Chem. Res. Toxicol. 1989,2, 334-340
334
DNA Adducts from Carcinogenic and Noncarcinogenic Enantiomers of Benzo[ a Ipyrene Dihydrodiol Epoxide Shee Chau Cheng,t Bruce D. Hilton,t John M. Roman,$ and Anthony Dipple**t BRI-Basic Research Program and Program Resources, Inc., NCI-Frederick Cancer Research Facility, Frederick, Maryland 21 701 Received July 27, 1989
The characterization of eight benzo[a]pyrene-deoxyribonucleoside adducts derived from reaction of the anti-dihydrodiol epoxide and deoxyguanylic and deoxyadenylic acids is described. It is reported that the epoxide ring is opened by the purine amino groups to yield similar amounts of both cis and trans products. NMR data show that the 7- and 8-hydroxyl groups are pseudodiaxial in the cis products and pseudodiequatorial in the trans products, and we suggest that these products arise from reaction with the diaxial and diequatorial conformers of the dihydrodiol epoxides, respectively. The chiral nature of the interactions of these metabolites with DNA restricts the range of products formed with this macromolecule, and a trans product with deoxyguanosine is the major product formed with either enantiomer of the anti-dihydrodiol epoxide.
Introduction The potent carcinogen benzo[a]pyrene (BP)' is a widespread environmental pollutant that is metabolically activated to bay region 7,8-dihydrodiolg,lO-epoxides (1-3). The binding of these metabolites to DNA is thought to be a key step in tumor initiation ( 4 ) , and the major DNA adduct formed in vivo is a deoxyguanosine adduct (5) that arises from trans opening of the anti-dihydrodiol epoxide (wherein the epoxide oxygen and benzylic hydroxyl group are trans) by the amino group of guanine in DNA (6). Essentially all the structural work has been done on ribosides, however, and none of the deoxyriboside adducts have been fully characterized. Even minor adducts may be biologically important. For example, it has been shown that 36% of the activation of ras protooncogene in vitro by anti-BP dihydrodiol epoxide occurs at adenine residues (7), despite a low overall reaction at this purine base. In this paper, we report results of a detailed characterization of adducts formed from the reaction of enantiomeric anti-BP dihydrodiol epoxides with DNA. We have characterized four deoxyadenosine and four deoxyguanosine adducts. The NMR spectra of these adducts indicate their conformations and offer some understanding of the mechanism of reaction of dihydrodiol epoxides with DNA. Experimental Section Optically active and racemic anti-BP 7,8-dihydrodiol 9,lOepoxides were purchased from NCI Chemical Carcinogen Reference Standard Repository and the Chemsyn Science Laboratories, Lenexa, KS, respectively. Calf thymus DNA, deoxyadenylic and deoxyguanylic acids, bacterial alkaline phosphomonoesterase, and venom phosphodiesterase were all obtained commercially. Circular dichroism (CD) spectra were measured on a Jasco Model J500A spectropolarimeter equipped with a data processing system for signal averaging. CD spectra of nucleoside adducts in methanol were normalized to 1.0 absorbance unit at . ,A
