Identification and Quantification of Stable DNA Adducts Formed from

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Chem. Res. Toxicol. 2005, 18, 984-990

Identification and Quantification of Stable DNA Adducts Formed from Dibenzo[a,l]pyrene or Its Metabolites in Vitro and in Mouse Skin and Rat Mammary Gland Rosa Todorovic, Prabu Devanesan, Eleanor Rogan, and Ercole Cavalieri* Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805 Received November 15, 2004

The stable adducts of dibenzo[a,l]pyrene (DB[a,l]P) formed by rat liver microsomes in vitro were previously quantified, whereas the depurinating adducts were both identified and quantified [Li, et al. (1995) Biochemistry 34, 8043]. In this article, we report the identification and quantification of the stable DNA adducts obtained from DB[a,l]P and DB[a,l]P-11,12dihydrodiol activated by rat liver microsomes and from reaction of (()-anti-DB[a,l]P-11,12dihydrodiol-13,14-epoxide (DB[a,l]PDE) and (()-syn-DB[a,l]PDE with DNA in vitro. In addition, the stable DNA adducts were identified and quantified following treatment of mouse skin with DB[a,l]P, DB[a,l]P-11,12-dihydrodiol, (()-anti-DB[a,l]PDE, or (()-syn-DB[a,l]PDE in vivo and treatment of rat mammary gland with DB[a,l]P in vivo. The DNA adducts were analyzed by the 32P-postlabeling method, and the major adducts were identified by comparison with standards. The six stable adducts of DB[a,l]P formed by rat liver microsomes in vitro were either guanine or adenine adducts of anti-DB[a,l]PDE or syn-DB[a,l]PDE. About 43% of the detected stable adducts from microsomes were with guanine and 44% were with adenine. The pattern of adducts formed from DB[a,l]P-11,12-dihydrodiol with microsomes was very similar to that from DB[a,l]P. Reaction of (()-anti-DB[a,l]PDE with DNA in vitro formed higher levels of stable adducts (55% from guanine and 39% from adenine) than (()-syn-DB[a,l]PDE did (about 44% with guanine and 47% with adenine). In mouse skin treated with DB[a,l]P, 1% of the total adducts detected were stable adducts, comprised of 51% guanine adducts and 46% from adenine; with DB[a,l]P-11,12-dihydrodiol, 54% of the total were stable adducts, with a pattern of adducts similar to those formed from DB[a,l]P. Treatment of mouse skin with (()-syn-DB[a,l]PDE formed 68% stable adducts, mostly at guanine. With (()-anti-DB[a,l]PDE, mouse skin contained almost exclusively (97%) stable adducts: 61% guanine adducts and 33% adenine adducts. In rat mammary gland treated with DB[a,l]P, 2% of the total adducts were stable, with 42% guanine adducts and 55% adenine adducts. Approximately equal to or greater amounts of stable guanine adducts were formed in all systems, except for rat mammary gland. In contrast, the majority of depurinating adducts were adenine adducts. The carcinogenic potencies of these compounds in mouse skin, published earlier, do not qualitatively or quantitatively correlate with stable adducts, but rather with depurinating adducts [Cavalieri, et al. (2005) Chem. Res. Toxicol. 18, xxx-xxx].

Introduction Dibenzo[a,l]pyrene (DB[a,l]P), an environmental contaminant, has been detected in soil and sediment samples (1, 2), coal tar extracts (2), cigarette smoke condensate (3), and in particulate matter formed during combustion of smoky coal (2, 4, 5). It is the most potent carcinogen among polycyclic aromatic hydrocarbons (PAH) (6-9) and therefore may pose a serious carcinogenic risk to humans. The carcinogenicity of DB[a,l]P in rat mammary gland and mouse skin was found to be significantly stronger than that of 7,12-dimethylbenz[a]anthracene (DMBA), previously considered to be the most potent PAH, as well as benzo[a]pyrene (BP), previously considered to be the most potent carcinogenic environmental PAH (7, 8). The proximate metabolite DB[a,l]P-11,12-dihydrodiol is almost as potent a carcinogen as DB[a,l]P (7), whereas (()* To whom correspondence should be addressed. Tel: 402-559-7237. Fax: 402-559-8068. E-mail: [email protected].

