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[Cancer Research 60, 5688-5695, October 15, 2000]
© 2000 American Association for Cancer Research

Carcinogenesis

Both (±)syn- and (±)anti-7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxides Initiate Tumors in Mouse Skin That Possess -CAA- to -CTA- Mutations at Codon 61 of c-H-ras1

Moon-shong Tang2, Suryanarayana V. Vulimiri, Aurora Viaje, James X. Chen, Deepa S. Bilolikar, Rebecca J. Morris, Ronald G. Harvey, Tom J. Slaga and John DiGiovanni

Department of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987 [M-s. T., J. X. C., D. S. B.]; Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science, [S. V. V., J. D.]; Department of Cancer Causation and Prevention, AMC-Cancer Research Center, Denver, Colorado 80214 [A. V., T. J. S.]; Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096 [R. J. M.]; and Ben May Institute, University of Chicago, Chicago, Illinois 60637 [R. G. H.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have determined the tumor-initiating activity of (±)syn- and (±)anti-7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide (syn- and anti-DMBADE), the two metabolically formed bay-region diol epoxides of DMBA, and we have also analyzed mutations in the H-ras gene from tumors induced by these compounds. Using a two-stage, initiation-promotion protocol for tumorigenesis in mouse skin, we have found that both syn- and anti-DMBADE are active tumor initiators, and that the occurrence of papillomas is carcinogen dose dependent. All of the papillomas induced by syn-DMBADE (a total of 40 mice), 96% of those induced by anti-DMBADE (a total of 25 mice), and 94% of those induced by DMBA (a total of 16 mice) possessed a -CAA- to -CTA- mutation at codon 61 of H-ras. No mutations in codons 12 or 13 were detected in any tumor. Topical application of syn- and anti-DMBADE produced stable adducts in mouse epidermal DNA, most of which comigrated with stable DNA adducts formed after topical application of DMBA. Further analysis of the data showed that levels of the major syn- and anti-DMBADE-deoxyadenosine adducts formed after topical application of DMBA are sufficient to account for the tumor-initiating activity of this carcinogen on mouse skin. Previously, we showed that both the syn- and anti-DMBADE bind to the adenine (A182) at codon 61 of H-ras. Collectively, these results indicate that the adenine adducts induced by both bay-region diol epoxides of DMBA lead to the mutation at codon 61 of H-ras and, consequently, initiate tumorigenesis in mouse skin.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DMBA3 is a potent PAH carcinogen (1, 2, 3) . The metabolically activated bay-region syn- and anti-diol epoxides of DMBA react with purines in DNA, and it has been suggested that the DNA adducts formed by activated DMBA trigger tumorigenesis and carcinogenesis (4, 5, 6, 7, 8, 9, 10, 11, 12) . However, the relative contribution of the adducts induced by these two diastereomeric bay-region diol epoxides in mutagenesis and carcinogenesis by DMBA remains unclear.

Two distinct molecular characteristics associated with DMBA-initiated skin tumorigenesis are as follows: (a) oncogenic activation exclusively occurs in the ras family of proto-oncogenes; and (b) the mutations are almost exclusively the A-to-T transversion (-CAA- to -CTA-) at the middle adenine in codon 61 of the H-ras gene (12, 13, 14, 15, 16) . These results raise the intriguing question of why most, if not all, DMBA-induced skin papillomas have a mutation in codon 61 but not in codons 12 or 13. This is particularly interesting because both the syn- and anti-DMBADE can react with purine residues in codons 12, 13, and 61 of this gene, and furthermore, mutations in codons 12, 13, or 61 of the H-ras gene can all trigger tumorigenesis (3 , 17, 18, 19, 20) . If the A-to-T transversion mutation at codon 61 in DMBA-induced mouse skin papillomas is caused by the adduction of metabolically activated DMBA to the adenine residue at this codon, then it is likely that both syn- and anti-DMBADE are tumorigenic because we have found previously that they both bind to the adenine at codon 61 (21 , 22) . Although many studies have demonstrated the formation of stable DMBA-DNA adducts derived from syn- and anti-DMBADE in mouse skin and other tissues (2 , 4 , 5 , 9 , 10) , it has been reported recently that DMBA is metabolically activated primarily via one electron oxidation at the 12-methyl position, giving rise to unstable purine adducts (23 , 24) . It has been hypothesized that the depurinated sites caused by these unstable adducts, rather than the stable bay-region diol epoxide purine adducts, induce transversion mutations including the A-to-T transversion in codon 61 of the H-ras gene that triggers tumor initiation by DMBA (25) . Conversely, Melendez-Colon et al. (26) have reported recently that stable DNA adducts could account for the carcinogenic actions of several PAHs, including DMBA.