Ultraviolet absorption spectra were recorded with a HewlettPackard P450A spectrophotometer and were also monitored on-line on a Hewlett-Packard Model 1090 high-pressure liquid chromatograph equipped with a diode array detector. Proton NMR spectra of acetylated tetraols and adducts were recorded in acetone-d6 with a 5-mm fixed tuned proton probe at 300 MHz on a GE-NMR NT-300 spectrometer equipped with a Nicolet 1280 data system. Sufficient signal to noise was obtained by signal averaging for 1-24 h while maintaining probe temperature at 25 "C. COSY (homonuclear 2D) experiments were performed by acquiring 512 blocks of 1K data points to yield a 512 by 512 symmetrized 2D data set. Presaturation of the residual HOD solvent resonance with low-power CW decoupling, and use of a 16-bit external AID, proved useful in overcoming dynamic range problems in some samples. The proton NMR spectrum of unacetylated tetraol was recorded in acetone-d6 by using a 5-mm fixed tuned proton probe at 500 MHz on a Varian VXR 500 spectrometer equipped with a Sun 41110 workstation. Mass spectra for peracetylated adducts were obtained by thermal desorptionjelectron impact ionization by using a VG 70-250 spectrometer (VG Analytical, Altrincham, U.K.). Full-scan nominal mass spectra were made from m/z lo00 to m/z 35 at 3 s/decade. Accurate mass measurements were made on the molecular ions via peak matching by using tris(perfluorohepty1)-s-triazineas the source of reference peaks a t 5000 resolving power. DNA Adduct Formation a n d Isolation. Calf thymus DNA (1 mg/mL) in Tris-HC1 buffer (0.1 M, pH 7) was mixed with an acetone solution (0.1 volume, 1mg/mL) of optically active anti-BP 7,8dihydrodiol9,10-epoxide. The reaction solution was incubated overnight at 37 "C, after which time the dihydrodiol epoxide hydrolysis products were extracted with water-saturated 1-butanol ( 3 X 1volume) followed by ether (2 X 1volume). The pH of the aqueous phase was then adjusted by addition of 1 volume of Tris-HC1buffer (0.2 M, pH 9),and venom phosphodiesterase was then added to hydrolyze the DNA to nucleotides at 37 "C for 48 h. Conversion of nucleotides to nucleosides was accomplished with alkaline phosphomonoesterase a t 37 "C for 24 h. The nucleoside adducts were recovered on Sep Pak Cartridges as described before (8). Deoxyadenosine and Deoxyguanosine Adducts. Solutions of deoxyadenylic (10 mg/mL) and deoxyguanylic acids (10 mg/mL) in Tris-HC1 buffer (0.1 M, pH 7) were separately mixed
'
BRI-Basic Research Program, NCI-Frederick Cancer Research Facility. *Program Resources, Inc., NCI-Frederick Cancer Research Facility.
0893-228Xj 8912702-0334$01.50/0
Abbreviations: BP, benzo[a]pyrene; CD, circular dichroism; dAdo, deoxyadenosine; dGuo, deoxyguanosine; dAMP, deoxyadenosine 5'phosphate; dGMP, deoxyguanosine 5'-phosphate.
0 1989 American Chemical Society
Benzo[a]pyrene-DNA
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 335
Adducts
op&Q
a. DNA
il 1
I
I
I
I
n
Ill
( +)-anti
n n (-)-anti
I
I
A
b. dGMP
I
c. dAMP
(+)-dA,
I ( + )-dA2 I
\i I
I
I
I
I
I
I
10
20
30
40
50
60
Time (min)
F i g u r e 1. HPLC elution profiles for BP-deoxyribonucleoL.,, adducts eluted from a reverse-phase Ultrasphere ODS column in a methanol/water gradient according to Pruess-Schwartz et al. (9). (a) Nucleoside adducts from the reaction of the (+)-anti enantiomer of the 7,8-dihydrodiol9,10-epoxideof BP with calf thymus DNA. (b and c) Nucleoside adducts from the reactions of the dihydrodiol epoxide with deoxyguanylic and deoxyadenylic acids, respectively. with an acetone solution of optically active or racemic anti-BP 7,8-dihydrodiol9,10-epoxide. The reaction solutions were incubated a t 37 "C overnight, and dihydrodiol epoxide hydrolysis products were then extracted with water-saturated 1-butanol (3 x 1volume) followed