Characterization of DNA adducts formed in vitro by ... - ACS Publications

Axel Paehler,, Janique Richoz,, John Soglia,, Paul Vouros, and, Robert J. Turesky ... H. Leveson,, Karen H. Dingley,, Kenneth W. Turteltaub, and, Laur...
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Chem. Res. Toxicol. 1992,5, 479-490

479

Characterization of DNA Adducts Formed in Vitro by Reaction of N-Hydroxy-2-amino-3-methylimidazo [4,5 -4quinoline and N-Hydroxy-2-amino-3,8-dimethylimidazo[4,5-flquinoxaline at the C-8 and N2 Atoms of Guanine Robert J. Turesky,*’tSusan C. Rossi,? Dieter H. Welti,+Jackson 0. Lay, Jr.,t and Fred F. Kadlubart Nestec Ltd., Nest16 Research Centre, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland, and National Center for Toxicological Research, Jefferson, Arkansas 72079 Received January 20, 1992

The covalent binding of the carcinogenic N-hydroxy metabolites of 2-amino-3-methylimidazo[4,5-flquinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-flquinoxaline (MeIQx) to deoxynucleosides and DNA was investigated in vitro. Two major adducts were formed by the reaction of the N-acetoxy derivatives of IQ and MeIQx with deoxyguanosine (dG); however, no adducts were formed with deoxycytidine, deoxyadenosine, or thymidine. From proton NMR and mass spectroscopiccharacterization the adducts were identified as 5-(deoxyguanosin-W-yl)-2-amino3-methylimidazo[4,5-flquinoline (dG-N2-I&), N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-flquinoline (dG-C8-IQ), 5-(deoxyguanosin-W-yl)-2-amino-3,8-dimethylimidazo[4,5-flquinoxaline (dG-N2-MeIQx), and N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5fl quinoxaline (dG-C8-MeIQx). The level of dG-C8adducts was approximately 8-10 times greater than the amount of dG-N2adducts formed from the reaction of dG with the N-acetoxy derivatives of IQ and MeIQx. The C-&substituted dG adduct was also the major adduct formed from reactions of DNA with N-acetoxy-IQ and N-acetoxy-MeIQx. Approximately 60-80 % of the bound carcinogens were recovered from DNA as dG-C8 adducts upon enzymatic digestion. The dG-N2 adducts also were detected and accounted for approximately 4% of the bound IQ and 10% of the bound MeIQx. These results suggest that the relative contributions of the nitrenium and carbenium ion resonance forms as well as DNA macromolecular structure are major determinants for DNA adduct substitution sites. Investigations on adduct conformation of 1H NMR spectroscopy revealed that the anti form is preferred for the dG-N2 adducts of IQ and MeIQx, while the syn form is preferred for the dG-C8 adducts. The possible role of these adducts in the initiation of carcinogenesis is discussed.

Introduction

amino-3,&dimethylimidazo[4,5-flquinoxaline(MeIQx)are two of the more abundant HAAs formed in cooked foods, Heterocyclic aromatic amines (HAAs)l are formed in and they are structurally representative of this class of proteinaceous foods during ordinary cooking practices (1, contaminants. I& and MeIQx are potent bacterial mu2). Seventeen HAAs have been identified thus far in a tagens and rodent carcinogens (3, 41, and I& is also a variety of foods at the low parts per billion level (1-3). powerful liver carcinogen in monkeys (5). 2-Amino-3-methylimidazo[4,5-flquinoline (I&) and 2The covalent modification of DNA by genotoxins is recognized as an important factor in the initiation of car+ Nestec LM. cinogenesis (6). HAAs, like many carcinogens, must be National Center for Toxicological Research. metabolically activated in order to exert their genotoxic 1 Abbreviations: dR, S’-deoxyrib,dG, 2’-deoxyguanosine;G, guanine, dA, 2/-deoxyadenosine; dT, thymidine; dC, 2/-deoxycytidine; HAAs, effects. Metabolic activation occurs through N-oxidation heterocyclic aromatic amines; IQ, 2-amino-3-methylimidazo[4,5-fl quiof the exocyclic amine group and is catalyzed primarily by noline;dG-N2-IQ,5-(deoxyguanosin-NZ-yl)-2-amino-3-methylimidazo[4,~ cytochrome P450 1A2 (7-10). The N-hydroxy metabolites flquinoline; dGC&IQ,N-(deoxyguanosin-&yl)-2-amino-3-methylim[4,5-flquinoline;MeIQx, 2-amino-3,&dimethylimidazo[4,5-flquinoxaline; may directly react with nucleophiles such as protein or dG-Nz-MeIQx,5-(deoxguanosin-hn-yl)-2-amino-3,&d~ethy~~idazo[4,5flquinodine; dG-C8-MeIQx, N-(deoxyguanosin-8-yl)-2-amino-3,8-di- DNA (11 ) ;the ultimate carcinogenic species, however, are methyliiidazo[4,5-flquinoxaliie; AAF, 2-(acetylamino)fluorene;dG-C& believed to be highly reactive N-acetoxy or N-(sulfonyAAF, N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene; dG-N2-AAF, 3loxy) esters (12-14). (deoxyguanosin-W-yl)-AAF;2-AF, 2-aminofluorene; dG-C8-AF, N (deoxyguanosin-8-yl)-2-aminofluorene; MAB; N-methyl-l-aminoAromatic amines, in general, form DNA adducts priazobenzene; dG-C&MAB, N-(deoxyguanosin-&yl)-MAB; dG-NZ-MAB, marily at the C-8 and N2atoms of guanine and to a lesser 3-(deoxyguanpein-hn-yl)-MAB AB, 4-aminoazobenzene;dG-C8-AB,N(deoxyguanosm-&yl)-ABPhIP, 2-amino-l-methyl-6-phenylimidazo[4,5- extent at the C-8 and N6 positions of adenine (15-23). For bl pyridine; Trp-P-2, 3-amino-l-methyl-5H-pyrido[4,3-b]indole; Glu-Pthe HAAs I&, Glu-P-1, and Trp-P-2, however, only C-8 1,2-amino-6-methyldipyrido[1,2-a:3’,2’-d] imidazole; 2-NA, 2-naphthylguanine adducts have been identified (24-26). A linear amine;FAB, fast atom bombardment;TMS, trimethylsilyl; NOE,nuclear Overhausereffect; COSY,homonuclear proton correlation spectroscopy; relationship has been shown between MeIQx dose adDMSO, dimethylsulfoxide;EDTA,ethylenediaminetetraaceticacid; D l T , ministered and DNA adduct formation in liver, a target dithiothreitol;RA, relative abundance;HPLC,high-pressureliquid chromatography. organ for tumorigenesis in the rodent (27,28). Analysis f

0 1992 American Chemical Society

480 Chem. Res. Toxicol., Vol. 5, No. 4, 1992

of hepatic DNA by acceleratormass spectrometryrevealed that adducts of MeIQx were formed in rodents even at dose levels approachinghuman daily exposure (28).Thus, despite the occurrence of HAAs only in trace amounts in many foods, daily exposure may be significant, and an association between nutritionally linked cancers that are commonin the western world,such as colorectaland breast cancers, and exposure to HAAs may exist (29). In the present study, we examined adducts that were produced by the reaction of deoxynucleosides or DNA with the Nhydroxy derivatives of IQ and MeIQx or their respective N-acetoxy derivatives generated in situ.

Experimental Procedures Chemicals. Caution: The heterocyclic aromatic amines ZQ, MeIQx, and several of their derivatives are carcinogenic to rodents and should be handled carefully. The following chemicals were obtained from Sigma Chemical Co. (St Louis, MO): 2'-deoxyguanosine,thymidine, 2'-deoxyadenosine, 2'-deoxycytidine, alkaline phosphatase (type 111-S),deoxyribonuclease I (type IV), phosphodiesterase I (type VII), alkaline phosphatase (type 111-S), Bis-Tris, and calf thymus DNA. Acetyl coenzyme A and dithiothreitol were obtained from Boehringer Mannheim (Rotkreuz, Switzerland). Sodium nitrite and ascorbic acid were obtained from Fluka (Buchs, Switzerland). DMSO-de (99.95% isotopic purity) was obtained from Dr. Glaser AG, Basel, Switzerland. [5-3H]IQ was obtained from Chemsyn (Lenexa, KS) a t a specific activity of 2.43 Ci/mmol and >95% radiochemical purity. The following chemicals were obtained from Toronto Research Chemicals (Downsview, Ontario, Canada): unlabeled MeIQx and [2J4C] MeIQx, specific activity 10mCi/mmol, radiochemical purity > 9 6 % , and unlabeled IQ and [2-l4C1IQ, specific activity 10 mCi/mmol, radiochemical purity >96%. SepPak (2-18 cartridges were purchased from Waters, Division of Millipore (Milford, MA). Chromatography solvents were HPLC grade. All remaining chemicals were reagent grade unless specified. Chemical Syntheses. The nitro derivatives of IQ and MeIQx were synthesized by the method of Grivas (30) with minor modifications. The recrystallization step was omitted, and instead the nitro compounds, which precipitated from solution during the reaction, were washed 2-3 times with ice-cold water. The chemical purity exceeded 99%. N-Hydroxy-IQ and N-hydroxy-MeIQx were synthesized from the respective nitro derivatives by the method of Enomoto et al. (31),except that the reaction was conducted in DMSO rather than ethanol (32). N-hydroxy-MeIQx and N-hydroxy-IQ were purified with a C-18 Sep-Pak (32). The N-hydroxy compounds (ca. 5-10 nmollpl) were stored in DMSO/ethanol (4:l) under argon and kept in liquid nitrogen until use. The purity of both N-hydroxy derivatives exceeded 97 % as determined by HPLC (33). Synthesis of Nucloside Adducts. Nucleosides (dG, dA, dT, dC) were dissolved (10 mg/mL) in a 100 mM sodium phosphate buffer, pH 7.0, containing 100 pM EDTA. The nucleoside solutions were than purged with argon for 10 min and warmed to 37 "C. All subsequent additions were made under argon. The N-hydroxy derivatives of IQ or MeIQx were added a t an amount of 6 nmol/mL. Acetic anhydride was then added in a 30-fold molar excess relative to the N-hydroxy derivatives. The reaction mixture was quickly capped, mixed, and incubated at 37 "C for 2 h. Typical reactionvolumes were 0.5-1 mL. Analysisof reaction products wasconducted directly by HPLC. Large-scalesyntheses for dG adducts were performed by increasing the amount of Nhydroxy compounds to 600 nmol/mL and using a total volume of 40 mL. In some instances, a portion of the adductsprecipitated from solution. The precipitate was collected by centrifugation and then dissolved in a minimum volume of DMSO. The portion of the adducts which did not precipitate were adsorbed onto C-18 Sep-Paks. After washing with water, the adducta were eluted

Turesky e t al. with methanol. Further purification was done by the HPLC conditions described below.

