Covalent DNA Adducts Formed by Benzo[c]chrysene in Mouse

Chem. Res. Toxicol. 1997, 10, 1275-1284

1275

Covalent DNA Adducts Formed by Benzo[c]chrysene in Mouse Epidermis and by Benzo[c]chrysene Fjord-Region Diol Epoxides Reacted with DNA and Polynucleotides A. S. Giles,‡ A. Seidel,† and D. H. Phillips* Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, U.K., and Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany Received July 2, 1997X

The metabolic activation in mouse skin of benzo[c]chrysene (B[c]C), a weakly carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar and crude oil, was investigated. Male Parkes mice were treated topically with 0.5 µmol of B[c]C, and DNA was isolated from the treated areas of skin at various times after treatment and analyzed by 32P-postlabeling. Seven adduct spots were detected, at a maximum level of 0.89 fmol of adducts/µg of DNA. Four B[c]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 µmol of the optically pure putative proximate carcinogens (+)- and (-)-trans-benzo[c]chrysene9,10-dihydrodiols [(+)- and (-)-B[c]C-diols] led to the formation of adducts which comigrated on TLC and HPLC with some of those formed in B[c]C-treated mice. The major adduct formed in mouse skin treated with B[c]C coeluted on TLC and HPLC with an adduct formed in mouse skin treated with (-)-B[c]C-diol. These results suggested that the detected adducts were formed by the fjord-region B[c]C-9,10-dihydrodiol 11,12-epoxides (B[c]CDEs). To test this, the four optically pure synthetic B[c]CDEs were reacted in vitro with DNA and with synthetic polynucleotides and these samples were 32P-postlabeled. Cochromatography, both on TLC and HPLC, of in vitro and in vivo adducts indicated that B[c]C is activated in mouse skin through formation of the (-)-anti- and (+)-syn-B[c]CDE with 9R,10S,11S,12R- and 9S,10R,11S,12Rabsolute configuration, respectively, both of which formed two DNA adducts in vivo. However, the major adduct present in the B[c]C-treated skin DNA was not a fjord-region B[c]CDE adduct but was possibly derived from a bay region B[c]CDE at the 1,2,3,4-position. The extent of DNA adduct formation by B[c]C in mouse skin DNA was lower than that of moderately carcinogenic PAHs previously studied by this method, suggesting a correlation between extent of DNA adduct formation and carcinogenic potential.

Introduction Carcinogenic polycyclic aromatic hydrocarbons (PAHs)1 are activated metabolically to electrophilic species (1) capable of covalent reaction with DNA to form DNA adducts. DNA adduct formation is thought to be a critical event in the initiation stage of tumorigenesis (2). Studies of PAHs have identified bay region diol epoxides as ultimate carcinogenic metabolites, which in principle * To whom correspondence should be addressed. Phone: +44-181643-8901. Fax: +44-181-770-7290. E-mail: [email protected]. † University of Mainz. ‡ Present address: King’s College School of Medicine and Dentistry, Caldecor Rd., London SE5 9RW, U.K. X Abstract published in Advance ACS Abstracts, October 15, 1997. 1 Abbreviations: PAH, polycyclic aromatic hydrocarbon; poly(dA‚ dT), poly(deoxyadenylic-deoxythymidylic acid); poly(dG‚dC), poly(deoxyguanylic-deoxycytidylic acid); PEI, poly(ethylenimine); B[a]P, benzo[a]pyrene; B[c]Ph, benzo[c]phenanthrene; B[c]PhDEs, benzo[c]phenanthrene-3,4-dihydrodiol 1,2-epoxides; (-)-anti-B[c]PhDE, (-)benzo[c]phenanthrene-(3S,4R)-dihydrodiol (1R,2S)-epoxide; (+)-synB[c]PhDE, (+)-benzo[c]phenanthrene-(3R,4S)-dihydrodiol (1R,2S)epoxide; B[g]C, benzo[g]chrysene; (-)-anti-B[g]CDE, (-)-benzo[g]chrysene-(11R,12S)-dihydrodiol (13S,14R)-epoxide; (+)-syn-B[g]CDE, (+)-benzo[g]chrysene-(11S,12R)-dihydrodiol (13S,14R)-epoxide; B[c]C, benzo[c]chrysene; (-)-B[c]C-diol, (-)-trans-benzo[c]chrysene-(9R,10R)dihydrodiol; (+)-B[c]C-diol, (+)-trans-benzo[c]chrysene-(9S,10S)-dihydrodiol; B[c]CDEs, benzo[c]chrysene-(9,10)-dihydrodiol 11,12-epoxides; (-)-anti-B[c]CDE, (-)-benzo[c]chrysene-(9R,10S)-dihydrodiol (11S,12R)-epoxide; (-)-syn-B[c]CDE, (-)-benzo[c]chrysene-(9R,10S)-dihydrodiol (11R,12S)-epoxide; (+)-anti-B[c]CDE, (+)-benzo[c]chrysene(9S,10R)-dihydrodiol (11R,12S)-epoxide; (+)-syn-B[c]CDE, (+)-benzo[c]chrysene-(9S,10R)-dihydrodiol (11S,12R)-epoxide; DB[a,h]A, dibenz[a,h]anthracene; DMBA, 7,12-dimethylbenz[a]anthracene.