by ether (2 X 1 volume). For preparative reactions with the racemic dihydrodiol epoxide, separation of adducted nucleotides from unmodified nucleotides was achieved by loading the reaction mixtures onto Sep Pak Cartridges, eluting with water to remove the umodified nucleotides, and eluting with 100% methanol to recover the reacted nucleotides. Conversion of nucleotides to nucleosides was accomplished by using alkaline phosphomonoesterase, and adducts were purified on Sep Pak Cartridges as described above. Adduct Separation. Deoxyribonucleoside adducts from reactions with DNA or deoxyribonucleotides were separated chromatographically on an Altex Ultrasphere ODS column (5 bm, 4.6 X 250 mm) eluted with methanol/water mixtures according to Pruess-Schwartz et al. (9), or in some cases, with 23% acetonitrile. For NMR and mass spectral analyses, acetylated derivatives of deoxyadenosine and deoxyguanosine adducts were used. Typically, the methanol solutions of adducts purified from reverse-phase chromatography were evaporated to dryness, and the adducts were acetylated with acetic anhydride in pyridine (lA.5 v/v) overnight at room temperature. After evaporation to dryness and repeated dissolution in and evaporation of benzene to remove residual pyridine/acetic anhydride, the dry residues were taken up in the appropriate dichloromethane/ethyl acetate/methanol mixture and purified by normal-phase chromatography (Zorbax Sil, 5 bm, 4.6 X 250 mm) (1mL/min) in this solvent mixture. Solvents used for the deoxyadenosine adducts and deoxyguanosine were 160:30:1 and 16030:5 dichloromethane/ethyl acetate/ methanol, respectively.
Results T h e chromatographic elution profile of products obtained from t h e reaction of (+)-anti-BPdihydrodiol epoxide with calf t h y m u s D N A is shown in Figure la. As reported (51, the (+)-enantiomer reacted with DNA to give predominantly one adduct. Comparison of t h e retention t i m e a n d ultraviolet spectra of this a d d u c t with those of
Figure 2. HPLC elution profiles for BP-deoxyribonucleoside adducts from (a) reaction of the (-)-anti enantiomer of the 7,8dihydrodiol9,lC-epoxide of BP with calf thymus DNA and (b and c) reaction of the dihydrodiol epoxide with deoxyguanylic and deoxyadenylic acids, respectively.
+7
0
7
3
5E
-.:+
-
- 70
-70'
+
250
300
350 1 7'0Wavelength (nm)
Figure 3. Circular dichroism spectra for the deoxyribonucleoside adducts of BP. Spectra were recorded in methanol and were normalized by dividing each spectrum by absorbance at Am=. deoxyguanosine and deoxyadenosine adducts (panelsb and c of Figure 1)confirmed that the product formed in DNA was t h e deoxyguanosine a d d u c t labeled (+)-dG,. I n contrast, t h e (-)-enantiomer reacted with DNA to yield three main nucleoside products, as shown in Figure 2a. T w o of these products have retention times a n d ultraviolet spectra similar t o the two deoxyguanosine adducts labeled (-)-dG, a n d (-)-dG, (Figure 2b). T h e third D N A product had an ultraviolet spectrum similar t o that of t h e (-)-dA, though t h e retention times appeared to be different because of column deterioration during t h e t i m e interval between analysis of t h e DNA and dAMP reactions (Figure 2c). A peak eluted at 26 m i n (Figure 2a) was identified as a tetraol o n t h e basis of its retention t i m e a n d UV spectrum. Since tetraols formed from t h e hydrolysis of the dihydrodiol epoxide had been previously extracted with
Cheng et al.
