxyequilenin.

Introduction 32P-Postlabeling analysis is a powerful technique for detection of DNA adducts induced by endogenous and exogenous mutagens or carcinogens (1, 2). Polyethylenimine (PEI)1-cellulose TLC plates are generally used to separate 32P-labeled adducts two-dimensionally under several different buffer conditions. This technique is relatively simple and does not require any costly equipment (2). However, since only one 32P-labeled sample can be analyzed per plate, much time and labor are required to analyze large numbers of DNA samples. Since the migration of DNA adducts on a TLC plate can be variable, co-chromatography is required to identify the DNA adducts. To separate the targeted DNA adduct on a TLC plate, several buffer conditions are used depending on the individual adducts. Therefore, use of TLC plates for 32P-postlabeling analysis is inconvenient and timeconsuming.

* To whom correspondence should be addressed. Phone: 631-4448018. Fax: 631-444-3218. E-mail: [email protected]. 1 Abbreviations: PEI, polyethylenimine; PAG, polyacrylamide gel; PAGE, polyacrylamide gel electrophoresis; dG, 2′-deoxyguanosine; dN3′P, 2′-deoxynucleoside 3′-monophosphate; 5′PdG, 2′-deoxyguanosine 5′-monophosphate; 5′PdG3′P, 2′-deoxyguanosine 3′,5′-diphosphate; TAM, tamoxifen; TAM R-sulfate, tamoxifen R-sulfate; R-OHTAM, R-hydroxytamoxifen; dG-N2-TAM, R-(N2-deoxyguanosinyl)tamoxifen; dG3′P-N2TAM, 2′-deoxyguanosine 3′-monophosphate-N2-tamoxifen; BPDE, 7,8dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; dG-N2-BPDE, benzo[a]pyrene diol epoxide-N2-2′-deoxyguanosine; AAF, 2-acetylaminofluorene; AF, 2-aminofluorene; dG-C8-AAF, N-(deoxyguanosin-8-yl)2-acetylaminofluorene; dG-C8-AF, N-(deoxyguanosin-8-yl)-2-aminofluorene; 4-OHEN, 4-hydroxyequilenin; PNK, polynucleotide kinase.

We reported previously that a single different deoxynucleoside (dC, dA, dG, or dT) embedded in an oligodeoxynucleotide results in different migration on a polyacrylamide gel (PAG) (3, 4). Oligodeoxynucleotides containing a single DNA adduct such as N-(deoxyguanosin8-yl)-2-acetylaminofluorene (dG-C8-AAF) (5), benzo[a]pyrene diol epoxide-N2-2′-deoxyguanosine (dG-N2-BPDE) (6), or N-(deoxyguanosin-8-yl)-2-tamoxifen (dG-N2-TAM) (7) migrated slower than the unmodified oligodeoxynucleotides. Based on these observations, the modified monomeric deoxynucleotides were expected to be resolved from the unmodified nucleotides on a PAG. In the present study, we developed a 32P-postlabeling analysis coupled with nondenaturing 30% polyacrylamide gel electrophoresis (32P-postlabeling/PAGE analysis). To show the usefulness of this assay, detection of tamoxifen (TAM)-DNA adducts was performed. We also demonstrated that oligodeoxunucleotides modified site-specifically with dG-N2-TAM can be used as standards to quantify TAM adducts. The 32P-postlabeling/PAGE assay could also be used to detect other DNA adducts such as 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-, 2-acetylaminofluorene (AAF)-, and 4-hydroxyequilenin (4-OHEN)-derived DNA adducts. This technique is much more convenient and much less timeconsuming than 32P-postlabeling/TLC analysis.

Experimental Procedures Materials. Calf thymus DNA, micrococal nuclease, and potato apyrase were purchased from Sigma-Aldrich (St. Louis,

10.1021/tx010083c CCC: $22.00 © 2002 American Chemical Society Published on Web 02/14/2002