syn- and (()-anti-DB[a,l]P-11,12-dihydrodiol-13,14epoxide (DB[a,l]PDE) were found to be less active as tumor initiators in mouse skin than the parent compound (10). syn-DB[a,l]PDE was a more potent carcinogen than the coresponding anti diastereomer (10). Similarly to other environmental carcinogens (11), the lipophilic PAHs are stored in human fat (12), suggesting that breast tissue, as well as mouse skin, might be constantly exposed to these potential carcinogens. The presence in the human breast of even small amounts of the highly carcinogenic DB[a,l]P could represent a significant risk factor. The exceptionally high carcinogenic potency of DB[a,l]P, along with potential human exposure to this compound from the environment, make it essential that the pathways of metabolic activation of DB[a,l]P in target tissue and DNA damage be characterized. Carcinogenic PAHs, such as DB[a,l]P, DMBA, and BP, have been shown to be metabolically activated to form DNA adducts by two major mechanisms, one-electron

10.1021/tx049681s CCC: $30.25 © 2005 American Chemical Society Published on Web 04/30/2005

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Figure 1. Structures of stable DNA adducts formed by DB[a,l]PDEs.

oxidation to form radical cation intermediates (13, 14) and monooxygenation to form bay region diol epoxides (15). The adducts formed by these mechanisms can be either stable or depurinating, i.e., released from DNA by cleavage of the glycosyl bond between the purine base and the deoxyribose (14). Loss of adducts by depurination results in apurinic sites in the DNA, which, if not repaired, can be mutagenic (16-18). Identification and quantification of depurinating DB[a,l]P-DNA adducts and quantification of stable DB[a,l]PDNA adducts formed in vitro after 3-methylcholanthreneinduced rat liver microsomal activation of DB[a,l]P were reported previously (19). In vitro, DB[a,l]P was activated by both the one-electron oxidation and the diol epoxide pathways to form DNA adducts. Approximately 85% of the DB[a,l]P-DNA adducts formed in the microsomal system were depurinating adducts, of which 60% were formed by one-electron oxidation and 40% by the diol epoxide pathway. Over 70% of all adducts detected were depurinating adenine adducts. The pattern of mutations induced by different PAHs in mouse skin papillomas and preneoplastic skin correlates well with the profile of depurinating DNA adducts (17, 18). Both DB[a,l]P and DMBA, which predominantly produce depurinating adenine (Ade) adducts, were found consistently to induce CAA f CTA mutations in codon 61 of the c-Harvey-ras oncogene (17). Diol epoxides can be metabolically produced as two enantiomeric pairs of diastereomers, designated as (()syn-DB[a,l]PDE and (()-anti-DB[a,l]PDE. DB[a,l]P is stereoselectively metabolized in the human mammary carcinoma cell line MCF-7 to form one enantiomer of each diol epoxide diastereomer, (+)-syn-DB[a,l]PDE and (-)anti-DB[a,l]PDE, which then bind extensively to deoxyadenosine (dA) residues in DNA (20, 21). However, MCF-7 cultures treated with DB[a,l]P-11,12-dihydrodiol, the proximate metabolite of DB[a,l]P in the diol epoxide pathway, formed only anti-DB[a,l]PDE-DNA adducts (20). Different DNA-binding metabolites are produced depending on the experimental conditions, such as cell type

and species of the animal chosen, time of exposure, and other experimental conditions. Ralston et al. (22) found that DNA from Sencar mouse embryo cell cultures and from cultures of a mouse keratinocyte cell line contained the same seven stable DB[a,l]P-DNA adducts. In contrast, DNA from mouse epidermis had only one major DB[a,l]P-DNA adduct, with two other adducts present in much smaller amounts. All of these were antiDB[a,l]PDE-DNA adducts. By identifying and quantifying the adducts formed by DB[a,l]P in target tissues, we can obtain valuable information concerning the DNA damage that leads to tumor initiation. In this article, we report the identification and quantification of stable DNA adducts of DB[a,l]P (Figure 1) formed in vitro and in rat mammary gland and mouse skin, as well as adducts of DB[a,l]P-11,12-dihydrodiol, syn-DB[a,l]PDE, and anti-DB[a,l]PDE formed in vitro and in mouse skin. Depurinating adducts were formed by both mechanisms (23), whereas the stable adducts were derived from the diol epoxide pathway in all of the systems studied.