Preparation of IQ- and MeIQx-DNA Adducts in Vitro. One milliliter of calf thymus DNA (5 mg/mL) was dissolved in either 100 mM sodium phosphate or 100 mM sodium citrate buffer, pH 5.0 or 7.0, containing 100 pM EDTA, warmed a t 37 "C, and purged with argon for 10 min. All subsequent additions were carried out under argon. N-hydroxy-IQ or N-hydroxyMeIQx (150 nmol) was added, and the solution was briefly vortexed; then acetic anhydride was added a t varying molar excess relative to the N-hydroxy compounds. The mixture was incubated for 2 h a t 37 "C. Adducted DNA was separated from unbound reaction products immediately upon completion of the incubation by extraction twice with an equal volume of watersaturated butanol followed by two extractions with an equal volume of phenol/chloroform ( l : l ) , by one extraction with an equal volume of chloroform, and finally by an ethanol precipitation. The DNA was then pelleted by centrifugation a t 5000g, washed twice with 70% ethanol, dried under vacuum, and stored under argon a t -80 "C. O-Acetyltransferase-MediatedDNA Binding. Adult male Sprague-Dawley rats (250-300 g) were obtained from Iffa Credo L'Arbresle (France). Preparation of hepatic cytosols and assays for O-acetyltransferase-mediated DNA binding were performed as previously described (32-34). The assays were conducted a t 37 "C in argon-saturated 50 mM sodium pyrophosphate buffer (pH 7.4) containing 1mM DTT, 2 mg/mL calf thymus DNA, 3.0 mg/mL cytosol protein, 1mM acetyl CoA, and 10 pM N-hydroxy substrate. Assays were terminated after 15 min by the addition of 2 volumes of water-saturated l-butanol. Unbound N-hydroxy compounds were removed as described above. The concentration of recovered DNA was determined by UV absorbance a t 260 nm (1mg/mL DNA = 22), and the extent of covalent binding was determined by liquid scintillation counting. DNA Digestion Conditions. The adducted DNA was digested to nucleosides by a modification of a method previously described for the digestion of DNA adducted with IQ (24). Modified DNA was placed in 5 mM Tris buffer, pH 7.5 (at 25 "C), containing 10 mM MgClz a t a concentration of 0.5 mg/mL. DNase I was added a t a final concentration of 0.03 mg/mL. This mixture was incubated for 5 h a t 37 "C. Phosphodiesterase I was then added to the incubation mixture a t a final concentration of 0.012 unit/mL. Alkaline phosphatase then was added to give a final concentration of 1.25 units/mL, and the resulting mixture was incubated a t 37 OC for 18 h. The digests were added to 3 volumes of ethanol, and the precipitate was removed by centrifugation. The supernatant, which contained greater than 95 % of the radioactivity, was concentrated by rotary evaporation a t 40 "C and then stored a t -80 "C until HPLC analysis. Instrumentation. The HPLC system was a Hewlett Packard 1090M system containing a diode array detector or a Varian 5500 system. Radioactivity was measured online with a Berthold LB 506 C-1 radioactivity monitor or with an LKB 1219 Rackbeta liquid scintillation counter. The UV absorbance was monitored at 264 or 275 nm. A Supelco LC-18 deactivated base reversephase column (4.6 mm i.d. X 25 cm, 5-pm particle size) was used for all work except for large-scale purification of adducts where a semipreparative Supelco deactivated base reverse-phase column (10 mm i.d. X 25 cm, 5-pm particle size) was employed. The solvent conditions for the purification of nucleoside adducts were 30% methanol in 70 % ammonium acetate (50 mM, pH 6.8) with a linear gradient to 100% methanol in 40 min a t a flow rate of 1mL/min for the analytical column and 3 mL/min for the semiprep column. In the case of the dG-N2-IQ adduct, which precipitated from solution during chemical synthesis, a 50 mM ammonium formate buffer (pH 6.8) was used instead of ammonium acetate. The dG-NZ-MeIQxadduct was further purified using a 30-min linear gradient from 0.015% acetic acid to 100% methanol. The HPLC analysis of DNA adduct digests was carried out in 70% 50 mM KH2POd- (acidified with phosphoric acid to pH 3.5) and 30% methanol. A linear gradient to 80% methanol over 30 min was employed at a flow rate of 1 mL/min. These

Chem. Res. Toxicol., Vol. 5, No. 4, 1992 481

DNA Adducts of IQ and MeIQx conditions minimized peak tailing and significantly improved the chromatography. These analyses were conducted with the analytical deactivated base column. Chromatography without counterion buffers was difficult due to the strong interactions between the adducts and the column matrix. Proton NMR spectra were recorded on a 360-MHz Bruker spectrometer with a 5-mm 'H probehead. The sample temperature was 21.2 f 0.5 O C . The parameters for basic onedimensional spectroscopy, nuclear Overhauser effect (NOE) difference spectra, and homonuclear correlation experiments (COSY) were chosen as previously described (33). The purified adducts were placed under vacuum (0.02mbar) in order to remove HPLC solvents and organic acids. The samples were prepared in DMSO-&in a gloveboxunder nitrogen. Spectra were acquired on approximately0.75 pmol of adduct I, 3 pmol of adduct II,0.25 pmol of adduct I11and 3pmol of adduct IV per 0.5 mL of DMSOde. Chemical shifts are reported in ppm downfield from tetramethylsilane. Proton assignments were generally obtained from two-dimensional COSY spectra and are based on comparisonswith synthetic dG, IQ, and MeIQx and with literaturevalues reported for dG and aromatic amine DNA adducts (14-253537). It was obrved that the shiftsand linewidthsof many signals, but especiallythe exchangeableprotons, were rather sensitiveto the final HPLC conditions (pH, buffer) and to the degree of drying. Therefore, the shift values are reported to 0.01 ppm only. Coupling constants (in absolute values) were determined directly (withoutspectralsimulation)from both couplingpartners when possible and are generally accurate to 0.2 Hz. Coupling constants were in general equal before and after addition of DzO; when deviations occurred, both values are cited. Positive ion fast atom bombardment mass spectra (FAB/MS) were acquired using either a Kratos MS-50 MS with an M-scan FAB source and ion gun or a Finnigan TSQ-70 MS with an Ion Tech gun. FAB employed xenon atoms accelerated to 9-10 kV, a thioglycerol matrix, and a copper or stainless steel target. The mass spectrometry conditions have been previously described in more detail (38). Derivatization by silylation was accomplished using N-methyl-N-(trimethylsilyl)trifluoroacetamide/N,O-bis(trimethylsilyl)trifluoroacetamide/dimethylformamide (1:l:l) at 40 "C for 2 h (38-40).

Table I. IQ and MeIQx DNA Binding ae a Function of Incubation Conditione

IQ MeIQx (pmolbound/ (pmol bound/ mg of DNA) mg of DNA)

incubation conditionsa

*

100 mM sodium phosphate, pH 5.0 100 mM sodium phosphate, pH 7.0

682 48 414 i 76

100 & 18 39 f 6

3950 f 280

4800 i 810

189 i 36

184 i 24

16 i 7

lli8

100 mM sodium phosphate, pH 7.0,

+ acetic anhydride

50 mM sodium pyrophosphate, pH 7.4, rat hepatic cytosol,

+ acetyl CoA

50 mM sodium pyrophosphate, pH 7.4, + rate hepatic cytosol

0 Values represent the modification of calf thymus DNA (5 mg/ mL) by reaction of 150fiM N-hydroxy-IQand -MeIQx alone (pH 5.0 and 7.0), with a 30-fold molar excess of acetic anhydride (pH 7.0), or byreactionofcalfthymusDNA(2mg/mL) with l0pMN-hydroxyIQ and -MeIQxcontaining rat liver cytosol (3mg/mL) 1mM acetyl CoA ( N = 3 SD).

0

10

h

10000 -

-dG-CB-UeIOx

su

5000 -

I\

Results Reaction of N-Hydroxy-MeIQxand N-Hydroxy-IQ withcalf Thymus DNA. Preliminary experiments with N-hydroxy-MeIQs revealed that the binding of this carcinogen to calf thymus DNA in 100 mM sodium phosphate (pH 7.0) was 2-fold or greater than the DNA binding observed in 100 mM sodium citrate (pH 7.0). Therefore, all subsequent experiments were conducted in phosphate buffer. Differences were observed between the chemicalreactivity of N-hydroxy-MeIQx and N-hydroxyIQ with DNA under both acidic (pH 5.0) and neutral pH (7.0) (Table I). The reactivity of N-hydroxy-IQ withDNA was approximately 10-fold greater than that observed for N-hydroxy-MeIQx at pH 7.0 and about 7-fold greater at pH 5.0. The addition of acetic anhydride to the incubation medium at pH 7.0 enhanced DNA binding nearly 10-fold for IQ and 125-fold for MeIQx. Optimal DNA binding was observed with a 30-fold molar exceas of acetic anhydride over N-hydroxy compounds; the addition of more acetic anhydride resulted in decreased levels of DNA binding. The highest level of binding for IQ was 3.95 nmol bound/mg of DNA and accounted for 13.2% of the starting material bound. The highest levels of binding for MeIQx was 4.80 nmol boundlmg of DNA which accounted for 16.0% of the starting material bound. Rodent hepatic O-acetyltransferasescatalyzed the acetyl CoA-dependent DNA binding of both N-hydroxy com-

30

20 T1me (mln)

,

0

10

20

,

,

,

,

30

,

,

,

,

,

,

,

,

,

40

Time (nln)

Figure 1. HPLC analyses of enzymatic digest of calf thymus DNA modified with (A) N-acetoxy-IQand (B)N-acetoxy-MeIQx,

pounds at comparable levels (Table I). Our previous work showed that N-hydroxy-MeIQx was a better substrate for rodent hepatic O-acetyltransferase (32,33).However, in the present experiment the N-hydroxy substrate concentration was decreased from 100 to 10 pM and the protein cytosol content was increased 3- to 6-fold. Therefore, the enzymes may not have been saturated with substrate and reactions were not linear with time. The DNA modified with N-hydroxy derivatives in the presence of acetic anhydride was enzymatically digested and analyzed by HPLC (Figure 1).One major radioactive peak and several smaller peaks were observed for both IQand MeIQx-bound DNA. The peaks which have been confirmed as dG adducts are designated as I& adducts I and I1 and MeIQx adducts I11 and IV. Reaction of N-Hydroxy-IQand N-Hydroxy-MeIQx with Deoxynucleosides. Reaction of dG with [I4C]labeled N-hydroxy-IQ and N-hydroxy-MeIQx in the presence of acetic anhydride yielded two products for each N-hydroxy compound which, respectively, had identical retention times to adducts I and I1 for I&,and adducts I11

482 Chem. Res. Toxicol., Vol. 5, No. 4, 1992

Turesky et al.