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MO). Spleen phosphodiesterase was obtained from Worthington Biochemical Corp. (Freehold, NJ). Wild-type T4 polynucleotide kinase (PNK) was purchased from New England BioLabs Inc. (Beverly, MA). 3′-Phosphatase-free T4 PNK and nuclease P1 were from Roche Molecular Biochemicals (Indianapolis, IN). [γ-32P]-ATP (specific activity, >6000 Ci/mmol) was obtained from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). TAM R-sulfate and diastereoisomers (fr-1 and fr-2) of trans-forms and diastereoisomers (fr-3 and fr-4) of cis-forms of dG 3′-monophosphate-N2-tamoxifen (dG3′P-N2-TAM) were prepared as described previously (8, 9). Oligodeoxynucleotides containing a single stereoisomer of dG-N2-BPDE were provided by Dr. Nicholas E. Geacintov, New York University (6). Reaction of DNA with TAM r-Sulfate. Following an established protocol (10), commercially available calf thymus DNA was incubated with RNase A, RNase T1, and proteinase K, extracted with phenol/chloroform, and then precipitated by ethanol. These procedures were repeated to remove RNA and protein (10). The concentration of DNA was determined by UV absorbance (50 µg ) 1.0 OD260 nm). Calf thymus DNA (5 µg) was reacted at 37 °C for 17 h with TAM R-sulfate (0, 10, 30, or 100 µg) in 100 µL of 100 mM Tris-HCl (pH 7.5). Ice-cold ethanol (1.0 mL) was added to the reaction mixture to recover the DNA. The DNA was washed with 70% ice-cold ethanol and used for 32P-postlabeling analysis. Formation of TAM-DNA Adducts via O-Sulfonation of r-OHTAM. Calf thymus DNA (10 µg) was incubated at 37 °C for 1 h with 100 µΜ R-hydroxytamoxifen (R-OHTAM), 200 µΜ 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and rat hydroxysteroid sulfotransferase a (STa) (80 or 240 milliunits) in 50 µL of 0.25 M potassium phosphate (pH 7.5) containing 8.3 mM mercaptoethanol, as similarly described previously (9, 11). After the reaction, DNA was recovered by phenol/chloroform extraction and used for 32P-postlabeling/PAGE analysis. Digestion of DNA Samples. DNA sample (0.1-5.0 µg) was enzymatically digested at 37 °C for 2 h in 20 µL of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2, using micrococcal nuclease (30 units) and spleen phosphodiesterase (0.15 unit). The reaction mixture was incubated for another 1 h with nuclease P1 (1 unit). After the incubation, 100 µL of water was added. The reaction samples were then extracted twice with 100 µL of butanol. The butanol fractions were combined, back-extracted with 50 µL of distilled water, and evaporated to dryness. 32P-Postlabeling/PAGE Analysis. The DNA digests were incubated at 37 °C for 40 min with 20 or 30 µCi of [γ-32P]-ATP and either wild-type or 3′-phosphatase-free T4 PNK (20-30 units), and then incubated with apyrase (50 milliunits) for another 40 min, as described previously (9). A part of the 32Plabeled sample was electrophoresed for 4-5 h on a nondenaturing 30% polyacrylamide gel (35 × 42 × 0.04 cm) with 14001600 V/20-40 mA. A 30% polyacrylamide gel was prepared from 40% polyacrylamide solution (60 mL), 10× TBE buffer, pH 7.0 (10 mL), distilled water (10 mL), 10% ammonium persulfate (0.6 mL), and TEMED (35 µL). 10× TBE buffer (pH 7.0) was prepared from 1 M Tris-base, 2.24 M boric acid, and 25.5 mM EDTA. The position of 32P-labeled adducts was established by β-phosphorimager analysis (Molecular Dynamics Inc.). To determine the radioactivity of 32P-labeled products, integrated values are measured using a β-phosphorimager. Values ranging from 1 to 108 have a linear response (data not shown). When the radioactivity is beyond the range, the shorter exposure to 32P-labeled products was used to determine the radioactivity within the linear range. The relative adduct levels were calculated according to Levay et al. (12), for example, (total dpm in adducts)/(1.48 × 1011 dpm), assuming that 3 µg of DNA was 9.09 × 103 pmol of dN3′P and the specific activity of the [γ-32P]ATP was 1.63 × 107 dpm/pmol. The specific activity of the [γ-32P]-ATP was corrected by calculating the extent of decay. Quantification of dG-N2-TAM Adducts. Oligodeoxynucleotides containing diastereoisomers of trans-forms (fr-1 and fr-2) or cis-forms (fr-3 and fr-4) of dG-N2-TAM (5′TCCTCCTCXC-