336 Chem. Res. Toxicol., Vol. 2, No. 5, 1989
11
OAc
H
I
I --
7.5
C8-H
7.0
'
6.5
I
"
I
" "
6.0
I
5.5
7.5
6.5
7.0
(
6.0
5.5
- )-dG, Cp-H
ACO
7.5
7.0
6.5
6.0
5.5
y ' OAc
7.O
C,-H
Cio-HI
I ,
I
6.5
6.0
5.5
6 (PPm) Figure 4. Downfield region of the 300-MHz NMR spectra of peracetates of typical deoxyribonucleoside adducts of BP in acetone-& Table I. Chemical Shifts (ppm) and Coupling Constants (Hz) for the Methine Protons of the Peracetates of Tetraols and Deoxyribonucleoside Adducts of the anti-Dihydrodiol Epoxide of BPa methine protons Cg-H Cg-H C7-H G0-H 6.95 5.70 J g , g = 2.4 5.87 Jg,lo = 3.7 6.80 6.21 Jg,g = 2.1 Jg.10 = 3.2 6.26 5.86 6.80 6.15 6.27 Jg,lo = 3.2 5.88 J8,g = 2.0 6.81 6.70 6.08 Jg,g = 2.3 6.02 6.81 J9,lO = -3 6.70 6.02 6.09 J8,g = 1.9 6.81 J9,lO = -3 5.92 7.33 Jg,g = 2.6 Jg,lo = 4.3 5.67 6.62 6.05 6.88 Jg,10 = 5.6 5.70 Jg,g = 2.2 6.63 6.93 6.02 Jg,g = 2.3 Jg.10 = 5.7 5.69 6.66 5.99 7.20 Jg,lo = 5.3 5.76 Jg,g = 2.1 6.62 7.20 5.76 5.99 Jg,g = 2.2 Jg,lo = 5.4 6.67 a
In acetone-d,.
1-butanol, this tetraol may have arisen from some labile adduct during the enzymic digestion procedure. The CD spectra of the nucleoside adducts (Figure 3) were useful in the initial steps of adduct characterization because previous studies on other dihydrodiol epoxides (10-13) have shown (a) that cis and trans adducts for a given nucleoside and a single dihydrodiol epoxide enantiomer exhibit spectra that are almost, but not exactly, mirror images of one another and (b) that two adducts which differ only in that the hydrocarbon stereochemistry is enantiomeric in each case exhibit spectra which bear a mirror image relationship to one another. For the four deoxyadenosine adducts, the CD spectra indicated that (+)-dA, and (-)-dA, have the same configuration at the 9,lO-bond (i.e., they are either both cis or both trans) and that adducts (-)-dA, and (+)-dA, share the other configuration. For the deoxyguanosine adducts, it was clear that
(+)-dG, and (-)-dG, were configurationally identical at the 9,lO-bond. Similarly, the mirror image relationship between the spectra of (+)-dG, and (-)-dGz also indicated the same configuration, i.e., both are cis or both are trans about the 9,lO-bond. The actual assignments of cis or trans structure at the 9,lO-bond of the adducts were based on the proton NMR spectra of the peracetates recorded in acetone-d6 (Figure 4). Coupling constants, single frequency decoupling experiments, D20exchange experiments, and 2D COSY experiments were required to make the assignments. The 1" and 3" sugar resonances were assigned on the basis of their chemical shifts in various model compounds as well as by the coupling pattern apparent in each multiplet (Table I). Assignment of the C,-H was established by the observation of coupling to a D,O-exchangeable resonance or by a single-frequency decoupling experiment
Benzo[a]pyrene-DNA
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 337
Adducts
C,-H
0
I
OH
irradiating either the suspected N-H proton or the Clo-H. In favorable cases the entire connectivity pattern could be obtained from the 2D COSY spectrum (data not shown). For (+)-dAl and (-)-dA, the proton at one end of the connectivity chain is very broad (Figure 4c), suggesting either restricted rotation or a large unresolved coupling to the N-H proton, or both. This latter is consistent only with that proton being the Clo-H,which is geminal to N6 of adenine. Comparison of the adduct data with the chemical shifts and coupling constants of the cis- and trans-tetraols allowed assignment of each of the adducts to a particular configuration. Since no N-H resonance was present in the tetraol spectra, and no unambiguous long-range coupling assignment could be made from the 2D COSY spectrum, the protons C7-H and Clo-H could not be unequivocally assigned, but they were tentatively assigned according to the argument that the bay region benzylic proton in tetraols is usually further downfield than