Analysis of Highly Polar DNA Adducts Formed in SENCAR Mouse

At the same dose of 400 nmol per mouse, DB[a,j]A and its 3,4-diol were able to initiate papillomas in SENCAR mouse skin (3.08 ± 1.89 and 3.48 ± 2.72...
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Chem. Res. Toxicol. 1999, 12, 60-67

Analysis of Highly Polar DNA Adducts Formed in SENCAR Mouse Epidermis Following Topical Application of Dibenz[a,j]anthracene Suryanarayana V. Vulimiri,*,† Wanda Baer-Dubowska,†,‡ Ronald G. Harvey,§ Jin-Tao Zhang,§ and John DiGiovanni† Department of Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, P.O. Box 389, Smithville, Texas 78957, and The Ben May Institute, University of Chicago, Chicago, Illinois 60637 Received June 15, 1998

The formation of DNA adducts in mouse epidermis has been examined following topical application of dibenz[a,j]anthracene (DB[a,j]A) and its metabolites, i.e., DB[a,j]A-3,4-diol, DB[a,j]A-3,4-10,11-bis-diol, DB[a,j]A-3,4-8,9-bis-diol, 10-OH-DB[a,j]A-3,4-diol, or 11-OH-DB[a,j]A3,4-diol, using a 32P-postlabeling assay. At initiating doses (400-1600 nmol), DB[a,j]A produced at least 23 DNA adduct spots, including four less polar (derived from the bay-region syn- and anti-diol-epoxides) and 19 highly polar DNA adducts. DB[a,j]A-3,4-diol produced 13 DNA adduct spots, four less polar and nine highly polar DNA adducts, and DB[a,j]A-3,4-10,11-bis-diol produced nine highly polar DNA adducts. Eight and seven of the highly polar DNA adducts generated by DB[a,j]A-3,4-diol and DB[a,j]A-3,4-10,11-bis-diol, respectively, migrated in the chromatography system like the highly polar DNA adducts produced by the parent compound. Sufficient amounts of radioactivity were associated with highly polar adduct spots 11, 13, and 22 to confirm their chromatogaphic identity in DNA samples from DB[a,j]A-, DB[a,j]A-3,4diol-, and DB[a,j]A-3,4-10,11-bis-diol-treated mice. 10-OH-DB[a,j]A-3,4-diol and 11-OH-DB[a,j]A-3,4-diol did not produce any highly polar DNA adducts that could be detected under our experimental conditions. At an initiating dose of 400 nmol, DB[a,j]A, DB[a,j]A-3,4-diol, and DB[a,j]A-3,4-10,11-bis-diol produced 22.4 ( 13.0, 15.6 ( 10.1, and 5.5 ( 0.3 (mean ( SD) adducts/109 nucleotides, of which 77, 65, and 100%, respectively, represented highly polar DNA adducts. At the same dose of 400 nmol per mouse, DB[a,j]A and its 3,4-diol were able to initiate papillomas in SENCAR mouse skin (3.08 ( 1.89 and 3.48 ( 2.72 papillomas per mouse, respectively, after 16 weeks of promotion with 12-O-tetradecanoyl phorbol 13-acetate), while the 3,4-10,11-bis-diol of DB[a,j]A was inactive as a tumor initiator. A quantitative correlation (r ) 0.935; p ) 0.0196) between levels of less polar DNA adducts and tumor-initiating activity of DB[a,j]A, DB[a,j]A-3,4-diol, and anti-DB[a,j]ADE was observed. This study demonstrates that the highly polar DNA adducts formed from DB[a,j]A in mouse epidermis arise primarily from the DB[a,j]A-3,4-10,11-bis-diol. However, the contribution of this metabolite to the tumorinitiating activity of DB[a,j]A appears to be small.

Introduction Dibenz[a,j]anthracene (DB[a,j]A1), one of the three possible isomeric dibenzanthracenes, is a common environmental carcinogen (1, 2). Human exposure to this compound occurs primarily through cigarette smoking, inhalation of polluted air, and ingestion of food and water contaminated with combustion effluents (1). DB[a,j]A (Chart 1, structure I) exerts its effects in biological systems only after metabolic activation to reactive intermediates similar to other polycyclic aromatic hydrocarbons (PAHs) (2). The interaction between such reactive intermediates and the nucleophilic centers in nuclear DNA results in the formation of covalent DNA adducts, * To whom correspondence and reprint requests should be addressed. † University of Texas M. D. Anderson Cancer Center. ‡ Present address: Department of Pharmaceutical Biochemistry, K. Marcinkowski’s University Medical School, ul. Grunwaldzka 6, 60-780 Poznan, Poland. § University of Chicago.

