Chem. Res. Toxicol. 1992,5, 293-302
293
Model Adducts of Benzo[ a Ipyrene and Nucleosides Formed from Its Radical Cation and Diol Epoxide N. V. S. RamaKrishna,t F. Gao,t N. S. Padmavathi,? E. L. Cavalieri,*pt E. G. Rogan,t R. L. Cerny,S and M. L. Gross$ Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588 Received September 9, 1991 Reference adducts formed by reaction of deoxyribonucleosideswith the ultimate carcinogenic forms of benzo[a]pyrene (BP), BP radical cation and BP diol epoxide, are essential for identifying the structures of adducts formed in biological systems. Electrochemical oxidation of BP in the presence of dG or dA produces adducts from BP radical cation. When 8 equiv of charge are consumed, four adducts are formed with d G 7-(BPS-yl)Gua, 8-(BP-6-yl)Gua,W-(BP-6-yl)dG and 3-(BP-6-yl)dG. With 2 equiv of charge, however, only 74BP-6-yl)Gua and 8-(BP-6-yl)dG (BP-648dG) are formed. Anodic oxidation of BP-6-C8dG affords 8-(BP-6-yl)Guae Anodic oxidation of BP in the presence of dA produces 7-(BP-6-yl)AdeBReaction of BP diol epoxide with dG yields 10-(guanin-7-y1)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP, whereas reaction with dA affords three adducts, 10-(adenin-7-yl)-7,8,9-trihydroxy-7,8,9,lO-tetrahydroBP and two isomers On the basis of comparative of 10-(deoxyadenosin-~-yl)-7,8,9-tr~ydroxy-7,8,9,lO-tetrahydroBP. kinetic studies among adducts of aromatic hydrocarbons and dG or G, only BP-6-C8dG easily loses the sugar moiety, providing a basis for a mechanism of hydrolysis of the glycosidic bond.
Introduction The hypothesis that polycyclic aromatic hydrocarbons (PAH)l are metabolically activated by two major mechanisms, one-electron oxidation to form PAH radical cations and monooxygenation to form bay-region diol epoxides (1-4), is based on the chemistry of PAH, metabolism studies, and the catalytic properties of cytochrome P-450. The structures of DNA adducts formed by these compounds provide insight into the mechanism(s) of metabolic activation. Quantitation of the adducts yields information on the relative contribution of one mechanism with respect to the other (5). Two DNA adducts of benzo[a]pyrene (BPI have been reported to be formed in vitro in reactions catalyzed by cytochrome P-450. One is the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at its C-10 position with the 2-amino group of dG [10-(dGWyy1)-7,8,9-trihydroxy-7,8,9,l0-tetrahydroBP, BPDE-10N2dG] (6, 7);the second reported adduct is formed by reaction of the BP radical cation with the N-7 of Gua [7-(BP-6-yl)Gua, BP-6-N7Gua],which is lost from DNA by depurination (8). These two adducts have also been identified in vivo, BPDE-10-N2dGin mouse skin (9) and BP-6-N7Gua in the urine and feces of rats (10). Within the scope of identifying all of the adducts formed by these two mechanisms of activation, we extended the synthesis and identification of adducts formed by oneelectron oxidation of BP, by conducting electrochemical oxidation of BP in the presence of nucleosides as previously reported (11). By reaction of BPDE with dG and dA,we synthesized diol epoxide adducts that potentially could be formed in biological systems. The synthesis and structure determination of the adducts allowed us to identify new BP-DNA adducts formed in biological sys-
* To whom correspondence should be addressed. University of Nebraska Medical Center.
* University of Nebraska-Lincoln.
tems (5). Furthermore, a quantitative assessment of all the stable and depurination adducts of BP was made (5). In this paper we also report the kinetics of hydrolysis of the adduct of BP bound at C-6 to the C-8 of dG, 84BP6-y1)dG (BP-6-Ct3dG). Under very mild conditions this adduct tends to eliminate its sugar. These results led us to postulate that the &(BP-&yl)Gua (BP-6-CSGua)adduct will not be found in DNA, but rather as a depurination adduct free in solution (5),analogously to the BP-6-N7Gua adduct (8).
Experimental Section (A) General Procedures. (1) UV. UV absorbance spectra were recorded with a Waters 990 photodiode array (PDA)detector during high-pressure liquid chromatography (HPLC) using CH30H/H20or CH3CN/H20 gradiente. (2) NMR Proton and homonuclear twedimenaional chemical shift correlationspectroecopy (COSY) NMR spectza were recorded on a Varian XL-300 at 299.938 MHz in DMSO-ds at 30 OC. Chemical shifta are reported relative to tetramethyleilane, which was employed either as a primary internal reference or as a secondary reference relative to DMSO at 2.50 ppm, and the J Abbreviations: BP, benzo[a]pyrene; BPDE, (+-7,t-&dihydroxy-
t-9,lO-epoxy-7,8,9,lO-tetrahydrobenzo[a]pyrene (anti); BP-6-N7Gua, 7(benzo[a]pyren-6-yl)guanine;BP-6-C8Gua, 8-(benzo[a]pyren-&yl)-
~e; guanine; BP-&C8dG,& ( b e n z o [ a ] p y r e n - & y l ) d ~ ~BP-&N3dG, 3-(benzo[a]pyren-6-yl)deoryguanosine:BPB-N dG, M-(benzo[a]pyren6-y1)deoxyguanosine;BP-6-C8G, 8-(benzo[a]pyren-6-yl)guanosine;BP6-CHz-C8dC, &(benzo[a]pyren-6-ylmethyl)deoxyguanosina;BPDE-10N2dG, 10-(deoxyguanosin-~-y1)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BPDE-lO-N7Gua, IO-(guanin-7-yl)-7,8,9-trihydroxy7,8,9,10-tetrahydrobenzo[a]pyrene;BPDE-lO-N7Ade,D(adenin-7-yl)7,8,9-trihydroxy-7,8,9,lO-tetrahydrobenzo[a]pyrene; BPDE-10-N6dA, 10-(deoxyadenosin-~-yl)-7,8,9-trihydroxy-7,8,9,lO-~trahydrobeno[a]-
pyrene; CA, collisional activation; CAD, colliiionally activated decompasition;COSY, two-dimensional chemical shift correlation spectroecopy; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; FAB, fast atom bombardmenc HPLC, high-pressure liquid chromatography; 7-MBA12-CH,-C8dG, 8-[(7-methylbenz[a]anthracen-12-yl)methyl]deoxyguanosine; MS/MS, tandem mass spectrometry; PAH, polycyclic aromatic hydrocarbon(s); PDA, photodiode array detector.
0893-228x/92/2705-0293$03.00/00 1992 American Chemical Society
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Scheme I. Electrochemical Oxidation of BP in the Presence of dG or dA
ANODIC OXIDATION E
oa
= I.lOV, PI.
