Alkylation of 2'-Deoxynucleosides and DNA by Quinone Methides

Michael P. McCrane , Mark A. Hutchinson , Omer Ad , and Steven E. Rokita .... Willem F. Veldhuyzen, Anthony J. Shallop, Roger A. Jones, and Steven E. ...
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Chem. Res. Toxicol. 1996, 9, 1368-1374

Alkylation of 2′-Deoxynucleosides and DNA by Quinone Methides Derived from 2,6-Di-tert-butyl-4-methylphenol Mark A. Lewis, Darla Graff Yoerg, Judy L. Bolton,† and John A. Thompson* Department of Pharmaceutical Sciences, C238, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received July 9, 1996X

4-Alkylphenols, such as the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT), exhibit toxicities that appear to be mediated by their oxidative metabolism to electrophilic quinone methides. Reactions of these Michael acceptors with simple nucleophiles and proteins have been reported, but little information is available on quinone methide binding to the competing nucleophilic sites in DNA. In the present investigation, 2′-deoxynucleoside adducts generated in vitro with two BHT-derived quinone methides, 2,6-di-tert-butyl-4-methylenecyclohexa-2,5dienone and 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4-methylenecyclohexa-2,5-dienone (BHTOH-QM) were isolated and identified. Both quinone methides produced adducts at the 1- and N2-positions of deoxyguanosine (dG) and the N6-position of deoxyadenosine (dA). In addition, a labile adduct formed at the 7-position of dG, which degraded to the corresponding 7-alkylguanine derivative. Additional work was conducted with BHTOH-QM, the more reactive of the two quinone methides. This species also formed stable adducts at the N4-position of deoxycytosine (dC) and the 3-position of thymidine and formed a labile adduct at the 3-position of dC that underwent hydrolytic cleavage to regenerate dC. In mixtures of deoxynucleosides treated with [14C]BHTOH-QM, alkylation occurred primarily at the N2- and 7-positions of dG and the N6-position of dA and occurred secondarily at the 1-position of dG. Treatment of calf thymus DNA with this quinone methide yielded N6-dA and N2-dG adducts with the former predominating. The unstable 7-dG adduct was detected by analysis of the 7-alkylguanine product from depurination. These results demonstrate that quinone methides are most likely to damage DNA through alkylation of the exocyclic amino groups of purine residues and possibly also by attack at the 7-position of dG followed by depurination.

Introduction Quinone methides are intermediates in lignin biosynthesis and in the metabolic activation of cytotoxic and genotoxic agents (1-3). These species can be generated by peroxidase- or P4501-catalyzed oxidations of ortho- and para-alkylphenols (4, 5) and by the isomerization of orthobenzoquinones (3, 6). Quinone methides are Michael acceptors, adding nucleophiles at the exocyclic methylene group to form benzylic adducts as shown in Figure 1A (2, 7). Strong evidence implicates these reactive intermediates in the protein binding and cytotoxicity of alkylated phenols such as butylated hydroxytoluene (BHT) (7-9) and eugenol (4, 10). The intrinsic reactivity of a quinone methide is highly dependent on its specific structural features. For example, half-lives in the presence of a nucleophile are increased by substituents at the exocyclic methylene group (11), by extended π conjugation (12), and by the presence of bulky, hydrophobic groups at both the 2- and 6-positions of the cyclohexadienone ring (13). In the latter case, these substituents shield * Author to whom correspondence should be addressed. † Present address: Department of Medicinal Chemistry and Pharmacognosy (M/C 781), University of Illinois at Chicago, Chicago, IL 60612-7231. X Abstract published in Advance ACS Abstracts, November 1, 1996. 1 Abbreviations: BHT, 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene); BHT-QM, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone; BHTOH, 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4methylphenol; BHTOH-QM, 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4-methylene-2,5-cyclohexadienone; dG, 2′-deoxyguanosine; dA, 2′-deoxyadenosine; dC, 2′-deoxycytosine; dT, thymidine; FAB/MS, fast atom bombardment mass spectrometry; P450, cytochromes P450; DNase, deoxyribonuclease.