Terashima et al. CTCTC, where X is a diastereoisomer of dG-N2-TAM; 4761 Da) were prepared as described previously (7). Based on the extinction coefficient at 260 nm, the concentration of this oligomer was determined; 34.8 µg (7.31 nmol) ) 1.0 OD260 nm. Therefore, when 0.152-152 fmol (0.743-743 pg) of this oligomer is mixed with 5 µg of purified calf thymus DNA (15 200 pmol of dNs), the actual level of TAM adducts in the mixture is 1 adduct/108 dNs-1 adduct/105 dNs. Such standard DNA was used to determine the recovery of TAM adducts and to quantify dG-N2-TAM adducts. BPDE- and AAF-Modified Deoxynucleotides Used for 32P-Postlabeling/PAGE Analysis. To prepare dG -N2-BPDE, 3′p oligodeoxynucleotides (5 µg) containing a single DNA adduct induced by (+)-anti-BPDE or (-)-anti-BPDE (6) were digested at 37 °C for 3 h in 20 µL of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2 using spleen phosphodiesterase (1.0 unit). dG3′P-C8-acetylaminofluorene (dG3′P-C8-AAF) was prepared by reacting dG3′P (0.3 mg) with N-acetoxy-acetylaminofluorene (0.5 mg) in 100 µL of 10 mM citrate buffer (pH 6.8) (13). dG3′P-C8-AAF was converted to dG3′P-C8-aminofluorene (dG3′P-C8-AF) under an alkaline condition (13). These modified nucleotides were isolated on a reverse-phase column, µBondapak C18 (0.8 × 30 cm, Waters), using a linear gradient of 0.05 M triethylammonium acetate (pH 7.0), containing 25-45% acetonitrile; the elution time was 40 min at a flow rate of 1.0 mL/ min (13). dG3′P-N2-BPDE (10 ng), dG3′P-C8-AAF (50 ng), and dG3′P-C8-AF (50 ng) were incubated at 37 °C for 40 min with 5 µCi of [γ-32P]-ATP and 3′-phosphatase-free T4 PNK (5 units) and then incubated with 50 milliunits of apyrase for another 30 min (9). A part of the 32P-labeled nucleotides was developed on a 30% PAG. Detection of DNA Adducts Derived from 4-Hydroxyequilenin. 4-OHEN was prepared by treating equilin with Fremy’s salt (14). Calf thymus DNA (10 µg) was reacted at 37 °C for 7.5 h with 4-OHEN (0.2 or 1.0 mg in 10 µL of DMSO) in 1.0 mL of 25 mM potassium phosphate buffer (pH 7.4). After the reaction, DNA was recovered using a molecular filter, Centricon 100 (Amicon), washed twice with 70% ethanol solution, and then evaporated to dryness. DNA sample (1.5 µg) was enzymatically digested with micrococcal nuclease, spleen phosphodiesterase, and nuclease P1, as similarly described for the TAM-DNA adducts. The procedure of butanol extraction was omitted in this case. The DNA digests were incubated at 37 °C for 40 min with [γ-32P]-ATP (20 µCi) and wild-type T4 PNK (20 units) in 20 µL of 500 mM Tris-HCl buffer (pH 9.5) containing 100 mM MgCl2, 100 mM DTT, and 10 mM spermidine, and then incubated with 50 milliunits of apyrase for another 30 min. 4-OHEN (2 mg in 10 µL of DMSO) was also reacted at 37 °C for 7.5 h with dN3′P (0.5 mg) in 1.0 mL of 25 mM potassium phosphate buffer, pH 7.4. After the centrifugation, one-twentieth of the supernatant was evaporated to dryness and incubated at 37 °C for 1 h with nuclease P1 (1 unit) in 20 µL of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2. The reaction mixture was evaporated to dryness and labeled with 32P, as similarly described for the 4-OHEN-modified DNA samples. A part of the 32P-labeled samples was analyzed by 32Ppostlabeling/PAGE assay.

Results 32 P-Postlabeling/PAGE Analysis. Calf thymus DNA was reacted with varying amounts of TAM R-sulfate. The recovered DNA was digested using the nuclease P1 enrichment method, extracted with butanol, and then labeled with 32P. When the 32P-labeled samples were subjected to 30% nondenaturing PAG, three TAM adducts were resolved (Figure 1, lanes 8-10). The migration of each adduct was consistent with that of trans-dG3′PN2-TAM (fr-1, lane 12; fr-2, lane 13) or cis-dG3′P-N2-TAM (a mixture of fr-3 and fr-4, lane 14). When DNA reacted with 100 µg of TAM R-sulfate was analyzed (lane 10),

32P-Postlabeling/PAGE

Analysis

Figure 1. Determination of TAM adducts in the DNA treated with TAM R-sulfate. Calf thymus DNA (5 µg) was reacted with TAM R-sulfate (0 µg, lane 7; 10 µg, lane 8; 30 µg, lane 9; 100 µg, lane 10) at 37 °C for 17 h. The recovered DNA (0.5 µg) was digested using the nuclease P1 enrichment method. The TAM adducts were extracted with butanol and labeled with 32P by the 3′-phosphatase-free T4 PNK, as described under Experimental Procedures. 0.1 volume of the 32P-labeled sample was developed for 5 h on a 30% PAGE. Diastereoisomers (lane 12, fr-1; lane 13, fr-2) of trans-dG3′P-N2-TAM, diastereoisomers (lane 14, a mixture of fr-3 and fr-4) of the cis-form, and the mixture of these standards (lane 11) were subjected to PAGE. Each dN3′P was also labeled with 32P and subjected to PAGE (dA3′P, lane 2; dC3′P, lane 3; dG3′P, lane 4; dT3′P, lane 5; a mixture of all dN3′P, lane 6). The migration of free [γ-32P]-ATP is shown in lane 1.

