Reactions of Dihydrodiol Epoxides of 5-Methylchrysene and 5, 6

Characterization of DNA Adducts Derived from syn-Benzo[ghi]fluoranthene- 3,4-dihydrodiol-5,5a-epoxide and Comparative DNA Binding Studies with ...
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Chem. Res. Toxicol. 1999, 12, 347-352

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Reactions of Dihydrodiol Epoxides of 5-Methylchrysene and 5,6-Dimethylchrysene with DNA and Deoxyribonucleotides Jan Szeliga,*,† Shantu Amin,‡ Fang-Jie Zhang,§ and Ronald G. Harvey§ Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, Naylor Dana Institute for Disease Prevention, American Health Foundation, Dana Road, Valhalla, New York 10595, and Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 Received October 8, 1998

Both syn and anti dihydrodiol epoxides from 5-methylchrysene (5-MCDE) and 5,6dimethylchrysene (5,6-DMCDE) were reacted under the same conditions with native DNA, denatured DNA, and purine deoxyribonucleotides, and the products were quantified. The extents of reaction with the deoxyribonucleotides were consistently greater for 5,6-DMCDE than for 5-MCDE. The yield of adducts in the reaction with DNA ranged from being a few-fold to 50-fold greater than those found in the corresponding deoxyribonucleotide reactions for both 5-MCDE and 5,6-DMCDE. The DNA-dependent enhancement of product yield was greater for 5-MCDE than for 5,6-DMCDE with a few exceptions among cis and trans deoxyadenosine adducts. The most substantial differences in DNA-dependent enhancement were found for deoxyguanosine adducts; thus, steric hindrance between the 6-methyl group in the 5,6-DMCDE and the minor groove in the DNA double helix may account for the greater DNA-dependent enhancement found in the 5-MCDE reactions.

Introduction (PAH)1

Polycyclic aromatic hydrocarbons are among the most potent chemical carcinogens (1). They are formed from the incomplete combustion of organic matter, particularly from fossil fuels (i.e., crude oil and coal), and natural combustion of forest and prairie. PAH are also present in tobacco smoke and in meat cooked over charcoal (2). As PAH are found throughout the human environment, exposure to these hazardous compounds cannot be avoided. The carcinogenic activity of PAH has been found to depend on their metabolic conversion to vicinal dihydrodiol epoxides in which the epoxide is in a bay or fjord region (3). The epoxide group of the dihydrodiol epoxide then undergoes attack at the benzylic carbon by the amino groups of deoxyguanosine and deoxyadenosine residues in DNA which forms both cis- and trans-opened DNA adducts (3-6). The covalent binding of dihydrodiol epoxides to DNA is believed to be the first of a series of events leading to tumorigenesis. The chemical binding of a given PAH dihydrodiol epoxide with DNA is dependent on various structural features such as the shape of the molecule, the size, the substituent, and steric factors (1). The in vitro reactions of PAH dihydrodiol epoxides with DNA (7-17) were reviewed recently (18). When these reactive metabolites are derived from planar hydrocarbons, such as benzo[a]pyrene, they react predominantly with guanine residues in DNA (19), whereas †

NCI-Frederick Cancer Research and Development Center. American Health Foundation. University of Chicago. 1 Abbreviations: PAH, polycyclic aromatic hydrocarbons; 5-MCDE, 5-methylchrysene dihydrodiol epoxide; 5,6-DMCDE, 5,6-dimethylchrysene dihydrodiol epoxide. ‡ §