a process generally considered to lead to an initiating event in tumorigenesis (3, 4). For most PAHs, the ultimate carcinogenic metabolites are vicinal diol-epoxides, most frequently of the “bay-region” type (5). According to the bay-region theory (5), 3,4-diol-1,2epoxides (V and VI) metabolically generated from DB[a,j]A via the trans-3,4-diol (II) should be the ultimate carcinogenic metabolite(s) (6). Studies performed in our 1 Abbreviations: DB[a,j]A, dibenz[a,j]anthracene; DB[a,h]A, dibenz[a,h]anthracene; B[b]F, benzo[b]fluoranthene; PAH, polycyclic aromatic hydrocarbon; PEI, poly(ethyleneimine); (()-anti-DB[a,j]ADE, (()-trans3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,j]anthracene; (()-syn-DB[a,j]ADE, (()-trans-3,4-dihydroxy-syn-1,2-epoxy1,2,3,4-tetrahydrodibenz[a,j]anthracene; DB[a,j]A-3,4-diol, trans-3,4dihydroxy-3,4-dihydrodibenz[a,j]anthracene; DB[a,j]A-5,6-diol, trans5,6-dihydroxy-5,6-dihydrodibenz[a,j]anthracene; DB[a,j]A-3,4-10,11bis-diol, trans,trans-3,4,10,11-tetrahydroxy-3,4,10,11-tetrahydrodibenz[a,j]anthracene; DB[a,j]A-3,4-8,9-bis-diol, trans,trans-3,4,8,9-tetrahydroxy-3,4,8,9-tetrahydrodibenz[a,j]anthracene; 10-OH-DB[a,j]A-3,4diol, trans-3,4,10-trihydroxy-3,4-dihydrodibenz[a,j]anthracene; 11-OHDB[a,j]A-3,4-diol, trans-3,4,11-trihydroxy-3,4-dihydrodibenz[a,j]anthracene; PNK, polynucleotide kinase; dGuo, deoxyguanosine; dAdo, deoxyadenosine; TPA, 12-O-tetradecanoyl phorbol 13-acetate.

10.1021/tx980139b CCC: $18.00 © 1999 American Chemical Society Published on Web 12/11/1998

Dibenz[a,j]anthracene-DNA Adducts in Mouse Skin Chart 1. DB[a,j]A and the Metabolites Used in This Studya

a I, dibenz[a,j]anthracene (DB[a,j]A); II, DB[a,j]A-3,4-diol; III, DB[a,j]A-3,4-10,11-bis-diol; IV, DB[a,j]A-3,4-8,9-bis-diol; V, (()anti-DB[a,j]ADE; VI, (()-syn-DB[a,j]ADE; VII, 10-OH-DB[a,j]A3,4-diol; VIII, 11-OH-DB[a,j]A-3,4-diol; IX, DB[a,j]A-5,6-oxide; and X, DB[a,j]A-5,6-diol.

laboratory demonstrated that (()-anti-DB[a,j]A-diol-epoxide [(()-anti-DB[a,j]ADE] (V) was significantly more active than the parent compound as a skin tumor initiator, suggesting that the metabolic formation of this diol-epoxide and its covalent modification of DNA may be involved in the process of tumor initiation by DB[a,j]A (6, 7). The anti-diol-epoxide of DB[a,j]A binds extensively to both dGuo and dAdo residues (at a ratio of ∼3:1) when reacted in vitro with calf thymus DNA (8, 9). The major reaction products obtained with either racemic or pure (+)-anti-DB[a,j]ADE arise through trans addition of the exocyclic amino groups of dGuo and dAdo. Although treatment of cultured mouse keratinocytes with DB[a,j]A produced adducts with both dGuo and dAdo residues covalently bound to the (+)-enantiomer of the anti-diolepoxide, the dAdo adducts were predominant (10). The (+)-anti-DB[a,j]ADE-trans-N6-dAdo adduct was also found in mouse epidermal DNA after topical application of DB[a,j]A (11). The fact that c-Ha-ras mutations in skin tumors resulting from topical application of DB[a,j]A (12) revealed A182 f T transversions at codon 61 suggests an important role for dAdo adducts in tumorigenesis by this PAH. Finally, in studies in which the mutagenicity of site-specific DNA adducts was examined, the (+)-antiDB[a,j]ADE-trans-N6-dAdo adduct produced exclusively A f T transversions (13).

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 61

In earlier studies involving the analysis of DNA adducts formed in mouse epidermis after topical application of DB[a,j]A, we observed the formation not only of simple diol-epoxide DNA adducts but also of more polar covalent DNA adducts (11). These polar DNA adducts represented a significant proportion of the 32P-labeled material recovered in HPLC chromatograms and accounted for most of the DNA binding of DB[a,j]A. The existence of highly polar PAH-DNA adducts was also shown recently for another isomer of dibenzanthracene, DB[a,h]A (14-16), for several other PAHs (17), and for an N-heterocyclic aromatic (18). DB[a,h]A was shown to form highly polar DNA adducts derived from the 3,410,11-bis-diol-epoxide via 3,4-diol and 3,4-10,11-bis-diol formation (14-16). Benzo[b]fluoranthene (B[b]F), a ubiquitous environmental pollutant, forms major DNA adducts from the further metabolism of phenolic dihydrodiols, such as 5-OH-B[b]F-9,10-diol (19). Chrysene, another environmental carcinogen, has been shown to form polar DNA adducts, one of them being formed via the reaction of a triol-epoxide, i.e., 9-hydroxychrysene-1,2-diol 3,4oxide, with DNA (20). During a study with the Nheterocyclic PAH, dibenz[a,j]acridine, Talaska et al. (18) suggested that this carcinogen may be metabolically activated to dibenz[a,j]acridine-3,4-5,6-bis-diol-1,2-oxide via a bay-region diol-epoxide. In previous studies from our laboratory using HPLC (11), we showed that at least some of the polar DB[a,j]A-DNA adduct peaks formed in mouse skin had retention times similar to those of early-eluting DNA adduct peaks formed after topical application of DB[a,j]A-3,4-diol. This observation suggested indirectly that DB[a,j]A-bis-diol-epoxide metabolites may account for at least some of the polar DB[a,j]ADNA adducts formed in mouse epidermis. In this study, we examined the possibility that some or all of the highly polar DNA adducts formed in mouse skin after topical application of DB[a,j]A are derived from further metabolism of the bay-region bis-dihydrodiols or from the bay-region phenolic dihydrodiols of DB[a,j]A.