-
bH
KCIO, 0.5M In DMF Molar Rslio:
1
n
BP6C8dG
BP6-NMG
B P-6- NZdG
10
BP-6-CSGua
BP-6-N7Gua
ANODIC OXIDATION Eor = l.lOV, pt,
K C I , 0.5U In DUF Molar Aallo
: I
20
BP-6-N7Ade
values are given in hertz. Typical instrument parameters were as previously reported (11). (3) FAB MS/MS. Collisionally activated decomposition (CAD) spectra were obtained by using a VG ZAB-T, a four-aedor tandem mass spectrometer of BEBE design. MS1 is a standard high-resolution ZAB of reverse geometry. MS2, also capable of high resolution, is of a prototype Mattuch-Herzog design, incorporating a standard magnet and an inhomogeneousESA. This design allows the use of a PDA for simultaneous detection of ions over a variable mass range and a single point detedor for scanning experiments. For experiments reported here, however, a single point detector was sufficient. Samples were dissolved in 5-10 pL of DMSO, and a 1-pL aliquot was placed on the probe along with the dithiothreitol/dithioeryhitolmatrix. A Cs+ion gun operated at 33 keV was used to desorb the ions from the probe. The instrument accelerating voltage was 8 kV. CAD spectra were obtained at the third field-free region (between MS1 and MS2) by addition of helium into the collision cell to attenuate the ion beam by 50%. The object slit of MS2 was c l d to the point that the slit was fuUy illuminated by the beam; thus,ita resolving power was base. Ten to fifteen scans were signal averaged for each spectrum. Data acquisition and data workup were controlled using a VAX 3100 workstation equipped with OPUS software. (4) HPLC. HPLC was conducted on a Waters 600E solvent delivery system equipped with a Waters 700 WISP autoinjector. Effluents were monitored for UV absorbance (254 nm) with a waters 990 PDA detector, and the data were collected on an APC-IV Powermate computer. Analytical runs were conducted using a YMC ODS-AQ 5-w,120-A column (6.0 x 250 mm) (YMC, Overland Park, KS) with a flow rate of 1mL/min. Preparative HPLC was conducted using a YMC ODS-AQ bpm, 120-A column (20 X 250 mm) at a flow rate of 6 mL/min. Both CH30H/H20 and CH3CN/H20 gradients were used depending upon the experiment. (5) Materials. BP was available in our laboratory and was purified by column chromatography on aluminum oxide and elution with benzene/hexane (1:l).The product was recrystallized from benzene/hexane (mp 177-178 "C). r-7,t-8-Dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydroBP (anti) (BPDE) (mp 205-207 "C) was obtained from the National Cancer Institute Chemical Carcinogen Repository, Bethesda, MD. It was more than 98% pure and was used as such. The deoxyribonucleosides dG, dA, dC, and dT and the ribonucleosides G and A were purchased from
either Aldrich (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO) and were desiccated over Pz05under vacuum at 110 OC for 48 h prior to use. Commercially available dimethylformamide (DMF) (Aldrich) was purified by refluxing over CaH2,followed by vacuum distillation just prior to use, and was stored over 4-A molecular sieves under argon. KCIOl (Aldrich) was used as obtained. BP is a hazardous chemical and is handled according to NIH guidelines (12). (B) Electrochemical Synthesis of Adducts. Electrochemical syntheses were conducted with a previously described apparatus (EG&G Princeton Applied Research, Princeton, NJ) (11). The oxidation potential used for the synthesis of BP adducts was 1.10 V, slightly less than its anodic peak potential of 1.12 V, as measured by using cyclic voltammety (Model CV27, Bioanalytical Systems, Lafayette, IN). All of the individual deoxyribonucleosides have anodic peak potentials 21-31V. Thus, during adduct synthesis at 1.10 V, none of the nucleosides was oxidized. The electrochemical reaction of BP with various nucleosides was conducted as reported earlier (II), but with a few modifications. Both the output current (i) and the total charge (8)were monitored throughout the experiment. In the procedure reported earlier, the reaction was stopped when i had decreased to ca. 1/20th of the initial value and a charge 3 times the theoretical charge expected (for a two-electron transfer) had accumulated; these two conditions were usually achieved in ca. 90 min. In the modified procedure, various experiments were conducted in which the reaction was stopped when the Q consumed corresponded to 2, 3, 4, or 7 equiv. The purification of the reaction products was also modified. After the reaction was stopped, the DMF solvent was removed under vacuum. The solid residue (KC10J was extracted four times with a solvent mixture of ethanol/chloroform/acetone (21:1), and the resulting extract was fiitered through a Whatman No. 1fluted flter paper. The combined solvent mixture was evaporated under vacuum. The residue was dissolved in 3 mL of dimethyl sulfoxide (DMSO),passed through a 0.45-pm fiiter, and analyzed by HPLC using either a CH30H/H20or CH3CN/H20gradient. purification of the adducts was conducted by preparative HPLC in CH30H/H20,followed by a CH3CN/H20 gradient. (1) BP and dG. Electrochemicalreaction of BP with dG (1:lO molar ratio) yielded BP-6-C8dG, BP-6-C8Gua, BP-6-N7Gua, 3-(BP-6-yl)dG (BP-6-N3dG),and ArZ-(BP8-yl)dG (BP-6-WdG) (Scheme I). The type and amount of adducts depended upon equivalents of charge consumed. The products were analyzed by
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 295
Model Adducts of BP a n d Nucleosides
Scheme 11. Reaction of BPDE with dG or dA
OH
+ BP tetraol
no
NH2
BPDE-1O-N7Gua
+
BPDE-10-N7Ade