A

C

300

400

500

400

300

500

Wavelength (nm)

Wavelength (nn)

D

B

\

300

400

500

300

Wavelength (na)

400

500

Wavolength (na)

Figure 2. On-line HPLC UV spectra (50 mM ammonium acetate, pH 6.8/methanol) of (A) adduct I (dG-N2-IQ),(B)adduct I1 (dG-Ca-IQ),(C) adduct I11 (dG-N2-MeIQx),and (D) adduct IV (dG-C8-MeIQx). Table 11. dG-N2-and dG-CS-IQ and dG-N2-and dG-CS-MeIQx Adduct Formation from Reaction of N-Acetoxy Derivatives with Calf Thymus DNA and Deoxyguanosine

incubation conditions' 100 mM sodium phosphate, pH 7.0, + acetic anhydride (reaction with DNA) 100 mM sodium phosphate, pH 7.0,

+ acetic anhydride (reaction with dG)

pmol bound/ mg of DNA 3950 f 280

IQ adduct I adduct I1 (dG-N2-NQ) (dG-Ca-IQ) pmol bound/ (%) (%) mgofDNA 4.3 f 1.0

70.8 f 12.8

5.7 f 2.0

40.9 & 8.9

4800 f 810

MeIQx adduct I11

adduct IV (dG-N2-MeIQx) (dG-C8-MeIQx) (%)

(%)

11.6 f 3.8

60.4 f 13.2

4.7 f 0.3

52.6 f 4.5

Values represent enzymatic digest of calf thymus DNA (5 mg/mL) modified with 150 pM N-hydroxy-IQ and N-hydroxy-MeIQx with a 30-fold molar excess of acetic anhydride. Approximately 7-15% of the bound heterocyclic amines appear to be uncharacterized adducts or depurinated products. Reaction of dG (10 mg/mL) was done with 60 pM N-hydroxy derivatives and a 30-fold molar excess of acetic anhydride ( N = 3 f SD). a

and IV for MeIQx. However, no adduction products, and only degradation products of the N-hydroxy compounds, were observed when reactions were conducted with dA, dC, and dT. The on-line UV spectra of the dG reaction products acquired by HPLC as described in Experimental Procedures are presented in Figure 2. The maxima for I& adduct I at pH 6.8 were observed at 267,295 (s), and 375 nm (Figure 2A). The chromophore of IQadduct I1differed greatly and did not resemble either IQ or dG with maxima found a t 248, 293, 306, and 342 nm (Figure 2B). The spectrum of adduct I1 is in excellent agreement with that reported for N-(deoxyguanosin-8-yl)-IQ(24). The UV spectra of MeIQx adduct I11 displayed maxima at 225, 243 (s), 285, and 410 nm, and adduct IV displayed maxima at 225, 253, 272 (s), 307, and 347 (8) (Figure 2C,D). A hypsochromic shift of 7-10 nm was observed in the maxima for adducts I and I11under acidic pH conditions (KHzP04 buffer, pH, 3.51, while there were not noticeable shifts in maxima for adducts I1 and IV. The UV spectra of these dG reaction products were indistinguishable from the

respective adducts recovered from the calf thymus DNA digests. Partitioning of dG adducts of IQ and MeIQx as a function of pH was conducted as described by Moore and Koreedausing 30%l-butanol in ethyl ether (41). Ionizable groups were present at both acidic and basic pH for all four adducts as evidenced by increased aqueous solubility. The PKa near 9-10 for all four adducts precluded adduction a t the N1 or Oe position, because adducts modified at these positions of dG do not display an alkaline PKa (41) .

The yields of the dG-NZ and dG-C8 adducts of IQ from the reaction of N-hydroxy-IQ with a 625-foldmolar excess of dG in the presence of acetic anhydride were 5.7 f 2.0% (adduct I: dG-N2-IQ)and 40.9 f 8.9% (adduct 11: dGC8-IQ) (Table 11). When this reaction was conducted with [5JH]IQ instead of [14C]-labeledmaterial under the same conditions, there was the expected UV-absorbing peak for both adducts; however, only adduct I1 contained tritium. There was also a substantial amount of tritium at

Chem. Res. Toxicol., Vol. 5, No. 4, 1992 483

DNA Adducts of IQ and MeIQx

,

A2

Adduct IV

Figure 3. Proposed structures of IQ and MeIQx adducts. Adduct I, 5-(deoxyguanoein-N2-yl)-2-amino-3-methylimidazo[4,5-flqui-

noline (dG-Nz-IQ);adduct 11,N-(deoxyguanoein-S-yl)-2-amino-3-methylimidazo[4,5-~quinoline (dG-CS-IQ); adduct III,5-(deoxyguanosin-N2-yl)-2-amino-3,8-dimethylimidazo[4,5-flquinoxaline (dG-N2-MeIQx); and adduct IV, N-(deoxyguanosin-S-y1)-2-amino-3,8dimethylimidazo[4,5-flquinoxaline(dG-CS-MeIQx).

the solvent front (accounting for 11.6 f 0.5% of the starting material) which was apparently due to tritiated water since it was eliminated from the sample by rotary evaporation a t 37 OC. A radioactive peak at the solvent front was not observed with the [WI-labeled material. The amount of tritiated water produced was approximately twice the amount of dG-N2-IQformed using [l4C1-labeled material, which indicates nitrenium ion formation followed by solvolysis with OH-/H20 to form the 5-hydroxyderivative of I&. The presence of 5-hydroxy-IQ was not confirmed because it is labile and not readily isolated without employing a fully nonmetallic HPLC system (42). The relative yields of the two dG adducts formed by reaction of N-hydroxy-MeIQx with a 625-fold molar excess of dG in the presence of acetic anhydride paralleled the levels seen with N-hydroxy-IQ and were 4.7 f 0.3 % (adduct 111: dG-N*-MeIQx) and 52.6 f 4.5% (adduct I V dG-C8MeIQx). The relative amounts of dG-N2 versus dG-C8 adduct formation from the reaction of N-acetoxy derivatives of IQ and MeIQx with calf thymus DNA differed. The dG-C8-IQ adduct was formed in approximately 16fold greater amounts than dG-N2-IQ while the dG-CSMeIQx adduct was recovered in approximately 5-fold greater amounts than the dG-N2-MeIQxadduct (Table 11). Spectroscopic Characterization. The dG reaction products were prepared in sufficient quantities for characterization by proton NMR spectroscopy and FAB/MS. The proposed structures of the adducts are displayed in Figure 3. The 360-MHz ‘HNMR spectral parameters of the parent compounds are given in Tables 111-V, and the

Table 111. 360-MHz 1H NMR Spectral Parameters of

Deoxyguanosine (dG).

chemical shift (ppm) 10.64b 7.92 6.47b 6.11 5.2Ib 4.96b 4.33 3.80 ~3.52~ 2.50e 2.19

multiplicity (no. of protons) s (1)a1 br 8 (1) e (2) sl br dd (1) d (1) t (1) m (lIc [dtl td (1) m (2) [2 x ddl ddd (1) ddd (1)

assignments N’H (G) H-8 (G) 2-NHz H-l’dR OH-3’dR OH-5‘dR H-3’dR H-4‘dR H-5’- and H-5”dR H-2’dR H-2”dR

Coupling Constants (Hz) Ji,,z, Ji,,z,, J2,,21, J2’,3’ J2“,3’

7.9 6.0 13.1 5.7 3.0

J3,,4< J49,5, J4$,5!,

J5*,6”

2.7

JS,,OH-V -3.9

[ ~ 4 . 6 1 ~ Js,,oH-s,

-5.5

[-4.61d [11.8]

a Symbols and abbreviations: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad, v = very, el = slightly, “ “ approximate description, [ I = coupling pattern or constanta obtained from Dz0exchangedspectrum. * Exchangeable withD20. 6 lines; 5 linesafter DzO exchange. H-5’ and H-5” are distinguished but not unequivocally assigned in the DzO-exchangedspectrum (3.56 and 3.50 ppm). e Partly under DMSO-d6 signal.

360-MHz lH NMR spectral parameters of the adducts are given in Tables VI-IX. Spectral parameters for dG are cited because available literature values were not reported

484 Chem. Res. Toxicol., Vol. 5, No. 4, 1992

Turesky et al.