Materials and Methods Chemicals. Caution: DB[a,j]A and the derivatives of DB[a,j]A described within this paper have been determined to be carcinogenic in laboratory animals. Hence, protective clothing and appropriate safety procedures should be employed when working with these compounds. DB[a,j]A was provided by the NCI Chemical Carcinogen Reference Standard Repository, a section of the Division of Cancer Cause and Prevention, National Cancer Institute, NIH, Bethesda, MD. (()-DB[a,j]A-trans-3,4-diol was synthesized as described previously (21). DB[a,j]A-trans-5,6-diol (Chart 1, X) and DB[a,j]A-5,6-oxide (IX) were synthesized as previously described (22, 23). Syntheses of DB[a,j]A-3,4-10,11-bis-diol (III), DB[a,j]A-3,4-8,9-bis-diol (IV), 10-OH-DB[a,j]A-3,4-diol (VII), and 11-OH-DB[a,j]A-3,4-diol (VIII) have been published (24, 25). RNase A (EC 3.1.4.22) was obtained from Worthington Biochemical Co. (Freehold, NJ), and micrococcal nuclease (Staphylococcus aureus, EC 3.1.4.1), apyrase, calf spleen phosphodiesterase (EC 3.1.16.1), and 3′-phosphatase-free T4 polynucleotide kinase (PNK) (EC 2.7.1.78) were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Guanidine isothiocyanate was obtained from Bethesda Research Laboratories Inc. (Gaithersburg, MD). Carrier-free [γ-32P]ATP (specific activity of 6000 Ci/mmol) was purchased from Du Pont NEN (Boston, MA). Macherey-Nagel poly(ethylene)imine (PEI)-cellulose TLC plates were obtained from Bodman (Aston, PA). 12-O-Tetradecanoylphorbol 13-acetate (TPA) was obtained from LC Services (Wobum, NY).

62 Chem. Res. Toxicol., Vol. 12, No. 1, 1999 Tumor Experiment. Female SENCAR mice were obtained from the National Cancer Institute (Frederick, MD) and shaved on the dorsal side when they were 6-7 weeks of age. Mice were allowed to stabilize for at least 2 days, and only those mice in the resting phase of the hair growth cycle were used for the experiment. The mice (30 per group) were initiated topically with 400 nmol of either DB[a,j]A, DB[a,j]A-3,4-diol, or DB[a,j]A3,4-10,11-bis-diol in 0.2 mL of acetone or received only the vehicle. Two weeks after initiation, mice began receiving twice a week treatments with 3.4 nmol of TPA in 0.2 mL of acetone. The incidence and number of papillomas were observed and recorded weekly. Promotion was continued in all groups until the average number of papillomas per mouse reached a plateau. Statistical analyses of the difference between mean papilloma responses (i.e., papillomas per mouse) were performed using the Mann-Whitney U-test. The level of significance was set at p < 0.05. DNA Adduct Formation in Mouse Epidermis. Female SENCAR mice (three or four animals per group) 6-7 weeks of age received a single topical application of one of the following compounds: DB[a,j]A, DB[a,j]A-3,4-diol, DB[a,j]A-3,4-10,11-bisdiol, DB[a,j]A-3,4-8,9-bis-diol, DB[a,j]A-5,6-oxide, DB[a,j]A-5,6diol, 10-OH-DB[a,j]A-3,4-diol, or 11-OH-DB[a,j]A-3,4-diol in 0.2 mL of acetone or acetone/tetrahydrofuran (40:60, v/v). Control animals received only the treatment vehicle. DB[a,j]A and DB[a,j]A-3,4-diol were applied topically at several different doses within the dose-response range for initiation (400-1200 nmol/ mouse) as indicated. (()-syn-DB[a,j]ADE and (()-anti-DB[a,j]ADE were applied at a dose of 400 nmol/mouse (11), and the rest of the compounds were applied at a dose of 1600 nmol/ mouse. Twenty-four hours after treatment, animals were sacrificed and epidermal DNA was isolated as described previously (11). The DNA concentration was measured spectrophotometrically assuming 1 mg of DNA equals 20 A260 units. Typically, the DNA samples showed a 260/280 ratio of 1.8-1.9. The yield of epidermal DNA was ∼0.1-0.2 mg/mouse. The DNA was stored at -70 °C until it was used. 32P-Postlabeling and Chromatography of 32P-Labeled DNA Adducts. Approximately 5-10 µg of mouse epidermal DNA or calf thymus DNA reacted in vitro with (()-syn- or (()anti-DB[a,j]ADE was analyzed for DNA adducts by the nuclease P1-enhanced 32P-postlabeling assay (26). Briefly, DNA was digested in a volume of 10 µL to 3′-monophosphates using micrococcal nuclease (0.04 unit/µL) and spleen phosphodiesterase (0.4 µg/µL) in the presence of 10 mM CaCl2 and 30 mM sodium succinate buffer (pH 6) for 3.5 h. Normal nucleotides from the digest were dephosphorylated by digesting further with nuclease P1 (0.55 µg/µL) in the presence of 0.06 M sodium acetate (pH 5) and 0.11 mM ZnCl2 for 45 min. The adducted nucleotides were then labeled with 150 µCi of [γ-32P]ATP (specific activity of 6000 Ci/mmol) and 10 units of 3′-phosphatase-free T4 PNK using a labeling buffer [200 mM bicine, 100 mM dithiothreitol, 100 mM MgCl2, and 11 mM spermidine (pH 7.5)] for 45 min. Unreacted ATP was converted to 32Pi by incubating the labeled digest further with 60 milliunits of apyrase at 37 °C for 30 min. A 2 µL aliquot of the labeled digest was diluted to 500 µL with 20 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 9.5), and a 5 µL portion was spotted onto a 20 cm × 20 cm PEI-cellulose sheet and chromatograhed one-dimensionally in 0.28 M (NH4)2SO4 and 50 mM NaH2PO4 (pH 6.8) to determine (i) the complete removal of normal nucleotides and (ii) the presence of excess ATP in the labeling reaction. Calf thymus DNA reacted with (()-benzo[a]pyrene-7,8-diol 9,10-epoxide was used in each labeling experiment as a benchmark to control the labeling efficiency and normalization of data. The specific activity of [γ-32P]ATP was calculated by labeling 1 µM 2′-deoxyadenosine 3′-monophosphate with 1 µM [γ-32P]ATP (26). Chromatography of DNA adducts was performed using a solvent system previously described (27) with the following modifications. Briefly, 32P-labeled DNA digests were spotted onto the top right-hand corner of a 20 cm × 20 cm PEI-cellulose