Experimental Procedures Chemicals. DB[a,l]P and DB[a,l]P-11,12-dihydrodiol were obtained from the National Cancer Institute Chemical Carcinogen Repository (Bethesda, MD). They were used as received; analysis by reverse phase HPLC on a Waters Millennium 2010 chromatography system with a 996 photodiode array detector and a YMC (Morris Plains, NJ) ODS-AQ 5 µm, 120 Å (6 mm × 250 mm) column with a CH3CN/H2O gradient showed that their purity was >99%. (()-anti-DB[a,l]PDE and (()-syn-DB[a,l]PDE were obtained from ChemSyn Science Laboratories (Lenexa, KS). Standard stable adducts [anti-DB[a,l]PDE-14-N2dGMP (specific structure not established), anti-cis-DB[a,l]PDE-14-N2dGMP, syn-cis-DB[a,l]PDE-14-N2dGMP, syn-trans-DB[a,l]PDE14-N2dGMP, syn-cis-DB[a,l]PDE-14-N6dAMP, syn-trans-DB[a,l]PDE-14-N6dAMP, anti-trans-DB[a,l]PDE-14-N6dAMP, and anticis-DB[a,l]PDE-14-N6dAMP] (Figure 1) were either available in the laboratory or were synthesized as previously described (24). Binding of DB[a,l]P to DNA in Rat Mammary Gland. Groups of 20 seven week old female Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) were treated with

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Table 1. Profile of Stable DB[a,l]P-DNA Adducts Formed in Vitroa amount of adduct, µmol/mol DNA-P (% of stable adducts) microsome-activated adduct spot anti-DB[a,l]PDE-dGMP anti-DB[a,l]PDE-dGMP anti-cis-DB[a,l]PDE-dGMP syn-trans-DB[a,l]PDE-dGMP syn-trans-DB[a,l]PDE-dGMP + syn-cis-DB[a,l]PDE-dGMP total DB[a,l]PDE-dGMP adducts anti-trans-DB[a,l]PDE-dAMP syn-trans-DB[a,l]PDE-dAMP + anti-cis-DB[a,l]PDE-dAMP syn-cis-DB[a,l]PDE-dAMP total DB[a,l]PDE-dAMP adducts unidentified adduct unidentified adduct unidentified adduct unidentified adduct total unidentified adducts total stable adducts a

syn-DB[a,l]PDE

anti-DB[a,l]PDE

DB[a,l]P

DB[a,l]P-11,12-dihydrodiol

296 (33) 149 (17) 46 (5)

0.75 (16)

8.4 (19)

0.53 (11)

3.2 (7)

0.79 (16) 2.07 (43)

3.8 (8) 15.4 (34) 13.2 (29) 2.8 (6) 16.0 (35) 7.1 (16) 1.5 (3) 5.6 (12) 14.1 (31) 45.5 (100)

52 (17) 83 (27) 135 (44)

491 (55) 121 (14)

34 (11) 110 (36) 144 (47)

219 (25) 340 (39)

1.63 (34) 0.45 (10) 2.08 (44) 0.32 (7) 0.12 (2)

12 (4) 15 (5) 27 (9) 306 (100)

53 (6) 53 (6) 884 (100)

0.17 (4) 0.61 (13) 4.76 (100)

The stable adducts are 15% of the total adducts with DB[a,l]P and 81% with DB[a,l]P-11,12-dihydrodiol (19).