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Figure 1. (A) Quinone methide formation and the addition of water. (B) The cyclohexadienone and aromatic resonance forms of quinone methides, and charge stabilization in BHTOH-QM.

the carbonyl oxygen from solvent interactions, thereby preventing stabilization of the ionized form shown in Figure 1B and providing an explanation for differences in the half-lives in aqueous media for para-quinone methides derived from BHT (BHT-QM, t1/2 51 min) and from an analog with methyl groups in place of both tertbutyl groups (t1/2 0.4 min) (13). Cytotoxicity has been related to quinone methide reactivity in isolated rat hepatocytes (13), rat liver slices (14), and a cell line © 1996 American Chemical Society

Quinone Methide-DNA Adducts

derived from transformed murine keratinocytes (15). Interactions of quinone methides with simple sulfur-, nitrogen-, and oxygen-centered nucleophiles have been examined (2, 7, 11), but little information is available on the reactions of these electrophiles with the competing nucleophilic sites in DNA. Quinonoid antitumor drugs such as mitomycin C, anthracycline antibiotics, and related compounds are believed to form covalent linkages with DNA bases through quinone methide intermediates (16-19). Binding occurs mainly at the exocyclic amino groups of guanine and adenine, but the specific binding sites of these complex, multiring species are most likely influenced by stereoelectronic factors associated with their ability to intercalate into DNA. Structurally simple quinone methides should also interact covalently with DNA and are potentially capable of inducing genotoxicity. For example, it was proposed recently that the carcinogenic effects of tamoxifen may be due to DNA adducts resulting from the formation of a quinone methide (20). In order to determine the preferred binding sites within DNA for simple quinone methides, we chose to work with a metabolite of BHT shown in Figure 1A that has been linked to its pneumotoxicity and tumor-promoting properties, 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4methylene-2,5-cyclohexadienone (BHTOH-QM). This compound, produced by two successive P450-catalyzed oxidations (5), is a more reactive electrophile than BHTQM due to charge stabilization provided by intramolecular hydrogen bonding (Figure 1B) (7). The rate of water addition to BHTOH-QM (t1/2 6.6 min) is intermediate between the values mentioned above for BHT-QM and the 2,6-dimethyl analog, suggesting its potential as a model for in vitro studies. Stable solutions of BHTOHQM have been prepared, and reactions of this species with nucleophilic sites in DNA compete effectively with the addition of water. The reactions of quinone methides are greatly influenced by the medium (21-23), so these studies were conducted under aqueous conditions and at physiologic pH. Adducts formed between BHTOH-QM and the individual 2′-deoxynucleosides were identified, and the relative rates of formation were determined. In addition, adducts of BHT-QM were investigated in order to compare regioselectivities for the binding of quinone methides with differing reactivities. These data facilitated an investigation of the principal sites of DNA alkylation by BHTOH-QM in vitro.

Experimental Section Chemicals. All chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) unless otherwise indicated, and BHT was recrystallized from hexane before use. [ring-14C]BHT (65.1 mCi/mmol) was prepared by Chemsyn Science Laboratories (Lenexa, KS), purified by semi-preparative HPLC, and diluted to 3.3 mCi/mmol prior to use. BHTOH was synthesized by a published procedure (24) to yield 0.74 g of the pure product as a white crystalline solid with spectral and chromatographic properties identical to those reported previously (25). The synthesis of [14C]BHTOH was carried out by substituting [l4C]methyl iodide (20 mCi/mmol) (DuPont NEN, Boston, MA) for methyl iodide in the synthesis of BHTOH. The product was purified by flash chromatography on silica gel (15:85 ethyl acetate-hexane), and its purity was established to be >98% by HPLC with radiochemical detection. The specific activity was determined to be 19.7 mCi/mmol. Deoxynucleosides, guanine, N6-benzyladenosine, and calf thymus DNA were obtained from Sigma Chemical Co. (St. Louis, MO). Quinone methides were prepared by chemical oxidation of the respective phenols (7, 13). Briefly, solutions containing 1.4 mg of the phenol/mL of dry