the amounts of fr-1, fr-2, and fr-3&4 were 1.3, 15.4, and 5.4 adducts/105 nucleotides, respectively. No DNA adducts were detected in the control DNA sample (lane 7). The TAM-DNA adducts migrated much slower than unmodified deoxynucleoside 3′-monophosphates (dN3′P) (lanes 2-5) and free [γ-32P]-ATP (lane 1). Four 32P-labeled dN3′P were also resolved on the PAG (lane 6). Electrophoresis of the 32P-labeled samples on the PAG took only 4-5 h. Effect of the 3′-Phosphatase Activity of T4 PNK on 32P-Labeling. Since wild-type T4 PNK has a 3′phosphatase activity, 32P-labeled nucleoside 3′,5′-diphosphates can be converted to the nucleoside 5′-monophosphates during the 32P-labeling reaction. For example, when dG3′P was labeled with 32P at pH 5.5 to pH 9.5 using a wild-type T4 PNK (Figure 2, lanes 6-10), the upper and lower bands representing 32P-labeled dG and dG3′P, respectively, was observed. When dG3′P was labeled at pH 9.5, only 32P-labeled dG3′P was detected (lane 6). Similar phenomena were observed when dC3′P, dA3′P, or dT3′P were used (data not shown). To avoid this conversion, alkaline conditions (>pH 9.5) were generally used when labeling DNA adducts with 32P, as reported earlier by Randerath et al. (15, 16). However, dG3′P-N2-TAM (fr2) was resistant to the 3′-phosphatase activity of T4 PNK

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Figure 2. Effect of the 3′-phosphatase activity of T4 PNK on the 32P-labeling efficiency of dG3′P and dG3′P-N2-TAM. 1 pmol of dG3′P-N2-TAM (fr-2, lanes 1-5 and 11-15) or dG3′P (lanes 6-10 and 16-20) was labeled with 32P using either 5 units of wildtype T4 PNK (lanes 1-10) or 0.5 unit of 3′-phosphatase-free T4 PNK (lanes 11-20) and subjected to 30% PAGE, as described under Experimental Procedures. The 32P-labeling reactions were carried out under the following pH conditions: pH 9.5 (lanes 1, 6, 11, and 16); pH 8.5 (lanes 2, 7, 12, and 17); pH 7.5 (lanes 3, 8, 13, and 18); pH 6.5 (lanes 4, 9, 14, and 19); pH 5.5 (lanes 5, 10, 15, and 20).

under all pH conditions examined (lanes 1-5). On the other hand, when 3′-phosphatase-free T4 PNK was used, only 32P-labeled dG3′P was formed under pH 5.5-9.5 buffer conditions (lanes 11-15), as similarly observed for dG3′P-N2-TAM (lanes 16-20). Preparation of TAM Adduct-Enriched Sample. To isolate the adducted nucleotides efficiently from the DNA digests, the nuclease P1 enrichment method (16) is widely used for 32P-postlabeling analysis. When TAM R-sulfatereacted DNA was digested without nuclease P1 treatment, only a trace amount (total 0.07 adduct/105 dN) of dG3′P-N2-TAM adducts were detected (Figure 3, lane 1). However, nuclease P1 treatment increased the level of detection 100-fold (7.10 adducts/105 dN) (lane 2). The amount of normal dN3′P detected with nuclease P1 (lane 2) was much lower than that without nuclease P1 (lane 1). When the butanol extraction was performed after the DNA digest procedure, the level of TAM adducts detected was increased 33-fold (lane 4). Particularly, the nuclease P1 enrichment followed by butanol extraction enhanced the detection of dG3′P-N2-TAM adducts (33.4 adducts/105 dN) 480-fold (lane 3) above that without nuclease P1 treatment and butanol extraction. Only a small amount of normal dN3′P (2.8 adducts/105 dN) was observed; the level of dN3′P was estimated to be 0.0084 pmol, 36 000 times less than the dN3′P (approximately 303 pmol) in the DNA hydrolysate and at least 400 times less than the [γ-32P]-ATP (3.34 pmol) used for the 32P-labeling. Such a small amount of the dN3′P does not interfere with the detection of TAM adducts. Thus, the butanol extraction is an essential procedure in the detection of TAM-DNA adducts by the 32P-postlabeling analysis. Quantitative Detection of TAM-DNA Adducts. Oligodeoxynucleotides containing diastereoisomers of

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Figure 3. Effect of nuclease P1 treatment and butanol extraction on detection of the TAM-DNA adduct. Calf thymus DNA (10 µg) was reacted at 37 °C for 17 h with TAM R-sulfate (500 µg). The recovered DNA (0.1 µg) was digested using micrococcal nuclease and spleen phosphodiesterase followed by nuclease P1 treatment (lanes 2 and 3) or no treatment (lanes 1 and 4). The DNA digest was extracted with (lanes 3 and 4) or without (lanes 1 and 2) butanol. These samples were labeled with 32P and subjected to PAGE. A mixture of 32P-labeled isomers of dG3′PN2-TAM and dN3′P was also subjected (lane 5).