DB[a,l]P as described in the accompanying article reporting the depurinating adducts (23). The smaller tissue aliquot was used for isolation of DNA and analysis of stable adducts by the 32Ppostlabeling method (25), while the larger aliquot was used for isolation of depurinating adducts, as reported elsewhere (23, 26). Binding of DB[a,l]P, DB[a,l]P-11,12-Dihydrodiol, and syn- or anti-DB[a,l]PDE to DNA of Mouse Skin. Groups of eight eight week old female Swiss mice (Eppley Colony) were treated on a shaved area of dorsal skin with 200 nmol of the carcinogen as described in the accompanying article reporting the depurinating adducts (23). The smaller aliquot of tissue was used to purify DNA and analyze stable adducts by the 32Ppostlabeling method (25). The other was used for determination of depurinating adducts, as reported elsewhere (19, 23). Binding of DB[a,l]P and DB[a,l]P-11,12-Dihydrodiol to DNA by 3-Methylcholanthrene-Induced Rat Liver Microsomes in Vitro. DB[a,l]P and DB[a,l]P-11,12-dihydrodiol were bound to calf thymus DNA in 15 mL reaction mixtures. The DNA samples were purified and processed as described previously (19). Binding of (()-anti-DB[a,l]PDE or (()-syn-DB[a,l]PDE to DNA in Vitro. (()-anti- or (()-syn-DB[a,l]PDE (80 µM) was bound to 3 mM calf thymus DNA (Pharmacia, Piscataway, NJ) in 15 mL reaction mixtures incubated at 37 °C for 30 min. At the end of the reaction, a 1 mL aliquot of the mixture was used to analyze stable adducts by the 32P-postlabeling method (25). The DNA from the remaining 14 mL was precipitated with two volumes of ethanol, and the supernatant was used to analyze depurinating adducts (19, 23). Identfication and Quantification of Stable Adducts. The stable adducts were identified and quantified by 32P-postlabeling analysis (25). The mobilities of the unknown adducts and the specific standard adducts (24) on TLC plates were compared under the same experimental conditions to identify the adduct spots. The mobility of individual adduct spots mixed with the appropriate standards was also used for identification. The recovery of adducts is limited to those adducts present in tissue at the end of the experiment, 4 h in mouse skin and 24 h in rat mammary gland (27, 28).

Results and Discussion Stable DNA Adducts Formed in Vitro. Detection and quantification of the stable adducts were accomplished by the 32P-postlabeling method (25). The stable adducts formed by reaction of syn-DB[a,l]PDE with DNA in vitro represented 97% of all of the adducts detected.

Figure 2. Autoradiogram of 32P-postlabeled DNA adducts formed by reaction of syn-DB[a,l]PDE with DNA. The film was exposed at room temperature for 2 min.

The level of binding was high, 306 µmol/mol DNA-P. Roughly equal amounts of guanine adducts (44%) and adenine adducts (47%) were identified (Table 1). Two adducts of syn-trans-DB[a,l]PDE-14-N2dGMP were detected by TLC; one was detected in a spot by itself, but the other ran in a second spot, mixed with syn-cis-DB[a,l]PDE-14-N2dGMP (Figure 2). The major adenine adduct was syn-cis-DB[a,l]PDE-14-N6dAMP, while the minor one was syn-trans-DB[a,l]PDE-14-N6dAMP. The reaction of anti-DB[a,l]PDE with DNA formed almost exclusively stable adducts, and the level of binding, 884 µmol/mol DNA-P, was even higher (Table 1). More guanine adducts (55%) were formed than adenine adducts (39%). Two forms of anti-DB[a,l]PDE-14-N2dGMP (specific structure unidentified) were detected, as well as a small amount of anti-cis-DB[a,l]PDE-14-N2dGMP (Figure 3). Both anti-cis-DB[a,l]PDE-14-N6dAMP and anti-trans-DB[a,l]PDE-14-N6dAMP were observed (Figure 3). The stable adducts formed by activation of DB[a,l]P by microsomes in vitro accounted for 15% of all adducts detected, the rest (85%) being depurinating adducts (23). When DB[a,l]P or DB[a,l]P-11,12-dihydrodiol was acti-

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Figure 3. Autoradiogram of 32P-postlabeled DNA adducts formed by reaction of anti-DB[a,l]PDE with DNA. The film was exposed at room temperature for 0.5 min.

Figure 5. Autoradiogram of 32P-postlabeled DNA adducts formed by activation of DB[a,l]P-11,12-dihydrodiol by rat liver microsomes in the presence of DNA. The film was exposed at room temperature for 6 min.

Figure 4. Autoradiogram of 32P-postlabeled DNA adducts formed by activation of DB[a,l]P by rat liver microsomes in the presence of DNA. The film was exposed at room temperature for 20 min.