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1369 acetonitrile were treated with a 30-fold molar excess of silver oxide (Sigma). These mixtures were stirred in the dark at 25 °C for 25 min (BHTOH) or 3 h (BHT) and passed through a 0.45-µm Teflon syringe filter. BHT-QM and BHTOH-QM have been fully characterized previously (7), and each new preparation was verified by HPLC. Quinone methide concentrations in acetonitrile were determined by UV analysis at λmax 287 nm and  of 2.82 × 104 M-1 cm-1 (11). Preparation and Characterization of Adducts. The general procedure employed for adduct formation is as follows. A 10 mM solution of a deoxynucleoside was prepared in 50 mM phosphate buffer at pH 7.4. A quantity of the quinone methide solution in acetonitrile (approximately 6-7 mL) was added to 30 mL of the deoxynucleoside solution to produce a final quinone methide concentration of 1.0 mM. This solution was maintained at 30 °C for 2 h and then extracted with 2 vol of ethyl acetate or 1-butanol. After the solvent was evaporated, the residue was redissolved in methanol/water (1:1) and subjected to semipreparative HPLC on a Beckman (Fullerton, CA) Ultrasphere ODS column (10 × 250 mm) at a flow rate of 2.5 mL/min. The mobile phase composition consisted of 50% methanol in 0.1 M aqueous ammonium phosphate for 5 min and increased to 90% methanol over the next 25 min. Adducts were isolated from the column effluent by evaporating the methanol under a stream of nitrogen at 25 °C, adding the aqueous solutions to C-18 extraction cartridges (J. T. Baker, Phillipsburg, NJ), washing the cartridges with 1 mL of water, and eluting the adducts with 1 mL of methanol. The unstable adduct 7-(BHTOH)dG was allowed to decompose to the 7-alkylguanine adduct in the HPLC mobile phase by incubating the solution at 37 °C for 20 h. The guanine adduct was then extracted with ethyl acetate for analysis. UV spectra were recorded at pH 6.5 in methanol0.1 M aqueous phosphate (80:20) with a Hewlett Packard (Palo Alto, CA) Model 1040 diode array spectrophotometric detector. FAB/MS data were obtained in glycerol on a VG 7070 instrument (Micromass, Beverly, MA), and 1H NMR spectra were recorded in Me2SO-d6 with a Varian (Palo Alto, CA) VXR-300 spectrometer. Analytical HPLC was conducted with a Hewlett Packard 1090 instrument and a 4.6 × 250 mm Ultrasphere ODS column (Beckman) with the same mobile phase described above, but at a flow rate of 1 mL/min. Products were detected with a photodiode array detector monitoring of 260 ( 30 nm or with a radioactivity flow detector (β-Ram, IN/US, Sarasota, FL). Spectral data for N2-(BHT)dG are as follows: UV λmax 253, 278 (sh); FAB/MS m/z (ion, % relative abundance), 486 (MH+, 2.3), 370 (MH+ - sugar, 27), 219 (BHT+, 100), 152 (guanine + H, 95); 1H NMR (Me2SO-d6) δ 1.34 (s, 18H, CH3), 2.16 (m, 1H, 2′-H), 2.50 (m, 1H, 2′-H), 3.79 (m, 1H, 4′-H), 4.11 (m, 1H, 3′-H), 4.31 (d, 2H, 5′′-H), 6.18 (t, 1H, 1′-H), 6.84 (bs, 1H, N2-H, exchangeable), 7.11 (s, 2H, 1′′-H), 7.90 (s, 1H, 8-H). Analysis of 1-(BHT)dG yielded the following data: UV λmax 252, 274 (sh); FAB/MS m/z (ion, % relative abundance), 486 (MH+, 0.5), 370 (MH+ - sugar, 4.5), 219 (BHT+, 37), 152 (guanine + H, 100); 1H NMR (Me SO-d ) δ 1.29 (s, 18H, CH ), 2.16 (m, 1H, 2′-H), 2 6 3 2.50 (m, 1H, 2′-H), 3.79 (m, 1H, 4′-H), 4.31 (m, 1H, 3′-H), 5.06 (d, 2H, 5′′-H), 6.10 (t, 1H, 1′-H), 7.01 (s, 2H, N2-H, exchangeable), 7.04 (s, 2H, 1′′-H), 7.93 (s, 1H, 8-H). Spectral data for N6-(BHT)dA are the following: UV λmax 270; FAB/MS m/z (ion, % relative abundance), 470 (MH+, 2.1), 352 (MH+ - sugar, 4.0), 219 (BHT+, 6), 152 (adenine + H, 12); 1H NMR (Me2SO-d6) δ 1.34 (s, 18H, CH3), 2.22 (m, 1H, 2′-H), 2.70 (m, 1H, 2′-H), 3.83 (m, 1H, 4′-H), 4.38 (m, 1H, 3′-H), 4.30 (d, 2H, 5′′-H), 6.30 (t, 1H, 1′-H), 7.04 (s, 2H, 1′′-H), 8.10 (s, 1H, 8-H), 8.20 (s, 1H, 2-H). Reactions with DNA. A solution of calf thymus DNA (1 mg) in 1 mL of water was treated with 150-µL portions of an acetonitrile solution of BHTOH-QM (approximately 1.5 mM) at times 0, 30, and 60 min. After a total incubation time of 90 min at 30 °C, the acetonitrile was evaporated under a stream of nitrogen, and the DNA was precipitated by adding 750 µL of ethanol and cooling at -20 °C for 30 min. DNA was isolated, washed three times with 2-mL portions of 80% ethanol, redis-