Figure 4. Comparison of experimental and actual frequencies of TAM-DNA adducts. Variable amounts (152, 15.2, 1.52, and 0.152 fmol) of 15-mer oligodeoxynucleotides containing diastereoisomers of trans-forms (fr-2, A) or cis-forms (fr-4, B) of dG-N2-TAM (5′TCCTCCTCXCCTCTC, where X is dG-N2-TAM) were mixed with 5 µg of purified calf thymus DNA (equivalent to 15 200 pmol of dNs) for preparing a DNA standard containing 1 adduct/105 dNs, 1 adduct/106 dNs, 1 adduct/107 dNs, and 1 adduct/108 dNs, respectively. These DNA standards were digested using the nuclease P1-enrichment method followed by butanol extraction as described under Experimental Procedures. The DNA digests were labeled with 32P using 3′-phosphatasefree T4 PNK (30 units) and [γ-32P]-ATP (30 µCi), and then incubated with apyrase (50 milliunits). The 32P-labeled samples were electrophoresed for 4-5 h on a nondenaturing 30% polyacrylamide gel (35 × 42 × 0.04 cm). Comparing the radioactivity of standard [γ-32P]-ATP (0.0167 pmol), the level of TAM adducts was determined by a β-phosphorimager analysis. Data from two or three samples are plotted.

trans- (fr-1 and fr-2) or cis-dG-N2-TAM (fr-3 and fr-4) were used as standards to demonstrate the quantitative analysis using 32P-postlabeling/PAGE assay. As shown in Figure 4, variable amounts (0.152-152 fmol) of 15mer oligomers containing dG-N2-TAM were mixed with

Terashima et al.

calf thymus DNA, and the level of the TAM adducts was determined using the nuclease P1-enrichment method followed by butanol extraction. The radioactivity of TAM adducts was determined by comparison with the intensity of a known amount of [γ-32P]-ATP. The amount of TAM adducts detected increased linearly depending on the amounts of oligodeoxynucleotide used (Figure 4A,B). As illustrated in Figure 4 by the dotted line, the recovery of TAM adducts was approximately 56%. When more than 15 units of micrococcal nuclease was used for DNA digestion, the recovery of TAM adducts was not significantly changed. The minimum amount detected (7 adducts/109 dNs) was observed when 5 µg of DNA (1.52 × 107 fmol of dN) mixed with 15-mer dG-N2-TAM-modified oligomers (0.11 fmol) was examined (data not shown). Using the standard curve of each diastereoisomer of the dG-N2-TAM adduct (Figure 4A for fr-2; Figure 4B for fr4), the actual level of TAM-DNA adducts in the following in vitro experiment was determined. Similar linearity was observed for fr-1 and fr-3 of dG-N2-TAM adducts (data not shown). Since the two-diastereoisomers (fr-3 and fr-4) of cis-dG-N2-TAM are not resolved, the level of the cis-dG-N2-TAM adducts was represented as an average of values obtained separately from standards fr-3 and fr-4. Formation of TAM-DNA Adducts via O-Sulfonation of r-OHTAM. To demonstrate the formation of TAM-DNA adducts via O-sulfonation of R-OHTAM, calf thymus DNA that had been incubated with R-OHTAM, PAPS, and STa (11) was analyzed by 32P-postlabeling/ PAGE (Figure 5). In the presence of 240 milliunits of STa, fr-2 of the trans-dG3′p-N2-TAM was a major adduct (56.8 adducts/106 dN); the levels of other trans-dG3′p-N2-TAM (fr-1) and cis-dG3′p-N2-TAM (a mixture of fr-3 and fr-4) were 7.90 and 5.29 adducts/106 dN, respectively. No TAM-DNA adducts were observed in the DNA incubated without STa or in the control DNA. Detection of BPDE- and AAF-Derived DNA Adducts. Four stereoisomers of dG3′P-N2-BPDE, dG3′P-C8AAF, and dG3′P-C8-AF were labeled with 32P and run for 5 h on a 30% nondenaturing PAG. All 32P-labeled dG3′PN2-BPDE adducts (Figure 6A, lanes 3-6) migrated much more slowly than the trans-form (fr-2) of dG3′P-N2-TAM (lane 2). The migration of dG3′P-C8-AAF (lane 7) and dG3′P-C8-AF (lane 8) was faster than fr-2 of dG3′P-N2TAM. Therefore, TAM adduct can be resolved from other DNA adducts such as dG3′P-N2-BPDE and AAF-derived adducts. Detection of 4-OHEN-Derived DNA Adducts. When dN3′P was reacted with 4-OHEN under neutral buffer conditions and labeled with 32P, 4-OHEN-modified dA3′P, dC3′P, and dG3′P (Figure 6B, lanes 3-5) were detected by the 32P-postlabeling/PAGE analysis. No adducts were detected with dT3′P (lane 6). When calf thymus DNA (10 µg) was reacted with 4-OHEN (0.2 or 1.0 mg), dC and dA adducts were detected as the major adducts (lanes 1 and 2); the levels of dC and dA adducts were 1.46-1.49 adducts/104 dN and 0.62-0.72 adduct/104 dN, respectively. Several unknown adducts were also detected as minor products. No adducts were detected in the control DNA sample (data not shown). Our results were different from the earlier report (14) where 4-OHEN-dG and 4-OHEN-dC were observed in hydrolyzed DNA by electrospray mass spectroscopy. As demonstrated here, 32Ppostlabeling/PAGE analysis can be used to determine what nucleotides are reactive to the activated metabolite.