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can be metabolically formed as a mixture of four stereoisomers classified as enantiomeric pairs of two diastereomers termed as syn- and anti-forms (1, 3). Planar PAHs such as benzo[a]pyrene (B[a]P), benz[a]anthracene, and chrysene form bay region diol epoxides which bond almost exclusively with guanine bases in DNA (4). However, some PAHs have a sterically hindered bay region which causes a distortion from planarity of the parent molecule and thus of its bay region diol epoxide derivatives (5). This distortion from planarity of the diol epoxides appears to render them more biologically active than planar diol epoxides. An example of a nonplanar PAH is benzo[c]phenanthrene (B[c]Ph), which possesses a sterically hindered bay region in its structure, termed a fjord region. The presence of a fjord region in B[c]Ph enforces a preferential diequatorial orientation of the hydroxyl groups in all four stereoisomeric diol epoxides, a conformation which has been found to be associated with high biological activity (6, 7). Metabolism studies have shown that B[c]Ph is stereoselectively activated via two fjord-region benzo[c]phenanthrene-3,4-diol 1,2-epoxides (B[c]PhDEs), namely, (-)-anti- and (+)-syn-B[c]PhDE with 1R,2S,3S,4R- and 1R,2S,3R,4S-configuration, respectively (8, 9). Furthermore, these fjord-region B[c]PhDEs bind to a much greater extent to adenine residues in DNA than the bay region diol epoxides of B[a]P (4, 10, 11). Benzo[g]chrysene (B[g]C), a moderately carcinogenic PAH on mouse skin, is another PAH possessing © 1997 American Chemical Society

1276 Chem. Res. Toxicol., Vol. 10, No. 11, 1997

Giles et al.

Scheme 1. Possible Pathways of Metabolic Activation of B[c]C to Reactive Fjord-Region Diol Epoxidesa

a

EH, epoxide hydrolase.

a fjord region in its structure and can be considered, therefore, as a higher homologue of B[c]Ph. In a previous study (10), we examined the metabolic activation of B[g]C in vivo and found it to occur very similarly to that of B[c]Ph, with two stereoisomeric fjord-region benzo[g]chrysene-11,12-diol 13,14-epoxides (B[g]CDEs), (-)-antiand (+)-syn-B[g]CDE with 11R,12S,13S,14R- and 11R,12S,13R,14S-configuration, respectively, responsible for DNA adduct formation. However, the extent of adduct formation following treatment with B[g]C was greater than that following treatment with B[c]Ph which correlates with the relative carcinogenic potentials of the two PAHs (12). Benzo[c]chrysene (B[c]C) is a PAH which occurs in coal tar and crude oil (13) and has been shown to be a weak carcinogen on mouse skin (14). The parent molecule is nonplanar due to the presence of a fjord region in its structure. By analogy with B[c]Ph and B[g]C, it is proposed that the metabolic activation of B[g]C in vivo proceeds through formation of the fjord-region benzo[c]chrysene-9,10-dihydrodiol 11,12-epoxides (B[c]CDEs), as shown in Scheme 1. Thus, the (9S,10R)-dihydrodiol (11R,12S)-epoxide [(+)-anti-B[c]CDE] and the (9S,10R)dihydrodiol (11S,12R)-epoxide [(+)-syn-B[c]CDE] are ex-

pected to be formed via the (+)-trans-benzo[c]chrysene(9S,10S)-dihydrodiol [(+)-B[c]C-diol], whereas the (-)trans-benzo[c]chrysene-(9R,10R)-dihydrodiol [(-)-B[c]Cdiol] is the expected metabolic precursor of the (9R,10S)dihydrodiol (11S,12R)-epoxide [(-)-anti-B[c]CDE] and the (9R,10S)-dihydrodiol (11R,12S)-epoxide [(-)-syn-B[c]CDE]. Previous investigators (15, 16) have shown that racemic mixtures of the syn- and anti-B[c]CDEs have higher chemical reactivities compared with the corresponding racemates of the B[g]CDEs and B[c]PhDEs. The B[c]CDEs were found to have also a higher mutagenic potency in bacterial and mammalian cells than the B[c]PhDEs (15). A racemic mixture of anti-B[c]CDE had 1.5-2.5 times greater mutagenic activity than the racemate of syn-B[c]CDE, which showed a similar mutagenic activity to the racemic B[g]CDEs (15). The extents of adduct formation in Chinese hamster V79 cells by racemates of the B[c]CDEs, B[g]CDEs, and B[c]PhDEs were also determined (16), and it was found that (()anti-B[c]CDE formed higher levels of DNA adducts than (()-syn-B[c]CDE and the racemic B[g]CDEs, which correlates with the observed mutagenic activities of these isomers in V79 cells (16).

Metabolic Activation of Benzo[c]chrysene

The present study was undertaken to elucidate the bioactivation of B[c]C to the fjord-region B[c]CDE in vivo and to characterize the DNA adduct profile formed in mouse skin, a tissue frequently used to investigate PAH carcinogenesis. Thus, we applied B[c]C and the two enantiomeric dihydrodiol precursors of the fjord-region diol epoxides, (-)-B[c]C-diol and (+)-B[c]C-diol, to mouse skin and analyzed the isolated DNA by 32P-postlabeling using a combination of TLC and HPLC. In addition, the reactions of the four optically pure synthetic B[c]CDEs with nucleic acids in vitro were studied. Comparison of the chromatographic properties of the adducts formed in vivo with those formed in vitro has allowed identification of some of the in vivo adducts.