To better understand the role of metabolically formed syn- and anti-DMBADE in DMBA-induced tumorigenesis and the role of stable DNA adducts from these metabolites, we have determined their tumor-initiating activity using the standard two-stage, initiationpromotion protocol of mouse skin tumorigenesis. In addition, we have examined the DNA adducts formed in epidermal DNA and the mutations at codon 61 as well as at codons 12 and 13 in all of the tumors generated by initiation with these two compounds and the parent compound, DMBA. We have found that both the syn- and anti-DMBADE possess tumor-initiating activity, and furthermore, all of the tumors generated by the initiation with these compounds have the same A-to-T transversion (-CAA- to -CTA-) of codon 61 of the H-ras gene. Importantly, the data show that the levels of the major dAdo adducts produced from metabolically formed syn- and anti-DMBADE after topical application of DMBA can account for the tumor-initiating activity of this PAH.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
DMBA was purchased from Eastman Kodak (Rochester, NY). Racemic syn- and anti-DMABDE were synthesized as described previously (27) . RNase A (E.C. 3.1.4.22) was obtained from Worthington Biochemical Co. (Freehold, NJ), and micrococcal nuclease (Staphylococcus aureus, E.C. 3.1.31.1), proteinase K, and apyrase were purchased from Sigma Chemical Co. (St. Louis, MO). Calf spleen phosphodiesterase (E.C. 3.1.16.1), 3'-phosphatase-free T4 polynucleotide kinase (E.C. 2.7.1.78), and XbaI were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Carrier-free [{gamma}-32P]ATP (specific activity, 6000 Ci/mmol) and [{alpha}32P]dCTP were supplied by DuPont NEN Research Products (Boston, MA). Macherey-Nagel poly(ethylene)imine-cellulose TLC plates were supplied by Bodman Chemicals (Aston, PA). TPA was obtained from LC Services (Wobum, NY).

Determining the Tumor-initiating Activity of syn- and anti-DMBADE.
The standard two-stage tumor initiation-promotion protocol was followed. Female SENCAR mice were obtained from the National Cancer Institute. The backs of the mice, 7–9 weeks of age, were shaved, and 2 days later, animals in the resting phase of the hair cycle were selected for treatment. Two-tenths ml (in acetone) of 10 nmol of DMBA and 10, 50, and 100 nmol of syn- and anti-DMBADE were applied to the shaved backs of the mice. Promotion with TPA began 2 weeks after treatment with the initiator. TPA (3.24 nmol in 0.2 ml of acetone) was applied topically to the backs of the mice twice a week. The number of mice with tumors and the number of tumors/mouse were counted weekly. Promotion of the tumors with TPA continued up to 20 weeks. The mice were sacrificed, and the tumors were collected. Tumors having diameters more than or equal to 3–10 mm were excised. Half of each tumor was processed for mutational analysis. The remaining half of the tumor and any surrounding nontumorous skin were fixed in 10% buffered formalin and processed for paraffin-embedding. Five-µm sections were cut perpendicular to the skin and stained with H&E. Round, finger-like or warty projections from the epithelial surface were identified as papillomas. Squamous cell carcinomas, identified by their broad base, elevated margin, and intracutaneous infiltration, were verified at autopsy and confirmed histopathologically. Keratoacanthomas were identified as endophytic, dome-shaped lesions with a central keratin-filled plug, imparting a crater-like topography. A total of 40 tumors from mice initiated with syn- DMBADE, 25 tumors from mice initiated with anti-DMBADE, and 16 tumors from mice initiated with DMBA were analyzed.