Table IV. 360-MHz IH NMR Spectral Parameters of IQa chemical shift (ppm) 8.72 8.54 7.71 7.57 7.43 6.56b 3.64

multiplicity (no. of protons) dd (1) ddd (1) d (1) dd (1) dd (1) s (2) br s (3)

assignments H-7 H-9 H-4 H-5 H-8 2-NHz W-CHB

Coupling Constants (Hz)

8.8

543 J5,9

0.7 4.2

51.8

1.8 8.3

57,9 58,s

0 Symbols and abbreviations: see footnote a, Table 111. Exchangeable with DzO.

Table V. 360-MHz ‘H NMR Spectral Parameters of MeIQx. chemical shift (ppm) 8.78 7.88 7.76 7.48b 3.71 2.74

multiplicity (no. of protons) s (1) d (1) d (1) s (2) s (3) s (3)

assignments H-7 H-4 H-5 2-NHz N3-CH3 8-CHa

Table VI. 360-MHz IH NMR Spectral Parameters of Adduct I: 5-(Deoxyguanosin-iV-yl)-2-aminoj-methylimidazo[ 4,5-flquinolinea chemical shift (ppm) 11.6gb 10.01b 9.02 8.81 8.60 8.09 7.59 6.51b 6.41 5.36b,c 4.96b 4.37 3.90 3.65 3.53e 2.62 2.40

multiplicity (no. of protons) (1)br s (1) s (1)

dd (1) dd (1) s (1)

dd (1) s (2)

dd (1) (1) br (1)br m (Ud [dtl td (1) s (3) m (2) br [2 X dd] ddd (1) ddd (1)

Deoxyribose Coupling Constants (Hz) 7.9 [7.7] 5.9 13.2 -5.7 -2.8

Ji,.z, J1,,2,, 528.2” J2‘,3’

-

J z v

8.8

Symbolsand abbreviations: see footnote a,Table 111. Exchangeable with DzO. 0

at the same temperature (35) or deviated from our results (36,37). The nomenclature of the dR atoms is that used by Evans et al. (35). The ‘H NMR spectra of dG-N2-I& and dG-C8-IQ (adducts I and 11)are presented in Figures 4 and 5. The FABlMS spectral parameters are presented in Table X. Adduct I: 5-(Deosyguanosin-NL-yl)-2-amino-3-methylimidazo[4,S-flquinoline.The positive ion FABIMS of adduct I exhibited prominent (about 70% RA) ions at mlz 680 and 608 in a ratio of approximately 1:l. These ions are attributed to the protonated bis- and tris-TMS derivatives. This result is consistent with other reports on the behavior of TMS derivatives of arylamine-DNA adducts analyzed by FABlMS (38,391. The expected BH2+ fragment ions from the two parent protonated molecules were also observed at mlz 420 and 348 (base peak), again in a ratio of about 1:l. These ions are attributed to loss of a bis-TMS derivatized deoxyribose moiety from mlz 680 and 608, the third TMS group in the tris-TMS derivative being located on the base moiety (38,39). These data are consistent with a TMS-derivatized nucleoside I& adduct of dG and exclude both the presence of an acetylated adduct and a ring-opened structure (19). The proton NMR of adduct I is shown in Figure 4A, and the resonances of the dR proton after D2O exchange are shown in Figure 4B. The H-8 proton of the deoxynucleoside was detected as a singlet a t 8.09 ppm. The H-4 and -5 protons of I&, expected as an AB system near 7.71 and 7.57 ppm, J = 8.8 Hz, were missing and indicated that substitution occurred on the I& ring moiety and not on the exocyclic amino group. The contaminant seen at 8.44 ppm was identified as formate signal by a spiking experiment. A sharp singlet at 9.02 ppm was detected which was not found in the spectrum of I& or dG. A very

[-2.61 [4.8Ie [4.7]e [11.81

J3f.4,

54’3 J4,,5,, J5,,5,, ~~~

Coupling Constant (Hz) 543

assignments N*H (G) [or 2-NH(G)] 2-NH (G) [or N*H(G)l H-4 (IQ) H-7 (IQ) H-9 (IQ) H-8 (G) H-8 (IQ) 2-NH2 (IQ) H-l‘dR OH-3‘dR OH-5’dR H-3‘dR H-4‘dR N3-CH3 (IQ) H-5’- and H-5”dR H-2‘dR H-2”dR

IQ Coupling Constants (Hz) 57,s

4.2

57,9

1.7

Je,s

8.3

Symbolsand abbreviations: see footnotea , Table 111. Exchangeable with DzO. Assignment from a COSY correlation between H3’dR and OH-3’dR. 6 lines; 5 lines after D20 exchange, quintet = dt (see Figure 4B). e H-5’ and H-5” can be distinguished (-3.57 and 3.52 ppm) but not unequivocally assigned in DpO-exchangedspectrum (see Figure 4B).

weak correlation was found in a COSY spectrum between this signal and the P - C H 3 group of the I& moiety. The characteristic long-rangeH-5,9 coupling normally observed for I&was missing. Upon irradiation of the W-CH3 signal, a small NOE of ca. -1.5% was observed on the signal at 9.02 ppm and permitted tentative assignment of this proton as H-4. This suggested that the 5 position of IQ was the site of adduction. Synthesis of this adduct with N-hydroxy-IQ [3Hl-labeled in the 5 position resulted in complete elimination of the label (vide supra) and thus confirmed the C-5 position of I&as the site of adduction. Only one NH2 resonance was detected (6.51 ppm). Two single-protonDzO-exchangeable resonances were observed at 10.01 and 11.68 ppm. These signals were tentatively assigned to the remaining 2-NH and the deshielded NIH of dG. The protons of the dR moiety were readily assignable through COSY experiments in the absence of and followingaddition of D2O. On the basis of these proton NMR and mass spectroscopic data, adduct I was determined to be an N2-substituted dG adduct at the 5 position of I&. Adduct 11: N-( Deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-flquinoline.The FABlMS spectrum of TMS-derivatized adduct I1 shows a prominent ion at m/z 680 (20% RA) corresponding to a protonated molecule for the tris-TMS derivative, whereas the corresponding bis derivative at mlz 608 was only about 2% RA. Consistent with the formation of primarily a tris derivative, the base peak was observed at mlz 420, presumably via loss of a bis-TMS-derivatized dR moiety from mlz 680,

Chem. Res. Toxicol., Vol. 5, No. 4, 1992 485

DNA Adducts of IQ and MeIQx

Table IX. 360-MHz IH NMR Spectral Parameters of Adduct IV: N-(Deoxyguanosin-8-yl)-2-amino3,&dimethylimidazo[4,5-flquinoxalinea

Table VII. 360-MHz IH NMR Spectral Parameters of Adduct 11: N-(Deoxyguanosin-8-yl)-2-amino3-methylimidazo[4,5-flquinolinea chemical shift (ppm) -11.15b 8.82 8.67 7.90 7.79 7.59 6.~55~ 6.53 -5.7-4.76 4.51 3.82 3.78 3.70d 3.56d 3.35 2.08

multiplicity (no. of protons) (1)br dd (1) ddd (1) d (1) dd (1) dd (1) 8 (2) ‘t” (1) (-2) v br m (W td (1) s (3) dd (1) dd (1) ddd (1) ddd (1)

assignments NIH (G) or 2-NH (IQ) H-7 (IQ) H-9 (I$) H-4 (IQ) H-5 (IQ) H-8 (IQ) 2-NHz (G) H-l‘dR OH-3’- and OH-5’dR H-3’dR H-4’dR N3-CH3 (IQ) H-5’- or H-5”dR H-5”- or H-5’dR H-2’dR H-2”dR

chemical shift (ppm) -l2.0-10.Ob 8.79 7.94 7.86 6.56 6.45* -6.4-4.0b 4.50 3.83 3.74 3.7lC 3.56c 3.32 2.80 2.07

multiplicity (no. of protons) (-2) v br s (1) d (1) d (1) dd (1) s (2) br (>1)v br dt (1) td (1) s (3) dd (1) dd (1) ddd (1) s (3) ddd (1)

assignments NIH ( G ) and 2-NH (MeIQx) H-7 (MeIQx) H-4 (MeIQx) H-5 (MeIQx) H-l’dR 2-NHz (G) OH-3’- and OH-5’dR H-3’dR H-4‘dR W-CH3 (MeIQx) H-5’- and H-5”dR H-5”- or H-5’dR H-2’dR 8-CH3 (MeIQx) H-2”dR

Deoxyribose Coupling Constants

Deoxyribose Coupling Constants (Hz) Ji,,z,

JY,Y J2’,2” J2’,3’ J2“.3’

-7.6 -6.6 13.0 -6.2 3.0

-

3.0 -5.1 [ - 4 . w -5.5 [-5.lId 11.5 [11.7]

J3,,4, J4,,5, J4,,5“ J5,,5“

~~

MeIQx Coupling Constant (Hz)

IQ Coupling Constants (Hz) 54.5

8.9 J5,9 0.7 J T , ~ 4.2 J7,g 1.7 Js,e 8.4 Symbolsand abbreviations: see footnote a, Table 111. Exchangeable with DzO. 5 lines before (fast exchange of OH-3’dR) and after DzO addition, quintet = dt. H-5’- and H-5“dRcannot be individually assigned. 54-5

Table VIII. 360-MHz IH NMR Spectral Parameters of Adduct 111: 5-(Deoxyguanosin-iV-yl)-2-amino3.8-dimeth~limidazol4,5-flauinoxaline~ ~

chemical shift

multiplicity (no. of protons)

assignments

-lMb -9.756 8.97 8.69 8.05 6.4tib 6.39 5.34b9c -5.0Sb 4.37 3.89 3.65 -3.53d 2.74 2.63 2.38

(>1)v br (51)br 8 (1) 8 (1) s (1)sl br s (2) br dd (1) (1)br (1)br (1)br [dtl td (1) s (3) m (-2) [2 X dd] 8 (3) m (-1) [ddd] m (-1) [ddd]

NIH (G) or 2-NH (G) 2-NH (G)or N1H (G) H-4 (MeIQx) H-7 (MeIQx) H-8 (G) 2-NHz (MeIQx) H-l’dR OH-3’dR OH-5’dR H-3‘dR H-4‘dR W-CH3 (MeIQx) H-5’- and H-5”dR 8-CH3 (MeIQx) H-2‘dR H-2”dR

Deoxyribose Coupling Constants (Hz) Ji,.z, Ji,,z,, Jz,,zi, J2‘,3’ J2”.3’