Vulimiri et al. TLC sheet (5 cm each the from top and right edges) and chromatographed in the first direction (D1, top to bottom) overnight in 1.7 M NaH2PO4 (pH 6) onto a 5 cm × 20 cm Whatman 17 Chr wick. After D1, chromatography sheets were trimmed 3 cm each above and to the right side of the origin and washed for 10 min in 1 L of deionized water. After air-drying, the chromatography sheets were turned upside down and run (bottom to top) in D3 solvent [4 M lithium formate and 7 M urea (pH 3.35)]. This was follwed by development (left to right) in D4 solvent [0.5 M Tris-HCl, 0.5 M boric acid, 0.5 M MgCl2, and 5 M urea (pH 8)]. In both D3 and D4 dimensions, the chromatography sheets were predeveloped in deionized water 1 cm above the bottom edge before the actual solvent development. The chromatography sheets were washed twice for 7 min in 1 L of deionized water after D3 and D4 and airdried. Finally, the chromatography sheets were developed in D5 (left to right) in 1.7 M NaH2PO4 (pH 6) overnight onto an 8 cm × 17 cm Whatman 17 Chr wick. After D5, wicks were cut off and chromatography sheets were soakded in deionized water for 10 min and air-dried. Chromatography sheets were exposed to Kodak X-Omat X-ray film with intensifying screens at -70 °C for 48 h. Chromatography sheets were scanned using Packard Instant Imager (Downers Grove, IL), and DNA adduct levels were calculated by dividing the adduct counts per minute with the specific activity of [γ-32P]ATP and the amount of DNA (picomoles of DNA-P) used (26). Re- and Cochromatography of Selected Highly Polar Adducts from DB[a,j]A, DB[a,j]A-3,4-diol, and DB[a,j]A3,4-10,11-bis-diol. For re- and cochromatography, 32P-labeled adduct spots (11, 13-15, and 22) from two-dimensional TLC maps from DB[a,j]A-, DB[a,j]A-3,4-diol-, and DB[a,j]A-3,4-10,11bis-diol-treated mouse epidermal DNA were extracted with 2-propanol/6 N ammonia as described previously (28). Aliquots (∼50-100 cpm) of extracts of selected spots to be compared between the parent compound and the 3,4-diol and 3,4-10,11bis-diol were applied individually or mixed in a 1:1 proportion with PEI-cellulose sheets and cochromatographed in three solvents: 0.56 M NaH2PO4, 0.35 M Tris-HCl, and 5.95 M urea (pH 8.2) (solvent I); 0.49 M NaH2PO4 and 4.9 M urea (pH 6.4) (solvent II); and 2-propanol/4 N ammonia (1:1) (solvent III). The adduct spots were detected by screen-enhanced autoradiography.

Results Analysis of DNA Adducts Formed Following Topical Application of DB[a,j]A and Its Metabolites to SENCAR Mouse Epidermis. Twenty-four hours following topical treatment of SENCAR mice with either DB[a,j]A or its metabolites, epidermal DNA was isolated, digested, and subjected to 32P-postlabeling analysis. Thinlayer chromatograms of the DNA adduct spots obtained from these digests are shown in Figure 1. On the basis of their migration in polar solvents, the adduct spots were designated as either less polar adducts (spots 1-4 migrating close to the origin; primarily the bay-region diol-epoxide DNA adducts) or highly polar adducts (spots 5-26 migrating farther away from the origin). The parent compound produced 23 DNA adduct spots (panel B), four of which were less polar adducts (1-4) and 19 of which were highly polar adducts (5-23). The epidermal DNA from vehicle-treated controls is shown in panel A. DNA from mice treated with DB[a,j]A-3,4-diol consistently gave rise to 13 DNA adduct spots (panel C), four of which were less polar adducts (1-4) and nine of which were highly polar adducts (6, 11, 13-16, 21, 22, and 24). Twelve of these DNA adduct spots, i.e., 1-4, 6, 11, 1316, 21, and 22 (circled in panel C), corresponded to the DNA adduct spots in the profile generated by the parent compound (panel B). Analysis of adducts in DNA samples