the non bay region benzylic proton, as has been found in previous studies (10-13). The structures of cis- and trans-tetraols have been reported earlier (14). Accordingly, the coupling are and constants for the tram-tetraol, Le., J7,8,J8,9, 9.3,2.4, and 3.7 Hz, respectively,whereas for the cis-tetraol, they are 4.2, 2.6, and 4.3 Hz,respectively (Table I). These assignments are confirmed by consideration of long-range couplings evident in the two tetraols after resolution enhancement (Figure 5). In the cis-tetraol a coupling of -0.5 Hz is observed between C8-H and Clo-H. This is consistent with the fact that these protons, in the cis form of the compound, are in the "W" conformation. In the trans-tetraol a coupling of -0.5 Hz was observed between C6-H and C7-H. Spectral simulation was used to confirm the presence of coupling to C7-H because of overlap with the AB multiplet of C4-H and C5-H. The coupling constants of individual adducts were used, therefore, to assign the specific configurations shown in Figure 6. The coupling constants exhibited by all the cis adducts and by the cis-tetra01indicated that the tetrahydro benzo ring system is in a half-chair conformation with the C7-H and C8-Hbeing pseudcdiequatorial and the purine moiety being pseudoaxial. In contrast, the coupling constants of the trans adducts were consistent with a half-chair conformation with the C7-Hand C8-H being pseudodiaxial and the purine again being pseudoaxial. In order to determine whether the same conformation would arise in unacetylated adducts, we have obtained the 500-MHz lH NMR spectrum of the unacetylated trans-tetraol (Figure 7). This shows that acetylation shifts all four methine protons downfield through a deshielding effect. In contrast, the C6-H and the Cll-H both shift upfield upon acetylation, suggesting that these protons are shielded. Despite this anisotropic nature of the acetyl groups, the coupling constant pattern of the methine protons, large, small,small (for J7,8,J8,g,and J9,10, i.e., 8.7, 2.2, and 3.3 Hz, respectively), was consistent with that of the acetylated
7.0
1
6.0
II .
II 111
8.5
6.5
8.0
7.5
7.0
6.5
6.0
6 (PPm)
Figure 5. Downfield region of sine bell resolution-enhanced 300-MHz NMR spectrum of peracetates of the trans-tetraol of the anti-dihydrodiol epoxide of BP in acetone-d6. Inset is the correspondingpartial spectrum of the peracetate of the cis-tetraol. Table 11. Accurate Mass Measurements for the Peracetates of Tetraols and Deoxyribonucleoside Adducts of anti-Dihydrodiol Epoxides of BP observed error, comobserved error, comDounds mass.amu mmu Dounds mass, amu mmu trans-tetraol" 488.1455 (-)-dA, 763.2551 -6.1 1.6 cis-tetraol 488.1472 -0.1 (+)-dG,' 779.2468 -2.9 (+)-dAlb 763.2462 -4.5 (+)-dG, 779.2458 -1.9 5.8 (-)-dGI 779.2381 (+MA, 763.2493 -0.3 779.2484 -4.5 (-)-dG, (-MA1 763.2548 -5.8
" The empirical formula for the peracetates of tetraols is C ~ H U Og, with a calculated mass of 488.1470 amu. bThe empirical formula for the pentaacetates of deoxyadenosine adducts is C4H9,NSOll, with a calculated mass of 763.2488 amu. CTheempirical formula for the pentaacetates of deoxyguanosine adducts is C4H3,N5OI2,with a calculated mass of 779.2437 amu. Table 111. Percentage of Total Products for Cis and Trans Deoxyadenosine (dAdo) and Deoxyguanosine (dGuo) Adducts from Reactions of anti-Dihydrodiol Epoxides of BP with DNA, with Deoxyadenylic Acid (dAMP), and with Deoxyguanylic Acid (dGMP) dihydrodiol dAdo dGuo dAMP dGMP epoxide cis trans cis trans cis trans cis trans (+)-anti 1 4 1 94 51 49 47 53 (-)-anti 15 22 63 47 53 48 52
derivative, indicating that acetylation did not drastically change conformation. The two tetraols and the adducts were subjected to mass spectral analyses by using the thermal desorption/electron impact ionization mode. The results of the accurate mass measurements are summarized in Table 11. As shown, the findings agree with the assigned structures to within *7 PPm.