D.81 i5.91 [13.31 [--5.71

[-3.01

J3~,4/ J4!,5’ J4,,5,1 J5,,5“

[-2.71 [ -4.7Id [-4.9Id [ 11.91

-

a Symbolsand abbreviations: see footnote a,Table 111. b Exchangeable with DzO (for integrals see text). ‘Assignment from a COSY correlation between H-3’dR and OH-3’dR. H-5’ and H-5” can be distinguished (-3.56 and 3.51 ppm) but not unequivocally assigned in DZO-exchanged spectrum.

whereas only a minor signal (10% RA) was observed at mlz 348 via this same loss from mlz 608. That the ion a t

8.9

Symbolsand abbreviations: see footnote a, Table 111. Exchangeable with DzO. H-5’- and H-V’dR cannot be individually assigned. a

ml2 420 is not due to loss of a TMS moiety from mlz 608 may be inferred from the similar ratios for mlz 6801420 and mlz 608/348. Furthermore, loss of a TMS group in FAB mass spectra of TMS-derivatized arylamine-nucleoside adducts was not reported from the BH2+ ion (or the protonated molecule) in unimolecular (38) or even in collision-induced fragmentation (39). The proton NMR spectrum of adduct I1 is shown in Figure 5A, and the expanded signals of the dR protons before DzO exchange are shown in Figure 5B. All 5 aromatic protons and the W-CH, group of the IQ molecule were detected, which indicated that substitution occurred through the exocyclic nitrogen and not through the aromatic carbon atoms. The H-8guanine proton, normally seen at ca. 8.0 ppm, was missing, and ita absence clearly demonstrates that adduction occurred through the C-8 position of dG (14-16,18-26). A broad D2O-exchangeable resonance which integrated to 1 proton was detected at 11.15ppm and tentatively assigned to the remaining NIH of IQ. The 2-NH signal of dG was presumably so broad (note the broadening of the HDO signal at ca. 3.3 ppm) that it was not detected. The OH-3’ and OH-5’ signals of the dR moiety were also observed as very broad signals between ca. 5.7 and 4.4 ppm. The assignmentof all coupled protons was confirmed by COSY experiments. The chemical shift of the dR H-2’ proton was increased by 0.85 ppm to 3.35 ppm (H-2’ of dG was observed a t 2.50 ppm) while the H-2” was shifted by -0.11 ppm to 2.08 ppm. In contrast, the H-2’ and H-2’’ protons of adduct I, dG-N2I&, were shifted by 0.12 and 0.21 ppm relative to dG. A further unique feature of adduct I1 was the previously noted (24) strong downfield shift of the H-9 (IQ) proton upon addition of D20 (measured difference 0.43 ppm). Shift changes of all other protons were notable but less than 0.1 ppm. This shift of the H-9(IQ) may be caused

.

486 Chem. Res. Toxicol., Vol. 5, No.4, 1992 A

12.0

1

10.0

11:o

m

Turesky et al.

I'

I/H@

1 ---1.-1-.7-----

6.50

640

3.90

4.40

H-5'dR H-WR

3.60 3.50

2.65

2.45

PPM

Figure 4. (A) 360-MHz lH NMR spectrum of adduct I (dG-N2-IQ)in DMSO-&. (*) Indicates signals due to contamination. (B) Expanded dR signals of D2O-exchanged spectrum of adduct I.

-

A

li.0

11.0 PPM

10.0

r

f H-~(M)

NaCY(lQ)

H - 5 ~ H-7(loI

H-8$Q)

H-9(K)l

OH-3'dR OH-SUR

9.0

0.0

ZO

6.0

PPM

H3'dR

5.0

4.0

3.0

-----6.50

4.50

3.80

3.70 PW

3.55

3.35

2.10

Figure 1. (A) 360-MHz 1H NMR spectrum of adduct I1 (dG-Cs-IQ) in DMSO-& (B) Expanded dR signals of adduct I1 (no D2O exihange).

~~

Table X. Relative Abundance of Major Ions Obrerved in the FAB Mnrr S m t m of Trimethvlrilvleted Adducts I-IV relative abundance (% ) at m/z for ion ITMSL adduct1 adduct11 adduct111 adductIV 100at767 A 4 9 9 MH+ 9at696 12at696 MH+ 3 70at680 20at680 MH+ 2 66 at 608 2 at 608 10 at 623 16 at 623 BHz+ 2 9 9 100at507 A BHz+ 1 95 at 420 100 at 420 26 at 436 100 at 436 BHz+ 0 100at348 lOat348 20 at 363 0

*

Not detected. Not resolved from the matrix.

by DzO-mediated hydrogen bonding, and it is not seen with adduct I, IQ, or other IQ metabolites.2 Adduct 111: B(Deoxyguanorin-NL-y1)-2-amino-3,8-

cli"iaazo[4,bfJcpinoxaline. The FAB/MS spectrum of TMS-derivatizsd adduct III showed two prominent ions at m/z 767 and 507 (both about 100% RA)attributed to the protonated molecule for a tetrakis-TMS-derivatized adduct and the fragment corresponding to loss of a

bis-derivatized dR,namely, the TMSrBHz+ ion. Weak ions were also observed for the protonated molecules of 2 D.

H. Welti, unpublished rwulte.

the tris- and bis-derivatized adducta at mlz 695 and 623. The poor signal-to-noise level somewhat obscured the additional fragments expected from loss of the bis-TMS deoxyribose moiety, although a signal was observed at ml z 435 above the matrix background. Interestingly,tetrakisTMS derivatives have not been observed for simple C&substituted arylamine-nucleoside adducta via FABIMS under similar conditions (38, 39). The presence of the fourth TMS group (the second attached to the carcinogen1 base moiety) must be attributed to a stable (non-matrixreactive) TMS-derivatized site on the carcinogen moiety or present on an N2- but not on a C-8=substitutedguanosine adduct. The proton NMR spectrum of adduct I11 displayed a similarpattern to that of IQ adduct I in addition to several contaminant signals. The H-7, H-9,8-CHs, and NS-CHs protons of MeIQx were present. However, the orthocoupled H-4,5 protons were missing, and instead, a sharp singlet was observed at 8.97 ppm. Thus, substitution occurred either at the C-4 or C-5 atom of MeIQx. The H-8 proton of guanine was observed at 8.05 ppm. Only one NH2 resonance was detected at 6.45 ppm. T w o broad additional D2O-exchangeable resonances which could not

DNA Adducts of IQ and MeIQx

be precisely integrated were tentatively attributed to the NIH and the remaining 2-NH of guanine at ca. 11.8 and 9.75 ppm. A COSY experiment confirmed the identity and proton assignments of the dR moiety. Unfortunately, the limited amount of material prevented NOE difference spectra, and [5-3H]MeIQx was not available to conduct syntheses to determine the possible absence of tritium in this adduct. Therefore, the site of substitution is not unequivocal and may be at the 4 or 5 position of MeIQx. On the basis of analogy with the dG-N2-IQand resonance delocalization of the nitrenium ion at the 5 position, it is most likely that adduction also occurred at the C-5 atom of MeIQx. The shifts of the H-2’ and H-2” dR protons relative to dG were 0.13 and 0.19 ppm, similar to those for adduct I. Adduct IV: N-(Deoxyguanosin-8-y1)-2-amino-3,8dimethylimidazo[4,5-5-flquinoxaline.The FAB/MS spectrum of TMS-derivatized adduct IV showed weak ions at m / z 695 (12 % ) and 623 (4 5% ) for the protonated molecules of the tris- and bis-TMS-derivatized adducts. Major ions at m/z 435 (BP) and 363 (20% RA) are attributed to loss of the bis-TMS-derivatized deoxyribose from each protonated molecule. NMR spectroscopic analysis of adduct IV revealed that all the aromatic protons and the 8-CH3andW-CHs groups of MeIQx were present. The assignment of the H-4 and H-5 protons of MeIQx were obtained from an NOE of -5.5% and -1.5%, respectively, upon irradiation of the W-CH3signal. The assignmentof the H-7 and H-4 protons of the MeIQx was also supported by weak COSY correlation signals with the 8-CH3 and W-CH3 signals, respectively. No signal for the H-8 guanine proton was detected near 8.0 ppm, indicating adduction at the C-8 position. Only one NH2 signal was found (6.45 ppm), while a broad DzO-exchangeable resonance integrating to 2 protons was detected over the region from 12.0 to 10.0 ppm and tentatively assigned to the NIH dG and the remaining 2-NH proton of MeIQx. All of the dR protons were accounted for in the spectrum and confirmed by a COSY experiment. As seen for the dG-C8-IQ (adduct 111, the H-2’ proton of the dR moiety was shifted 0.82 ppm downfield and the H-2” was shifted upfield by-0.12 ppm relative to dG. An NOE of -6.8% was observed a t the 8.79 ppm singlet upon irradiation of the 8-CH3 signal of MeIQx, which confirmed the assignment of this signal as the H-7 proton of MeIQx and thus excluded 7-(deoxyguanosinN2-yl)-2-amino-3,8-dimethylimidazo[4,5-flquinoxaline as a hypothetical structure (the H-8 proton of guanine then would be shifted downfield to 8.79 ppm). Thus, the mass and lH NMR spectra of adduct IV were consistent with those expected for N-(deoxyguanosin-8-yl)-MeIQx.