HPLC by eluting the column with 30% CH30H in HzO for 5 min, followed by a linear gradient to 100% CH30H in 75 min at a flow rate of 1mL/min. The adducts were then purified by preparative HPLC and their structures determined by NMR and MS. The structures of the three adducts BP-6-C8dG, BP-6-C8Gua, and BP-6-N7Gua were confirmed by NMR and MS and are consistent with data reported earlier (11). BP-6-N3dG UV, A, (nm) 254,266,286,302,357,374,395, 408; ‘H NMR, 6 1.75 (m, 2 H, 2’-Hz), 3.45-3.53 (m, 3 H, 4’-H, 5’-Hz), 3.80 (m, 1 H, 3’-H), 5.42 (m, 1 H, 1’-H), 6.80 [bs, 2 H, ~ - N H ~ ( G w )7.33 ] , (d, 1H, 5-H), 7.53 (d, 1H, 7-H), 7.81 (t, 1 H, 8-H), 7.92 (t, 1H, 9-H), 8.05-8.11 (m, 2 H, 4-H, 2-H), 8.26 (d, 1 H, 3-H), 8.30 [s, 1H, 8-H(Gua)], 8.45 (d, 1H, 1-H), 8.57 (d, 1H, 12-H),9.31-9.40 (m, 2 H, 10-H, 11-H);MS, (M + H)+ C30HaN504 calcd 518.1828, found 518.1817. BP-6-N2dG: UV, A,, (nm) 254,266,286,302,357,374,395, 408;‘H NMR,6 1.65-1.85 (m, 2 H, 2’-H& 3.50-3.62 (m, 2 H, 5’-Hz), 3.65-3.95 (m, 2 H, 3’-H, 4’-H), 5.39 (t, 1H, 1’-H), 6.65 [bs, 1 H, 2-NH(Gua)], 7.09 [bs, 1H, 1-NH(Gua)],7.38 (d, 1H, 5-H), 7.56 (d, 1H, 7-H), 7.85 (t, 1H, 8-H), 7.96 (t, 1H, 9-H), 8.08-8.13 (m, 2 H, 4-H, 2-H), 8.30 (d, 1 H, 3-H), 8.43 [s, 1 H, 8-H(Gua)], 8.50 (d, 1H, 1-H),8.63 (d, 1H, 12-H),9.35-9.45 (m, 2 H, 10-H, 11-H); MS, (M + H)+ C3,,HZ4N5O4calcd 518.1828, found 518.1836. (2) B P and G. The reaction between BP and G was also conducted at a molar ratio of l:lO, with consumption of 12.4 C of charge (3.2 equiv). It afforded two products, BP-6-C8G (20%) and BP-&N7Gua (3%),and 78% of u n r d BP. The structures of these two adducts were confiied by N M R and MS. The data were consistent with those reported earlier (11). (3) B P and dA. The reaction procedure for the coupling of BP‘ with dA was the same as that of BP’ and dG. When the reaction was conducted at a molar ratio of BPdA equal to l : l O , no appreciable amounts of adducts were formed, even with the consumption of 5 equiv of charge. The reaction was successfully carried out, however, at a molar ratio of BP to dA of 1:20, and the charge consumed was 5.8 equiv (22.2 C). This afforded only one adduct, 74BP-6-yl)Ade (BP-6-N7Ade) (5%) (Scheme I). BP-6-N7Ade: UV, A, (nm) 256,267,288,302,357,377,396, 407; ‘H NMR, 6 5.70 [bs, 2 H, 6-NHz(Ade)],7.32 (d, 1 H, 5-H), 7.44 (d, 1H,7-H), 7.85 (t, 1H, BH),7.99 (t, 1H, 9-H), 8.14-8.18 (m, 2 H, 4-H, 2-H), 8.33 (d, 1 H, 3-H), 8.39 [a, 1 H, 2-H(Ade)], 8.54 (d, 1 H, 1-H), 8.65 (d, 1 H, 12-H), 8.68 [s, 1 H, 8-H(Ade)], calcd 9.43 (m, 2 H, 10-H, 11-H); MS, (M + H)+ CZ5Hl8N5O4 386.1406, found 386.1411. (C)Reaction of B P D E with either dG o r dA. (1) BPDE and dG. A mixture of 5 mg (0.016 mmol) of BPDE and 50 mg (0.187 “01) of dG was dissolved in 5 mL of dry DMF and stirred at 100 “C for 2 h under a stream of dry argon. The solution was then cooled to room temperature, 5 mL of DMSO was added, and +
+
BP tetraol
BPDE-IO-NBdA
the solution was fitered through a 0.45-~mfiter. HPLC analysis of an aliquot of the solution using a solvent system of 20% CH3CN in HzO for 5 min, followed by a linear gradient to 100% CH3CN in 80 min, showed the complete disappearanceof BPDE and the presence of two products. These products were purified by preparative HPLC in CH3CN/Hz0(gradient as above with a flow rate of 6 mL/min), followed by a CH30H/Hz0 gradient (as described for BP-dG adducts with a flow rate of 6 mL/min). Their structureswere established by N M R and fast atom bombardment (FAB) MS. One product was BP tetraol, and the other was the adduct 10-(Gua-7-y1)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP (BPDE-lO-N7Gua) (64% yield) (Scheme 11). B P D E ~ O - N ~ G UUV, : & (nm) 246,278,330,346; ‘H NMR, 6 3.76 (dd, 1 H, 8-H, J 8.6 Hz), 4.36 (bs, 1 H, 9-H), 4.86 (bs, 1 H, OH), 4.95 (d, 1 H, 7-H, J,,8 = 8.6 Hz), 5.63 (bs, 1H, OH), 6.17 (bs,1H, OH), 6.30 [bs,2 H, ~-NHZ(GW)],6.80 (d, 1H, 10-H, J = 5.7 Hz), 7.83 (d, 1 H, 11-H, J = 9.2 Hz), 7.99 (t, 1H, 2-H), 8.06-8.33 [m, 6 H, 1-H, 3-H, 4-H, 5-H, 12-H, &H(Gua)], 8.57 (s, 1 H, 6-H); MS, (M H)+ Cz5HZoN504 calcd 454.1515, found 454.1527. (2) BPDE and dA. The above reaction conditions were also used for the reaction of BPDE with dA. HPLC analysis showed the disappearance of BPDE and the presence of four products (Scheme 11). They were purified by preparative HPLC and identified as 10-(Ade-7-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP (BPDE-lO-N7Ade) (36%), two isomers of lO-(dA-@-yl)-7,8,9trihydroxy-7,8,9,10-tetrahydroBP(BPDE10-PdA) (fmt eluting, 25%, and second eluting, 28%), and BP tetraol (10%). BPDE-lO-N7Ade: UV, ,A (nm) 246,279,318,331,346; ‘H NMR, 6 3.78 (dd, 1 H, 8-H), 4.52 (dd, 1H, 9-H), 4.91 (bs, 1 H, OH), 5.01 (d, 1H, 7-H, J = 7.0 Hz), 5.90 (bs, 1H, OH), 6.40 (bs, 1 H, OH), 6.75 (d, 1H, 10-H), 7.24 [bs, 2 H, 6-NHz(Ade)],7.72 (d, 1H, 11-H, J = 9.0 Hz),7.98-8.41 [m, 8 H, aromatic, &H(Ade) and 2-H(Ade)],8.56 (a, 1H, 6-H); MS, (M + H)+ C a & i s 0 3 d c d 438.1566, found 438.1556. BPDE-10-WdA isomers: UV, ,A, (nm) 245,280,316,331, 346, ‘H NMR, 6 2.34 (m, 1H, 2’-H), 2.74 (m, 1H, 2’-H), 3.57-3.68 (m, 2 H, 3’-H, 4’-H), 3.85 (dd, 1H, 8-H), 3.90 (bs, 1H, OH), 4.19 (m, 2 H, 5’-Hz), 4.39 (dd, 1 H, 9-H), 4.98-5.52 (m, 2 H, 3’-OH, 5’-OH), 5.39 (d, 1H, 7 H, J = 7.4 Hz), 5.78 (bs,1H, 7-OH), 6.06 (bs, 1H, OH), 6.27 (t, 1 H, l‘-H), 6.55 (bs, 1 H, lO-H), 7.26 [a, 1H, 2-H(Ade)],7.89-8.28 (m, 6 H, 1-H, 2-H, 3-H, 4 H , 5 H , 12-H), 8.39 [s, 1 H, 8-H(Ade)], 8.52 (a, 1 H, 6-H), 9.13 [m, 2 H, 11-H, J = 10.2 Hz, 6-NH(Ade)];NMR (CD30D),downfield region, 5.19 (d, 1 H, 7-H), 6.31 (t, 1H, 1’-H), 6.80 (d, 1 H, 10-H), 7.81-8.10 [m, 7 H, 1-H, 2-H, 3-H, 4-H, 5-H, 12-H, 2-H(Ade)],8.21 [s, 1H, 8-H(Ade)],8.40-8.50 (m, 2 H, 11-H, 6-H); MS, (M + H)+ CWH a 5 0 6d c d 554.2040, found (firsteluting) 554.2049 and (second eluting) 554.2043.