those derived from nonplanar hydrocarbons, such as 7,12-dimethylbenz[a]anthracene, are distributed more evenly over both adenine and guanine residues in DNA (20). It is conceivable that the adducts formed with the two purine residues in DNA have different biological potencies. Therefore, a better understanding of the factors that determine site selectivity in dihydrodiol epoxide-DNA reactions is desired. To approach this objective, we have compared the reactions with DNA of syn- and anti-dihydrodiol epoxides of a planar hydrocarbon, 5-methylchrysene, with those of the dihydrodiol epoxides of a structurally related nonplanar hydrocarbon, 5,6-dimethylchrysene (21). The DNA adducts formed from the two diastereomeric dihydrodiol epoxides of both 5-methylchrysene (5-MCDE) (9, 10, 22) and 5,6-dimethylchrysene (5,6-DMCDE) (17, 23) are well-characterized, and methods for their separation and quantification have been identified. Additionally, these hydrocarbon derivatives differ only in the presence or absence of the methyl group at the 6-position. We have found that for each of the four dihydrodiol epoxides, the extents of the reaction with deoxyadenosine residues and deoxyguanosine residues in purine deoxyribonucleotides at neutral pH are comparable. This indicates that all of these dihydrodiol epoxides are similarly reactive toward the two purine residues. However, in both native and denatured DNA, the relative extent of reaction with these two purines changes substantially. In particular, the native DNA structure promotes reaction of one enantiomer of the anti-5-MCDE with deoxyguanosine residues to a much greater extent than it promotes this reaction for the analogous enantiomers of 5,6-DMCDE.

10.1021/tx980228o CCC: $18.00 © 1999 American Chemical Society Published on Web 03/12/1999

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Materials and Methods Caution: 5-Methylchrysene and 5,6-dimethylchrysene dihydrodiol epoxides are a carcinogenic hazard and should be handled appropriately. Racemic syn- and anti-5-methylchrysene 1,2-dihydrodiol 3,4epoxide and racemic syn- and anti-5,6-dimethylchrysene 1,2dihydrodiol 3,4-epoxide were synthesized as described previously (24-26). Calf thymus DNA, 2′-deoxyguanosine 5′-monophosphate and 2′-deoxyadenosine 5′-monophosphate (as sodium salts), deoxyribonuclease I (type IV, from bovine pancreas), phosphodiesterase I (type VII, from crotalus atrox venom), and Escherichia coli alkaline phosphatase [suspension in a 2.5 M (NH4)2SO4 solution] were obtained from Sigma (St. Louis, MO). Sep-Pak C18 cartridges were obtained from Waters (Milford, MA), and an Ultrasphere ODS reverse-phase column (5 µm, 4.6 mm × 250 mm) was purchased from Beckman (Fullerton, CA). Ultraviolet absorption spectra were monitored on-line with a Hewlett-Packard 1090 Series II HPLC system equipped with a diode array detector and were also measured with a Milton Roy Spectronic 3000 diode array spectrophotometer. Circular dichroism spectra were measured on a Jasco J500A spectropolarimeter in methanol, and normalized to 1 absorbance unit at the wavelength of maximum absorbance of the adduct (260262 nm). Reactions with DNA and Adduct Analysis. Native DNA or denatured DNA (obtained by heating the native DNA solution in a boiling water bath for 10 min and subsequently cooling rapidly in an ice bath) (1 mg/mL) in Tris/HCl buffer (pH 7) was treated with 0.1 mL of an acetone solution (1 mg/mL) of the racemic syn- or anti-5-methyl- or 5,6-dimethylchrysene dihydrodiol epoxides, followed by incubation at 37 °C for 48 h. Before each experiment, the dihydrodiol epoxide purity was checked by reaction with mercaptoethanol (5 µL of a dihydrodiol epoxide was quenched with 40 µL of 1.5 M aqueous 2-mercaptoethanol in 0.5 M NaOH and, after 10 min, was neutralized with 40 µL of 0.5 M HCl) and analyzed by reverse-phase HPLC. DNA samples were extracted with water-saturated 1-butanol (4 × 1 volume) followed by ether (3 × 1 volume) and were then purged with N2. DNA was enzymatically hydrolyzed to deoxyribonucleosides using DNase I, venom phosphodiesterase, and alkaline phosphatase (8). The deoxyribonucleoside adducts were recovered on a Sep-Pak cartridge that was eluted sequentially (the eluent was monitored on-line by an UV monitor) with water, 5% methanol, and finally 100% methanol to recover carcinogendeoxyribonucleoside adducts. The methanol solution was evaporated to dryness and then dissolved again in a known volume of 50% methanol. Aliquots (corresponding to 10 µg of dihydrodiol epoxide and 100 µg of DNA) from each sample were injected onto a Beckman Ultrasphere ODS column (5 µm, 0.46 cm × 25 cm) that was eluted at a flow rate of 1 mL/min. All adducts were quantified from peak areas at 260 nm. Averages of at least five separate injections per sample from two parallel reactions were used, and extinction coefficients were assumed to be the same for deoxyribonucleotide adducts and deoxyribonucleoside adducts. Deoxyribonucleoside adducts derived from DNA (and some from deoxyribonucleotide reactions) were separated as follows. The syn-5,6-dimethylchrysene 1,2-dihydrodiol 3,4-epoxide adducts were eluted isocratically for 10 min with 23% acetonitrile followed by a gradient that decreased the acetonitrile level to 0% and increased the methanol level to 50% over the next 50 min, and then further increased the methanol level to 65% over the next 40 min. The adducts derived from the anti5,6-dimethylchrysene dihydrodiol epoxide were eluted isocratically with 40% methanol for 10 min, followed by a linear increase to 56% methanol over the course of 80 min, and then eluted isocratically for a further 10 min. The syn-5-methylchrysene 1,2-dihydrodiol 3,4-epoxide adducts were eluted isocratically for 20 min with 20% acetonitrile followed by a linear gradient to 25% acetonitrile over the next 55 min and isocratically for another 15 min. The adducts from the anti-5-methylchrysene derivative were eluted isocratically for 45 min with 20% acetonitrile, followed by a linear gradient to 25% acetoni-