Discussion Metabolic activation of HAAs through N-oxidationleads to metabolites which can form DNA adducts. Although N-hydroxy derivatives of IQ and MeIQx react directly with DNA, the ultimate carcinogenic species are believed to be N-acetoxy, N-(sulfonyloxy), or N-(prolyloxy) esters (11-16). These esters are highly unstable and are transformed through heterolytic fission into highly reactive nitrenium ions which are then attacked by nucleophiles such as protein or DNA (11,12). Studies with N-hydroxy-1naphthylamine and N-hydroxy-2-naphthylaminerevealed selective reaction of these compoundswith polynucleotides but not with low molecular weight nucleophiles. DNA

Chem. Res. Toxicol., Vol. 5, No. 4, 1992 487 adduct formation was dependent on the concentrations of both DNA and the N-hydroxyarylamine (I 7,18).Thus, macromolecular DNA facilitated adduct formation through several forces including hydrophobicity, intercalation, hydrogen bonding, van der Waals attractions, and nucleophilicity (11,12, 17, 18). In contrast to the lipophilicarylaminesmentioned above, the hydrophilic N-hydroxy-IQ and N-hydroxy-MeIQx readily reacted with dG to produce both C-8 and N2 adducts. The formation of ring-substituted dG-N2adducts of IQ and MeIQx demonstrates that nitrenium ion formation occurred and suggests that the reaction mechanism is an s N 1 or an intermediate sN2 mechanism (43). The amount of dG-C8 adducts was approximately 8- to 10-foldgreater than the amount of dG-N2adducts formed by the reaction of dG with N-acetoxy-IQ and N-acetoxyMeIQx. The lower yield of the N2adducts may reflect the relative contribution of each resonance form of the nitrenium ion. The ratio of the dG-N2 to dG-C8 adduct formation in double-stranded DNA from reaction with N-acetoxy-IQwas lower than that observed from reaction of N-acetoxy-IQ with dG. In the case of N-hydroxyMeIQx, the ratio of dG-N2 to dG-C8 adduct formation from reaction of the N-acetoxy-MeIQx with DNA was higher than with dG. Therefore, macromolecular DNA structure, in addition to the relative contribution of the nitrenium ion and carbocation resonanceforms, influences the site of adduct substitution. Chemical syntheses of arylaminedG-C8adducts through reaction of N-acetoxyarylamineswith dG are documented (14-16,21,44), and there are two reports on syntheses of dG-N2 adducts of AAF and 2-NA (45, 46). Our results demonstrate for the first time that dG-N2adducts of IQ and MeIQx and the adduct dG-C%MeIQx are produced from the in vitro reaction of N-acetoxy derivatives of Nhydroxy-IQ and N-hydroxy-MeIQx with dG or DNA; however, no adducts were found after reaction of the Nacetoxy derivatives with other deoxynucleosides. The chemical reactivity of N-hydroxy-IQ with DNA and the synthesis and spectroscopic characterization of the dGC8-IQ adduct were previously reported (24). However, the investigators used [5-3HlIQ instead of [14C]-labeled material, and therefore, they did not the detect dG-N2-IQ adduct since the [3H]-label is completely removed from this adduct. The conformation of the glycosidic linkage of adducted DNA appears to be an important factor in adduct persistence in vivo and may influence the toxicological properties of DNA adducts (47-50). The sterochemical terminology used in this discussion is that of Davies and Danyluk (51)and references therein. The two preferential conformationsof the deoxynucleosidetermed syn and anti are in rapid exchange, and their relative proportions are dependent upon the substituents on either the sugar or the base. The chemical shift of the sugar 2’ proton is regarded as a useful indicator of the preferred conformation of the glycosyl bond because of the deshielding effect of the nearby guanine N-3 atom in the syn conformation (35). Proton NMR studies revealed a conformational change about the glycoside bond of the adduct dG-C8AAF from the preferentially existing anti structure of dG to the alternative syn form. In contrast, the anti form is the preferred conformation for the dG-C8-AF adduct (35). The dG-C8-AAF adduct appears to induce a greater distortion of the DNA helix than the dG-Ca-AF adduct,

488 Chem. Res. Toxicol., Val. 5, No. 4, 1992

which results in a more rapid removal of dG-C8-AAF in vivo and a greater persistence of dG-C8-AF (47-50). The H-2’ signals of the IQ and MeIQx dG-CS adducts I1 and IV are found near 3.3 ppm, while the shifts for the dG-N2 adducts I and I11 are ca. 2.6 ppm, which is very near that of dG (2.5 ppm). This suggests that the preferred conformation of the dG-C8 adducts of IQ and MeIQx is the syn form while the dG-N2adducts are preferentially in the anti form. Our structural formulae for these adducts have been drawn accordingly (Figure 3). However, this conformation theory was developed and tested on dG and dA derivatives substituted at the C-8 position of the base (35,52-54). In our case, the adduction products of I& and MeIQx occur at two different positions of guanine, the C-8 and N2 atoms. Furthermore, adduction occurs through the exocyclic heterocyclic amine group for the dG-CS adducts and through the C-5 atom for the dG-N2 adducts. The spatial arrangement of the HAAs in the four adducts is not known. In the case of proximity to the dR moiety, aromatic ring current effects may influence the shifts of the dR proton signals. Additional evidence which supports the postulate of the dG-C8 adducts in the syn geometry comes from the ribose ring conformation. The C-2’ endo population, , ~ JJ3’,4! coupling constants, determined mainly from J I ~ and clearly increased in the C-8-NH-substituted (deoxy)nucleosides present in the anti conformation (35, 37, 54). However, either there was an increase of the C-3’ endo population or the ribose puckering was not significantly altered in secondary amine C-8 adducts having the syn form ( 3 5 , 5 4 ) . In the case of the IQ and MeIQx dG adducts I-IV, coupling constants are very similar to those for dG (Tables I11and VI-IX), which suggests that the dR ring does not strongly interact with the heterocyclic aromatic substituents. The fact that the dR ring geometry of the adducts is not changed also reinforces the hypothesis of dG-C8adducts existing mainly in the syn conformation. The similarity of the J4’,5tand J4,5” coupling constants with those of dG shows that there are no significant changes in the conformation of the C-4’, C-5’ bond of the sugar moiety in dG adducts I-IV. This conformation is predominantly gauche,trans (gt)/trans,gauche(tg). Previous studies have demonstrated that C-BNH-substituted adducts in the anti form often display a clearcut increase in the gauche,gauche (gg) population (35, 37, 54). The increase of the gg population in dG-C-BNH adducts in the anti conformation brings the (2-5’ oxygen in close proximity to the purine C-BNH and has led to the postulate of an intramolecular hydrogen bond (52,54,55). This hydrogen bond was also proposed by Evans and coworkers in the dG-C&AF adduct where a downfield shift of the remaining C-BNH proton (8.77 ppm) compared to the AF 2-NHz protons (5.74 ppm) was found (35). Inspection of these data also shows a marked downfield shift of the OH-5’ signal to near 6.0 ppm. We suggest that a downfield shift of the OH-5’dR signal relative to the OH-3’dR signal, together with preferred gg conformation about the C-4’, C-5‘bond, may be regarded as an indicator of hydrogen bridging in primary aromatic amine dG-C8 adducts in the anti conformation (the OH-3’dR signal remains within its normal range of 5.0-5.4 ppm). This postulate is supported by the resonance signal of the OH5’dR signal (6.06 ppm) of dG-C&AB which is believed to be in the anti form (21). In the case of dG-C8-MAB, which exists preferentially in the syn conformation, and where

Turesky et al.

the C-8-NH proton is replaced by a methyl group that obstructs hydrogen bridge formation, the signal of the OH-5’dR proton is upfield from the OH-3’dR signal (56). In the case of dG-N2adducts, where no OH-5’dR hydrogen bridging would be expected for either the syn or anti form, on the basis of examination of Dreiding models, the OH5’dR signal is also found upfield from the OH-3’dR resonance (45, 56). Accordingly, the OH-5’ signal of IQ and MeIQx dG-N2 adducts I and I11 are upfield relative to the OH-3’ signals. For the dG-C8 adducts I1 and IV, no exchangeable proton signals were detected near 6.0 ppm. Spectral analysisof dG-CSIQ in more dilute solution resulted in separation of the broadened OH-3’ and OH-5’ signals below 5.3 ppm, with the OH-5’ signal being slightly upfield. These findings further support the preference of IQ and MeIQx dG-C8 adducts for the syn form. Several of the major dG-CS aromatic amine adducts formed in rodents have been correlated with induction of mutations in bacteria and mammalian cells (44,57,58). The dG-CS adduct of 4-aminobiphenyl, a recognized human urinary bladder carcinogen found in cigarettes, was recently identified in biopsy samples of human urinary bladder of cigarette smokers and suggests that this adduct may be involved in the initiation of bladder cancer (59). Although dG-CS adducts of aromatic amines are the predominant adducts formed in rodents (11,441,the dGN2 adducts of several arylamines formed in low levels, including AAF (dG-N2-AAF) and N-methyl-4-aminoazobenzene (dG-N2-MAB),are persistent lesions (45,56, 60) and may be toxicologically significant. The dG-C8 guanine adduct of IQ was estimated by the [32Pl-postlabelingtechnique (61)to account for more than half of the total adduct level in liver, a target tissue for tumorigenesis, in mice, rats, and monkeys (62). A similar adduct profile was observed after postlabeling of DNA adducted in vitro with N-hydroxy-IQ (24). As many as 5-8 other adducts which have not been characterized also were detected by postlabeling; it is not known whether these products are other unique adducts, oligonucleotides, or degradation products. Four major adducts of MeIQx were detected by [32Pl-postlabelingin liver of mice treated with MeIQx or in vitro by reacting azido-MeIQx with calf thymusDNA (63). Another study reported 7 adducts (27). More recent studies using [32P]-postlabelinghave shown that there are two or three major DNA adducts formed from N-hydroxy-IQ and N-hydroxy-MeIQx (64,65) and are consistent with our results obtained in vitro. The detection and estimation of DNA adducts by postlabeling assume that all adducts are labeled with equal efficiency by the T4 polynucleotide kinase. However, without the dG-N2 3’-monophosphate standards, the actual labeling efficiency of the dG-N2adducts is uncertain and, consequently, their identification and their contribution to DNA adduct formation in vivo are unknown. Several additional adducts of IQ and MeIQx whose identities are unknown also appear to be formed in vitro with DNA in minor amounts (Figure 1). Treatment of modified DNA with RNase A and RNase TIprior to DNase treatment did not result in removal of these products. Therefore, it is unlikely that these adducts are RNA derivatives attributed to calf thymus DNA contaminated with low levelsof RNA (22). A partialdepurination during enzyme hydrolysis could contribute to part of the uncharacterized material; however, mock digests with purified adducts indicate that hydrolysis of the glycosidic linkage

DNA Adducts of ZQ and MeIQx

does not occur under these digestion conditions. The low amount of material and background contamination prevented UV spectral comparison of these unknown derivatives with adducted guanyl derivatives of IQ and MeIQx. Identification of these other derivatives would require reacting gram quantities of DNA with N-hydroxy-IQ and MeIQx. Both IQ and MeIQx are metabolically transformed by human tissues to genotoxic species (9, 10, 32). Furthermore, another ubiquitous food-borne heterocyclic aromatic amine, PhIP, has been implicated as a major DNAdamaging agent by PPI-postlabeling of urinary mutagens from individuals who smoke black tobacco (65).Thus, HAAs may have a role in the etiology of human cancers, and the role of dG-C8 and dG-NZ adducts in carcinogenesis merits study. In order to evaluate the DNA-damaging activity of heterocyclic amines, a rigorous characterization of DNA adducts and their physiochemical properties and reliable analytical methodologies of detection must be developed. These in vitro studies provide a basis for a better understanding of the chemistry of heterocyclic aromatic amine DNA adducts.