Determining the H-ras Mutations of DMBADE-induced Papillomas.
Papillomas induced by DMBADE treatment were obtained surgically. DNAs were isolated from tumor tissue samples (28) . To identify the -CAA- to -CTA- mutation at codon 61 of the H-ras gene, this region of DNA underwent two rounds of amplification. An aliquot containing 1 µg of mouse genomic DNA was amplified for 30 cycles with a first set of primers (5'-GGTGTAGGCTGGTTCTGTGGATTCTC- and 5'-GCACACGGAACCTTCCTCAC-, 25 pmol each). The amplified DNA bands were isolated and then purified by Bio-Rad PCR Kleen Spin column and quantified by FluoroImager SI (Vistra DNA Systems). An aliquot containing 50 ng of the first round-amplified DNA was further amplified with a second set of primers (5'-TGTGGATTCTCTGGTCTGAGGAGAG- and 5'-CATAGGTGGCTCACCTGTACTGATG-, 25 pmol) in the presence of {alpha}-[32P]dCTP. The first round of amplification yielded 329-bp DNA fragments. The second round of amplified DNA was then digested with XbaI enzyme; this treatment converted the amplified 269-bp fragments to 124- and 145-bp fragments only in samples with the -CAA- to -CTA- mutation at codon 61 of the H-ras gene (12 , 29 , 30) . The digested DNAs were separated by 7% PAGE. The sequences of exon I and exon II (encompassing codons 12, 13, and 61) of the H-ras gene were determined by standard PCR sequencing technique according to the manufacturer’s specifications (United States Biochemical Corp., Cleveland, OH).

DNA Adduct Analysis by 32P-Postlabeling.
Female SENCAR mice, 6–7 weeks of age, were shaved on their backs 2 days before carcinogen treatment. Mice that were in the resting phase of the hair cycle were used for the experiment. Mice (15/group) received topical applications of DMBA (10 and 100 nmol), syn-DMBADE (50 and 100 nmol), or anti-DMBADE (50 and 100 nmol) in 0.2 ml of acetone, and control animals received 0.2 ml of acetone alone. Five mice from each group were sacrificed by cervical dislocation at 3, 6, and 12 h after the carcinogen treatment, the skin was removed, and the epidermis was scrapped. DNA was isolated from mouse epidermis as described previously (31) . DNA adduct analysis was done by the nuclease P1 method of 32P-postlabeling (32) following the chromatographic conditions of Schmeiser et al. (33) .

Cochromatography of DNA Adducts Formed in Mouse Epidermis with DMBA Diol Epoxide Marker Adducts.
The identity of some of the DMBA-DNA adducts formed in the epidermal DNA of SENCAR mice (D1–D3 and D5–D7) was determined by cochromatography with the corresponding marker adducts obtained by reacting calf thymus DNA with anti- or syn-DMBADE in vitro. For this purpose, 32P-labeled adduct spots from two-dimensional TLC maps from the parent compound (D1–D3 and D5–D7) and corresponding marker adducts from calf thymus DNA reacted with anti- and syn-DMBADE were extracted with isopropanol:4 N ammonia as described (34) . The following five solvents were used for cochromatography of DNA adducts: solvent I: 0.56 M LiCl, 0.35 M Tris-HCl, and 5.95 M urea (pH 8.2); solvent II: 0.35 M Tris-HCl, 0.35 M H3BO3, 7 mM EDTA, 0.91 M NaCl, and 5.6 M urea (pH 8.0); solvent III: isopropanol:4 N ammonia (1:1); solvent IV: 2 M ammonium formate (pH 3.5); and solvent V: 0.49 M NaH2PO4 and 4.9 M urea (pH 6.4).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Both (±)syn- and (±)anti-DMBADE Possess Skin Tumor-initiating Activity.
The tumor-initiating activity of syn- and antiDMBADE on SENCAR mouse skin, in comparison with DMBA, was determined by using the standard two-stage, initiation-promotion protocol. Seven groups of 15 SENCAR mice each were treated with 10 nmol of DMBA; 10 nmol, 50 nmol, and 100 nmol of syn-DMBADE; and 10, 50, and 100 nmol of anti-DMBADE. Five mice were treated with 0.2 ml of acetone only as a control group. Subsequently, mice in all groups were treated with 3.24 nmol of TPA given twice weekly. A repeat experiment was performed in a group of 15 SENCAR mice initiated with 100 nmol of syn-DMBADE. The development of skin papillomas in the various groups of mice is shown in Fig. 1Citation . Whereas 10 nmol of syn-DMBADE produced no papillomas, 67% of mice initiated with 50 nmol and 85% of mice initiated with 100 nmol of syn-DMBADE developed skin papillomas after 17 weeks of promotion (Fig. 1A)Citation . Similar results were observed in mice initiated with anti-DMBADE; 63% of mice treated with 50 nmol and 81% of mice treated with 100 nmol of anti-DMBADE developed papillomas (Fig. 1B)Citation . In comparison, 100% of mice initiated with 10 nmol of DMBA developed papillomas (Fig. 1)Citation .