Szeliga et al. trile over the course of 35 min, and by isocratic elution for 5 min. Reactions with Deoxyribonucleotides and Adduct Analysis. Solutions of deoxyguanylic and deoxyadenylic acids were reacted with the same dihydrodiol epoxides and in the same buffer used for DNA reactions under conditions ranging from nucleotide/dihydrodiol epoxide ratios by weight of 1/1 to 1/1000. The time needed for complete reaction was established by determining the amount of residual dihydrodiol epoxide with 2-mercaptoethanol. This enabled a rapid, direct measurement of tetraols, deoxyribonucleotide adducts, and unreacted dihydrodiol epoxide as the mercaptoethanol adduct. Since adduct levels were low in these nucleotide reactions, graphical extrapolation was required to obtain data at the ratio used for DNA reactions (adducts from 5 µg of dihydrodiol epoxide and 50 µg of deoxyguanylic acid and from 5 µg of dihydrodiol epoxide and 50 µg of deoxyadenylic acid). The deoxyribonucleotide adducts were either enzymatically hydrolyzed to deoxyribonucleoside adducts with alkaline phosphatase prior to analysis or analyzed directly by HPLC. The deoxyribonucleotide adducts derived from the reactions of the racemic syn- and anti-dihydrodiol epoxides of 5-methyl- and 5,6-dimethylchrysene with deoxyguanylic and deoxyadenylic acids were separated using 0.05 M K2HPO4/KH2PO4 (pH 7) buffer in combination with organic solvents, as follows. Separation of the syn-5,6-dimethylchrysene dihydrodiol epoxide-deoxyguanylic acid adducts required isocratic elution with 17% acetonitrile in buffer for 45 min, followed by a linear increase to 30% acetonitrile over the next 45 min. The deoxyadenylic acid adducts were eluted with 15% acetonitrile in buffer for 5 min, followed by a linear increase to 25% acetonitrile over the next 55 min, and then to 30% acetonitrile over the following 17.5 min. The anti-5,6-dimethylchrysene dihydrodiol epoxidedeoxyguanylic acid adducts were eluted isocratically with 35% methanol for 15 min, followed by a linear gradient to 45% methanol over the course of 65 min, and then to 50% methanol over the next 20 min. The anti-dihydrodiol epoxide-deoxyadenylic acid adducts were separated using isocratic elution with 15% acetonitrile in buffer for 5 min, followed by a linear increase to 25% acetonitrile over the course of 55 min, and to 30% acetonitrile over the following 17.5 min. The syn-5-methylchrysene dihydrodiol epoxide-deoxyguanylic acid adducts were separated isocratically with 15% acetonitrile in buffer for 70 min, followed by a linear gradient to 25% acetonitrile over the course of 55 min, and isocratic elution for another 5 min. The deoxyadenylic acid adducts were eluted isocratically with 15% acetonitrile for 5 min, followed by a linear gradient to 22% acetonitrile for 55 min, and then isocratically for 10 min, and again with a linear gradient to 30% acetonitrile for 17.5 min. Separation of the anti-5-methylchrysene dihydrodiol epoxide adducts derived from reactions with the deoxyguanylic acid was achieved by isocratic elution with 38% methanol in buffer for 60 min, followed by a linear increase to 42% methanol over the course of 15 min. The deoxyadenylic acid adducts were eluted isocratically with 15% acetonitrile in buffer for 5 min, followed by a linear gradient to 20% acetonitrile over the course of 55 min, and then to 30% acetonitrile over the following 17.5 min.