Acknowledgment. We thank Dr. Cynthia D. Leaf for valuable critiques of the manuscript and Mrs. Francia Arce Vera for technical assistance with NMR spectroscopy. References (1) Felton, J. S., Knize, M. G., Shen,N. H., Andresen, B. D., Bjeldanes,

L. F., and Hatch, F. T. (1986) Identification of the mutagens in cooked beef. Enu. Health Perspect. 67, 17-24. (2) Felton, J. S., and Knize, M. G. (1990) Heterocyclic-Aminemutagens/ carcinogens in foods. In Handbook of Experimental Pharmacology (Cooper, C. S., and Grover, P. L., Eds.) Vol. 94/1, pp 471-502, Springer-Verlag, Berlin and Heidelberg. (3) Sugimura, T. (1986) Studies on environmental chemical carcinogenesis in Japan. Science 233, 312-318. (4) Sugimura, T. (1988)Successful use of short-time tests for academic purposes: their w e in identification of new environmental carcinogens with possible risk for humans. Mutat. Res. 205, 33-39. (5) Adameon, R. H., Thorgeirsson, U. P., Snyderwine, E. G., Reeves, J., Dalgard, D. W., Takayama, S., and Sugimura, T. (1990) Carcinoin nonhuman genicity of 2-amino-3-methylimidazo[4,5-flquinoline primates: induction of tumors in 3 macaques. Jpn. J.Cancer Res. 81,10-14. (6) Miller,J. A. (1970)Carcinogenesisby chemicals: an overview. Cancer Res. 30, 559-576. (7) Yamazoe,Y., Shimada, M., Shinohara, A., Saito, K., Kamataki, T., and Kato, R. (1983) Microsomal activation of 2-amino-3-methylimidazo[4,5-flquinoline,a pyrolysate of sardine and beef extracts, to a mutagenic intermediate. Cancer Res. 43, 5768-5774. (8) Yamazoe,Y., Abu-Zeid, M., Manabe, S., Toyama, S., and Kato, R. (1988) Metabolic activation of a protein pyrolysate promutagen 2amino-3,8-dimethylimidazo[4,5-flquinoxaline by rat liver microsomes and purified cytochrome P450. Carcinogenesis 9, 105109. (9) Shimada, T., Iwasaki, M., Martin, M. V., and Guengerich, F. P. (1989)Human liver microsomal cytochrome P-450 enzymes involved in the bioactivationof procarcinogensdetected by umu generesponse in Salmonella typhimurium TA1535/pSk1002. Cancer Res. 49, 3218-3228. (10) Butler, M. A,, Iwasaki, M. Guengerich, F. P., and Kadlubar, F. F. (1989) Human cytochrome P-450PA (P-450IA2),the phenacetin 0deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenicarylamines. Proc. Natl. Acad. Sci. U.S.A. 86,7696-7700. (11) Beland, F. A., and Kadlubar, F. F. (1985)Formation and persistence of arylamine DNA adducts in vivo. Enuiron. Health Perspect. 62, 19-30. (12) Kadlubar, F. F., and Beland, F. A. (1985) Chemical properties of ultimate carcinogenic metabolites of arylamines and arylamides. In Polycyclic Hydrocarbons and Carcinogenesis (Harvey, R. G., Ed.) ACS Symposium Series 283, pp 331-370, American Chemical Society, Washington, DC.

Chem. Res. Toxicol., Vol. 5, No. 4, 1992 489 (13) Kato, R. (1986) Metabolic activation of mutagenic heterocyclic amines from protein pyrolysates. CRC Crit. Reu. Toxicol. 16,308347. (14) Kriek, E., Miller, J. A., and Miller, E. C. (1967) 8-(N-2-Fluorenylacetamido)guanosine, an arylamidation reaction product of guanosine and the carcinogen N-acetoxy-N-2-fluorenylacetamide in neutral solution. Biochemistry 6, 177-182. (15) Bartsch, H., Dworkin, M., Miller, J. A., and Miller, E. C. (1972) Electrophilic N-acetoxyaminoarenes derived from carcinogenic Nhydroxy-N-acetylaminoareneby enzymatic deacetylation and transacetylation in liver. Biochim. Biophys. Acta 286, 272-298. (16)Lin, J.-K., Schmall, B., Sharpe, I. D., Miura, I., Miller, J. A., and Miller, E. C. (1975) N-Substitution of carbon 8 in guanosine and deoxyguanosine by the carcinogen N-benzoyloxy-N-methyl-4aminoazobenzene in vitro. Cancer Res. 35,832-843. (17) Kadlubar, F. F., Miller, J. A., and Miller, E. C. (1978) Guanyl Osarylamination and @-arylation of DNA by the carcinogen N-hydroxy-1-naphthylamine. Cancer Res. 38,3628-3638. (18) Kadlubar, F. F., Unruh, L. E., Beland, F. A., Straub, K. M., and Evans, F. E. (1980)In vitro reaction of the carcinogen, N-hydroxy2-naphthylamine, with DNA at the C-8 and NZ atoms of guanine and at the N6 atom of adenine. Carcinogenesis 1, 139-150. (19) Beland, F. A., Allaben, W. T., and Evans, F. E. (1980) Acetyltransferase-mediated binding of N-hydroxyarylamides to nucleic acids. Cancer Res. 40, 834-840. (20) Martin, C. N., Beland, F. A,, Roth, R. W., and Kadlubar, F. F. (1982) Covalent binding of benzidine and N-acetylbenzidine to DNA at the C-8 atom of deoxyguanosine in vivo and in vitro. Cancer Res. 42,2678-2696. (21) Delclos, K. B., Tarpley, W. G., Miller, E. C., and Miller, J. A. (1984) 4-Aminoazobenzeneand Nfl-dimethyl-4aminoazobenzeneas equipotent hepatic carcinogensin male C57BL/6 X C3H/HeF1mice and as characterization of N-(deoxyguanosin-8-yl)-4-aminoazobenzene the major persistent hepatic DNA-bound dye in these mice. Cancer Res. 44, 2540-2550. (22) Flammang, T. J., Westra, J. G., Kadlubar, F. F., and Beland, F. A. (1985)DNAadducts formed from theprobableproximate carcinogen, N-hydroxy-3,2’-dimethyl-4-aminobiphenyl, by acid catalysis or Sacetyl coenzyme A-dependent enzymatic esterification. Carcinogenesis 6, 251-258. (23) Westra, J. G., Flammang, T. J., Fullerton, N. F., Beland, F. A,, Weis, C. C., and Kadlubar, F. F. (1985) Formation of DNA adducts in vivo in rat liver and intestinal epithelium after administration of the and its hydroxamic acid. carcinogen 3,2’-dimethyl-4-aminobiphenyl Carcinogenesis 6, 37-44. (24) Snyderwine,E. G.,Roller,P.P., Adamson,R. H., Sato, S., andThorgeirsson, S. S. (1988) Reaction of the N-hydroxylamine and Nacetoxyderivativesof 2-amino-3-methylimidazo[4,5-flquinoline with DNA. Synthesis and identification of N-(deoxyguanosin-8-yl)-IQ. Carcinogenesis 9, 1061-1065. (25) Hashimoto, Y., Shudo, K., and Okamoto, T. (1982) Modification of DNA with potent mutacarcinogenic 2-amino-6-methyldipyrido[l,2a:3’,2’-dlimidazole isolated from a gluatamic acid pyrolysate structure of the modified nucleic acid base and initial chemical event caused by the mutagen. J.Am. Chem.SOC.104, 7635-7640. (26) Hashimoto, Y., Shudo, K., and Okamoto, T. (1980)Activation of a Identification mutagen,3-amino-l-methyl-5H-pyrido[4,5-blindole. and its reaction of 3-hydroxyamino-l-methyl-5H-pyrido[4,3-blindole with DNA. Biochem. Biophys. Res. Commun. 96,355-362. (27) Yamashita, K., Adachi, M., Kato, S., Nakagama, H., Ochiaia, M., Wakabayashi,K., Sato, S., Nagao, M.,and Sugimura,T. (1990)DNA adducts formed by 2-amino-3,8-dimethylimidazo[4,5-flquinoxaline in rat liver: doseresponseon chronicadministration. J p n .J . Cancer Res. 81, 470-476. (28) Turteltaub, K. W., Felton, J. S., Gledhill, B. L., Vogel. J. S., Southon, J. R., Caffee, M. W., Finkel, R. C., Nelson, D. E., and Davis, J. C. (1990)Acceleratormaas spectrometry in biochemicaldosimetry Relationship between low-level exposure and covalent binding of heterocyclic amine carcinogens to DNA. Proc. Natl. Acad. Sci. U.S.A. 87, 5288-5292. (29) Weisburger, J. H. (1987) Mechanisms of nutritional carcinogenesis associated with specific human cancers. ISI Atlas Sci.: PharmaC O ~ .1, 162-167. (30) Grivas, S. (1988) Synthesis of 3,8-dimethyl-Z-nitro-3H-imidazo[4,5flquinoxaline, the 2-nitro analogue of the food carcinogen MeIQx. J . Chem. Res. (S), 84. (31) Enomoto, M.,Sato,K.,Miller, E. C.,and Miller,J. A. (1968)Reactivity of the diacetyl derivative of the carcinogen4-hydroxyaminoquinoline1-oxidewith DNA, RNA, and other nucleophiles. Life Sci. 7,10251032. (32) Turesky, R. J., Lang, N. P., Butler, M. A., Teitel, C. H., and Kadlubar, F. F. (1991) Metabolic activation of carcinogenicheterocyclic aromatic amines by human liver andcolon. Carcinogenesis 12,18391845.