32P-Postlabeling/PAGE

Analysis

Figure 5. Formation of TAM-DNA adducts via STa-catalyzed sulfonation of R-OHTAM. Calf thymus DNA (10 µg) was incubated at 37 °C for 1 h with 200 µM PAPS, STa (0, 0.8, or 2.4 µg), and 100 µM R-OHTAM. The recovered DNA (3.0 µg) was digested using the nuclease P1 enrichment method. The TAM adducts were extracted with butanol and labeled with 32P, as described under Experimental Procedures. 0.25 volume of the 32P-labeled sample was developed for 5 h on a 30% PAGE.

Discussion The advantage of the PAGE system for 32P-postlabeling analysis was demonstrated by detection of dG-N2-TAM adducts. Previously, when a TLC plate was used to analyze 32P-labeled TAM adducts in our laboratory (9, 11), three different buffer conditions were required to separate the targeted DNA adducts. During this procedure, TLC plates were washed with distilled water and dried when the development buffer was changed. At least 28 h was required to separate the TAM-DNA adducts using a TLC plate. In contrast, using the 32P-poslabeling/ PAGE system, TAM adducts can be resolved in 5 h. Unlike TLC, large numbers of 32P-labeled samples can be loaded concomitantly on the PAGE with standard markers. For example, many factors involved in the 32Pposlabeling assay such as 3′-dephosphorylation by nuclease P1 (data not shown) or T4 PNK (Figure 2) and the efficiency of adduct extraction (Figure 3) can be examined quickly by this PAGE system. Therefore, the PAGE system is much more convenient and much less time-consuming than the TLC system. Since large amounts of highly radioactive [γ-32P]-ATP are used for 32P-postlabeling assay, careful handling is required during labeling and analysis of adducted nucleotides. In our laboratory, to subject 5 µL of 32P-labeled sample on the TLC plate at least 3 min is required per sample for spotting and drying. In contrast, it takes only