Materials and Methods Caution: Diol epoxides and 32P are hazardous materials. For both, contamination must be avoided by wearing impermeable laboratory gloves, laboratory coats, and protective goggles. Exposure to ionizing radiation should be minimized by working with Perspex shielding and by monitoring with appropriate dosimeters (Geiger counters and personal film badges). Hazardous wastes must be collected in approved containers and stored for disposal according to local safety rules. General. Reagents and materials for 32P-postlabeling were obtained from the suppliers mentioned previously (10). HPLC grade methanol was purchased from BDH Ltd. (Poole, Dorset, U.K.), and Zorbax phenyl-modified reversed-phase columns were from Hichrom (Reading, Berks, U.K.). B[c]C was obtained from the Community Bureau of Reference (Brussels, Belgium) and was 99.5% pure. The dihydrodiol and fjord-region diol epoxide derivatives of B[c]C were synthesized as described (A. Seidel et al., manuscript submitted for publication). In brief, racemic benzo[c]chrysene9,10-dihydrodiol was synthesized from the corresponding oquinone by known methods (15, 17, 18). Resolution of the optically active B[c]C-diols was accomplished as described for benzo[c]phenanthrene-3,4-dihydrodiols via preparative HPLC separation of the diastereomeric bis-(-)-menthoxy acid esters (19). The enantiomerically pure fjord-region B[c]CDEs were prepared from enantiomerically pure B[c]C-diols according to published procedures (17, 19). Poly(deoxyadenylic-deoxythymidylic acid) [poly(dA‚dT)] and poly(deoxyguanylic-deoxycytidylic acid) [poly(dG‚dC)] (both alternating copolymers) were obtained from Sigma Chemical Co. Ltd. (Poole, Dorset, U.K.). Topical Treatment of Mice. Male Parkes mice (6-8 weeks old) were purchased from the MRC National Institute for Medical Research (Mill Hill, London, U.K.). B[c]C was applied to the shaved dorsal skin of mice (0.5 µmol/mouse in 150 µL of acetone); control mice received acetone only. Groups of four animals were killed by cervical dislocation 6 h and 1, 2, 4, 7, and 21 days after treatment, and the treated areas of skin were removed and frozen. Groups of mice were also treated as above with 0.5 µmol of (+)-B[c]C-diol or (-)-B[c]C-diol in 150 µL of acetone. Control mice received acetone only. For each treatment, groups of four mice were killed 24 h after treatment and the treated areas of skin removed and frozen. DNA Isolation. The dermal surface of the frozen skin was scraped with a scalpel blade, and the remaining frozen epidermal layer was powdered in liquid nitrogen (20). The powdered skin samples were then thawed in 10 mM EDTA, and after homogenization, a 10% solution of SDS (0.1 vol) was added and the DNA isolated and purified using a previously published phenol extraction method (21). Reaction of Diol Epoxides with DNA, Poly(dA‚dT), and Poly(dG‚dC). Aliquots (100 µL) of solutions of either DNA, poly(dA‚dT), or poly(dG‚dC) (1 mg/mL) in 0.1 M Tris-HCl (pH 7.4) were mixed with solutions of the isomeric diol epoxides of B[c]C (10 µg) in THF/ethanol (1:8, v/v; 22.5 µL). The reaction

Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1277 mixtures were kept in the dark at room temperature overnight and then extracted with water-saturated diethyl ether (6 × 130 µL). The aqueous solutions were stored at -20 °C prior to analysis. 32P-Postlabeling Analysis. 1. Nuclease P1 Enhancement Method. DNA samples from mouse skin (4 µg) were digested with micrococcal nuclease and spleen phosphodiesterase with further digestion with nuclease P1, as described previously (22). Samples were then 32P-labeled according to previously published methods (23). 2. Standard Method. DNA samples from in vitro treatments (1 µg) were digested with micrococcal nuclease and spleen phosphodiesterase, as described previously (24). Samples were then 32P-labeled using the postlabeling procedure essentially as described by Gupta et al. (23). Thin-Layer Chromatography. Resolution of 32P-labeled adducts was performed on poly(ethylenimine) (PEI)-cellulose TLC sheets (20 × 20 cm) using a four-directional anion-exchange solvent system as described previously (24). Adduct spots on the chromatograms were visualized by autoradiography at -70 °C for 1 h (in vitro samples) or for between 4 h and 2 days (in vivo samples) using screen intensifiers. Quantitation of Adducts. The adduct spots were excised from the chromatograms and levels of radioactivity determined by Cerenkov counting. Appropriate blank areas of the chromatogram were counted to obtain background levels, which were subtracted. The mean of the results of at least three 32Ppostlabeling experiments was determined in each case. 1. Standard Method. The levels of adducts present were determined by relating the cpm in normal nucleotides to the cpm in adduct nucleotides, as previously described (23). 2. Nuclease P1 Enhancement Method. The specific activity of [γ-32P]ATP was determined as described previously (10, 22). The levels of adducts were calculated from the formula: fmol of adducts/µg of DNA ) [adduct radioactivity (dpm/µg of DNA)]/[specific activity of [γ-32P]ATP (dpm/fmol)]. HPLC Analysis. Adducts were eluted from TLC plates as described previously (24). HPLC analysis of the eluted material was carried out with the apparatus described by Pfau and Phillips (25), consisting of two Waters 501 HPLC pumps, a Waters 712 WISP autosampler, a Waters 440 280-nm absorbance detector, and a Berthold LB 507 HPLC radioactivity monitor. Gradient control and other data processing were achieved with Waters Baseline-810 software. Phenanthrenecis-9,10-diol was used as an internal marker, with a retention time of between 40 and 50 min, depending on the HPLC column.

Results Formation and Persistence of B[c]C-DNA Adducts in Mouse Skin. When digests of DNA from B[c]C-treated mouse skin were 32P-postlabeled and chromatographed on 20 × 20-cm PEI-cellulose plates, the autoradiogram shown in Figure 1A was obtained. Four principal adduct spots (a, c, d, and f) and three minor adduct spots (b, e, and g) were consistently detected in DNA isolated 24 h after treatment. Thirty percent of the radioactivity on the TLC plate was contained in adduct spot a, with adduct spots c, d, and f accounting for 10%, 25%, and 17%, respectively; adduct spots b, e, and g each accounted for between 2% and 6% of the total radioactivity on the TLC plate. In DNA from mice killed 2 days after treatment, adduct spot g could not be detected on the TLC plates and, by 21 days following treatment, neither could adduct spots b and e. The pattern of formation and removal of total DNA adducts formed by B[c]C metabolites in mouse skin is shown in Figure 2A. A maximum adduct level of 0.89 fmol/µg of DNA was attained 24 h after treatment. There was a loss of 65% of the total adducts by day 4, followed by a slower removal of adducts over subsequent days.


Giles et al. Table 1. HPLC Retention Times of Adducts Formed in Mouse Skin Treated with B[c]C, (-)-B[c]C-dihydrodiol, or (+)-B[c]C-dihydrodiol adduct lettera a b c d e f g j k m

Figure 1. Autoradiograms of PEI-cellulose TLC maps of 32Plabeled digests of DNA from mouse skin treated with (A) B[c]C; (B) (-)-B[c]C-dihydrodiol; (C) (+)-B[c]C-dihydrodiol; (D) acetone (control). Mice were treated topically with 0.5 µmol of each compound. DNA was isolated and 32P-postlabeled using the nuclease P1 enhancement method as described in the text. The directions in which the chromatograms were developed with solvent were D1, top to bottom; D2, bottom to top; D3 and D4, left to right. The origin is located near the bottom left-hand corner of each map and was excised prior to autoradiography, which was for 2 days. Adduct spots are assigned letters based on their chromatographic mobilities.