Dibenz[a,j]anthracene-DNA Adducts in Mouse Skin

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 63

Figure 1. Typical autoradiograms showing 32P-labeled DNA adducts formed in SENCAR mouse epidermis following a single treatment with DB[a,j]A or its metabolites: (A) vehicle, (B) DB[a,j]A (1600 nmol), (C) DB[a,j]A-3,4-diol (800 nmol), (D) DB[a,j]A-3,4-10,11-bisdiol (1600 nmol), (E) DB[a,j]A-3,4-8,9-bis-diol (1600 nmol), (F) 10-OH-DB[a,j]A-3,4-diol (1600 nmol), (G) 11-OH-DB[a,j]A-3,4-diol (1600 nmol), (H) DB[a,j]A-5,6-diol (1600 nmol), and (I) DB[a,j]A-5,6-oxide (1600 nmol). 32P-labeled DNA digests were chromatographed as described in Materials and Methods. The origin is located at the bottom left-hand corner of each chromatogram. Chromatogram gels were exposed to Kodak X-Omat X-ray film with intensifying screens for 48 h at -70 °C.

from DB[a,j]A-3,4-10,11-bis-diol-treated mice revealed nine DNA adduct spots (panel D), and seven of these spots, i.e., 11, 13-15, 20, 22, and 23 (circled in panel D), migrated in the chromatography system like DNA adduct spots produced by the parent compound in mouse epidermis. One of the highly polar adduct spots (20) produced by the parent compound (panel B) was not detected in DNA from mice treated with the 3,4-diol (panel C), while a minor adduct (spot 26) formed by the 3,4-10,11bis-diol (panel D) was not produced by the 3,4-diol (panel C). The presence of spot 26 in the chromatogram of DNA from DB[a,j]A-treated mice (panel B) could not be determined due to higher background in all of these chromatograms. The DB[a,j]A-3,4-8,9-bis-diol produced rela-

tively few DNA adduct spots (panel E). 32P-Postlabeling analyses following treatment with either 10-OH-DB[a,j]A-3,4-diol or 11-OH-DB[a,j]A-3,4-diol revealed no detectable polar DNA adduct spots from either phenolic dihydrodiol (panels F and G, respectively). However, 11OH-DB[a,j]A-3,4-diol generated some less polar DNA adducts that migrated like adduct spots produced following topical application of the parent compound (Figure 1B) and DB[a,j]A-3,4-diol (Figure 1C). In other experiments, two other potential metabolites of DB[a,j]A were examined: DB[a,j]A-5,6-diol and DB[a,j]A-5,6-oxide. These metabolites were also applied topically to SENCAR mice and epidermal DNA samples isolated 24 h later. Following application of DB[a,j]A-5,6-diol, two major polar DNA

64 Chem. Res. Toxicol., Vol. 12, No. 1, 1999

Vulimiri et al. Table 1. Formation of DNA Adducts in Epidermis following Topical Application of DB[a,j]A and Its Metabolites to SENCAR Mice DNA adducts/109 nucleotidesa treatment

less polar adductsb

highly polar adductsc

total adducts

acetone DB[a,j]A DB[a,j]A-3,4-diol DB[a,j]A-3,4-10,11-bis-diol DB[a,j]A-3,4-8,9-bis-diole 10-OH-DB[a,j]A-3,4-diole 11-OH-DB[a,j]A-3,4-diole (()-anti-DB[a,j]ADE (()-syn-DB[a,j]ADE

5.1 ( 2.0 5.5 ( 4.1 NDf ND ND 2.1 70.0 ( 18.3 11.0 ( 0.1

17.3 ( 11.2 10.1 ( 6.1 5.5 ( 0.3 0.6 ND ND ND ND

22.4 ( 13.0d 15.6 ( 10.1 5.5 ( 0.3 0.6 ND 2.1 70.0 ( 18.3 11.0 ( 0.1

a Mean ( SD of two or three experiments determined at a dose of 400 nmol. b Less polar DNA adducts, located closer to the origin of the thin-layer chromatogram that are considered primarily the bay-region diol-epoxide-DNA adducts (1-4). c Highly polar adducts, adducts which move away from the origin (5-26). d DB[a,j]A produced 44.4 ( 23.1 adducts/109 nucleotides at a dose of 1600 nmol. e Single determination at a dose of 1600 nmol/mouse. f ND, not detected.

Figure 2. Typical autoradiograms showing 32P-labeled DNA adducts formed in vivo (SENCAR mouse epidermis) and in vitro (calf thymus DNA) by DB[a,j]A-diol-epoxides: (A) (()-anti-DB[a,j]ADE and mouse epidermis, (B) (()-syn-DB[a,j]ADE and mouse epidermis, (C) (()-anti-DB[a,j]ADE in vitro, and (D) (()syn-DB[a,j]ADE in vitro. For the in vivo experiments, mice received a single topical application of 400 nmol of either diolepoxide. Chromatography conditions are described in Materials and Methods. The origin is located at the bottom left-hand corner of each chromatogram. Chromatograms were exposed to Kodak X-Omat X-ray film with intensifying screens either for 48 h at -70 °C (panels A and B) or for 5 min at room temperature (panels C and D). A1-A6 denote anti-DB[a,j]ADEDNA adducts; S1-S4 denote syn-DB[a,j]ADE-DNA adducts.