Discussion In accord with earlier published reports (6),our studies showed that reaction of the (+)-anti-dihydrodiol epoxide
338 Chem. Res. Toxicol., Vol. 2, No. 5, 1989
Cheng et al. Scheme I
1
OH
(+)-anti 7,8-dihydrodiol 9,lO-epoxide
H OH
(+)-d4
s H 6H
dG
conformer I
( - ) - a n t i 7,8-dihydrodiol
9,lO-epoxide
cis product
6H (-)-d4
Figure 6. Partial structures of deoxyribonucleosideadducts from anti-dihydrodiolepoxides of BP. c,,,-n
1
0 0.
"o*, HO+' OH
c,,-n
9.0
8.5
II
8.0
7.5
70
6.5
6.0
5.5
5.0
4.5
6 (ppm)
Figure 7. Downfield region of the 500-MHz NMR spectrum of the trans-tetraol of the anti-dihydrodiolepoxide of BP in acetone-d6. of BP with DNA yielded predominantly one product, viz., a trans deoxyguanosine adduct (Table 111). In contrast, the (-)-enantiomer reacted with DNA to give three products. Two of these products arose from cis and trans opening of the epoxide ring by the exocyclic amino group of guanine, and the third product was a trans deoxyadenosine adduct. Although the (-)-enantiomer reacted proportionately more with the adenine residues in DNA than the (+)-enantiomer, the major products for the (-)-enantiomer were deoxyguanosine adducts. This finding is somewhat at variance with the report by Meehan and Straub (15) which showed that the major DNA adduct for the (-)-anti-BP dihydrodiol epoxide was a deoxyadenosine adduct. However, DNA polymerase was mostly stopped at guanines during replication of rat H-ras DNA that had been treated with either the (+)- or (-)-enantiomer of BP dihydrodiol epoxide (16),suggesting that both enantiomers
conformer
(b)
trans product (R,
I1
= H, R, =
purine)
cis product (R, = H, R, = purine)
react mostly with guanine residues in DNA in line with expectations from the present observations. One possible reasons for the discrepancy between this study and that by Meehan and Straub may lie in the fact that some BPDNA adducts are unstable (17, 18) and tetraol can be liberated from the labile adducts. In this study, tetraol was released from the (-)-anti-BP dihydrodiol epoxide modified DNA but not from DNA reacted with the (+)enantiomer. Thus, the loss of labile deoxyguanosine adducts (19) could explain the discrepancy in the yields of deoxyadenosine and deoxyguanosine adducts for (-)-BP dihydrodiol epoxide reported in the literature. Although the structures of the majority of the anti-BP dihydrodiol epoxide-DNA adducts have been inferred previously from comparison with the corresponding ribosides (5, 18, 20) the present work represents the first characterizations of the deoxyribosides themselves. By reacting anti-dihydrodiol epoxides of BP with deoxyguanylic and deoxyadenylic acids, we were able to obtain sufficient material to determine proton NMR spectra. These spectra showed that the trans and cis adducts have different conformations for C7-Hand C8-H,analogous to those reported earlier for several nucleophilic addition products of the anti-dihydrodiol epoxide (14). Since acetylation did not change the conformation of the trans-tetraol, it is reasonable to assume that the unacetylated BP products are also in the same conformation as their acetylated counterparts. The preferred conformers of the anti-dihydrodiol epoxides of BP adopt a pseudodiaxial conformation for C7-H and C8-H (conformer I1 in Scheme I) (14). The fact that these two protons are pseudodiequatorial in cis adducts and pseudodiaxial in trans adducts suggests that either (a) the cis adducts were formed from conformer I and the trans adducts were formed from conformer I1 or (b) both cis and trans adducts arose from the preferred conformer I1 and this conformation changed only in the case of the cis adducts. For both cis and trans adducts, the purine deoxyriboside moiety is pseudoaxial, reducing any steric interactions in the bay region. This contrasts with cis adducts of the anti-dihydrodiol epoxides of 5-methyl-