Figure 2. Formation and persistence of DNA adducts in B[c]Ctreated mouse skin. DNA was extracted from the skin and 32Ppostlabeled as described in the text. Quantitation was performed after TLC resolution of the adduct spots: (A) total adduct levels (adducts a-g); (B) individual adducts.

Twenty-one days after treatment, only 3% of the damage detected at 24 h remained. The overall pattern of formation and removal of DNA adducts of B[c]C metabolites in mouse skin was analogous to that seen for the formation and removal of adducts formed by metabolites of B[g]C in mouse skin (10). Those adducts which could still be detected after 21 days (adducts a, c, d, and f in Figure 1A) each followed the same pattern of removal as that in Figure 2A, as indicated in Figure 2B for adducts a, c, and d. Formation of DNA Adducts by B[c]C-diols in Mouse Skin. When the DNA isolated from mouse skin that had been treated with (-)-B[c]C-diol or (+)-B[c]Cdiol was analyzed by 32P-postlabeling, the autoradiograms shown in Figure 1B,C, respectively, were obtained. Adduct spots have been assigned the same letters as those in Figure 1A, where they appeared to be chromatographically equivalent. However, stereochemical considerations dictate that (-)- and (+)-B[c]C-diols cannot

a

B[c]C

retention times (min) (-)-B[c]C-dihydrodiol (+)-B[c]C-dihydrodiol

28.0

28.0

48.3 45.8

45.4 45.9

48.1 53.3

52.0

50.9 43.3

49.8 41.9 42.4

38.6 73.7 40.0

Adduct letters correspond to those in Figure 1.

form an identical adduct. (-)-B[c]C-diol gave an adduct pattern of nine spots (Figure 1B). Two major spots, f and d, accounted for 36% and 18%, respectively, of the total radioactivity on the TLC plate, adducts j, c, and e accounted for 13%, 8%, and 8%, respectively, and four other spots each accounted for between 0.4% and 2% of the total radioactivity. Thus, adduct spot a, which was the major (30% of the total binding) adduct spot detected in skin DNA from mice treated with B[c]C (Figure 1A), was only a minor adduct spot detected in the DNA of mice treated with (-)-B[c]C-diol (Figure 1B). Adduct spots h, j, and k formed in the skin DNA of mice treated topically with (-)-B[c]C-diol (Figure 1B) did not have similar chromatographic mobilities to any adducts in the DNA from mice treated with the parent compound (Figure 1A). Twenty-four h after treatment of the skin with (-)-B[c]Cdiol, the total DNA adduct level was 13.96 fmol/µg of DNA. This value is 16-fold greater than the maximum adduct level attained in the epidermal DNA of mice treated topically with B[c]C. Treatment of mouse skin with (+)-B[c]C-diol gave an adduct pattern shown in Figure 1C, consisting of four major spots, d, m, n, and g, accounting for 18%, 15%, 10%, and 14%, respectively, of the total radioactivity on the TLC plate. The remaining six adduct spots accounted for between 2% and 6% of the total radioactivity each. Adduct spots m, n, o, p, and q did not have similar chromatographic mobilities to any of the adducts formed by the parent compound. A total adduct level of 3.49 fmol/µg of DNA was obtained 24 h after topical treatment with (+)-B[c]C-diol, 4-fold lower than the level of adducts formed by (-)-B[c]C-diol metabolites but 4-fold higher than the total level of adducts formed following treatment of mouse skin with B[c]C. HPLC Analysis of the B[c]C-DNA Adduct Spots in Mouse Skin. Areas containing major adduct spots from postlabeled samples of DNA treated with B[c]C or one of the two B[c]C-diols were excised from the TLC plates and eluted. Aliquots of eluates of spots assigned the same letter, containing approximately the same level of radioactivity, were then examined both separately and by coinjection on HPLC. Table 1 gives the HPLC retention times of those adduct spots formed by B[c]C in mouse skin which could be detected by the HPLC method employed and the retention times of the material in the corresponding B[c]C-diol adduct spots. This procedure was applied to the analysis of adducts a, c, d, and f (Figure 1). The small amount of radioactivity that could be recovered from adduct spots b, e, and g was insufficient to permit further analysis by HPLC. With the


Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1279

Figure 3. HPLC analysis of DNA adduct spots a, eluted from the TLC plates shown in Figure 1, panels A and B, formed by B[c]C and (-)-B[c]C-dihydrodiol, respectively. The profiles are (A) B[c]C adduct a; (B) (-)-B[c]C-dihydrodiol adduct a; (C) cochromatography of a mixture of A and B. The arrow marks the position of the UV marker.

Figure 4. Autoradiograms from 32P-postlabeled digests of the B[c]CDEs reacted with DNA (left-hand column), poly(dG‚dC) (middle column), and poly(dA‚dT) (right-hand column): (A-C) (-)-anti-B[c]CDE; (D-F) (-)-syn-B[c]CDE; (G-I) (+)-anti-B[c]CDE; (J-L) (+)-syn-B[c]CDE. 32P-Postlabeling was by the standard method. Adduct spots have been numbered the same where they have been shown to be the same using cochromatography on TLC. A suffix (b) is given where this adduct spot comigrates in the standard TLC solvent system with the corresponding spot from reaction of the diol epoxide with DNA and poly(dA‚dT). The directions of the solvents were as described in the legend to Figure 1.