+
296 Chem. Res. Toxicol., Vol. 5, No. 2, 1992
RamaKrishna et al.
(D) Kinetics of Hydrolysis of the Glycosidic Bond of Selected Adducts. The adducts chosen for this study were BP-6-C8dG, 8-(BP-6-yl)G (BP-&CSG), 8-(benzo[a]pyren-6-ylmethy1)dG (BP-6-CH2-C8dG),and 8-[(7-methylbenz[a]anthracen-12-yl)methyl]dG (7-MBA-12-CH2-C8dG),and the parent compound dG was studied under similar conditions. The synthesis of BP-6-CH2-C8dG will be published elsewhere, and the 7MBA-12-CH2-C8dGwas prepared as previously reported (13). The kinetics of hydrolysis of these selected adducts were determined at several pHs and temperatures. For pH 3,4, and 5,O.l M citric acid/O.l M sodium citrate buffers were used; for pH 7.5 and 9.0,0.2 M NaH2P04/0.2M Na2HP04buffers were used; and for pH 11, a 0.05 M Na2HP04/0.1M NaOH buffer was used. The general procedure involved 5-10 pg of the adduct in 25 pL of DMSO added to 400 pL of the buffer solution and incubated for 0.5, 4,or 12 h at 37, 55, 70, or 90 "C. At the end of the incubation, the reaction mixture was cooled in ice and the produds were extracted with CHC13(1mL) four times. The pooled CHC13 extracts were evaporated to dryness under argon, and the residue was dissolved in 50 pL of DMSO/CH30H (1:l)and analyzed by HPLC. Either a CH30H/H20or an CH3CN/H20 gradient was used for analysis of the hydrolysis products of the adducts and dG. The products were analyzed by using a Whatman Partisil lOSCX column (4.6 X 250 mm) with 10 mM NaH2PO4, pH 7.02, at a flow rate of 0.7 mL/min.
Results and Discussion (A) Synthesis and Structure Determination of B P Adducts Formed with Nucleosides by Electrochemical Oxidation, Anodic oxidation of BP and dG yielded four primary adducts, BP-6-N7Gua, BP-6-C8dG, BP-6N3dG, and BP-6-N2dG, and one secondary adduct, BP6-C8Gua (Scheme I). When the reaction consumed 2 electron equiv, anodic oxidation of BP in the presence of dG yielded two adducts, BP-6-C8dG (7%) and BP-6N7Gua (20%), with 68% unreacted BP. Upon consumption of 2.2 electron equiv, the reaction yielded three products, BP-6-C8dG (6701, BP-6-C8Gua (6%), and BP6-N7Gua (31%), with 41% unreacted BP. With 8 electron equiv consumed, the reaction produced four adducts, BP-6-CSGw (14%), BP-6-N2dG (6%), BP-6-N3dG (3%), and BP-6-N7Gua (46%), and 10% unreacted BP. The analytical chromatograms of the products obtained under these three different reaction conditions are shown in Figure 1A-C. In going from 2 (Figure 1A) to 2.2 electron equiv (Figure lB), a new product, BP-6-C8Gua, is formed, suggesting that this secondary product arises from oxidation of BP-6-CSdG. In fact, upon consumption of 8 equiv (Figure IC), BP-6-C8dG is totally oxidized to BP-6-C8Gua. To demonstrate that this oxidation occurs, purified BP-6-C8dG was electrochemically oxidized under the conditions in which the adducts were prepared. After 30 min of reaction, 55% of the BP-6-C8dG had been converted to BP-6-C8Gua. Upon consumption of 3.2 equiv of charge, oxidation of BP in the presence of the ribonucleoside G afforded BP6-C8G (20%) and BP-6-N7Gua (3%). These data are similar to those previously reported (11)and show that the preponderant adduct is BP-6-C8G, not the BP-6-N7Gua obtained from anodic oxidation of BP in the presence of dG (Figure 1A-C). Furthermore, BP-6-C8G is not further electrochemically oxidized to BP-6-C8Gua. No appreciable amounts of adducts were formed by anodic oxidation of BP in the presence of dA at a molar ratio of l:lO, respectively. When a molar ratio of BP:dA equal to 1:20 and 5-8 electron equiv were used, the reaction produced BP-6-N7Ade ( 5 % yield) (Scheme I). No products were detected by anodic oxidation of BP in the presence of dC, dT, or A at a molar ratio of BP to nucleoside of 1:20.
t
B
A
t
f
i
P
!/ ' I1 I I1 I/
i i 4 1 10
II I
t
I1 I l l 1 1 ill1
1 1
1
E3 '
2 '0
"
30'
'
40
o '- L 5
Time, min
Figure 1. HPLC separation of the products obtained by (A-C) electrochemical oxidation of BP in the presence of dG and (D and E) reaction of BPDE with dG or dA.
The structure elucidations of BP-6-C8dG, BP-6-C8G, BP-6-C8Gua, and BP-6-N7Gua were reported previously (11). The evidence for the structure determinations of BP-6-N2dG,BP-6-N3dG, and BP-6-N7Ade was obtained by a combination of UV, NMR, and FAB MS/MS. The bathochromic shift of 8-10 nm above 300 nm in the UV spectra of all of these adducts compared to BP is characteristic of substitution of BP at C-6 (14). (1) BP-6-N3dG. The NMR spectrum of BP-6-N3dG (Figure 2A) shows the absence of the singlet proton resonance of the BP moiety at C-6 (8.72 ppm) and the characteristic shift upfield of the 5-H and 7-H compared to BP (II), indicating that substitution is at C-6. The remaining protons of the BP moiety are assigned by comparing the chemical shifta with those of the parent compound BP and by using COSY. COSY data for the aliphatic region allow us to assign unequivocally the 1'-H, 2'-H2, and 3'-H protons of the dG moiety. The remaining protons, 4'-H and 5'-H2, are thought to be under the broad signal of H 2 0 (figure not shown). With D20 exchange (figure not shown) the H20 signal moves downfield and these proton resonances,4'-H and 5'-H2, are clearly assigned. The presence of the sharp singlet of the proton at C-8 of Gua (8.30 ppm) indicates that Gua is not substituted at C-8. In addition, the two protons that give a resonance at 6.80 ppm (exchangeable with D20) demonstrate that there is not substitution at the amino group. Substitution at the N-7 of Gua is excluded by the presence of the sugar moiety in the adduct. Thus, it appears that adduct formation with the Gua moiety occurs a t the N-3 position. The presence of deoxyribose provides further evidence for an N3 adduct, as substitution at this position cannot destabilize the glycosidic bond. The chemical shifts of the protons of the BP moiety in this adduct are similar to those of the BP-6-
Chem. Res. Toxicol., Vol. 5, No. 2,1992 297
Model Adducts of BP and Nucleosides A
a
11H,lOH
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Figure 2. NMR spectra of (A) BP-6-N3dG, (B) BP-6-N2dG, and (C)BP-6-N7Ade.