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Table 1. HPLC and UV Data for the Adducts of BHTOH-QM adduct

HPLC tR a (min)

λmaxb (nm)

N2-dG 1-dG 7-dG 7-guanine N6-dA N4-dC 3-dC 3-dT

29.7 27.3 27.7 29.1 34.0 28.8 27.4 33.0

256, 278 256, 272(sh) 258, 284(sh) 282 270 276 280 268

a Retention times under the conditions described in the Experimnental Section. The benzyl alcohol derivative of BHTOH elutes at 32.0 min. b Spectra recorded at pH 6.5.

Table 2. Mass Spectral Data for Adducts of BHTOH-QMa adduct

MH+

MH+ - sugar

ArCH2+

(base + H)+

N2-dGb 1-dGc 7-guanine N6-dA N4-dC 3-dC 3-dT

501 (2.1) 502 (0.6) 386 (5.6) 486 (1.0) 462 (5.1) 462 (16) 478 (25)

385 (1.4) 386 (3.5)

235 (0.6) 235 (18) 235 (100) 235 (10) 235 (9.6) 235 (100) 235 (100)

151 (13) 152 (100) 152 (60) 136 (37) 112 (20) 112 (90) 127 (75)

370 (9.0) ndd nd nd

Table 3. NMR Data for Purine Adducts of BHTOH-QM protons(s)a

N2-dG

chemical shift (ppm) 1-dG 7-guanine

N6-dA

Base 1 10.35 (s, 1H) N2 or 2 6.69 (t, 1H) N6 8 7.90 (s, 1H) 1′ 2′R 2′β 3′ 4′ 5′

6.18 (t, 1H) 2.16 (m, 1H) 2.58 (m, 1H) 4.35 (m, 1H) 3.79 (m, 1H) 3.54 (m, 2H)

1′′

7.10 (s, 2H)

2′′ 3′′ 4′′ 5′′

7.02 (bs, 2H) 7.95 (s, 1H)

nd ndb 7.09 (s, 2H) 8.33 (s, 1H) 8.22 (bs, 1H) 8.05 (s, 1H) 8.22 (bs, 1H)

Sugar 6.12 (t, 1H) 2.19 (m, 1H) 2.50 (m, 1H) 4.33 (bs, 1H) 3.79 (bs, 1H) 3.50 (m, 2H)

ArCH2 7.05 (s, 1H); 7.06 (s, 1H) 1.33 (s, 9H) 1.29 (s, 9H) 1.31 (s, 6H) 1.25 (s, 6H) 3.54 (s, 2H) 3.50 (s, 2H) 4.31 (d, 2H) 5.06 (bs, 2H)