Results and Discussion syn- and anti-dihydrodiol epoxides, derived from 5-methylchrysene, a planar hydrocarbon, and 5,6-dimethylchrysene, a nonplanar hydrocarbon (21), were reacted with native DNA, denatured DNA, and deoxyribonucleotides under identical conditions. The products were analyzed to uncover the basis for known differences in the reactions of these two types of dihydrodiol epoxides with DNA. The structures of the adducts formed in DNA and the abbreviations used to refer to them in this paper are shown in Figure 1. These adducts, as nucleosides, were separated by HPLC, identified by comparison with standards of deoxyadenosine and deoxyguanosine ad-

Dihydrodiol Epoxide-DNA Reaction Chemistry

Figure 1. Structures of the four configurational isomers of the 5-methylchrysene dihydrodiol epoxides (YdH) or 5,6-dimethylchrysene dihydrodiol epoxides (YdCH3) and their possible products of reaction with DNA or purine deoxyribonucleotides. The labels denote the absolute stereochemistry of the adducts at C4 (S or R), deoxyguanosine adducts (G) and deoxyadenosine adducts (A), and the direction of the epoxide ring opening as (t) trans and (c) cis.

ducts, and also compared with the known chromatographic properties of the DNA adducts established previously in this laboratory (9, 10, 17). Chromatographic profiles of adducts obtained from reactions of the syn- and anti-dihydrodiol epoxides of both 5-methyl- and 5,6-dimethylchrysene with DNA are shown in Figure 2. The two minor dGuo cis adducts [(R)Gc and (S)Gc], derived from the anti-5-methylchrysene dihydrodiol epoxide (see Figure 2D), were not found previously (9). Here, they were identified by comparison of their retention times and UV spectra with those of adducts obtained from reaction with deoxyguanylic acid. In turn, the latter were obtained in quantities that allowed configurations of the adducts to be established from an empirical relationship between the absolute configuration at the site of attachment of the hydrocarbon to the deoxyribonucleoside and its CD spectrum (13). The major adducts formed in DNA from the syn- and anti-dihydrodiol epoxides of both 5-methyl- and 5,6dimethylchrysene result from reaction with purine residues (18). For this reason, only reactions with the 5′-phosphates of dGuo and dAdo were studied. Adducts were quantitatively analyzed by HPLC of the deoxyribonucleotide reaction mixtures (profiles not shown). We reported previously that most of the syn- and anti-5,6dimethylchrysene dihydrodiol epoxide-deoxyribonucleotide adducts (17) were eluted from HPLC in the same sequence as the deoxyribonucleoside adducts, except that the elution sequence for the cis and trans adducts from