adduct spots were observed, which did not correspond to any of the spots produced by the parent compound in vivo (panel H). Treatment with the DB[a,j]A-5,6-oxide produced relatively few, less polar adducts (panel I). The less polar DNA adducts produced by the parent compound, i.e., spots 1-4, displayed chromatographic mobilities similar to those of the corresponding DB[a,j]ADE adducts generated either in vitro or in vivo, as shown in Figure 2. The (()-anti-DB[a,j]ADE produced three major DNA adducts (spots A1, A3, and A4) and three minor adducts (spots A2, A5, and A6) in SENCAR mouse epidermis after topical application (panel A), five of which had a pattern of mobility similar to those of adducts generated by reacting calf thymus DNA with (()anti-DB[a,j]ADE in vitro (panel C). The (()-syn-DB[a,j]ADE produced two major adducts (S3 and S4) and two minor adduct spots (S1 and S2) in mouse epidermis (panel B). This profile was similar to the adduct profile generated by reacting calf thymus DNA with the (()-synDB[a,j]ADE (panel D). Notably, the enantiomeric bayregion diol-epoxides of DB[a,j]A did not produce any highly polar adducts either in vitro or in vivo (again, see Figure 2). Quantitation of DNA Adduct Formation by DB[a,j]A or Its Metabolites in SENCAR Mouse Epidermis. As shown in Table 1, at an initiating dose of 400 nmol, DB[a,j]A, DB[a,j]A-3,4-diol, and DB[a,j]A-3,4-10,11bis-diol produced 22.4 ( 13.0, 15.6 ( 10.1, and 5.5 ( 0.3 (mean ( SD) adducts/109 nucleotides, of which 77, 65, and 100%, respectively, represented highly polar DNA

adducts. At a dose of 1600 nmol/mouse, DB[a,j]A-3,4-8,9bis-diol produced very low levels of covalent DNA adducts (0.6 adduct/109 nucleotides), all of which were highly polar DNA adducts, while the total adducts produced by the parent compound at this dose were 44.4 ( 23.1 adducts/109 nucleotides. In contrast, 10-OH-DB[a,j]A-3,4diol did not produce any detectable DNA adducts, while 11-OH-DB[a,j]A-3,4-diol produced only very low levels of less polar DNA adducts (2.1 adducts/109 nucleotides). For comparison, the bay-region diol-epoxides, i.e., (()-antiand (()-syn-DB[a,j]ADE, were also applied topically at a dose of 400 nmol. Total covalent DNA adduct levels from these compounds were 70.0 ( 18.3 and 11.0 ( 0.1 adducts/109 nucleotides, respectively, and all of the adducts detected were less polar in nature. Thus, (()anti-DB[a,j]ADE produced ∼6.4-fold higher levels of less polar DNA adducts than (()-syn-DB[a,j]ADE and an ∼13.7-fold higher level of less polar DNA adducts compared to the parent compound, DB[a,j]A. Re- and Cochromatography of Highly Polar DNA Adducts from DB[a,j]A-, DB[a,j]A-3,4-diol-, and DB[a,j]A-3,4-10,11-bis-diol-Treated Mouse Epidermal DNA. Five of the highly polar DNA adducts (11, 13-15, and 22) formed following treatment with DB[a,j]A, also present in the epidermal DNA of mice treated with the 3,4-diol and 3,4-10,11-bis-diol, were further analyzed. To confirm that the chromatographic properties of these adducts were identical, selected 32P-labeled DNA adduct fractions were extracted from thin-layer chromatograms of DNA from DB[a,j]A, DB[a,j]A-3,4-diol, and DB[a,j]A3,4-10,11-bis diol-treated mice and subjected to re- and cochromatography after mixing two adduct samples at an ∼1:1 ratio. As shown in Figure 3, two of the adduct fractions, viz., spots 11 and 13, formed by the parent compound were chromatographically identical to the corresponding fractions generated by the 3,4-diol (columns A and C, respectively) and the 3,4-10,11-bis-diol (columns B and D, respectively) in three different solvents (rows I-III). Two other adduct fractions, spots 14 and 15, were chromatographically identical between the parent compound and the 3,4-diol (columns E and F, respectively). However, the chromatographic identity of these fractions could not be demonstrated between the parent compound and the 3,4-10,11-bis-diol due to insufficient radioactivity. Another highly polar adduct fraction

Dibenz[a,j]anthracene-DNA Adducts in Mouse Skin

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 65

Figure 4. Tumor initiating activity of DB[a,j]A or its metabolites in SENCAR mouse skin. Female SENCAR mice (30 per group) 7-9 weeks of age were initiated with a single topical application of vehicle alone (0.2 mL of acetone) or 400 nmol of DB[a,j]A, DB[a,j]A-3,4-diol, or DB[a,j]A-3,4-10,11-bis-diol in acetone. After 2 weeks, all of the groups were promoted with 3.4 nmol of TPA in acetone applied twice a week.

(spot 22) produced by the parent compound in vivo was also chromatographically identical with the corresponding adduct spot generated by the 3,4-diol and the 3,410,11-bis-diol (data not shown). Analysis of the Tumor-Initiating Activity of DB[a,j]A-3,4-10,11-bis-diol. Since the 3,4-10,11-bis-diol produced several polar DNA adducts similar to those found in DNA samples from mice treated with either the parent compound or the 3,4-diol, the tumor-initiating activity of 3,4-10,11-bis-diol was examined at a single dose (400 nmol) in SENCAR mouse epidermis. Additional groups were initiated with either the DB[a,j]A-3,4-diol or the parent compound DB[a,j]A at the same dose. As shown in Figure 4, the tumor-initiating activity of DB[a,j]A and DB[a,j]A-3,4-diol was approximately equal (3.08 ( 1.89 vs 3.48 ( 2.72 papillomas/mouse, respectively). These data are very similar to our previously published findings (6). Notably, at 400 nmol, DB[a,j]A3,4-10,11-bis-diol did not possess any detectable skin tumor-initiating activity in SENCAR mice.