490 Chem. Res. Toxicol., Vol. 5, No. 4, 1992 (33) Turesky, R. J., Bracco-Hammer, I., Markovic, J., Richli, U., Kappeler, A,-M.,and Welti, D. H. (1990)The contribution of N-oxidation to the metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-flquinoxalinein rat hepatocytes. Chem. Res. Toxicol. 3, 524-535. (34) Flammang, T. J., and Kadlubar, F. F. (1986) Acetyl coenzyme Adependent metabolic activation of N-hydroxy-3,2’-dimethyl-4aminobiphenyl and several carcinogenic N-hydroxy arylamines in relation to tissue and species differences, other acetyl donors, and arylhydroxamic acid-dependent acyltransferases. Carcinogenesis 7,919-926. (35) Evans, F. E., Miller, D. W., and Beland, F. A. (1980) Sensitivity of the conformation of deoxyguanosine to binding at the C-8 position by N-acetylated and unacetylated 2-aminofluorene. Carcinogenesis 1, 955-959. (36) Slessor, K. N., and Tracey, A. S. (1973) Conformational studies on the 2’-deoxy nucleosides. Carbohydrate Res. 27,407-414. (37) Meier, C., and Boche, G. (1991) The modification of guanine nucleosides and nucleotides by the borderline arylamine carcinogens 4-methyl- and 4-methoxyaniline: chemistry and structural characterization. Carcinogenesis 12, 1633-1640. (38) Bryant, M. S.,Chiarelli, M. P.,and Lay,J. O., Jr. (1991)Development of fast atom bombardment mass spectral methods for the identification of carcinogen-nucleoside adducts. Journal of the American Society of Mass Spectrometry and Allied Topics (in press). (39) Annan, R. S., Giese, R. W., and Vouros, P. (1990) Detection and structural characterization of amino polyaromatic hydrocarbondeoxynucleosideadducts using fast atom bombardment and tandem mass spectrometry. Anal. Biochem. 191, 86-95. (40) Mitchum, R. K., Freeman, J. P., Beland, F. A., and Kadlubar, F. F. (1985) Mass spectrometric identification of DNA adducts formed by carcinogenic aromatic amines. In Mass Spectrometry in the Health and Life Sciences (Burlingame, A. L., and Castagnoli, N., Jr., Eds.) pp 574-580, Elsevier Science Publishers B. V., Amster~~. aam. Moore, P. D., and Koreeda M. (1976) Application of the change in partition coefficientwith pH to the structure determination of alkyl substituted guanosines. Biochem.Biophys. Res. Commun.73,459464. Vavrek, M. T., and Weisburger, J. H. (1990) Efficient method for the isolation by HPLC of a major metabolite of the carcinogen 2amino-3-methylimidazo[4,5-flquinoline (IQ),the 5-hydroxy derivative. Cancer Lett. 51, 79-83. Spectrum. Bentley, T. W.,and Schleyer,P. v. R. (1976)The SNZ-SN~ 1. Role of nucleophilic solvent assistance and nucleophilically solvatedion pair intermediates in solvolyseaof primary and secondary arenesulfonates. J. Am. Chem. SOC.98, 7658-7666. Beland, F. A., Beranek, D. T., Dooley, K. L., Heflich, R. H., and Kadlubar,F. F. (1983)Arylamine-DNA adducts in vitro and in vivo: Their role in bacterial mutagenesis and urinary bladder carcinogenesis. Enuiron. Health Perspect. 49, 125-134. Westra, J. G., Kriek, E., and Hittenhausen, H. (1976)Identification of the persistently bound form of the carcinogen N-acetyl-2aminofluorene to rat liver DNA in vivo. Chem.-Biol. Interact. 15, 149-164. Famulok,M.,Bosold,F.,andBoche,G.(1989)SynthesisofN-acetoxy2-aminonaphthaline, an ultimate carcinogen of the carcinogenic 2naphthylamine, and its in vitro reactions with (bio)nucleophiles. Tetrahedron Lett. 30, 321-324. Yamasaki, H., Pulkrabek, P., Grunberger, D., and Weinstein, I. B. (1977)Differential excision from DNA of the C-8 and NZguanosine adducts of N-acetyl-2-aminofluoreneby single strand-specific endonucleases. Cancer Res. 37,3756-3760. Moore, P. D., Rabkin, S. D., Osborn, A. L., King, C. M., and Straws, B. S. (1982)Effect of acetylated and deacetylated 2-aminofluorene adducts on in vitro DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 79,7166-7170. Bichara, M., and Fuchs, R. P. P. (1985)DNA binding and mutation spectra of the carcinogenN-2-aminofluorene in Escherichia coli. A correlation between conformation of the premutagenic lesion and the mutation specificity. J. Mol. Biol. 183, 341-351. 3

~

Turesky et al. (50) Pierce, J. R., Case, R., and Tang, M.-S. (1989)Recognitionand repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease. Biochemistry 28,5821-5826. (51) Davies,D. B., and Danyluk, S. S. (1974)Nuclear magnetic resonance studies of 5’-rib0 and deoyribonucleotide structures in solution. Biochemistry 13, 4417-4434. (52) Evans, F. E., and Kaplan, N. 0. (1976) 8-Alkylaminoadenylnucleotides as probes of dehydrogenase interactions with nucleotide analogsof different glycosyl conformation. J.Biol. Chem. 251,67916797. (53) Jordan, F., and Niv, H. (1977)C&aminopurine nucleosides. A welldefined steric determinant of glycosyl conformational preferences. Biochim. Biophys. Acta 476,265-271. (54) Evans, F. E., and Wright, J. M. (1980) Proton and phosphorus-31 nuclear magnetic resonance study on the stabilization of the anti conformation about the glycosyl bond of 8-alkylamino adenyl nucleotides. Biochemistry 19, 2113-2117. (55) Pohorille, A., Perahia, D., and Pullman, B. (1978)Molecular orbital studies on the conformation of 8-amino- and 8-dimethylaminoadenosine 5’-monophosphate Biochim. Biophys. Acta 517, 511516. (56) Beland, F. A., Tullis, D. L., Kadlubar, F. F., Straub, K. M., and Evans, F. E. (1980) Characterization of DNA adducts of the carcinogen N-methyl-4-aminoazobenzenein vitro and in vivo. Chem.-Biol. Interact. 31, 1-17. (57) Beranek, D. T., White, G. L., Heflich,R. H., and Beland, F. A. (1982) Aminofluorene-DNA adduct formation in Salmonella typhimurium exposed to the carcinogen N-hydroxy-2-acetylaminofluorene. h o c . Natl. Acad. Sci. U.S.A. 79, 5175-5178. (58) Lasko, D. D., Basu, A. K., Kadlubar, F. F., Evans, E. E., Lay, J. O., Jr., and Essigmann, J. M. (1987)A probe for the mutagenic activity of the carcinogen 4-aminobiphenyl: synthesis and characterization of an Ml3mplO genome containing the major carcinogen-DNA adduct at a unique site. Biochemistry 26, 3072-3081. (59) Talaska,G.,Al-Juburi,A.Z.S.S.,andKadlubar,F.F. (1991)Smoking related carcinogen-DNA adducts in biopsy samplesof human urinary bladder: Identification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl as a major adduct. Proc. Natl. Acad. Sci. U.S.A. 88,535Ck 5354. (60) Beland, F. A., Dooley, K. L., and Jackson, C. D. (1982) Persistence of DNA adducts in rat liver and kidney after multiple doses of the carcinogenN-hydroxy-2-acetylamiiofluorene. CancerRes. 42,13481354. (61) Randerath,K. E., Randerath,E.,Agrawal,H. P., Gupta, R. C.,Schurdak, M. E., and Reddy, M. V. (1985) Postlabelling methods for carcinogen-DNA adduct analysis. Environ. Health Perspect. 62, 57-65. (62) Schut, H. A. J., Snyderwine, E. G., Zu, H.-X., and Thorgeimon, S. S. (1991) Similar patterns of DNA adduct formation of 2-amino3-methylimidazo[4,5-flquinoline in the Fischer 344 rat, CDFl m o w , cynomolgus monkey and Salmonella typhimurium. Carcinogenesis 12, 931-934. (63) Turteltaub, K. W., Watkins, B. E., Vanderlan, M., and Felton, J. S. (1990) Role of metabolism on the DNA binding of MeIQx in mice and bacteria. Carcinogenesis 11, 43-49. (64) Peluso, M., Castegnaro, M., Malaveille, C. Talaska, G., Vineis, P., Kadlubar, F., and Bartach, H. (1990) 32P-Postlabelinganalysis of DNAadducted withurinarymutagensfromsmokersofblacktobacco. Carcinogenesis 11, 1307-1311. (65) Peluso, M., Castegnaro, M., Malaveille, C., Friesen, M., Garren, L., Hautefeuille, A., Vineis, P., Kadlubar, F., and Bartach, H. (1991) 32Postlabelinganalysis of urinary mutagens from smokers of black tobacco implicates 2-amino-l-methyl-6-phenylimidazo[4,5-blpyridine (PhIP) as a major DNA-damagingagent. Carcinogenesis 13, 713-7 17.

Registry No. N-OH-IQ, 77314-23-9; N-OH-8-MeIQ, 14203828-6;dG,961-07-9; dA,958-09-8; dT,50-89-5; dC,951-77-9; dGN*-IQ,142038-29-7;dG-Ca-IQ,115747-35-8;dG-N*-MeIQx, 142038-30-0; dG-C8-MeIQx, 142038-31-1; N-OAc-IQ, 115722-786;N-OAc-8-MeIQx, 142038-32-2; 0-acetyltransferase,9012-300.