6.34 (t, 1H) 2.24 (m, 1H) 2.71 (m, 1H) 4.40 (m, 1H) 3.87 (m, 1H) 3.60 (m, 2H) 7.22 (s, 2H) 7.13 (s, 2H) 1.30 (s, 9H) 1.27 (s, 6H) 3.51 (s, 2H) 5.23 (s, 2H)

1.30 (s, 9H) 1.27 (s, 6H) 3.51 (s, 2H) 4.54 (d, 2H)

a Proton assignments correspond to structures shown in Figure 2. b Not detected due to traces of H2O in the sample.

a Unless indicated otherwise, each m/z value (and the % abundance) was obtained by positive ion FAB/MS in glycerol. In some cases, glycerol-related ions produced the most intense peaks. b Negative ion mode, the ions are not protonated. c A sodium adduct ion also was present at m/z 524 (0.3%). d No ion detected.

solved in 1 mL of DNase buffer (50 mM sodium acetate, 2 mM CaCl2, and 10 mM MgCl2, pH 6.5), and incubated with DNase I (4.4 units) for 20 h at 37 °C. Then 50 µL of 0.5 M Tris buffer (pH 9.0) and 0.013 unit (20 µL) of snake venom phosphodiesterase (contaminated with nucleotide pyrophosphatase) were added, and the mixture was incubated at 37 °C for 4 h. This treatment resulted in complete hydrolysis to the deoxynucleoside level. Adducts were isolated as described above and dissolved in methanol for HPLC analysis. To determine 7(BHTOH)guanine from depurination, DNA was treated with BHTOH-QM as described above, incubated at 20 °C, precipitated, and washed. The isolated DNA was redissolved in 0.1 M phosphate buffer containing 25% methanol (to simulate the depurination conditions described above), incubated at 37 °C for 20 h, and extracted with ethyl acetate. Extracts were dried, and a solution was prepared in methanol for HPLC analysis. In order to optimize separation of the 7-(BHTOH)guanine peak from other adduct peaks, the mobile phase was changed to 30% acetonitrile in water for 5 min, increasing to 55% acetonitrile over 10 min, and maintaining this composition for an additional 10 min.

Figure 2. Structures of the BHTOH-dG adducts.

Results Quinone Methide Reactions with dG. Two stable adducts were detected by HPLC-photodiode array analysis of the products (Table 1) resulting from incubations of BHTOH-QM with dG. Structures were deduced from their UV, FAB/MS (Table 2), and 1H NMR data (Table 3) with reference to published spectra of fully characterized 1-, N2-, O6-, 7-, and 8-p-methoxybenzyl adducts of guanosine (26). The major adduct resulted from quinone methide attack at the exocyclic amino group forming N2(BHTOH)dG shown in Figure 2. The spectral data are in close agreement with those reported for the p-methoxybenzyl analog with particular attention to NMR signals (Figure 3) corresponding to the C-8, N-1, and N2protons (26). The benzylic (5′′) protons are highly sensitive to the site of attachment, as the analogous protons of the 1-, O6-, and 7-p-methoxybenzyl adducts of guanosine occur considerably further downfield. Additional

Figure 3. 1H NMR spectrum of N2-(BHTOH)dG recorded in Me2SO-d6. Peak assignments are summarized in Table 3, and those indicated with an asterisk disappeared upon the addition of D2O. Peaks at 4.86, 5.25, 7.17, and 10.65 ppm are assigned to OH groups, and those at 6.69 and 10.35 ppm to NH groups.

evidence for the structure of N2-(BHTOH)dG was provided by the addition of D2O, which resulted in exchange of the N2-proton and collapse of the benzylic protons to a singlet. The minor dG adduct displayed UV and NMR spectra (Tables 1 and 3) consistent with the addition

Quinone Methide-DNA Adducts

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1371

Figure 5. Structures of the dA, dC, and dT adducts of BHTOHQM. The Ar-CH2 group is defined in Figure 2.