N7Gua adduct (11). The complexity of the signals corresponding to the 5-H and 7-H protons is characteristic of the presence of two diastereomersgenerated by hindered rotation of dG at the C-6 bond of BP. This was previously observed for BP-6-C8dG and BP-6-C8G (11). The sharp doublets of the 5-H and 7-H signals (Figure 2C) suggest that for BP-6-N7Ade rotation is free. Similar results were obtained in the NMR spectrum of BP-6-N7Gua (11). FAB MS of BP-6-N3dG produces an (M + H)+ ion of m/z 518 and an abundant fragment ion of m/z 402. This fragment ion is formed by hydrogen transfer from the sugar to the base with subsequent elimination of C5H8O3. Upon collisional activation (CA), the (M + H)+ ion predominantly fragments to yield an ion of m / z 402. CA of the source-produced m / z 402 ion by using a three-sector instrument (11) gives a spectrum of product ions (not shown) that is similar to that of BP-6-N7Gua (Figure 3A). An abundant and unresolved ion cluster is found in the region of m/z 252 and a less abundant cluster is seen at m/z 277 [apparently (BP - NC)'+]. The abundance ratio of the ions at m/z 252 and 277 is indicative of substitution at nitrogen and not a t carbon. We had insufficient material to repeat the experiment with the four-sedor tandem instrument.
(2) BP-6-N2dG. The NMR spectrum of BP-6-N2dG (Figure 2B) is consistent with the assigned structure. The absence of the sharp singlet at 8.72 ppm indicates that substitution is at the C-6 position of the BP moiety. This observation is further substantiated by the shielding effect of 5-H and 7-H due to the peri effect of Gua substituted at (2-6. The remaining protons of BP are assigned by comparing the chemical shifts with those of the parent compound BP and by using COSY. The presence of the sharp singlet of the proton at C-8 of Gua (8.43 ppm) indicates that dG was not substituted at C-8. The chemical shifts corresponding to the 5-H and 7-H protons are a combination of two doublets of the diastereomers generated by hindered rotation of dG at the c-6 bond of BP. This was observed for BP-6-N3dG, as well as for BP-6-CEklG and BP-6-C8G (11). In addition, the presence of the deoxyribose proton resonances suggests that subatitution did not occur at the N-7 position of Gua. The proton with resonance at 6.65 ppm, which is exchangeable with D20, integrates to only one, suggesting that substitution is at the N2 position of dG. The presence of deoxyribose proton resonances provides further evidence for an N2dG adduct, because substitution at this position cannot destabilize the glycosidic
~
~
1
1
RamaKrishna et a1.
298 Chem. Res. Toxicol., Vol. 5,No. 2, 1992 252
277
303 315
343
360
I
sponds to the resonance of the two protons of the 6-NH2 of Ade, demonstrating that no substitution occurred at the amino group. Furthermore, the absence of proton resonances in the aliphatic region indicates that this molecule does not contain the deoxyribose moiety. This is consistent with substitution of Ade at N-7, which destabilizes the glycosidic bond, resulting in elimination of the sugar moiety. The absence of the characteristic singlet of the proton at C-6 of BP at 8.72 ppm suggests that the covalent bond is between the N-7 of Ade and C-6 of BP. This conclusion is further substantiated by the shielding effect of 5-H and 7-H due to substitution of Ade at C-6 of the BP moiety. The remaining aromatic proton resonances are assigned by using COSY and by comparing their chemical shifts with those of the parent compound BP. The CAD spectrum of the source-produced m/z 386 ion from BP-6-N7Ade is expected to resemble that of the corresponding m / z 402 ion of BP-6-N7Gua (Figure 3A). Because of differences in the six-membered ring of Ade compared to Gua, the spectrum (Figure 3D) is different and more informative than those of the Gua adducts. The expected fragment ions of m / z 277, (BP-NC)'+, and of m/z 252, BP'+, are seen, as is the (M + H - NH,) of m/z 369. In comparison to the spectra of compounds with adduction to N-7 of Gua, however, there are notable differences: CZ$Il,,' at m/z 250, C19Hg+at m/z 237, ClgHll+ at m/z 239, and C18H8'+ at m/z 224. The unique series of high-mam ions of m/z 369,357,342, 330,315,303, and 228 are due to parallel fragmentations of the Ade six-membered ring to eliminate increasingly larger portions of the ring: NH3, CH,NH, HNCHNHz, CHZNCHNH, NHCHNCHNHZ, CH,NCHNCHNH, and HNCHNCHNCHNH,, respectively. If this chemistry persists for other adducts of Ade, shifts in the pattern will reveal adduction at sites in the six-membered ring. (B) Synthesis and Structure Determination of BPDE Adducts with dG and dA. Reactions of BPDE and dG afforded the adduct BPDE-lO-N7Gua and BP tetraols (Figure 1D). Reaction between BPDE and dA produced BPDE-lO-N7Ade (eluting at 30.5 min in Figure lE), two isomers of BPDE-10-N6Ade (eluting at 36.2 and 37.8 min), and BP tetraols (35.6 min). The structure determination of the adducts was made by using a combination of UV, NMR, and FAB MS/MS. Initial evidence of adduction was provided by the characteristic 8-10-nm bathochromic shifta of the absorptions above 300 nm for all four of the adducts compared to BP tetraol. A typical UV absorption spectrum of the adduct BPDE-lO-N7Gua vs BP tetraol is illustrated in Figure 4. ( 1 ) BPDE-lO-N7Gua. The aromatic protons of BPDE-lO-N7Gua (Figure 5A) exhibited resonance at chemical shifts between 8.06 and 8.33 ppm, with the exception of 2-H, which appeared as a triplet at 7.99 ppm. The doublet at 7.83 ppm (J = 9.2 Hz) has been tentatively assigned as 11-H. The upfield shift of the 11-H resonance by 0.5 ppm compared to BP tetraol (not shown) and BPDE-10-N6dA (see below and ref 15) suggests that substitution of the N7Gua moiety is equatorially at (2-10. The sharp singlet at 8.57 ppm is assigned as 6-H. The proton giving the broad singlet at 6.30 ppm, which is exchangeable with D20,is assigned as the 2-NH, of Gua, suggesting that the NH, was not substituted. After D20 exchange, the aromatic region of the spectrum (inset in Figure 5A) became less complex, and the sharp singlet at 8.11 ppm is assigned as the C-8 of Gua. The absence of the proton resonances of the sugar moiety (Figure 5A) indicates that this adduct does not contain the deoxyribose moiety. Thus, it is reasonable to postulate that the substitution +
Figure 3. Partial CAD spectra of source-producedBP-base ions: (A) BP-6-N7Gua ( m / z 402), (B) BP-6-C8Gua ( m / z 402), (C) BP-6-N2dG (m/z 402), and (D) BP-6-N7Ade ( m / z 386).