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Figure 2. HPLC profiles of the deoxyribonucleoside products from reactions of the racemic syn-5,6-DMCDE (A), syn-5-MCDE (B), anti-5,6-DMCDE (C), and anti-5-MCDE (D) with DNA. Peak labels are defined in Figure 1.

the syn-dihydrodiol epoxide and deoxyguanylic acid was the reverse of that for the deoxyguanosine adducts. The same relation in elution order was found for the 5-methylchrysene dihydrodiol epoxide-deoxyribonucleotide and -deoxyribonucleoside adducts by comparing the CD spectra of known deoxyribonucleoside adducts with those from the deoxyguanylic and deoxyadenylic acid reactions. In reactions of the deoxyadenylic and deoxyguanylic acids with syn-dihydrodiol epoxides of both hydrocarbons, cis opening of the dihydrodiol epoxides was more extensive than trans opening, whereas for the anti-dihydrodiol epoxides, trans opening predominated. The yields of all nucleotide reaction products were consistently greater for the 5,6-dimethylchrysene dihydrodiol epoxides than for the 5-methylchrysene dihydrodiol epoxides (lower panel in Figure 3). Furthermore, all four dihydrodiol epoxide stereoisomers gave greater total adduct yields with deoxyadenylic acid than with deoxyguanylic acid. In reactions with denatured DNA, the syn-5,6-dimethylchrysene dihydrodiol epoxide gave greater adduct yields than the syn-5-methylchrysene dihydrodiol epoxide as was the case in the deoxyribonucleotide reactions. This was also the case for some of the products in the antidihydrodiol epoxide reactions, but much more trans deoxyguanosine adducts were formed with the antidihydrodiol epoxide from 5-methylchrysene than with that from 5,6-dimethylchrysene dihydrodiol epoxide. Product yields in denatured DNA reactions for both deoxyguanosine and deoxyadenosine adducts (middle panels in Figure 3), for each hydrocarbon dihydrodiol

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Figure 3. HPLC peak areas for deoxyribonucleoside adducts derived from 10 µg of dihydrodiol epoxide and 100 µg of DNA and for deoxyribonucleotide adducts derived from 5 µg of dihydrodiol epoxide and 50 µg of deoxyguanylic acid together with those derived from 5 µg of dihydrodiol epoxide and 50 µg of deoxyadenylic acid. The reaction products are from native DNA (upper panels), from denatured DNA (middle panels), and from deoxyribonucleotides (lower panels), reacted with each optically pure dihydrodiol epoxide isomer (RSRS, first column; SRSR, second column; SRRS, third column; and RSSR, fourth column) from 5-MCDE (white bars) and 5,6-DMCDE (black bars).

epoxide, were greater than the yields of comparable deoxyguanosine and deoxyadenosine adducts from the deoxyribonucleotide reactions (lower panels in Figure 3). In relation to denatured DNA, the secondary structure of native DNA enhanced trans-deoxyguanosine adduct formation only with the (SRRS)- and (RSSR)-5-methylchrysene dihydrodiol epoxide isomers. This secondary structure also enhanced trans-deoxyadenosine adduct formation in reactions with the (SRSR)- and (RSSR)isomers of dihydrodiol epoxides of both 5-methyl- and 5,6dimethylchrysene. Otherwise, the native DNA structure decreased adduct yields relative to yields of the denatured DNA reaction, and this was particularly notable for the (RSRS)- and (SRSR)-isomers of 5,6-dimethylchrysene dihydrodiol epoxide (upper panels in Figure 3). Because all reaction conditions were the same, the 6-methyl group in 5,6-dimethylchrysene, which contributes a second electron-donating group to 5-methylchrysene and is responsible for the distortion from planarity in 5,6-dimethylchrysene, must be responsible for the difference in adduct yields and adduct distributions found for DNA reactions of comparable dihydrodiol epoxides of the planar hydrocarbon, 5-methylchrysene, and nonplanar hydrocarbon, 5,6-dimethylchrysene. To establish the basis for the difference in DNA adduct yields in reactions of dihydrodiol epoxides of these two hydrocarbons, the yield of any adduct in reaction with

Szeliga et al.