Figure 4. Time courses for quinone methide depletion and adduct formation. Incubation conditions are described in the Experimental Section. (A) BHTOH-QM incubated with dG: BHTOH-QM (9), N2-dG (2), 1-dG (b), and 7-dG adducts ((). (B) BHT-QM incubated with dG: BHT-QM (9) and 7-dG adduct ((). (C) BHTOH-QM incubated with dC: BHTOH-QM (9), N4dC (2), and 3-dC adducts (().

of the quinone methide to the 1-position forming 1(BHTOH)dG (Figure 2). A signal corresponding to the N-1 proton of guanine was absent, and the chemical shifts of the C-8 and benzylic protons as well as the exchangeable N2-protons closely match the analogous signals from 1-(p-methoxybenzyl)guanosine. On the other hand, the published spectra of O6- and 7-(p-methoxybenzyl)guanosine adducts (26) clearly do not match the spectrum of this product. When the reaction of [14C]BHTOH-QM with dG was analyzed by HPLC at early time points, a third adduct was detected. This species was unstable, disappearing from the reaction mixture after depletion of the quinone methide (Figure 4A). Although attempts to isolate quantities of pure material sufficient for NMR analysis were unsuccessful, small amounts of the adduct were isolated from the HPLC effluent after short-term incubations. The UV spectrum (Table 1) is nearly identical to that of 7-(p-methoxybenzyl)guanosine (26), strongly suggesting that this adduct is 7-(BHTOH)dG (Figure 2), which undergoes cleavage of the glycosidic linkage as is

common for N-7 adducts of dG (27). When the separated adduct was allowed to decompose in the HPLC effluent at 37 °C, a stable product resulted that readily partitioned into ethyl acetate. The FAB/MS data (Table 2) confirms that this compound is a guanine adduct of the quinone methide, and the UV and 1H NMR data (Tables 1 and 3) are constant with the spectra of 7-alkylguanines (28-30). In particular, the C-8 proton at 8.05 ppm falls within the 7.90-8.48 ppm range for some other 7alkylated derivatives. Finally, treating guanine with BHTOH-QM resulted in several adducts, and one of these had the same HPLC, UV, and 1H NMR characteristics as the decomposition product. These data strongly support the structure of the unstable dG adduct as 7-(BHTOH)dG, which depurinates to form the corresponding 7-guanine derivative. BHT-QM is substantially less reactive than BHTOHQM due to differences in electron distribution (Introduction), and as the regioselectivity of electrophilic attack on dG can be influenced significantly by the nature of the electrophile (31), it was of interest to determine if qualitative differences in adduct formation occur with these two quinone methides. As expected, BHT-QM reacted much more slowly than the hydroxylated analog, but spectral data of the major and minor products (Experimental Section) demonstrated that both the N2dG and 1-dG adducts were produced with the former predominating. In addition, an unstable product was generated and tentatively assigned the structure of the 7-dG adduct based on its UV spectrum and decomposition profile (Figure 4B), which are nearly identical to those of 7-(BHTOH)dG. These results demonstrate that only quantitative differences in adduct formation occurred with the two quinone methides. Adducts with dA, dC, and dT. One stable adduct was detected in each case when [14C]BHTOH-QM was incubated with either dA, dC, or dT. These products were isolated and confirmed to be mono-adducts by FAB/MS (Table 2). Spectra of the dA adduct are consistent with the structure of N6-(BHTOH)dA (Figure 5), as the UV λmax at 270 nm (Table 1) and the NMR spectrum (Table 3) closely agree with the spectra of an authentic sample of N6-benzyladenosine. These data exclude the possibility of adduct formation at the other most likely site, the N-1 position of dA, as those products exhibit λmax values in the 257-260 nm range (32) and a downfield shift of the NMR signal corresponding to the C-2 proton of adenosine (33). The N6-dA adduct also was generated with BHT-

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Table 4. NMR Data for Pyrimidine Adducts of BHTOH-QM chemical shift (ppm)

a

proton(s)a

N4-dC

3-dT

N4 5 6

Base 7.93 (t, 1H) 5.77 (d, 1H) 7.73 (d, 1H)