Discussion Figure 3. Re- and cochromatography of selected DNA adduct spots formed by DB[a,j]A, DB[a,j]A-3,4-diol, and DB[a,j]A-3,410,11-bis-diol in vivo. Approximately 50-100 cpm of each adduct spot or mixture of two spots generated by mixing the adduct spot from DB[a,j]A (Figure 1B) with the corresponding adduct fraction produced in mouse epidermis from either DB[a,j]A-3,4diol (Figure 1C) or DB[a,j]A-3,4-10,11-bis-diol (Figure 1D) was chromatographed one-dimensionally in various solvents. Row I, 0.56 M NaH2PO4, 0.35 M Tris-HCl, and 5.95 M urea (pH 8.2). Row II, 0.49 M NaH2PO4 and 4.9 M urea (pH 6.4). Row III, 2-propanol/4 N ammonia (1:1). Columns A and B, spot 11; columns C and D, spot 13; column E, spot 14; column F, spot 15. In each column, the spotting is as follows: (a) adduct fraction from DB[a,j]A (all columns), (c) adduct fraction from either DB[a,j]A-3,4-diol (columns A, C, E, and F) or DB[a,j]A-3,4-10,11bis-diol (columns B and D), and (b) contents of lanes a and c mixed at a ratio of 1:1 and spotted. Autoradiography was carried out for 64-72 h at -70 °C using Kodak X-Omat X-ray film with intensifying screens. O denotes the origin; F denotes the solvent front.

This study has further examined the nature of the polar DNA adducts produced in mouse epidermal DNA following topical application of DB[a,j]A. In previous studies from our laboratory, topical application of DB[a,j]A to mouse epidermis produced a complex profile of DNA adducts (11). In this study, we analyzed a series of potential metabolites of DB[a,j]A for their ability to produce DNA adducts to further determine the origin of both less polar and highly polar DNA adducts derived from DB[a,j]A. The major findings of this study are as follows. (i) The highly polar DNA adducts produced in mouse epidermis following topical application of DB[a,j]A were tentatively identified as arising primarily from the 3,4-10,11-bis-diol. (ii) DB[a,j]A-3,4-8,9-bis-diol and the 10OH- and 11-OH-diols produced only low levels of DNA adducts when topically applied at doses equivalent to that of DB[a,j]A. (iii) Topical application of the 5,6-diol of DB-

66 Chem. Res. Toxicol., Vol. 12, No. 1, 1999

[a,j]A produced two major highly polar DNA adduct spots in TLC chromatograms that did not correspond to any of the highly polar DNA adducts derived from the parent compound. (iv) DB[a,j]A-3,4-10,11-bis-diol did not possess skin tumor-initiating activity at a dose of 400 nmol in SENCAR mice. (v) An excellent correlation was observed between levels of less polar DNA adducts (i.e., those derived from bay-region diol-epoxides) and skin tumorinitiating activity of DB[a,j]A, DB[a,j]A-3,4-diol, DB[a,j]A3,4-10,11-bis-diol, and (()-anti-DB[a,j]ADE. Overall, these results indicate that while a significant proportion of the polar DNA adducts detected in mouse skin appear to arise from DB[a,j]A-3,4-10,11-bis-diol, the contribution of these adducts to the tumor-initiating activity of DB[a,j]A may be relatively small. Recent studies have implicated bis-diol-epoxide metabolites in the metabolic activation of DB[a,h]A to DNA binding species in mouse skin (14-16). In this regard, topical application of the 3,4-10,11-bis-diol of DB[a,h]A to mouse skin produced a single major DNA adduct spot (assessed by 32P-postlabeling) that comigrated on TLC with the major DNA adduct spot produced by topical application of either DB[a,h]A or DB[a,h]A-3,4-diol. In contrast to that of DB[a,h]A, topical application of DB[a,j]A produced a complex profile of DNA adducts in mouse epidermis as detected with a 32P-postlabeling assay (Figure 1). DB[a,h]A, on the other hand, has been shown to produce relatively few DNA adducts in mouse skin as assessed by 32P-postlabeling analysis (14). This may be due to the fact that DB[a,j]A is extensively metabolized in microsomes compared to the other two isomers, i.e., DB[a,h]A and dibenz[a,c]anthracene (29, 30). A majority of the DNA adducts (∼77%) formed following topical application of DB[a,j]A were qualitatively (Figure 1) and quantitatively (Table 1) polar in nature which is consistent with our earlier work (11). In our previous study, we noted that a significant portion of the polar DNA adducts derived from DB[a,j]A comigrated with DNA adducts derived from DB[a,j]A-3,4-diol. As shown in Figure 1, we found that 12 DNA adduct spots formed from DB[a,j]A-3,4-diol comigrated with similar DNA adduct spots derived from the parent compound. Notably, further analysis at three highly polar DNA adduct spots formed from the 3,4-10,11-bis-diol showed chromatographic identity with the highly polar adducts derived from the parent compound. Thus, although the DNA adduct profiles are more complex than that with DB[a,h]A, it appears that a significant proportion of the DNA adducts produced in mouse epidermis after topical application of DB[a,j]A do arise from the 3,410,11-bis-diol metabolite, i.e., the bay-region bis-diol similar to DB[a,h]A. As part of this study, we also examined the possibility that other metabolites, including other potential bis-diol metabolites, were involved in the metabolic activation of DB[a,j]A to DNA binding species. Earlier studies on the metabolism of DB[a,j]A by Lecoq et al. (30) suggested that the major polar metabolite of DB[a,j]A formed in 3-methylcholanthrene-induced rat liver microsomal preparations was a 3,4-8,9-bis-diol which is probably formed via a 3,4diol or a 5,6-diol of DB[a,j]A. In studies from our laboratory in primary cultures of mouse keratinocytes, both the 3,4-diol and the 5,6-diol of DB[a,j]A were formed, the latter being the major intracellular metabolite produced, supporting this possibility (31). However, analysis of this potential in vivo metabolite revealed the formation