Figure 6. Autoradiogram of 32P-postlabeled DNA adducts formed following treatment of mouse skin with DB[a,l]P. The film was exposed at room temperature for 20 h.

vated by rat liver microsomes in the presence of DNA, the amount of adducts detected with the dihydrodiol was 10-fold higher (46 µmol/mol DNA-P) than with the parent compound (5 µmol/mol DNA-P), and at least six stable adducts of anti- and syn-DB[a,l]PDE with Ade and Gua were detected (Figures 1, 4, and 5 and Table 1). Comparison of the mobility of the stable adducts with that of authentic adduct standards (Figure 1) revealed similar adduct patterns from the parent compound and the dihydrodiol metabolite. The specific structure for the standard of anti-DB[a,l]PDE-14-N2dGMP was not conclusively established by fluorescence line-narrowing spectroscopy (24). This adduct represented 16% of the stable adducts from DB[a,l]P and 19% from the dihydrodiol (Table 1). anti-cis-DB[a,l]PDE-14-N2dGMP and a mixture of the trans and cis isomers of syn-DB[a,l]PDE-14-N2dGMP were also detected (Figures 4 and 5). The predominant adduct spot is a mixture of syn-trans-DB[a,l]PDE-14-N6dAMP and anti-cis-DB[a,l]PDE-14-N6dAMP adducts (34% from the parent compound and 29% from the dihydrodiol, Table 1). syn-cis-DB[a,l]PDE-14-N6dAMP was a minor adduct in both cases. The abundance of one of the unidentified adducts was found to depend on the age of the DNA sample. In fresh DNA, the amount of this adduct is very small, but it increased with aging of

the DNA. It appeared to arise from progressive decomposition of syn-trans-DB[a,l]PDE-14-N6dAMP or anti-cisDB[a,l]PDE-14-N6dAMP. One changing DB[a,l]P-DNA adduct was also detected by Hughes and Phillips (29). Equal amounts of the stable adducts formed by microsomal activation of DB[a,l]P (43-44%) or DB[a,l]P11,12-dihydrodiol (34-35%) were Ade and Gua adducts. Equal amounts of stable Ade and Gua adducts in the microsomal system were also reported by Arif and Gupta (30). Stable Adducts Formed in Mouse Skin. When mouse skin was treated with DB[a,l]P, stable adducts were 1% of the total adducts detected (23). We observed two major stable adducts, anti-cis-DB[a,l]PDE-14-N2dGMP and a mixture of syn-trans-DB[a,l]PDE-14-N6dAMP and anti-cis-DB[a,l]PDE-14-N6dAMP, accounting for 87% of total stable adducts (Figure 6 and Table 2). Only 3% of the stable adducts were not identified. Similar results were observed by Hughes and Phillips (29) in mouse skin, where two adducts were present in larger amounts than the other four, although Ralston et al. (22) detected only one major DB[a,l]P-DNA adduct, with two other adducts present in much smaller amounts, and all were from the anti isomer of DB[a,l]PDE (22). However, it can still be concluded that the stable adducts in mouse

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Table 2. Profile of Stable DB[a,l]P-DNA Adducts Formed in Mouse Skina amount of adduct, µmol/mol DNA-P (% of stable adducts) adduct spot anti-DB[a,l]PDE-dGMP anti-DB[a,l]PDE-dGMP anti-cis-DB[a,l]PDE-dGMP syn-trans-DB[a,l]PDE-dGMP syn-trans-DB[a,l]PDE-dGMP + syn-cis-DB[a,l]PDE-dGMP total DB[a,l]PDE-dGMP adducts anti-trans-DB[a,l]PDE-dAMP syn-trans-DB[a,l]PDE-dAMP + anti-cis-DB[a,l]PDE-dAMP syn-cis-DB[a,l]PDE-dAMP total DB[a,l]PDE-dAMP adducts unidentified adducts total stable adducts

DB[a,l]P

DB[a,l]P-11,12-dihydrodiol

0.01 (5) 0.09 (45)

syn-DB[a,l]PDE

0.24 (5) 0.71 (15) 1.43 (31)

anti-DB[a,l]PDE 5.2 (13) 11.2 (27) 8.9 (21)

8.3 (30) 0.10 (50)

0.15 (3) 2.53 (54)

0.08 (40) 0.01 (5) 0.09 (45) 0.01 (5) 0.20 (100)

1.78 (38) 0.10 (2) 1.88 (40) 0.27 (6) 4.68 (100)

11.9 (43) 20.2 (73)

25.3 (61) 0.8 (2)

2.1 (8) 13.1 (31) 4.1 (15) 6.2 (23) 1.2 (4) 27.6 (100)

13.9 (33) 2.5 (6) 41.7 (100)

a The stable adducts are 1% of the total adducts with DB[a,l]P, 20% with DB[a,l]P-11,12-dihydrodiol, 68% with syn-DB[a,l]PDE, and 97% with anti-DB[a,l]PDE (23).