1.82 (s, 3H) 7.78 (s, 1H)

1′ 2′R 2′β 3′ 4′ 5′

Sugar 6.17 (t, 1H) 1.95 (m, 1H) 2.05 (m, 1H) 4.17 (m, 1H) 3.75 (m, 1H) 3.54 (bs, 2H)

6.21 (s, 1H) 2.18 (m, 1H) 2.18 (m, 1H) 4.23 (m, 1H) 3.77 (m, 1H) 3.57 (m, 2H)

1′′ 2′′ 3′′ 4′′ 5′′

ArCH2 7.02 (s, 2H) 1.34 (s, 9H) 1.30 (s, 6H) 3.54 (s, 2H) 4.30 (d, 2H)

7.08 (s, 2H) 1.30 (s, 9H) 1.25 (s, 6H) 3.50 (bs, 2H) 3.50 (s, 2H)

Proton assignments correspond to structures shown in Figure

5.

QM, as the spectra of this product (Experimental Section) were analogous the those of the BHTOH-QM adduct. BHTOH-QM addition to dC also occurred at the exocyclic amino group forming N4-(BHTOH)dC (Figure 5). The NMR spectrum (Table 4) contains a doublet centered at 4.30 ppm corresponding to the two benzylic protons and a triplet centered at 7.93 ppm due to a single N4-proton that exchanged with D2O, resulting in the collapse of the benzylic protons to a singlet. The adduct formed by treating dT with BHTOH-QM was assigned the structure 3-(BHTOH)dT shown in Figure 5. The UV spectrum (Table 1) is essentially identical to that of 3-(2-carboxyethyl)thymidine formed by the addition of β-propiolactone (34). NMR data (Table 4) also are consistent with this structure, especially the shift of the benzylic protons at 3.50 ppm, as this signal would be expected to occur considerably further downfield if alkylation had taken place on oxygen. No additional adducts were detected with dA or dT, but incubations with dC resulted in another product that decomposed over time as shown in Figure 4C. A small quantity of this compound was successfully isolated by HPLC, and the resulting UV and FAB/MS data are shown in Tables 1 and 2. As with the unstable adduct of dG, however, we were unable to isolate quantities of pure material sufficient for NMR analysis. Decomposition of this adduct occurred more rapidly under basic conditions, generating dC and the benzylic alcohol derivative of BHTOH as determined by comparisons of the HPLC and UV properties with authentic standards. The identity of this unstable dC adduct cannot be assigned with certainty, but the most likely structure is that of 3-(BHTOH)dC (Figure 5), which is supported by the fact that several R,β-unsaturated carbonyl compounds are known to undergo Michael additions at the 3-position of dC (35). Relative Rates of Adduct Formation from Deoxynucleosides and DNA. [14C]BHTOH-QM was incubated with a mixture of dG, dA, and dC until the quinone methide had completely reacted. Thymidine was omitted due its relatively slow reaction with the quinone methide. The products were analyzed by radiochromatography and the relative quantities of the 1-dG, N2-dG, 7-dG, and N6dA adducts were 1.0:5.5:2.8:3.5, respectively, and no dC

Figure 6. HPLC analysis of DNA adducts. Calf thymus DNA was treated with [14C]BHTOH-QM, and the deoxynucleosides were prepared and analyzed with an acetonitrile/water mobile phase as described in the Experimental Section. Chromatography was conducted with tandem (A) UV detection at 260 ( 30 nm and (B) radiochemical detection. The hydrolyzed sample was spiked with unlabeled standards of 1-(BHTOH)dG (peak 1), N2(BHTOH)dG (peak 2), and N6-(BHTOH)dA (peak 3) to confirm retention times of the 14C-labeled adducts. Peak 4 is due to the benzyl alcohol always formed as a side product during reactions of the quinone methide in aqueous media.