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  		Fig. 1. Time course of papilloma formation in mice treated with syn- and anti-DMBADE and DMBA. Female SENCAR mice initiated with syn-DMBADE (10, 50, and 100 nmol) and DMBA (10 nmol) (A) and anti-DMBADE (10, 50 and 100 nmol) and DMBA (10 nmol) (B) treatment were followed by TPA treatment as described in "Materials and Methods." The percentages of mice that developed papillomas versus the time of TPA treatment are presented. No papillomas were found in mice with mock treatments.

 
The number of papillomas/mouse induced by syn- and antiDMBADE, however, was significantly lower than that induced by DMBA. Whereas an average (two experiments) of 14 papillomas/mouse were induced after 17 weeks of promotion by a 10-nmol initiating dose of DMBA, only 1.5–1.7 papillomas/mouse were induced by 50 nmol of syn- and anti-DMBADE treatment, and 2.5–2.7 papillomas/mouse were induced by 100 nmol of syn- and antiDMBADE treatment at 17 weeks of promotion with TPA (Fig. 2)Citation .



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  		Fig. 2. Number of papilloma formations in mice treated with syn- (A) and anti-DMBADE (B) and DMBA followed by TPA treatment. Female SENCAR mice initiated with syn-DMBADE (10, 50, and 100 nmol) and DMBA (10 nmol) anti-DMBADE (10, 50 and 100 nmol) treatment were followed by TPA treatment as described in "Materials and Methods." The average number of papillomas/mouse versus the time of TPA treatment is presented. No papillomas were found in mice with mock treatments.

 
All of the Papillomas Induced by (±)syn- and (±)antiDMBADE Have an A-to-T Transversion Mutation (-CAA- to -CTA-) at Codon 61 of H-ras.
It has been reported that >90% of DMBA-induced papillomas in mouse skin have an A-to-T transversion mutation (-CAA- to -CTA-) at codon 61 of the H-ras gene. If this mutation in papillomas is a result of the DMBA-induced bay-region syn- and anti-diol epoxide DNA adducts formed at this codon, then we would expect the papillomas induced by syn- and anti-DMBADE to have the same mutation as those induced by DMBA. To test this possibility, we determined whether the genomic DNA isolated from papillomas induced by either syn- or anti-DMBADE treatment possessed the -CAA- to -CTA- mutation at codon 61 of the H-ras gene using the PCR-RFLP method. A region of 269 nucleotides in length encompassing codon 61 of the H-ras gene was amplified with a nest of four primers in the presence of [{alpha}-32P]dCTP. The amplified DNA fragments were then digested with XbaI, and the resultant DNA fragments were separated by electrophoresis in a 7% polyacrylamide gel. Only DNA fragments with the -CAA- to -CTA- mutation at codon 61 of the H-ras gene will create an XbaI site in the 269-bp fragment, which, after XbaI digestion, will result in two DNA fragments of sizes 124 and 145 bp. The schematic representation of this method is shown in Fig. 3Citation , and a typical experimental result is shown in Fig. 4Citation . We have found that all of the papillomas (in a total of 40 mice) induced by syn-DMBADE, 96% of anti-DMBADE-induced papillomas (in a total of 25 mice), and 94% of papillomas (in a total of 16 mice) induced by DMBA, contained an XbaI site at codon 61 of the H-ras gene, indicating a sequence change from -CAA- to -CTA- at this codon. This conclusion was confirmed by DNA sequencing (data not shown). No such sequence change was observed in genomic DNA isolated either from the skin of mice treated with acetone only at the time of initiation or from normal skin of mice with tumors (two from DMBA and four from syn-DMBADE). Also, mutations were not detected in codons 12 or 13 of H-ras in any tumor examined.



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  		Fig. 3. Schematic representation of the method of detection of the -CAA- to -CTA- mutation at codon 61 of the H-ras gene. Because the DNA fragments are evenly labeled with 32P, the ratio of radioactive counts from 124 bp plus 145 bp over the total count represents the fraction of cells within the papilloma with the mutation.