Figure 7. Autoradiogram of 32P-postlabeled DNA adducts formed following treatment of mouse skin with DB[a,l]P-11,12-dihydrodiol. The film was exposed at room temperature for 1 h.

skin arise exclusively from DB[a,l]PDE, in particular from anti-DB[a,l]PDE. Treatment of mouse skin with DB[a,l]P-11,12-dihydrodiol produced similar results, although relatively more stable adducts (20%) (23) were formed and additional guanine adducts were detected (Figure 7 and Table 2). With DB[a,l]P, the stable Gua adducts were formed with the metabolically produced anti-DB[a,l]PDE diastereomer, although a small amount of the syn-DB[a,l]PDE diastereomer was seen with the dihydrodiol. The major dAMP adduct spot (38% of stable adducts) contained a mixture of syn-trans-DB[a,l]PDE-14-N6dAMP and anticis-DB[a,l]PDE-14-N6dAMP. With the parent compound or the dihydrodiol, slightly more guanine adducts (50 and 54%, respectively) were observed than adenine adducts (46 and 40%, respectively). However, the major difference between treatment of mouse skin with DB[a,l]P or DB[a,l]P-11,12-dihydrodiol was in the total amount of adducts produced. The dihydrodiol resulted in about a 25fold higher level of adducts than DB[a,l]P. When mouse skin was treated with syn-DB[a,l]PDE, stable adducts comprised 68% of the total adducts detected (23). Over 70% of the stable adducts formed following treatment of mouse skin with syn-DB[a,l]PDE were with guanine (Figure 8 and Table 2), whereas treatment with anti-DB[a,l]PDE formed almost exclu-

Figure 8. Autoradiogram of 32P-postlabeled DNA adducts formed following treatment of mouse skin with syn-DB[a,l]PDE. The film was exposed at room temperature for 5 min.

Figure 9. Autoradiogram of 32P-postlabeled DNA adducts formed following treatment of mouse skin with anti-DB[a,l]PDE. The film was exposed at room temperature for 1 min.

sively stable adducts (23), with about twice as much guanine adducts as adenine adducts (Figure 9 and Table 2). Treatment with syn-DB[a,l]PDE resulted in two guanine adducts not found following treatment with the parent compound, although one of them is detected after treatment with the dihydrodiol (Figures 6-8 and Table 2). anti-DB[a,l]PDE resulted in 50% more total stable

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reported previously that DB[a,l]P was stereoselectively metabolized in MCF-7 cells to form only one enantiomer of each of the diol epoxide diastereomers, (+)-syn- and (-)-anti-DB[a,l]PDE (20, 21).

Conclusions

Figure 10. Autoradiogram of 32P-postlabeled DNA adducts formed following treatment of rat mammary gland with DB[a,l]P. The film was exposed at room temperature for 15 h. Table 3. Profile of Stable DB[a,l]P-DNA Adducts from Rat Mammary Glanda amount of adduct, nmol/mol DNA-P (% of stable adducts) adduct spot

DB[a,l]P

anti-DB[a,l]PDE-dGMP anti-cis-DB[a,l]PDE-dGMP syn-trans-DB[a,l]PDE-dGMP + syn-cis-DB[a,l]PDE-dGMP total DB[a,l]PDE-dGMP adducts syn-trans-DB[a,l]PDE-dAMP + anti-cis-DB[a,l]PDE-dAMP syn-cis-DB[a,l]PDE-dAMP total DB[a,l]PDE-dAMP adducts unidentified DB[a,l]PDE-DNA adducts total stable adducts

12 (5) 54 (24)

a

32 (14) 98 (43) 99 (43) 26 (11) 125 (54) 6 (3) 229 (100)

The stable adducts are 2% of the total adducts detected (23).