Vulimiri et al.

of only very low levels of adducts (∼74-fold lower than that of DB[a,j]A at a dose of 1600 nmol), none of which comigrated with DNA adduct spots from the parent compound, the 3,4-diol or the bis-diol. Thus, the 3,4-8,9bis-diol of DB[a,j]A appears to be of little importance in the metabolic activation of DB[a,j]A to DNA binding species in mouse epidermis in vivo. Triols have also been shown to be intermediates in the metabolic activation of certain PAHs (17). For example, chrysene, an environmental contaminant and weak carcinogen, has been shown to form a triol epoxide in a rat hepatic microsomal metabolizing system in the presence of chrysene-1,2-diol or 3-hydroxychrysene (18). We tested the possibility of the involvement of two potential phenolic dihydrodiol metabolites in the metabolic activation of DB[a,j]A to DNA binding species in mouse epidermis. In this regard, 10-OH-DB[a,j]A-3,4-diol and 11-OH-DB[a,j]A-3,4-diol yielded very low or undetectable levels of DNA adducts, and neither compound yielded highly polar DNA adducts in mouse epidermis. Finally, we also examined the possible formation of DNA adducts following topical application of either DB[a,j]A-5,6-diol or DB[a,j]A-5,6oxide. The 5,6-diol was of interest because of the possible further metabolism of this metabolite as noted above. The 5,6-oxide was of interest because earlier work from our laboratory using cultured mouse keratinocytes showed that a major DNA adduct formed from DB[a,j]A in these cells arose from this metabolite (10). However, although DB[a,j]A-5,6-diol generated two major highly polar DNA adducts in mouse epidermis, these adducts were totally different from the highly polar DNA adducts generated by the parent compound or any of the other DB[a,j]A metabolites tested (Figure 1). In addition, the 5,6-oxide produced only less polar DNA adducts at very low levels which did not appear to correspond to any less polar DNA adducts derived from DB[a,j]A (Figure 1). Thus, all of the above results suggest little involvement of the 3,48,9-bis-diol, 10-OH-3,4-diol, 11-OH-3,4-diol, 5,6-diol, and 5,6-oxide in the metabolic activation of DB[a,j]A to DNA binding species in mouse epidermis in vivo. As part of this study, we examined the tumor-initiating activity of DB[a,j]A-3,4-10,11-bis-diol at a dose equivalent to that of both DB[a,j]A and DB[a,j]A-3,4-diol. Previous work from our laboratory demonstrated that the bayregion 3,4-diol of DB[a,j]A was approximately equipotent with the parent compound as a tumor initiator in SENCAR mice (6). In contrast, the (()-anti-DB[a,j]ADE was 2.8-5.8 times more active as a tumor initiator than the parent compound DB[a,j]A, depending on the dose and solvent used for initiation (6). As shown in Figure 4, the 3,4-10,11-bis-diol was essentially inactive as a skin tumor initiator when tested at a dose of 400 nmol in SENCAR mice. These data suggest that the DNA adducts formed from the 3,4-10,11-bis-diol metabolite may not play a major role in the tumor-initiating activity of DB[a,j]A. To further explore this possibility, we correlated the levels of less polar versus highly polar DNA adducts in Table 1 with the tumor-initiating activity of DB[a,j]A, DB[a,j]A-3,4-diol, and DB[a,j]A-3,4-10,11-bis-diol shown in Figure 4; papilloma data for the (()-anti-DB[a,j]ADE were taken from ref 6. For this analysis, the average number of papillomas per mouse at 16 weeks was used. This analysis revealed the interesting observation that the level of less polar DNA adducts (i.e., those adducts migrating near the origin in TLC chromatograms; those primarily derived from the bay-region diol-epoxides)

Dibenz[a,j]anthracene-DNA Adducts in Mouse Skin

positively correlated (r ) 0.935; p ) 0.0196) with the tumor-initiating activity of the four compounds while levels of the highly polar adducts did not (r ) 0.041; p ) 0.9484). This latter analysis supports the hypothesis that the less polar DNA adducts (i.e., those derived from simple bay-region epoxides) are primarily responsible for the tumor-initiating activity of DB[a,j]A. In conclusion, DB[a,j]A is extensively metabolized in mouse epidermis to form a number of both less polar and highly polar DNA adducts. The majority of these DNA adducts appear to arise from the bay-region diol-epoxides and the bay-region bis-diol-epoxides, respectively. However, further analysis of the data obtained in this study suggests that the latter DNA adducts play a minor role in the tumor-initiating activity of DB[a,j]A in SENCAR mice. These observations support our earlier hypothesis that the bay-region diol-epoxides are the ultimate carcinogenic metabolites of DB[a,j]A (6, 7).

Chem. Res. Toxicol., Vol. 12, No. 1, 1999 67

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Acknowledgment. This research was supported by U.S. Public Health Service Grant CA36979 (J.D.), NIEHS Center Grant P30 ES07784-02, MDA Core Grant CA 16672, NCI Grant CA67937 (R.G.H.), and ACS Grant CN-22 (R.G.H.). A portion of this work was presented at the 88th Annual Meeting of the American Association for Cancer Research (abstract 2289) held in San Diego, CA, in April 1997.

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