bond. The assignment of all of the deoxyribose proton resonances is achieved by comparing the chemical shifts with those of the parent compond dG (11) and by using COSY. FAB MS of BP-N2dGgives an (M + H)+ ion of m/z 518 and the ion of m/z 402 that results from elimination of the sugar moiety from (M + H)+. The CA spectrum of the m/z 518 ion is dominated by the fragment of m/z 402. Upon CA (Figure 3C) the source-produced fragment ions of m/z 402 further decompose to m/z 385 (M + H NH3)+,m/z 277 (BP - NC)'+, m/z 252 B P + , and m/z 239 (ClgHll)+. The CA spectrum closely resembles but is not identical to that of m/z 402 ions produced from BP-6N7Gua (Figure 3A). The smaller abundance of the m/z 277 ions with respect to that of the m / z 252 is characteristic of nitrogen rather than carbon substitution as shown by BP-6-C8Gua (Figure 3B) in which m/z 277 is more abundant than m/z 252. For this compound, the formation of (BP - NC)' is viewed as excision of the ring carbon of the base moiety in a manner similar to the loss of CO from the phenol molecular ion. The appearance of the m / z 268 ion, albeit small in abundance, is significant because it is likely to be BP NH3+,a product not expected by substitution at other nitrogen atoms of the base. (3) BP-6-N7Ade. The NMR spectrum of BP-6-N7Ade (Figure 2C) contains the two sharp singlets at 8.39 and 8.68 ppm of the C-2 and C-8 protons of Ade, indicating that these positions are not substituted. The broad singlet at 5.70 ppm, which disappears after D20 exchange, corre+
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 299
Model Adducts of BP and Nucleosides
TT-r--r-, 200
250
. I .r
1-
300
r
7
r
-
T
w
-
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T
-
v 400
Wavelength (nm)
Figure 4. UV spectra of BPDE-lO-N7Gua and BP tetraols recorded in CH30H/H20by a photodiode array detector during HPLC separation.
took place at the N-7 of Gua. All other protons are assigned by using COSY and D20 exchange experiments. The FAB MS of BPDE-lO-N7Gua shows an (M H)+ ion of m/z 454. Upon CA, the protonated species produces several characteristic ions (Figures 6a). The ions of m / z 436 and 418 arise from successive loss of water molecules from the (M + H)+ ion. The ion of m/z 303 arises by hydrogen rearrangement, followed by loss of neutral Gua from the (M H)+ ion, and is formally (BP triol)+. The ion of m/z 152 is protonated Gua. A series of ions of m/z 285, 257, and 239 results from losses of H20, CO, and a second H20 from the triol portion of the molecule. This fragmentation is characteristic of BP triol. These data, together with those from NMR, are in accord with the assigned structure, BPDE-lO-N7Gua, for this adduct. (2) BPDE-lO-N7Ade.The absence of the sugar proton resonances in the NMR spectrum (Figure 5B) indicates that this molecule does not have the deoxyribose moiety. The broad singlet at 7.24 ppm, exchangeable with D20, corresponds to the NH2 protons of Ade, demonstrating that no substitution exists at the amino group. The sharp singlet at 8.26 ppm is assigned as the 2-H of Ade by comparing its chemical shift with that of the corresponding proton in dA (not shown). The 8-H proton of Ade has been tentatively identified as the sharp singlet at 8.2 ppm. Because the adduct does not contain the deoxyribose moiety, it is logical to postulate that the C-10 position of the BP triol is linked to the N-7 of Ade. Furthermore, the sharp singlet at 8.56 ppm is assigned as 6-H of the BP triol moiety. The doublet a t 7.72 ppm is assigned as 11-H of BP triol. The upfield shift of this proton is similar to that for the 11-H proton of BPDE-lO-N7Gua, suggesting that the N7-Ade substitution at C-10 of the BP triol is presumably equatorial. All other aromatic proton resonances appear close together, making it difficult to assign them independently, even with COSY. The aliphatic protons in the upfield area are assigned by COSY and D20 exchange experiments. The FAB mass spectrum shows the desorption of the (M + H)+ species of m / z 438. The CAD spectrum of (M + H)+ is similar to that of the Gua-containing species. The only differences are the successive losses of H20molecules to produce fragments at different m/z values: 420 and 402. Elimination of the base (with H-transfer) gives the expected mlz 303 ion. The (M + H)+ of the base at m/z 136 is indicative of the presence of Ade. The ions in the m/z range 200-310 are nearly identical to those in the spectrum
+
+
of the Gua adduct and should be regarded as a signature for the BP triol moiety. These data, along with those from NMR (Figure 5B), are consistent with the proposed structure. (3) BPDE-10-N'dA Isomers. The NMR spectrum (Figure 5C) of the isomer of BPDE-10-N'dA eluting at 37.8 min (Figure 1E) was compared to that of dA (figure not shown). All of the proton resonances corresponding to the deoxyribose moiety are assigned by using COSY and by comparing their chemical shifts with those of the parent compound dA. The two sharp singlets at 8.39 and 8.52 ppm are assigned as C-8 of Ade and 6-H of the BP triol moiety, respectively. The sharp singlet signal at 7.26 ppm is assigned as the C-2 proton of the dA moiety; this proton undergoes a 1 ppm upfield shift compared to the corresponding proton of dA (not shown). Because of the ring current effect of the PAH moiety in PAH-nucleoside adducts dissolved in DMSO, protons of the base sometimes are shifted upfield or downfield. This phenomenon has been observed for the C-2 of Ade in WdA and WA adducts of dibenzo[a,l]pyrene2 and for the C-8 proton in 12MBA-7-CH2-N7Gua (13). When the NMR spectrum of BPDE-10-N'dA is run in CD30D, the chemical shifts of the aromatic protons are identical to those of cis-BPDE10-N'A, with no upfield shift of the C-2 proton (15). The absence of the broad singlet at 7.40 ppm corresponding to NH2and the appearance of a D20-exchangeable proton at 9.13 ppm suggest that dA is substituted at the N6 position. Because of adduction at the (2-10 position of the BP triol moiety through the N6 of dA, 11-H is downfield at 9.13 ppm. All other aromatic proton resonances appear together, and it is difficult to assign them independently. The NMR spectrum of the isomer eluting at 36.2 min (Figure 1E) is identical to that described above. The (M + H)+ ion of m/z 554 fragments upon CA to produce several ions that are diagnostic of structure (see Figure 6C for the spectrum of the isomer eluting a t 37.8 min). The ion of m/z 438 results from elimination (with H-transfer) of the deoxyribose moiety. As for the other BPDE adducts, the ions in the range of m/z 200-310 are characteristic of the BP triol moiety. The ion of m/z 252, not observed from the other adducts, results from elimination (with H-transfer) of neutral BPDE to give protonated dA. This fragmentation ion indicates that the covalent bond between BPDE and dA occurs at the N6 of dA. The ion of m/z 136 is the expected protonated Ade. These data along with the NMR spectra in DMSO-d6 and CD30D indicate that the two diastereomers are cisBPDE-10-N'dA. (C) Kinetics of Hydrolysis of the Glycosidic Bond of Selected Adducts. The loss of the deoxyribose moiety of BP-6-C8dG by both anodic oxidation and hydrolysis under mild conditions led us to compare the stability of the glycosidic bond of this adduct to those of other selected adducts, including BP-6-CH2-C8dG, 7-MBA-12-CH2CSdG, and BP-6-CBG (Chart I). BP-6-CBG was chosen because the bond between BP and the nucleoside is identical to that in BP-6-C8dG, and the only structural difference is the presence of the 2'-OH in the sugar moiety of BP-6-C8G. BP-6-CH2-CSdG and 7-MBA-12-CH&SdG were chosen because the C-8 of the Gua moiety in these adducts is linked to the PAH via a methylene group, preventing conjugation between the PAH and Gua. If the instability of the BP-6-CBdG glycosidic bond is the result of the conjugation of BP with the Gua moiety, a similar instability of BP-6-CBG would be anticipated, whereas Unpublished results.