 


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  		Fig. 4. Detection of the occurrence of the -CAA- to -CTA- mutation at codon 61 of the H-ras gene in syn-DMBADE-induced (A), anti-DMBADE-induced (B), and DMBA-induced (C) papillomas in mouse skin. Genomic DNAs isolated from papillomas underwent two rounds of amplification using two sets of primers in the presence of [{alpha}-32P]dCTP. The amplified DNAs were digested with restriction enzyme XbaI and then separated by gel electrophoresis. The details are described in "Materials and Methods." Numbers at the bottom of the panel, represent tumors isolated from different mice. S27A and S49, are DNA isolated from the acetone-treated control mice.

 
(±)syn- and (±)anti-DMBADE Induce the Same DNA Adducts as DMBA in Mouse Epidermis.
It has been found that the major DNA adducts formed in mouse skin after topical application of DMBA are syn- and anti-diol epoxide-dGuo and -dAdo adducts; these results have led to the conclusion that syn- and anti-DMBADE are the major metabolites that interact with DNA and initiate mutagenesis and tumorigenesis by DMBA (4, 5, 6, 7, 8, 9, 10, 11, 12) . Our results showing that papillomas initiated by DMBA and both syn- and anti-DMBADE have the same mutation at codon 61 of the H-ras gene are consistent with this conclusion. To further test whether the DNA adducts formed by DMBA treatment arise from the interactions of syn- and antiDMBADE with mouse skin DNA, we analyzed the DNA adducts formed in epidermal DNA after topical application of DMBA, syn-DMBADE, and anti-DMBADE by the 32P-postlabeling technique (32 , 33) . Topical application of 10 nmol of DMBA and 100 nmol each of the syn- and anti-diol epoxides produced 13.4, 8.6, and 17.5 adducts/109 nucleotides, respectively, at the time of maximum DNA binding (24 h for DMBA and 6 h for the diol epoxides). Fig. 5Citation shows the TLC autoradiograms of DNA adducts at 24 h after topical application of DMBA (Fig. 5B)Citation and at 6 h after topical application of either anti-DMBADE (Fig. 5C)Citation or syn-DMBADE (Fig. 5D)Citation . TLC autoradiograms were similar at other time points (data not shown). As seen in Fig. 5Citation , 13 DNA adduct spots were detected after topical application of 10 nmol of DMBA (D1–D13). Twelve of the DMBA-DNA adduct spots migrated to positions similar to DNA adduct spots produced by either syn-DMBADE (adduct spots D2, D4, and D11–D13), anti-DMBADE (adduct spot D3), or both (adduct spots D1, D5–D7, D9, and D10). Adduct spot D8 did not comigrate with any syn- or anti-DMBADE-DNA adduct spot. Moreover, DMBA-DNA adduct spots D1–D3 were subjected to cochromatography analysis in several additional TLC solvent systems (Fig. 6)Citation and found to comigrate with either an anti-DMBADE-DNA adduct (D1-A1, D3-A3) or a syn-DMBADE-DNA adduct (D2-S2) spot. These spots were chosen for rechromatography because they migrated similarly to the three major deoxyribonucleoside adducts produced by DMBA in mouse epidermis as assessed by Schmeiser et al. (33) . On the basis of the cochromatography results and by analogy with the data of Schmeiser et al. (33) , we tentatively assigned these DNA adduct spots as follows: D1, anti-DMBADE-dGuo; D2, syn-DMBADE-dAdo; and D3, antiDMBADE-dAdo. Overall, the results of our adduct analyses confirm that the widely accepted diol epoxide metabolic pathways for DMBA activation account for the majority of the stable DMBA-DNA adducts detected with the methods used (9 , 10 , 33, 34, 35, 36, 37, 38, 39) .



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  		Fig. 5. Representative autoradiograms showing 32P-labeled DMBA-DNA adducts formed in SENCAR mouse epidermis and in vitro. Approximately 2–5 µg of DNA were analyzed by nuclease P1-enhanced 32P-postlabeling assay. Epidermal DNA adduct profiles from SENCAR mice treated with acetone (panel A), 100 nmol of DMBA (panel B), 100 nmol of (±)anti-DMBADE (panel C), and 100 nmol of (±)syn-DMBADE (panel D), 24 h (panels A and B) or 6 h (panels C and D) after treatment are shown. Panels E and F, adduct profiles from calf thymus DNA that was reacted with (±)anti-DMBADE and with (±)syn-DMBADE, respectively. TLC sheets were exposed to Kodak X-Omat X-ray films with intensifying screens at -70°C for 8 h (panels A–C) or 16 h (D) and for 3 min at room temperature (panels E and F). Origins were located in the lower left corner. IS, adduct spot derived from internal standard DNA (obtained from the skin of mice topically treated with dibenz[a,j]acridine). The IS-DNA was mixed with in vivo sample DNA prior to enzyme digestion to monitor labeling efficiency. b, background spots that were also seen in control DNA (panel A). Adducts with the prefixes D, A, and S represent DNA adducts formed in vivo by DMBA, (±)anti-DMBADE, and (±)syn-DMBADE, respectively. Adducts with prefixes a and s represent adduct markers generated by reacting (±)anti- and (±)syn-DMBADE, respectively, with calf thymus DNA. The adducts with same number (1–13) from different alphabets (D, A, S, a, and s) represent similar chromatographic mobility.