retention time of the UV marker standardized to 50 min, those B[c]C-DNA adducts which could be detected showed retention times of between 28 and 52 min. B[c]C adducts a, d, and f (Figure 1A) showed very similar retention times (28.9, 45.8, and 52.0 min, respectively) to the corresponding adducts from (-)-B[c]C-diol (Figure 1B) (Table 1). When these pairs of adducts were cochromatographed, they were found to coelute. Adduct f formed by (-)-B[c]C-diol eluted as two peaks on HPLC (Table 1), only one peak of which coeluted with adduct f formed in mouse skin treated topically with B[c]C, as described above. Similarly, adduct f formed by (+)-B[c]C-diol eluted as two distinct peaks on HPLC with retention times of 49.8 and 41.9 min (Table 1), but neither of these showed similar retention times to adduct f formed in mouse skin treated with B[c]C. Adduct d formed by (+)-B[c]C-diol (Figure 1C) eluted on HPLC with a significantly longer retention time than adduct d formed by B[c]C (Figure 1A), as shown in Table 1, despite the fact that these two adducts had similar chromatographic mobilities on TLC. When mixed and cochromatographed, this pair of adducts did not coelute. Adduct c formed in mouse skin that had been treated with B[c]C (Figure 1A) eluted with a retention time of 48.3 min which was similar to the retention time of adduct c formed by (+)-B[c]C-diol (Figure 1C), and these adducts were found to coelute (Table 1), whereas adduct c formed by (-)-B[c]C-diol (Figure 1B) eluted with a retention time of 45.4 min. As Figure 3A,B shows, respectively, the material from B[c]C-DNA adduct spot

a and (-)-B[c]C-diol-DNA adduct a both eluted at about 28 min. Figure 3C shows the coelution of these two adducts. Formation of B[c]CDE-DNA Adducts in Vitro. Solutions of the four B[c]CDEs were individually reacted with a solution of DNA. The modified nucleic acid samples were then digested and 32P-postlabeled using the standard procedure and chromatographed on PEI-cellulose plates. The TLC maps arising from samples of DNA reacted with (-)-anti-, (-)-syn-, (+)-anti-, and (+)syn-B[c]CDE are shown in Figure 4A,D,G,J, respectively. Adduct spots detected consistently have been assigned numbers. (-)-anti-B[c]CDE formed three principal adducts, spots 1, 5, and 6 (Figure 4A), and five minor adducts numbered 2, 3, 4, 7, and 8 in the same autoradiogram. (-)-syn- and (+)-anti-B[c]CDEs both formed a major adduct with DNA (adduct spots 12 and 15, Figure 4D,G, respectively) and a further five minor adducts each. (+)-syn-B[c]CDE formed a total of eight adducts detectable by 32P-postlabeling, shown in Figure 4J. The three adducts which are unnumbered but are indicated by arrows, one formed by (+)-anti-B[c]CDE (Figure 4G) and two formed by (+)-syn-B[c]CDE (Figure 4J), are discussed further below. The total levels of adducts formed by each B[c]CDE with DNA were determined and are given in Figure 5, expressed as pmol/µg of DNA. (+)-anti- and (-)-anti-B[c]CDEs were found to be equally reactive toward DNA under the reaction conditions employed, giving total adduct levels of 41 pmol/µg of DNA. The least reactive


Giles et al. Table 2. HPLC Retention Times of the Major Adduct Spots Resolved by PEI-Cellulose TLC of 32P-Labeled Digests of the B[c]CDEs Reacted with DNA, Poly(dG‚dC), and Poly(dA‚dT) diol epoxide (-)-anti

adduct no.a 1 3 4

6

36.3 34.9 39.4 41.4 40.0 43.5 26.5

7

36.4

8 9 + 10 11 12

60.7 39.5 49.9 64.0 57.6 37.8

13 15 16

33.0 36.8 38.9

17 18 19 20 + 21

37.3 53.9 37.8 45.1 58.9 37.4 34.7 33.0

5

Figure 5. Comparison of the relative levels of adducts formed with DNA by the B[c]CDEs, determined by 32P-postlabeling. TLC and quantitation were as described in the text.

B[c]CDE toward DNA was (+)-syn-B[c]CDE (27 pmol/µg of DNA). Reactions of the B[c]CDEs with Poly(dG‚dC) and Poly(dA‚dT). 1. TLC Analyses. The four B[c]CDEs were reacted with poly(dG‚dC) and poly(dA‚dT), which were then subjected to 32P-postlabeling analysis by the standard method. Figure 4 shows how the adducts formed by the B[c]CDEs with DNA (left-hand column) can be characterized with respect to the base that is adducted by comparison with the adducts arising from reaction of each isomer with either poly(dG‚dC) (Figure 4, center column) or poly(dA‚dT) (Figure 4, right-hand column). Each B[c]CDE formed between three and eight adducts with poly(dG‚dC) and between two and five adducts with poly(dA‚dT). (-)-syn- and (+)-syn-B[c]CDEs showed only weak reactivity toward poly(dG‚dC), as shown in Figure 4E,K, respectively, but the detected adduct spots have chromatographic equivalents in the adduct maps of (-)-syn- and (+)-syn-B[c]CDEs reacted with DNA (Figure 4D,J, respectively), as indicated by identical numbering. As was found for the adenine adducts of the B[g]CDEs (24), the adenine adducts of the B[c]CDEs generally showed greater mobilities than the guanine adducts in the TLC solvents employed in this study. Cochromatography of modified poly(dG‚dC) and poly(dA‚dT) samples indicated that those spots labeled with a suffix b in the poly(dG‚dC) samples (Figure 4B,E) comigrated with those corresponding spots (without a suffix) in the poly(dA‚ dT) samples (Figure 4C,F). The cochromatography results also indicated that (+)-syn-B[c]CDE gave rise to two DNA adduct spots (indicated by unnumbered arrows in Figure 4J) which were not present in the adduct maps of (+)-syn-B[c]CDE-modified poly(dG‚dC) (Figure 4K) or poly(dA‚dT) (Figure 4L). Similarly, DNA modified with (+)-anti-B[c]CDE showed one extra adduct spot (unnumbered in Figure 4G) not present in the modified polydeoxyribonucleotide adduct maps (Figure 4H,I). Also observed were two additional spots in the poly(dG‚dC) sample modified by (-)-anti-B[c]CDE (unnumbered spots, Figure 4B) which were not present in the DNA sample (Figure 4A). An additional adduct spot was observed in the poly(dA‚dT) sample modified by (+)-anti-B[c]CDE (Figure 4I) which was not present in the DNA sample modified by this diol epoxide (Figure 4G). 2. HPLC Analyses. Areas of interest on the TLC plates obtained following chromatography of the B[c]CDE-modified DNA, poly(dG‚dC), and poly(dA‚dT) samples (Figure 4A-L) were excised and eluted, and aliquots containing approximately the same quantity of radioac-