300 Chem. Res. Toricol., Vol. 5, No. 2, 1992 I
A
RamaKrishna et al. OH
II
B
CY
Table I. Kinetics of Hydrolysis of Selected Nucleoside Adducts hydrolysis of the glycosidic bond, % time, temp, BP-6- BP-6- 7-MBA-12BP-6pH h "C C8dG C8G CHyC8dG CH&8dG dG 0 0 0 0 3.0 0.5 37 100 0 4 37 0 0 0 0 12 37 0 0 0 4 55 0 0 21 12 55 7 16 0 4.0 0.5 37 79 0 0 0 0 1 37 100 0 0 4 55 0 19 12 55 4 8 0 5.0 0.5 37 70 0 0 0 0 7.5 0.5 37 27 0 0 0 0 31 0 0.5 55 0 41 0 0.5 70 0 48 0 0.5 90 0 42 0 9.0 0.5 37 0 0 53 0 11.0 0.5 37 0 0 0
BP-6-CHZ-C8dG and 7-MBA-12-CHz-C8dGWould display a stability similar to that of dG itself. The hydrolyses of these adducts were conducted at several pHs and temperatures (Table I). BP-6-C8dG hydrolyzed completely at pH 3 in 0.5 h at 37 "C and mostly at pH 4 and 5 at the same temperature. At pH 7.5, BP6-C8dG hydrolyzed to the extent of 27% in 0.5 h; however, this percentage did not increase dramatically for the same time period when the temperature was raised to 55,70, or 90 "C. This adduct also underwent partial elimination of the sugar moiety at pH 9 (42%) and pH 11 (53%). The BP-6-C8G adduct was very stable in both acidic and basic conditions, undergoing only marginal hydrolysis after 12 h at pH 3 or 4. The same results were obtained with 7-MBA-12-CHz-C8dG. In the adducta containing a bond between C-6 of BP and a nucleic acid base, the BP and base moieties are oriented in space perpendicular to one another (11, 14). By inspecting CPK space-filling models of BP-6-C8dG and
Chem. Res. Toxicol., Vol. 5, No. 2, 1992 301
Model Adducts of BP and Nucleosides
the N-7 position generates a carbenium ion at C-8 that is stabilized by the BP moiety, thereby favoring the loss of deoxyribose by &elimination. Under basic conditions, the formation of a carbanion at the C'-2 of BP-6-C8dG (but obviously not BP-6-C8G) obtained by removal of the acidic proton would presumably be stabilized by the BP moiety, thereby favoring again the loss of deoxyribose by 8-elimination.
Conclusions Various adducts formed by the ultimate carcinogenic intermediates BP radical cation and BPDE with dA and dG have been synthesized and their structures elucidated. Of these adducts, BP-6-N7Gua, BP-6-C8Gua, BP-6N7Ade, and BPDE-lO-N7Ade have been identified in biological systems ( 5 , 8 ) . It is possible that some of the other synthesized adducts containing the deoxyribose moiety will be identified as minor stable BP-DNA adducts (5). A study of the relative instability of BP-6-C8dG under oxidizing and mild hydrolytic conditions has been extremely useful in understanding how BP-6-C8Gua is formed electrochemically and directing us to identify BP-6-CSGua as a depurination adduct formed biologically
1' > I
303
1
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,
,
,
,
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,
450
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,
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Figure 6. CAD spectra of BPDE adducts: (A) BPDE-lO-N7Gua ( m / z 454), (B) BPDE-lO-N7Ade ( m / z 438), and (C) BPDE-10N6dA (m/z 554). Chart I. Structures of Selected Nucleoside Adducts and dG
(5).