Benzo[a]pyrene-DNA
Adducts
chrysene (10) and benzo[c]phenanthrene (11)where the purine substituent is pseudoequatorial, thereby increasing the steric crowding in the bay region. This conformation for cis adducts of 5-methylchrysene and benzo[c]phenanthrene suggets that the adducts cannot readily change conformation and that, therefore, the conformation of the adducts is most likely determined by the conformation of the dihydrodiol epoxide (i.e., pathway a in Scheme I is most probably followed). In concert with this interpretation, only low yields of cis adducts are obtained from the 5-methylchrysene and benzo[c]phenanthrene dihydrodiol epoxides where steric crowding in the bay region is very high. In the benzo[a]pyrene case, the two conformers of the anti-dihydrodiol epoxides can interconvert because there is no steric hindrance in the bay region, and this may explain the higher yield of cis product (Table 111) formed with deoxyguanosine and deoxyadenosine 5’-monophosphate as compared with bay region substituted dihydrodiol epoxides. Further support for pathway a in Scheme I arises from the fact that the conformer with diaxial OH groups, i.e., conformer I in Scheme I, is more reactive than conformer I1 (21). This increased reactivity could explain why roughly similar amounts of cis and trans products are formed with deoxyribonucleotides even though I, the cis adduct precursor, is the minor component in the conformational equilibrium. Second, Yagi et al. (21) showed that when the equilibrium is altered in favor of conformer I by the introduction of a fluorine substituent at C-6 in the antidihydrodiol epoxide of benzo[a]pyrene, the ratio of cis to trans products formed increases substantially over that found for the nonfluorinated analogue. Again, this is constitent with cis adduct arising from conformer I and trans adduct arising from conformer 11. Third, in the absence of any hydroxyl grops, i.e., for BP tetrahydroepoxide, where DNA should be similarly accessible to either conformer, cis adducts predominate over trans adducts (22). These considerations also offer some insights into the mechanism of the reaction of anti-BP dihydrodiol epoxides with DNA. In contrast to reactions with nucleotides to give similar amounts of cis and trans adducts with either enantiomer, the formation of only one major adduct in DNA for the (+)-enantiomer must be a consequence of some specific chiral interaction with DNA, presumably involving the 7- and 8-hydroxyl groups (22). If intercalation is a necessary prerequisite for DNA binding (23), it is conceivable that the predominant conformer, 11,cannot change conformation once it is in the DNA structure or that conformer I, the precursor of cis adducts, is too bulky to intercalate into DNA. This latter could partially explain why compounds such as the syn-dihydrodiol epoxide of BP (241, the anti-dihydrodiol epoxide of 6-fluorobenzo[alpyrene (25), and the dihydrodiol epoxides of benzo[elpyrene (26), all of which preferentially adopt conformation I (27, 28), are noncarcinogenic. This cis deoxyguanosine adduct is formed in DNA with the (-)-enantiomer, however, indicating that for this specific adduct intercalation may not be required. Acknowledgment. This research was sponsored by the National Cancer Institute, DHHS, under Contract N01CO-74101 with Bionetics Research Inc. and Contract N01-(30-74102 with Program Resources, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US.Government. By acceptance of this article, the publisher or re-
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 339 cipient acknowledges the right of the US.Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.
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