(-)-syn

(+)-anti

(+)-syn

DNA

23 24 25

retention times (min) poly(dG‚dC) poly(dA‚dT) 36.6 34.9 c

mixb 36.3 35.0

41.3 39.3 43.5 26.5 21.6 35.6 23.4 61.3 39.1 50.1

42.0 40.0 43.9 26.6 36.2

68.7 57.1 37.5 41.4 32.8 36.1 38.4 43.0 37.0

61.1 39.5 51.2 67.7 56.9 37.4 c 35.9 c

54.2 38.0

36.8 54.2 38.2

57.8

59.4

34.5 33.4

34.7 33.5

c c

a Adduct numbers correspond to those in Figure 4. b Mix refers to the coelution of the adduct formed with DNA and the corresponding adduct formed with either poly(dA‚dT) or poly(dG‚dC). c The eluted adduct spot did not contain sufficient radioactivity either to be detected by the HPLC detector or to be cochromatographed with another adduct.

tivity were examined both separately and together by HPLC. The retention times of the adducts shown in the autoradiograms in Figure 4 are given in Table 2, where they contained enough radioactivity to be detected by the HPLC detector, along with the retention times of mixes of pairs of adducts which were cochromatographed. The retention times have been normalized in this case to a UV marker retention time of 40 min. In Table 2, some pairs of spots are listed together (for example, spot 9 with spot 10) where they were eluted from their TLC plate together due to an unclear boundary between them. All of the numbered adduct spots detected in B[c]CDEmodified DNA were identified as adducts with either adenine or guanine or as a mixture of adducts with the two bases, as indicated in Table 2. Spots 6 and 7 of (-)anti-B[c]CDE-modified poly(dG‚dC) and spot 16 of (+)anti-B[c]CDE-modified poly(dG‚dC) each eluted as two peaks, one in each case with a retention time corresponding to that of the corresponding adduct formed by these diol epoxides in DNA, the other of which showed no equivalent in the DNA samples. Adduct spot 12 formed by (-)-syn-B[c]CDE with poly(dA‚dT) was found not to coelute with spot 12 from DNA which coeluted only with adduct 12b formed by this diol epoxide in poly(dG‚dC). A summary of the results of the in vitro study of the B[c]CDEs is given in Table 3. Where verification by HPLC of the base involved in adduct formation could not be made due to insufficient amounts of radioactivity in the samples, and the identity of the base has been deduced on the basis of TLC cochromatography experi-


Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1281

Table 3. Summary of the DNA Adducts Formed by Each B[c]CDE, Showing the Percentage Each Adduct Is of the Total Detected Reaction with DNA and the Identity of the DNA Base with Which the Adduct Was Formed diol epoxide (-)-anti

(-)-syn

(+)-anti

(+)-syn

adduct no.

% of total radioactivity

1 2 3 4 5 6 7 8 9 + 10 11 12 13 14 15 16 17 18 19 20 + 21 22 23 24 25

60 1 4 7 14 11 2 1 13 4 80 2 1 80 8 1 2 5 28 3 20 8 26

DNA base and % of total radioactivitya guanine adenine 60 1* 4 5*# 5# 11 2 1 7.5# 4 80

2# 9#

6.5# 2* 1*

80 8 1 26*# 3* 20*

2 5 2# 8 26

a An asterisk (*) indicates that the identity of the adduct has been postulated from the results of TLC cochromatography experiments only. A pound sign (#) indicates that the percentage of the total reaction with DNA represented by these adducts has been determined by comparison of the areas under their HPLC peaks.

ments alone, this has been indicated in Table 3 by an asterisk. The extent of reaction of each of the B[c]CDEs with adenine and guanine bases in DNA has been calculated as a percentage of the total reaction of each isomer with DNA, as was done for the B[g]CDEs in our earlier study (24). Where the adduct spot detected in the DNA samples was found to comprise an adenine adduct and a guanine adduct, the percentage that each base contributed to that adduct spot was estimated from the areas under the peaks of the two components when the material in that adduct spot was analyzed by HPLC. This is indicated in Table 3 by #. Thus it was found that (-)anti-, (-)-syn-, and (+)-anti-B[c]CDEs all reacted extensively with guanine bases in DNA, forming about 90% of total adducts with this base, compared with 10% of adducts with adenine bases (see also Figure 5). The reaction of (+)-syn-B[c]CDE with DNA was more evenly divided between the two bases, with at least 40% of total adducts formed with adenine residues. It should be noted that 15% of the total radioactivity detected on the (+)syn-B[c]CDE/DNA TLC plate and 4% of the total radioactivity on the (+)-anti-B[c]CDE/DNA TLC plate could not be assigned unequivocally to either guanine or adenine adducts from the chromatographic comparison of the modified DNA sample with the modified poly(dA‚ dT) and poly(dG‚dC) samples (Figure 5). Identification of the in Vivo B[c]C Adducts. Adducts formed in vitro which appeared to comigrate with adducts formed in DNA from mouse skin treated with B[c]C or one of the B[c]C-diols were chromatographed both singly and together on HPLC, and the results are summarized in Table 4. The retention times of adducts shown in Table 1 cannot be compared directly with those in Table 2 due to differences in column behavior in the two sets of analyses, demonstrated by changes in the

Table 4. Summary of the Nature of DNA Adducts Formed in B[c]C-Treated Mouse Skin and in Skin Treated with (-)- or (+)-B[c]C-dihydrodiols adduct letter