Acknowledgment. This work was supported by U.S. Public Health Service Granta R O 1 - C A M , RO1-CA25176, and Pol-CA49210 and Institutional Core Grant CA36727 from the National Cancer Institute. The four-sector tandem mass spectrometer was purchased with funds awarded by the former NSF regional instrumentation program (Grant CHE8620177) and the University of NebraskaLincoln. We especially thank Dr. Momcilo Miljkovic, Milton S. Hershey Medical Center, Pennsylvania State University, for valuable suggestions. References
on
6H
H
BP6-C8dG
AH
BP-6-CBG
BP-B-CH&BdG 0
dG
BP-6-C8G, one can see that a large partial conjugation of the aromatic ring system of BP and the Gua moiety can be better achieved in BP-6-C8dG than in BP-6-C8G. In BP-6-C8dG the 2'-OH prevents the BP from assuming an orientation more parallel to the plane of the purine ring. Under neutral and acidic conditions, the protonation of
(1) Cavalieri, E. L., and Rogan, E. G. (1985) One-electron oxidation and two-electron oxidation in aromatic hydrocarbon carcinogenesis. In Free Radicals in Biology (Pryor, W. A., Ed.) Vol. VI, pp 323-369, Academic Press, New York. (2) Cavalieri, E., and Rogan, E. (1985) Role of radical cations in aromatic hydrocarbon carcinogenesis. Enuiron. Health Perspect. 64,69-84. (3) Sims, P., and Grover, P. L. (1981) Involvement of dihydrodiols and diol epoxides in the metabolic activation of polycyclic hydrocarbons other than benzo[a]pyrene. In Polycyclic Hydrocarbons and Cancer (Gelboin, H. V., and Ts'o, P. 0. P., Eds.) pp 117-181, Academic Press, New York. (4) Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 4875-4917. (5) Devanesan, P. D., RamaKrishna, N. V. S., Todorovic,R., Rogan, E. G., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small,G. J. (1992) Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro. Chem. Res. Toxicol. (following paper in this issue). (6) Sims, P., Grover, P. L., Swaisland, A., Pal, K., and Hewer, A. (1974) Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature (London) 252, 326-328. (7) Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Beland, F. A., Harvey, R. G., Kasai, H., Miura, I., and Nakanishi, K. (1976) Benzo(a)pyrene-nucleic acid derivative found in vivo: Structure of a benzo(a)pyrenetetrahydrodiol epoxide-guanosine adduct. J. Am. Chem. SOC.98, 5714-5715. (8) Cavalieri, E. L., Rogan, E. G., Devanesan, P. D., Cremonesi, P., Cerny, R. L., Gross, M. L., and Bodell, W. J. (1990) Binding of benzo[a]pyrene to DNA by cytochrome P-450-catalyzed oneelectron oxidation in rat liver microsomes and nuclei. Biochemistry 29, 4820-4827. (9) Koreeda, M., Moore, P. D., Wislocki, P. G., Levin, W., Conney, A. H., Yagi, H., and Jerina, D. M. (1978) Binding of benzo[a]-
Chem. Res. Toxicol. 1992,5,302-309
302
pyrene 7,&diol-9,10-epoxides to DNA, RNA and protein of mouse skin occurs with high stereoselectivity. Science (Washington, D.C.)199, 778-781. (IO) Rogan, E. G., RamaKrishna, N. V. S., Higginbotham, S., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1990) Identification and quantitation of 7-(benzo[a]pyren-6-yl)guanine in the urine and feces of rata treated with benzo[a]pyrene. Chem. Res. Toxicol. 3, 441-444. (11) Rogan, E., Cavalieri, E., Tibbels, S., Cremonesi, P., Warner, C., Nagel, D., Tomer, K., Cerny, R., and Gross, M. (1988) Synthesis and identification of benzo[a]pyrene-guanine nucleoside adducta formed by electrochemical oxidation and by HRP catalyzed reaction of benzo[a]pyrene with DNA. J. Am. Chem. SOC.110, 4023-4029. (12) NIH Guidelines for the Laboratory Use of Chemical Carcinogens (1981) NIH Publication No. 81-2385, US. Government
Printing Office, Washington, DC. (13) RamaKrishna, N. V. S., Cavalieri, E. L., Rogan, E. G., Dolinkowski, G., Cerny, R. L., Gross, M. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992) Synthesis and structure determination of the adducts of the potent carcinogen 7,12-dimethylbenz[a]anthracene and deoxyribonucleosides formed by electrochemical oxidation: Models for metabolic activation by one-electron oxidation. J. Am. Chem. SOC.(in press). (14) Cavalieri, E., and Calvin, M. (1971) Photochemical coupling of benzo[a]pyrene with l-methylcytosine: Photoenhancement of carcinogenicity. Photochem. Photobiol. 14, 641-653. (15) Jeffrey, A. M., Grzeskowiak, K., Weinstein, I. B., Nakanishi, K., Roller, P., and Harvey, R. G. (1979) Benzo(a)pyrene-7,8-dihydrodiol 9,lO-oxide adenosine and deoxyadenosine adducts: Structure and stereochemistry. Science (Washington, D.C.)206, 1309-13 11.
Identification and Quantitation of Benzo[ a Ipyrene-DNA Adducts Formed by Rat Liver Microsomes in Vitro P. D. Devanesan,? N. V. S. RamaKrishna,t R. Todorovic,t E. G. Rogan,*9t E. L. Cavalieri,? H. Jeong,i R. Jankowiak,i and G. J. Small* Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Department of Chemistry and Ames Laboratory-USDOE, Iowa State University, Ames, Iowa 50011 Received September 9, 1991 The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) a t C-10 with the 2-amino group of dG (BPDE-10-N2dG)and the adduct formed by reaction of BP radical cation a t C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound a t C-6 to the C-8 of Gua (BP-6C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound a t C-10 to the N-7 of Ade (BPDElGN7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-inducedrat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BPDNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%),BP-6-N7Gua (lo%),BP-6-C8Gua (12%), and BPDE-lO-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dGand 20% of the depurination adduct BPDE-lO-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDElGN7Ade was obtained. Activation of BP by horseradish peroxidase afforded 32% of stable unidentified adducts and 68% of depurination adducts, with 48% of BP-6-N7Ade, 9% of BP-6-N7Gua, and 11% of BP-6-C8Gua These results show that with activation by cytochrome P-450 the BP-DNA adducts are predominantly formed by one-electron oxidation and lost from DNA by depurination.
Introductlon Covalent binding of polycyclic aromatic hydrocarbons (PAH)' to target cell DNA is thought to be the first event in the tumor-initiating process. The physicochemical properties of PAH and the catalytic properties of cytochrome P-450s suggest that PAH are generally activated by two major mechanisms, one-electron oxidation with formation of radical cations (1, 2 ) and monooxygenation to yield bay-region diol epoxides (3, 4). Some PAH are
* To whom correspondence should be addressed. University of Nebraska Medical Center.
* Iowa State University.
thought to be activated by the diol epoxide pathway alone,
others by one-electron oxidation alone, and some by a Abbreviations: BP, benzo[a]pyrene; BPDE, (+)-r-7,t-8-dihydroxy-
t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti); BP-6-N7Ade, 7(benzo[a]pyren-6-yl)adenine;BP-6-N7Gua, 7-(benzo[a]pyren-6-y1)guanine; BP-6-C8Gua, 8-(benzo[a]pyren-6-yl)guanine;BP-6-C8dG, 8(benzo[a]pyren-6-yl)deoxyguanosine;BPDE-10-N2dG, 10-(deoxyguanosin-~-y1)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BPDE-lO-N7Gua,lO-(guanin-7-yl)-7,8,9-trihydroxy-7,8,9,lO-te~~y~~ benzo[a]pyrene; BPDE-lO-N7Ade, 10-(adenin-7-yl)-7,8,9-trihydroxy7,8,9,10-tetrahydrobenzo[a]pyrene;clotrimazole, l-(o-chloro-a,a-di-
phenylbenzy1)imidazole;DMSO, dimethyl sulfoxide; DPEA, 2-[(4,6-dichlorobiphenyl-2-yl)oxy]ethylaminehydrobromide; FLN, fluorescence line narrowing;FLNS, fluorescence line narrowingspectroscopy;HPLC, high-pressure liquid chromatography; PAH, polycyclic aromatic hydrocarbon(a).
0 1992 American Chemical Society