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10 s to load a 32P-labeled sample on PAG. Therefore, the PAGE system minimizes exposure to 32P during handing. The trans-forms of dG-N2-TAM adducts can be resolved from the cis-forms by the PAGE system. Moreover, the two diastereoisomers of the trans-form of dG-N2-TAM adducts can be separated by PAGE while such diastereoisomers were not resolved by the TLC system, as shown previously (8, 9, 11). In addition, each stereoisomer of dG3′P-N2-BPDE migrated differently on PAG. When the mixture of four stereoisomers of dG3′P-N2-BPDE was run overnight on the PAG, all stereoisomers could be resolved (data not shown). Although the detection limit was similar to that reported for the 32P-postlabeling/TLC system (8), the resolution of DNA adducts by the PAGE system may be better than that by the TLC system. Since wild-type T4 PNK has 3′-phosphatase activity that removes the 3′-monophosphates from unmodified and/or adducted nucleotides (Figure 2), certain amounts of 32P-labeled adducts may be dephosphorylated. To avoid this activity, the 32P-labeling step is generally carried out under alkaline conditions (>pH 9.5) (16). If alkalinesensitive DNA adducts are examined, 3′-phosphatase-free T4 PNK can be used to label adducts under neutral pH conditions. When 32P-postlabeling/PAGE assay is used, 32 P-labeled sample can be developed under the neutral conditions. Thus, the 32P-postlabeling/PAGE system is useful for analysis of alkaline- or acid-sensitive DNA adducts. When DNA was digested using the nuclease P1 enrichment method, the DNA digest still contained 32P-labeled normal nucleotides that interfere with the detection of TAM-DNA adducts. To increase the sensitivity of detection, we introduced a butanol extraction step after the nuclease P1 digestion since the butanol efficiently extracts TAM adducts from the DNA digest and minimizes contamination by normal nucleotides (8, 17, 18). Using this procedure, TAM adducts can be labeled 5 times more efficiently with 32P. Thus, if DNA adducts similar to TAM adducts can be recovered efficiently by butanol extraction, this step is essential for the 32P-postlabeling assay. The relative adduct levels were calculated by using the specific activity of [γ-32P]-ATP-labeled adducted nucleotides (12) or by comparing the amounts of labeled adducts to labeled normal nucleotides (16). However, the 32Ppostlabeling assay may underestimate adduct level because of incomplete DNA digestion, inefficiency of adduct labeling by PNK, and loss of adducts during the enrichment procedure (19). Using site-specifically-modified oligomers with dG-N2-TAM as standards, the recovery of TAM adducts was also determined (Figure 4). The level of TAM adducts observed was approximately 56% of the actual amount present. The difference between the experimental and the actual frequency of TAM adducts reflects the efficiency of enzymatic DNA digestion, the efficiency of extraction of adducted nucleotides from the DNA digest, and the labeling efficiency of the TAM adducts with 32P. The amount of TAM adducts detected (between 1 adduct/108 dNs and 1 adduct/105 dNs) increased linearly depending on the amounts of the oligomer used. The actual level of TAM adducts can be obtained using the standard curve (Figure 4). Therefore, the actual TAM adduct level in our in vitro experiments (Figure 5) was approximately 90% higher than we reported (9). As demonstrated for 4-OHEN-derived DNA adducts, 32P-postlabeling/PAGE analysis can also be used to

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Figure 6. Detection of several DNA adducts by 32P-postlabeling/PAGE analysis. (A) Migration of BPDE- and AAF-derived deoxynucleotides on polyacrylamide gel. To show the position of unmodified dN3′P, calf thymus DNA (0.1 µg) was digested enzymatically without nuclease P1 treatment and labeled with 32P using 3′-phosphatase-free T4 PNK. 0.1 volume of the 32P-labeled sample was developed for 5 h on a 30% PAG (lane 1). 10 ng each of trans-form (fr-2) of dG3′P-N2-TAM (lane 2), dG3′P-N2-(+)-trans-BPDE (lane 3), dG3′P-N2-(+)-cis-BPDE (lane 4), dG3′P-N2-(-)-trans-BPDE (lane 5), and dG3′P-N2-(-)-cis-BPDE (lane 6) was labeled with 32P using 3′-phosphatase-free T4 PNK, as described under Experimental Procedures. 50 ng of dG3′P-C8-AAF (lane 7) or dG3′P-C8-AF (lane 8) was also labeled with 32P. One-fifth volume of the 32P-labeled standard was developed on a 30% PAG. (B) Detection of DNA adducts derived from 4-OHEN. Calf thymus DNA (10 µg) was reacted at 37 °C for 7.5 h with 0.2 mg (lane 1) or 1.0 mg (lane 2) of 4-OHEN in 1.0 mL of 25 mM potassium phosphate buffer, pH 7.4. The recovered DNA (1.5 µg) was enzymatically digested using nuclease P1 enrichment method and labeled with 32P, as described under Experimental Procedures. 4-OHEN (2 mg) was also incubated at 37 °C for 7.5 h with 0.5 mg of dA3′P (lane 3), dC3′P (lane 4), dG3′P (lane 5), or dT3′P (lane 6) in 1.0 mL of 25 mM potassium phosphate buffer, pH 7.4. The dN3′P sample (25 µg) was incubated for 1 h with nuclease P1 (1 unit) in 20 µL of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2 . The reaction mixture was evaporated to dryness and labeled with 32P. One-fifth volume of the 32P-labeled samples was developed for 5 h on a 30% PAG.

examine the reactivity of an activated metabolite with nucleotides and DNA. The 32P-postlabeling/PAGE assay could also be used to detect other DNA adducts such as dG-N2-BPDE- and AAF-derived DNA adducts. This convenient technique has significant advantages over the 32 P-postlabeling/TLC assay and could be applied to the detection of many other DNA adducts.

Acknowledgment. A preliminary report of this study was presented at the 92nd Annual Meeting of the American Association for Cancer Research, March 2001, in New Orleans, LA. We thank Dr. Kurt Randerath for

informative discussion about the 32P-postlabeling/PAGE analysis. This research was supported by Grants ES09418 and ES04068 from the National Institute of Environmental Health Sciences.