% of total B[c]C adducts

c d f fa gb ja ka mc

10 25 17 2

diol epoxide and DNA base with which adduct was formed (+)-syn (-)-anti (-)-anti (-)-syn (+)-syn (-)-anti (-)-anti (+)-syn

guanine/adenine guanine guanine/adenine guanine/adenine adenine guanine guanine guanine

a Adduct formed in mouse skin treated with (-)-B[c]C-dihydrodiol but not in skin treated with B[c]C. b The nature of the diol epoxide and the DNA base was deduced from the results of chromatography experiments involving (+)-B[c]C-dihydrodiol and (+)-syn-B[c]CDE. c Adduct formed in mouse skin treated with (+)B[c]C-dihydrodiol but not in skin treated with B[c]C.

elution time of the UV marker. When compared directly in coelution experiments, B[c]C adduct spots d and f (Figure 1A) were found to coelute with DNA adducts 1 and 5, respectively, formed by (-)-anti-B[c]CDE in vitro (Figure 4A). Thus, adduct d is formed with guanine and adduct spot f contains both guanine and adenine adducts. Adduct c formed in mouse skin treated with B[c]C was found to coelute with adduct 21 formed by (+)-syn-B[c]CDE in vitro, so it is a mixture of guanine and adenine adducts. Adduct a formed in mouse skin treated with B[c]C did not coelute with any of the adducts formed in vitro by the B[c]CDEs. Adduct g formed in mouse skin treated with (+)-B[c]C-diol was found to coelute with adduct 25 formed by (+)-syn-B[c]CDE in vitro, indicating that adduct g formed in skin treated with B[c]C may be due to the formation of this diol epoxide from the parent compound in vivo. Adduct f formed in mouse skin treated with (-)-B[c]C-diol eluted as two distinct peaks: one with a retention time of 52.0 min, which coeluted with (-)anti-B[c]CDE adduct 5 as described above, and the other with a retention time of 43.3 min, which coeluted with adduct 12 formed by (-)-syn-B[c]CDE. Adducts j and k formed in mouse skin treated with (-)-B[c]C-diol were found to coelute with adducts 6 and 8, respectively, formed by (-)-anti-B[c]CDE in vitro, and adduct m formed in skin treated with (+)-B[c]C-diol was found to coelute with adduct 23 formed by (+)-syn-B[c]CDE in vitro.

Discussion It is proposed that the weak carcinogenic potency of B[c]C on mouse skin, reported by Hartwell (14), is due to the metabolism of this compound predominantly to biologically unreactive metabolites. The results of the present study have shown that the maximum total level of adducts detectable in mouse epidermal DNA following topical treatment with B[c]C is very low (0.89 fmol/µg of DNA. In our previous study (10) it was observed that the maximum total level of adducts detectable in the DNA of skin from mice treated topically with B[g]C, a moderate carcinogen on mouse skin (14), was 6.55 fmol/ µg of DNA, a value 7-fold higher than that for B[c]Ctreated mice. Together, the results of the studies of B[g]C and B[c]C demonstrate a positive correlation between the carcinogenic potency of a PAH and the extent of DNA adduct formation, as initially reported by Brookes and Lawley (26). The maximum total level of DNA adducts formed in mouse skin by B[c]C is similar to that detected


in skin treated with B[c]Ph (0.24 fmol/µg of DNA) (10), which is also a weak carcinogen in mouse skin (12). The results of this study demonstrate that B[c]C is metabolically activated predominantly through formation of (-)-anti- (R,S,S,R-absolute configuration) and (+)-syn(S,R,S,R-absolute configuration) B[c]CDEs, indicating a high degree of stereoselectivity in the metabolism of the parent compound, as has been shown for B[g]C (10, 27), B[c]Ph (8), and dibenzo[a,l]pyrene (28), three other PAHs with fjord regions in their structures, and for 7,12dimethylbenz[a]anthracene (DMBA) (29) and B[a]P (1). However, adduct formation by (-)-B[c]C-diol in mouse skin was extensive (13.96 fmol/µg of DNA) when compared with the level of adducts formed following treatment with the parent compound (0.89 fmol/µg of DNA). Similarly, a high total level of DNA adducts formed in mouse skin treated topically with (+)-B[c]C-diol was observed (3.49 fmol/µg of DNA) in comparison with the total level of adducts formed in mouse skin treated with B[c]C. This latter observation may be explained, in part, by the absence of two adduct spots, m and n, from B[c]Ctreated skin DNA which accounted for 25% of the total adducts in the DNA from skin treated with (+)-B[c]Cdiol. The metabolism of the parent compound to the B[c]C-diols therefore appears to be limited, indicating that a different metabolic pathway to biologically unreactive metabolites predominates. However, a systematic study of the metabolic pathways of B[c]C has not, as yet, been carried out. An additional factor responsible for the low DNA adduct level formed by the parent hydrocarbon could be that B[c]C is only a weak inducer in mouse skin of the catalytically most competent cytochrome P450 isoforms for PAH metabolism, found in the cytochrome P450 1A and 1B families (30-33). This has recently been demonstrated to be the case for the structurally related PAH B[c]Ph in mouse epidermis (30), explaining the low level of adducts formed in this tissue (10, 34). Adduct spot a formed in the skin of mice treated topically with B[c]C was not identified in this study, as it did not cochromatograph with any of the B[c]CDE adducts being used as chromatography markers. It did, however, cochromatograph with adduct a formed in mice treated topically with (-)-B[c]C-diol. The observation that the parent compound was metabolized more extensively to the metabolite which formed adduct a than was (-)-B[c]C-diol is somewhat unusual and implies that these two adducts may not be the same despite the chromatography results. However, it is possible that both the parent compound and (-)-B[c]C-diol are metabolized to a common reactive metabolite that is not a fjord-region diol epoxide or is one that contains an additional metabolic modification. The latter was found to be the case for DNA adducts formed following metabolic activation of DB[a,h]A in mouse skin, where the major DNA-binding metabolite was found to be a bis(diol epoxide) (35). However, in the case of DB[a,h]A, these bis(diol epoxide) adducts were characteristically more polar and showed greater chromatographic mobility on PEI-cellulose than diol epoxide adducts (35). Since adduct a formed by B[c]C is less polar than the adducts formed by B[c]CDEs, the possibility that it is formed by a bis(diol epoxide) is less likely. Therefore, it seems more likely that adduct a formed from B[c]C represents a major adduct of a bay region B[c]CDE metabolically formed at the 1,2,3,4-position which comigrates with the faint spot a formed by the (-)-B[c]C-diol. This possibility is presently under investigation in our laboratory.