 


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  		Fig. 6. Cochromatography of selected 32P-labeled bisphosphate DMBADE-DNA adducts formed in vivo with anti-and syn-DMBADE adduct markers formed in vitro. Cochromatography was done (using solvent I, as described in "Materials and Methods") by mixing an equal number of counts from the adducts formed by DMBA in vivo (D1--D3; Fig. 1Citation B) with marker adducts formed by reacting calf thymus DNA with anti-DMBADE (a1 and a3; Fig. 1Citation E) or syn-DMBADE (s1 and s2: Fig. 1Citation F). The DNA adducts formed by DMBA in vivo were tentatively identified as follows: A, D1-a1; B, D2-s2; C, D3-a3. O, origin; F, solvent front. TLC sheets were exposed to Kodak X-Omat X-ray films with intensifying screens at -70°C for 60 h.

 
Relationship Between Formation of the Major DMBA Adducts and Tumor-initiating Activity.
The data obtained in the current study allow us to address an important question regarding DMBA-initiated tumorigenesis, i.e., can stable diol epoxide DNA account for the tumor-initiating activity of this PAH in mouse skin? To address this question, we compared the levels of specific dAdo adducts produced by DMBA with those produced by (±)syn- and (±)anti-DMBADE in relation to the tumor-initiating activity of these compounds. In Table 1Citation , the levels (adducts/109 nucleotides) of DMBA-DNA adducts D1–D13 (at 10-nmol initiating-dose) are given and then compared with the levels of corresponding anti- and syn-DMBADE-DNA adducts (100-nmol initiating dose). We have calculated the tumor-initiating potential of the two major dAdo adducts (D2 and D3) that are formed after topical application of DMBA. The calculation of tumor-initiating potential is based on the following three assumptions: (a) because we have found all of the tumors initiated by these compounds have a -CAA- to -CTA- mutation at codon 61 of the H-ras gene, we have assumed only adenine adducts are relevant to tumor initiation by DMBA; (b) we have further assumed that the two major dAdo adducts formed after topical application of DMBA (D2 and D3) account for the majority of its tumor-initiating activity on mouse skin; similarly the corresponding adducts formed from syn- or anti-DMBADE (S2 and A3, respectively) account for the majority of the tumor-initiating activity of these diol epoxides; and (c) the "tumor-initiating potential" for a given DMBA-dAdo adduct is extrapolated from the maximum number of tumors initiated by the same adduct produced by syn- (S2) and anti- (A3) DMBADE at the doses used (10 nmol for DMBA, 100 nmol for syn- and anti-DMBADE). Thus, 100 nmol of syn-DMBADE produced 0.4 adducts/109 nucleotides of S2, accounting for 2.5 papillomas/mouse, whereas 100 nmol of anti-DMBADE produced 3.4 adducts/109 nucleotides of A3, accounting for 2.7 papillomas/mouse. This analysis reveals the tumor-initiation potential for the DMBA-DNA adduct, D2, is 12.5 papillomas/mouse and 1.5 papilloma/mouse for the DMBA-DNA adduct, D3, for a total of 14.0 papillomas/mouse. This value is similar to the number of papillomas/mouse observed after initiation with 10 nmol of DMBA (Fig. 2)Citation . Thus, these results show that the levels of both of the major syn-dAdo (D2) and anti-dAdo (D3) adducts produced after topical application of DMBA are sufficient to account for the tumor-initiating activity of this PAH.