References (1) Anonymous (1994) DNA adducts: Identification and biological significance. IARC Sci. Publ. No. 125, 1-478. (2) Randerath, K., and Randerath, E. (1993) Postlabeling methodss an historical review. IARC Sci. Publ. No. 124, 3-9. (3) Shibutani, S., Takeshita, M., and Grollman, A. P. (1991) Insertion of specific bases during DNA synthesis past the oxidationdamaged base 8-oxodG. Nature 349, 431-434.

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(4) Shibutani, S. (1993) Quantitation of base substitutions and deletions induced by chemical mutagens during DNA synthesis in vitro. Chem. Res. Toxicol. 6, 625-629. (5) Shibutani, S., Suzuki, N., and Grollman, A. P. (1998) Mutagenic specificity of (acetylamino)fluorene-derived DNA adducts in mammalian cells. Biochemistry 37, 12034-12041. (6) Shibutani, S., Margulis, L. A., Geacintov, N. E., and Grollman, A. P. (1993) Translesional synthesis on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-BPDE (7,8dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). Biochemistry 32, 7531-7541. (7) Terashima, I., Suzuki, N., and Shibutani, S. (1999) Mutagenic potential of R-(N2-deoxyguanosinyl)tamoxifen lesions, the major DNA adducts detected in endometrial tissues of patients treated with tamoxifen. Cancer Res. 59, 2091-2095. (8) Dasaradhi, L., and Shibutani, S. (1997) Identification of tamoxifen-DNA adducts formed by R-sulfate tamoxifen and R-acetoxytamoxifen. Chem. Res. Toxicol. 10, 189-196. (9) Shibutani, S., Dasaradhi, L., Terashima, I., Banoglu, E., and Duffel, M. W. (1998) R-Hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts. Cancer Res. 58, 647-653. (10) Umemoto, A., Kajikawa, A., Tanaka, M., Hamada, K., Seraj, M. J., Kubota, A., Nakayama, M., Kinouchi, T., Ohnishi, Y., Yamashita, K., and Monden, Y. (1994) Presence of mucosa-specific DNA adduct in human colon: possible implication for colorectal cancer. Carcinogenesis 15, 901-905. (11) Shibutani, S., Shaw, P. M., Suzuki, N., Dasaradhi, L., Duffel, M. W., and Terashima, I. (1998) Sulfation of R-hydroxytamoxifen catalyzed by human hydroxysteroid sulfotransferase results in tamoxifen-DNA adducts. Carcinogenesis 19, 2007-2011.

Chem. Res. Toxicol., Vol. 15, No. 3, 2002 311 (12) Levay, G., Pongracz, K., and Bodell, W. J. (1991) Detection of DNA adducts in HL-60 cells treated with hydroquinone and p-benzoquinone by 32P-postlabeling. Carcinogenesis 12, 1181-1186. (13) Shibutani, S., Gentles, R., Johnson, F., and Grollman, A. P. (1991) Isolation and characterization of oligodeoxynucleotides containing dG-N2-AAF and oxidation products of dG-C8-AF. Carcinogenesis 12, 813-818. (14) Shen, L., Qiu, S., Chen, Y., Zhang, F., Breemen, R. B., Nikolic, D., and Bolton, J. L. (1998) Alkylation of 2′-deoxynucleosides and DNA by the Premarin metabolite 4-hydroxyequilenin semiquinone radical. Chem. Res. Toxicol. 11, 94-101. (15) Randerath, K., Reddy, M. V., and Gupta, R. C. (1981) 32P-labeling test for DNA damage. Proc. Natl. Acad. Sci. U.S.A. 78, 61266129. (16) Reddy, M. V., and Randerath, K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551. (17) Shibutani, S., Suzuki, N., Terashima, I., Sugarman, S. M., Grollman, A. P., and Pearl, M. L. (1999) Tamoxifen-DNA adducts detected in the endometrium of women treated with tamoxifen. Chem. Res. Toxicol. 12, 646-653. (18) Shibutani, S., Ravindernath, A., Suzuki, N., Terashima, I., Sugarman, S. M., Grollman, A. P., and Pearl, M. L. (2000) Identification of tamoxifen-DNA adducts in the endometrium of women treated with tamoxifen. Carcigonegesis 21, 1461-1467. (19) Phillips, D. H., Farmer, P. B., Beland, F. A., Nath, R. G., Poirier, M. C., Reddy, M. V., and Turteltaub, K. W. (2000) Methods of DNA adduct determination and their application to testing compounds for genotoxicity. Environ. Mol. Mutagen. 35, 222-233.

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