Giles et al.

The reactivities of the B[c]CDEs toward DNA in vitro have also been studied, and the total levels of DNA adducts measured were similar to those of the B[g]CDEs (24). As was the case for (-)-anti-B[g]CDE and (-)-antiB[c]PhDE (24), the (-)-anti-isomer of B[c]CDE was highly reactive toward DNA. However, the range of total adduct levels exhibited by the four B[c]CDEs was much lower than seen for the B[g]CDEs and B[c]PhDEs (24). Despite a comparable lower limit [27 pmol/µg of DNA, formed by (+)-syn-B[c]CDE], the highest level of adduct formation was significantly lower [41 pmol/µg of DNA, formed by both (-)-anti- and (+)-anti-B[c]CDEs] than that of the diol epoxides of B[g]CDE and B[c]PhDE (63 and 137 pmol/µg of DNA, respectively) (24). It is assumed from a great deal of previous work on PAH diol epoxides that adducts are formed overwhelmingly at the purines, rather than the pyrimidines, in poly(dG‚dC) and poly(dA‚dT) and hence in DNA. All four B[c]CDEs were found to react with both adenine and guanine residues in DNA, in keeping with the observations of sterically hindered diol epoxides of PAHs such as B[c]Ph (19, 36), B[g]C (24, 37, 38), 5,6-dimethylchrysene (39), and DMBA (40). The level of reaction with adenine bases was significantly lower than that seen for the B[g]CDEs and B[c]PhDEs. A study of the mutagenic potentials of the B[c]CDEs in the c-Ha-ras-1 protooncogene revealed that there was surprisingly little activation at the adenine residue of codon 61, in comparison with the extent of activation at guanine residues in codons 12, 13, and 61 (41). The authors suggested that this may be due to the fact that only activating mutations could be detected in the experimental system employed and that only one of the seven sites of ras activation is at an adenine residue. The results of the present study offer the explanation that reaction with guanine residues is preferential. The B[c]CDE isomer showing the greatest reaction with adenine in DNA, (+)-syn-B[c]CDE, shares the same stereochemistry, S,R,S,R-configuration, as the strongly adenine-reactive isomer of both B[g]CDE and B[c]PhDE (24). Interestingly, the study by Du et al. (41) of the mutagenic potentials of the B[c]CDEs in c-Ha-ras-1 demonstrated that the (+)-syn-B[c]CDE isomer was highly mutagenic. This contrasts with a study of the B[c]CDEs in Chinese hamster V79 cells, where the (()-antiB[c]CDEs were more mutagenic than the (()-syn-B[c]CDEs (15), but this may be due to an effect on mutagenic potential exerted by racemic mixtures. These authors related mutagenic potential to the extent of overall adduct formation, rather than to the formation of adducts with greater mutagenic potential. However, several investigators have noted that the mutagenic, and in particular the tumorigenic, potential of a diol epoxide correlates with the extent of reaction of the diol epoxide with adenine residues (28, 39, 42-44). This was particularly apparent in a recent study of the B[c]PhDEs (44), where (+)-syn-B[c]PhDE formed very few adducts with DNA when compared with the other three B[c]PhDE isomers, but 80% of these adducts were with adenine. Interestingly, this isomer was found to be similar in its mutagenic and tumorigenic activities to the two enantiomeric (+)- and (-)-anti-B[c]PhDEs which are among the most active PAH diol epoxides tested (6, 11, 15). However, the relationship between adenine adduct formation and mutagenic potential could only be made for the most adenine-reactive DE isomer of a PAH; otherwise, no correlation was noted (44).


The additional adducts detected either by TLC or by HPLC in samples of B[c]CDE-modified poly(dG‚dC) or poly(dA‚dT) not present in the DNA samples may be due to the sensitivity of the detection systems employed in this study. The detection of DNA adduct spots which could not be assigned to adducts in the modified polydeoxyribonucleotide samples is somewhat surprising. In the case of (+)-syn-B[c]CDE it may be explained by a poor reaction of the diol epoxide with poly(dG‚dC), as the detected adducts in this sample were all very weak. However, reaction of (+)-anti-B[c]CDE with poly(dG‚dC) was strong, and so its additional adduct spots cannot be explained in this way. From the results of the present study and those of our previous ones (10, 24), it would appear that it is the extent of metabolism of B[c]C and B[g]C to their fjordregion diol epoxides, via the precursor diols, that determines the subsequent extent of adduct formation in mouse skin, since the reactivities toward DNA of the B[g]CDEs and B[c]CDEs are comparable (24). In summary, we have shown that B[c]C is metabolically activated in mouse skin to the two fjord-region diol epoxides (-)-anti- and (+)-syn-B[c]CDEs, formed at the 9,10,11,12-position, and also to an unidentified metabolite, possibly a bay region diol epoxide formed at the 1,2,3,4-position. The lower overall level of DNA adducts detected in skin treated with B[c]C, as compared with that in skin treated with B[g]C, correlates with the lower carcinogenic potency reported for B[c]C in mouse skin compared with that for B[g]C (14).

Acknowledgment. A.S.G. gratefully acknowledges receipt of a Research Studentship from the Institute of Cancer Research. This work was supported, in part, by Grant EV5V-CT920213 from the Commission of the European Communities (DG XII).

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Covalent DNA Adducts Formed by Benzo[c]chrysene in Mouse

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