Figure 4. Each panel shows the results of comparison of the DNA factors for one isomeric dihydrodiol epoxide of both hydrocarbons calculated as adduct yield in DNA/adduct yield in deoxyribonucleotide for 5-MCDE: adduct yield in DNA/adduct yield in deoxyribonucleotide for 5,6-DMCDE labeled for deoxyguanosine cis adducts (Gc), deoxyguanosine trans adducts (Gt), deoxyadenosine cis adducts (Ac), and deoxyadenosine trans adducts (At). In cases where the DNA factors for the 5,6DMCDE were greater than those for the 5-MCDE, the inverse ratio was plotted on a downward pointing scale. Consequently, the lowest value of such a calculation is 1, and this indicated that DNA factors for 5-MCDE and 5,6-DMCDE were the same. The dotted lines cut off that part of the bars (between 0 and 1) where both DNA factors are equal. The black bars are for comparison of DNA factors for native DNA and shadowed bars for denatured DNA.

DNA was compared to the yield of the same adduct in reaction with the corresponding deoxyribonucleotide. This ratio, adduct yield in DNA/adduct yield in deoxyribonucleotide, was considered to reflect the contribution of DNA to the reaction, defined here as a DNA factor. To determine if this effect of DNA structure was greater for 5-MCDE than for 5,6-DMCDE, the corresponding DNA factors were compared. For example, the DNA factor for the deoxyguanosine cis adduct from the (RSRS)-isomer of 5-MCDE was compared with the DNA factor for the deoxyguanosine cis adduct from the (RSRS)-isomer of 5,6DMCDE. The result (how many times this DNA factor was greater for the 5-MCDE than for the 5,6-DMCDE) was plotted in Figure 4 [the closed bar labeled Gc in the upper panel for the syn-(RSRS)-dihydrodiol epoxide]. To distinguish those cases where the DNA factors for the 5,6-DMCDE were greater than those for the 5-MCDE, the results in the latter case were plotted on a downward pointing scale in Figure 4. Figure 4 shows that the DNA factors for the deoxyguanosine adducts for 5-MCDE were always higher than those for the 5,6-DMCDE. The DNA factors for the cisdeoxyguanosine adducts were found to be 5 times higher