adducts were detected (relative yield N6-(BHTOH)dA > 7-(BHTOH)dG > 1-(BHTOH)dG. Characterizations of dexoynucleoside-quinone methide adducts enabled determinations of adduct formation in calf thymus DNA. Hydrolysis of DNA treated with radiolabeled BHTOH-QM and HPLC analysis of the resulting nucleosides demonstrated that adduct formation occurred predominantly at the exocyclic amino group of dA. The relatively small amount of N2-dG adduct was unexpected from the results with individual deoxynucleosides, but a preference for dA over dG adducts also was observed during the Michael addition of 1,4-benzoquinone to DNA (36). The N6 group of dA lies in the major groove of the DNA double helix while N2 of dG is positioned in the minor groove, the widths of which are 12 and 6 Å, respectively, which may explain the preference for reactions of the sterically bulky BHTOH-QM with dA. No other stable deoxynucleoside adducts were found in quinone methide-treated DNA, and neither was the 7-dG adduct detected as this labile product apparently depurinates during the long incubation periods required for DNA hydrolysis. The formation of 7-(BHTOH)guanine was observed, but it appears that alkylation at the 7-position of dG is variable and highly sensitive to the reaction conditions. Additional work will be necessary to clarify the importance of depurination to DNA damage resulting from the binding of quinone methides to N-7 of dG. Although reactions of quinone methides with DNA have not been widely investigated, there is considerable information in the literature concerning adduct formation with other Michael acceptors. For example, malondialdehyde (37), acrolein, crotonaldehyde (38), and methylvinyl ketone (39) react mainly at the 1- or N2-position of guanine to form adducts that cyclize spontaneously. Analogous products are generated at the 1-, and N6positions of adenine (36, 39) and at the 3- and N4positions of cytidine (40). The alkylation of dG at N-7 does not seem to be an important pathway for simple R,βunsaturated carbonyl compounds, but such products have been reported in a few cases (37, 41), and their contributions may be underestimated because of the labile nature of N-7 adducts. The regioselectivity for akylation by this class of compounds, therefore, is similar to that determined for BHTOH-QM even though the latter species is considerably more reactive and requires only minutes rather than many hours for adduct formation in vitro. These findings are consistent with previous data demonstrating that soft nucleophiles react with dG preferentially at the N-7 and N2-positions (31, 42, 43). In addition, the more polarizable electrophiles generally favor N2 alkylation, and quinone methides surely fit into this category (Figure 1B). Our data indicate that BHTOH-QM primarily alkylates exocyclic amino groups of purines unless the reaction temperature is lowered to about 20 °C. Under these conditions, the formation of a 7-dG adduct is a more important pathway. The nature of this temperature dependence, however, has not been fully characterized, and the contributions of depurination to DNA damage under physiological conditions are uncertain. Although BHT-QM and BHTOH-QM were utilized in these studies mainly as models for predicting the sites of DNA alkylation by simple p-quinone methides, there

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1373

is a possibility that these electropiles may bind to DNA following exposures to BHT in vivo. Investigations into the genotoxicity of this food additive have generally yielded negative results (44, 45), but one study indicates carcinogenic activity in mouse skin (46) and another demonstrates a low level of DNA binding in livers of rats treated with BHT (47). It should be pointed out that the reactivity of BHT-QM may be too low to generate significant numbers of DNA adducts, and the two-step metabolic conversion of BHT to BHTOH-QM is speciesand tissue-dependent. For example, this pathway does not contribute significantly to BHT metabolism in rat tissues or in the livers of uninduced mice, but it is one of the main routes of biotransformation in mouse lung (5, 25). Testing for BHT genotoxicity generally has not been carried out under optimal conditions for the generation of BHTOH-QM, and no direct testing of the mutagenic potential of this electrophile has been carried out. In conclusion, the results described here emphasize the possibility that some alkylphenols can bind to DNA through quinone methide intermediates forming both stable and labile adducts at guanine and adenine residues. Additional attention should be focused, therefore, on the potential genotoxicity of phenolic compounds that are capable of being oxidized to quinone methides in vivo.

Acknowledgment. The authors thank Dr. Alban Allentoff for preparing the [14C]BHTOH. This research was supported by NIH Grant ES06216.

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