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  		Table 1 Comparison of DNA adducts induced by DMBA (±)anti- and (±)syn-DMBADEsa treatment

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present work attempted to address two fundamental questions regarding DMBA-initiated tumorigenesis in mouse skin: (a) the role of specific bay-region diol epoxides in DMBA-initiated tumorigenesis; and (b) why mutations observed in DMBA-induced papillomas almost exclusively occurred at codon 61 (-CAA- to -CTA- transversion) of the H-ras gene. Our current results demonstrate that both the syn- and anti-DMBADE possess tumor-initiating activity in SENCAR mouse skin, and more importantly, that the papillomas generated by initiation with these bay-region diol epoxides have the same mutation (-CAA- to -CTA-) at codon 61 of H-ras as those tumors generated by initiation with DMBA. Additionally, our results demonstrate that both the syn- and anti-DMBADE produce stable DNA adducts in mouse epidermis after topical application. Furthermore, almost all of the stable DNA adducts produced in epidermal DNA by topical application of DMBA arise from either the syn- or anti-DMBADE. Finally, we found that the levels of the major syn- and anti-DMBADE-dAdo adducts (adduct spots D2 and D3, respectively, in Fig. 5Citation ) produced in mice treated with 10 nmol of DMBA, when compared with the corresponding levels in mice treated with 100 nmol of the diol epoxides (DNA adduct spots S2 and A3, respectively), could account for the tumor-initiating activity of DMBA. Therefore, the current data indicate that both the anti- and syn-diol epoxides contribute significantly to the tumor-initiating activity of DMBA, and that the mutations at codon 61 of H-ras gene in DMBA-induced papillomas can result from adenine adducts derived from either diol epoxide.

Although the DNA adducts induced by metabolically activated bay-region diol epoxides of DMBA have long been believed to play a major role in DMBA tumorigenesis, recently this model has been challenged (24 , 25) . Considerable work with DMBA has shown that treatment of various mouse cells in culture and mouse epidermis in vivo produces a number of stable DNA adducts (4, 5, 6, 7, 8, 9, 10, 11, 12 , 26) . These DNA adducts arise primarily from the bay-region anti- and syn-3,4-diol 1,2-epoxides of DMBA (4, 5, 6, 7, 8, 9, 10, 11, 12) . In cells or tissues that are targets for DMBA-initiated tumorigenesis, three major deoxyribonucleoside adducts have been identified using HPLC methods (33 , 34) . These DNA adducts arise from reaction of the anti-DMBADE with dGuo and dAdo and from reaction of the syn-DMBADE with dAdo. Schmeiser et al. (33) showed that these three major DMBA-DNA adducts, identified as deoxyribonucleoside adducts in earlier studies, were present and could be identified as deoxyribonucleotide adducts in fetal mouse cells exposed to DMBA using the 32P-postlabeling method. In contrast, Devanesan et al. (24) reported that a very high dose of 200 nmol of DMBA produced few stable DNA adducts in mouse skin at 4 h after treatment, and they concluded that the stable DNA adducts accounted for <1% of the total DNA binding associated with DMBA in mouse skin. Chakravarti et al. (25) have further proposed that the major pathway for the metabolic activation of DMBA is via one-electron oxidation, which occurs at the 12-methyl position. These workers have hypothesized that apurinic sites arising from the reaction of this metabolite with the N7 position of adenine and the subsequent depurination occurs in the genomic DNA, leading to adenine insertion in the complementary strand of DNA during the subsequent round of DNA replication. Therefore, it was hypothesized that the depurination at dAdo caused by these unstable adducts, rather than the stable bay-region diol epoxide dAdo adducts, induce A-to-T transversion mutations in codon 61 of the H-ras gene, triggering tumor initiation by DMBA (25) . However, our results, shown in Fig. 5Citation of the current study, demonstrate that even a 10-nmol dose of DMBA produces a significant amount of stable DNA adducts in mouse epidermis 24 h after topical treatment, and furthermore, we show that these adducts arise primarily from the anti- and syn-DMBADEs. These results are very similar to the results reported by Schmeiser et al. (33) , although their study used cultured fetal mouse cells. We have also examined DNA samples from mice treated with higher doses of DMBA (including 100- and 200-nmol doses) and from mice treated for earlier time points (including 6 and 12 h). The TLC autoradiogams (data not shown) looked very similar to those shown in Fig. 5Citation , although the total adduct levels differed in these various samples, as expected.