adducts than syn-DB[a,l]PDE, nine times more than DB[a,l]P-11,12-dihydrodiol, and 200-fold more than DB[a,l]P itself. Nonetheless, DB[a,l]P is much more carcinogenic in mouse skin than either of the DB[a,l]PDEs (7, 8, 10). Stable Adducts Formed in Rat Mammary Gland. After intramammillary injection of rat mammary glands with DB[a,l]P, 2% of the total adducts detected were stable adducts (23). Slightly more stable adenine adducts (55%) were detected than guanine adducts (42%; Figure 10 and Table 3). The major adduct spot (43%; Figure 10) contained a mixture of syn-trans-DB[a,l]PDE-14-N6dAMP and anti-cis-DB[a,l]PDE-14-N6dAMP. The guanine adduct formed in the greatest amount (23%) was the anticis-DB[a,l]PDE-14-N2dGMP adduct. These adducts were also detected in high proportions in mouse skin treated with DB[a,l]P or the 11,12-dihydrodiol (Table 2). Similar results were reported by Arif et al. (31) following intramammillary injection of DB[a,l]P in Sprague-Dawley rat mammary gland. They also found a higher proportion of stable adducts at adenine than at guanine. The level of stable adducts that we report (0.2 µmol/mol DNA-P; Table 3) falls between their values at 6 h (0.09 µmol/mol DNA-P) and at 2 days (1.8 µmol/mol DNA-P). In human mammary carcinoma MCF-7 cell cultures, Ralston et al. (21) detected three major DNA adducts, with dAMP accounting for 80-90% of total stable adducts, two formed by reaction of (+)-syn-DB[a,l]PDE and the third by reaction of (-)-anti-DB[a,l]PDE. It was

We have identified and elucidated the structure of almost all of the stable adducts arising from DB[a,l]P in the target organs mouse skin and rat mammary gland. Our results show that the amount of stable adducts does not correlate with the observed carcinogenic potencies of these compounds in mouse skin. DB[a,l]P and DB[a,l]P-11,12-dihydrodiol are potent carcinogens in mouse skin (7), whereas (()-syn- and (()-anti-DB[a,l]PDE, which form significantly larger amounts of stable adducts than DB[a,l]P and DB[a,l]P-11,12-dihydrodiol (Table 2), were found to be less active as tumor initiators in mouse skin than the parent compound (7, 8, 10). syn-DB[a,l]PDE, which forms significantly more depurinating adducts (Table 2) (23), is a more potent carcinogen than anti-DB[a,l]PDE (10). Only a minor amount of DNA adducts arising from DB[a,l]P in mouse skin, rat mammary gland, and in vitro were stable adducts formed by the diol epoxide pathway. The overwhelming majority of the DNA adducts were depurinating adducts formed by both the one-electron oxidation and the diol epoxide pathways (19, 23). At least six stable DB[a,l]P adducts were detected in all of the samples of DNA (Figures 2-10 and Tables 1-3). The pattern of stable adducts was similar in mouse skin, rat mammary gland, and in vitro, but the amounts of individual adducts differ. Both anti- and syn-DB[a,l]PDEDNA adducts were detected as Ade and Gua adducts. DB[a,l]P-11,12-dihydrodiol produced 80% depurinating adducts (23) and 20% stable adducts in mouse skin, and the pattern of stable adducts was similar to that of DB[a,l]P (Figures 6 and 7 and Table 2). Equal proportions of Gua and Ade adducts were detected in vitro (Table 1), and a slightly larger proportion of Gua adducts was detected in mouse skin (Table 2). In contrast, syn- and anti-DB[a,l]PDE in mouse skin predominantly formed stable adducts (68 and 97%, respectively) (19, 23) and the majority of those were with Gua (Table 2). In summary, there is no correlation between the formation of stable DB[a,l]PDE-DNA adducts in mouse skin and the tumorigenic potency of the DB[a,l]P metabolites, DB[a,l]P-11,12-dihydrodiol, syn-DB[a,l]PDE, and anti-DB[a,l]PDE (7, 8, 10). In fact, DB[a,l]P, which produces the smallest proportion of stable adducts, is the most carcinogenic, whereas the anti-DB[a,l]PDE, which produces the highest amount and proportion of stable adducts, is the least carcinogenic in mouse skin.

Acknowledgment. This research was supported by U.S. Public Health Service Grants P01 CA49210 and R01 CA49917 from the National Cancer Institute. Core support in the Eppley Institute is provided by Grant P30 CA36727 from the National Cancer Institute.

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