Dihydrodiol Epoxide-DNA Reaction Chemistry

for the (SRSR)-5-MCDE, 8 times higher for the (RSRS)5-MCDE, and about 4 times higher for the trans-deoxyguanosine adducts for both syn-5-MCDE isomers. The same comparison for the anti-dihydrodiol epoxides showed that for the trans-deoxyguanosine adducts the DNA factors were 7 times higher for the (SRRS)-5-MCDE, 9 times higher for the (RSSR)-5-MCDE, and 3 and 4 times higher for the cis-deoxyguanosine adducts. The DNA factors for reactions with denatured DNA indicate for all deoxyguanosine adducts the same pattern as for the native DNA reactions, but the differences between DNA factors were about half of those seen in the reactions for native DNA. Overall, the data in each panel in Figure 4 present a similar trend of relations between DNA factors; however, for the syn-dihydrodiol epoxides (panels labeled syn RSRS and syn SRSR) the differences between DNA factors for both tested 5-MCDE and 5,6-DMCDE are greater for the deoxyguanosine cis adducts than for the deoxyguanosine trans adducts, whereas the reverse relation was found for the anti-dihydrodiol epoxides (labeled anti SRRS and anti RSSR). This trend follows the preferential epoxide ring opening that is cis for the syn5-methyl- and 5,6-dimethylchrysene dihydrodiol epoxides and trans for both anti-dihydrodiol epoxides. The native and denatured DNA structures increase the yield of reaction products derived from the 5-MCDE in reaction with the guanine amino group in DNA more substantially than they increase the yield of analogous products derived from the 5,6-DMCDE. As can be seen in Figure 4, the differences between DNA factors for adducts derived from the reactions of 5-MCDE versus 5,6DMCDE with the adenine amino group in native DNA and denatured DNA are small without a clear preference for either of the tested hydrocarbons. As has been mentioned earlier, 5,6-dimethylchrysene is distorted from planarity because the steric interaction between the 6-methyl group and the 5-methyl group does not allow an in-plane accommodation of the hindrance between the 5-methyl group and the hydrogen atom at C4. If the reaction of dihydrodiol epoxide with the amino groups in the purine residues in DNA occurs through a carbocation intermediate (3, 27), the carbocation of the 5,6-dimethylchrysene dihydrodiol epoxide would again encounter the same hindrance due to of the sp2 hybridization of the carbocation at C4. This hindrance would destabilize the carbocation more in 5,6-DMCDE than in 5-MCDE; however, the presence of the second electrondonating methyl group in 5,6-DMCDE should contribute to stabilization of this intermediate. Since these effects are expected to be similar whether the reactant is DNA or deoxyribonucleotide, it is unlikely that these effects are responsible for differences in 5-MCDE versus 5,6DMCDE reactions in DNA compared with those of deoxyribonucleotides. Although the 6-methyl group in the 5,6-dimethylchrysene dihydrodiol epoxide might cause steric hindrance, this is not the case in reactions with deoxyadenylic and deoxyguanylic acids because such an effect would decrease adduct yields for 5,6-dimethylchrysene dihydrodiol epoxides relative to those for the 5-methylchrysene dihydrodiol epoxides. The experimental data (Figure 3) suggest that the presence of the 6-methyl group in 5,6DMCDE favors adduct formation in the reactions with deoxyadenylic and deoxyguanylic acids, whereas the absence of the 6-methyl group in 5-MCDE reactions

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favors reactions with deoxyguanosine in DNA, but has little effect on reactions with deoxyadenosine in DNA (Figure 4). This suggests that the effect of the 6-methyl group in 5,6-DMCDE in giving a lower extent of reaction with deoxyguanosine in DNA than 5-MCDE may be attributed to steric hindrance between the hydrocarbon dihydrodiol epoxide and DNA structure, since the most affected reactions were those with the amino group in the minor groove and this effect was more apparent in native than in denatured DNA. Separate intercalative noncovalent binding of PAH-dihydrodiol epoxides to DNA (28, 29), with the proper orientation for forming adducts, is required for deoxyadenosine and deoxyguanosine adduct formation and is consistent with the present finding on 5-methyl- and 5,6-dimethylchrysene dihydrodiol epoxides in reactions with DNA in aqueous solutions. The basis for the difference in adduct distribution at deoxyguanosine and deoxyadenosine sites in DNA for 5,6-DMCDE versus 5-MCDE lies therefore probably in the greater reactivity of dihydrodiol epoxides from 5-methylchrysene for the amino group of deoxyguanosine residues, not in a greater affinity of the dihydrodiol epoxide derived from the nonplanar 5,6-dimethylchrysene for deoxyadenosine residues in DNA.

Acknowledgment. We are grateful to Dr. Anthony Dipple for helpful comments on the manuscript. The research was supported by the National Cancer Institute, DHHS, under contract with ABL and by grants from the National Cancer Institute (CA 67937) and the American Cancer Society (CN-22) to R.G.H. and Cancer Center Support Grant CA-17613 to S.A.

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