Chakravarti et al. (25) also reported that skin papillomas initiated with a derivative of DMBA with a partially saturated A-ring, THDMBA, possessed A-to-T transversion mutations in codon 61 of H-ras. The fact that this compound possesses tumor-initiating activity and, presumably, cannot be metabolically activated to bay-region diol epoxides was used as evidence to support one-electron oxidation at the 12-methyl group as the major metabolic pathway for DMBA. However, earlier work from one of our laboratories (40) showed that THDMBA was approximately 10–20-fold less potent than the parent hydrocarbon as a tumor initiator in the skin of SENCAR mice. These earlier data were interpreted to indicate that the metabolism in the A-ring of DMBA was essential for >90% of its tumor-initiating activity on mouse skin. These earlier data are consistent with the data in the current paper, showing that both the syn- and anti-DMBADEs possess tumor-initiating activity, and that the major dAdo adducts formed from both the syn- and anti-DMBADEs are present at levels consistent with their role in producing the majority of the tumor-initiating activity associated with DMBA in mouse skin. It remains intriguing as to what metabolic pathway(s) contribute to the metabolic activation of THDMBA and ultimately account for its much weaker tumor-initiating activity. Chakravarti et al. (25) have suggested that THDMBA is metabolically activated at the 12-methyl group via one-electron oxidation, thereby leading to the formation of unstable N7-dAdo adducts. Nair et al. (40) analyzed the tumor-initiating activity of five site-specifically fluorinated derivatives of THDMBA, an A-ring cyclopentano analogue of THDMBA, and a 6-fluoro-12-exo tautomer of 6F-THDMBA to address potential sites of metabolic activation of THDMBA. The results from these analyses, in conjunction with metabolism experiments (41) , suggested the possibility that hydroxylation at either C-1 or C-12 of THDMBA may be important for the biological activity of this PAH on mouse skin. Nevertheless, it is clear from the data in our current study that these pathways for the metabolic activation of THDMBA would account for only a small proportion of the biological activity of DMBA, if indeed they pertain to DMBA.

In comparison with DMBA, both the syn- and anti-DMBADE exhibited weaker tumor-initiating activity; the average number of papillomas/mouse was 5–9-fold lower than in mice initiated with DMBA (10 nmol; Figs. 1Citation and 2Citation ). We have shown in Table 1Citation that this can be explained by the levels of specific dAdo adducts produced from the syn- and anti-DMBADEs formed metabolically after topical application of DMBA. The reason why the diol epoxides are not more biologically active and do not produce higher DNA adduct levels may be related to their chemical reactivity. In this regard, because syn- and anti-DMBADE are highly reactive toward nucleophiles, a portion of the administered dose is very likely lost to chemical reactions, including hydrolysis, ultimately reducing the probability of interaction with genomic DNA. Furthermore, the reactive diol epoxides must pass through the stratum corneum and the granular and spinous layers of the epidermis before reaching the basal layer where epidermal target cells for initiation are believed to reside (42) . Thus, more DNA adducts may form in these layers than in the target cells in the basal layer. In contrast, DMBA must first be absorbed by cells before it gets metabolically activated, thereby increasing the probability that the diol epoxides formed may enter the nucleus and interact with genomic DNA of epidermal target cells in the basal layer. These factors may contribute to the weaker tumor-initiating activity of syn- and anti-DMBADE.

It has been found in many studies that the H-ras oncogene is the major oncogene activated in mouse skin papillomas initiated by PAHs, including DMBA. One important finding is that the site of mutation in the H-ras gene in skin papillomas is carcinogen dependent. Whereas mutations at codons 13 and 61 have been observed in the papillomas induced by benzo(a)pyrene (43 , 44) ,4 mutations occur almost exclusively at codon 61 in papillomas induced by DMBA. Because it has been shown that H-ras with mutations at codons 12, 13, and 61 are oncogenic and that bay-region diol epoxides formed from metabolically activated B(a)P and DMBA are able to react with adenine and guanine residues in genomic DNA (17, 18, 19, 20, 21) , these findings raise an intriguing question as to why the mutation in the H-ras gene induced by DMBA in mouse skin papillomas is confined to codon 61. It has been argued that DMBA-initiated epidermal cells that have a -CAA- to -CTA- mutation at codon 61 of the H-ras gene may have a growth advantage under the influence of the tumor-promoting agent, TPA, and undergo preferential clonal expansion; these cells consequently rise to benign papillomas (45) . However, this explanation cannot account for the results that B(a)P-induced papillomas have mutations at codon 13 as well as at codon 61 of the H-ras gene, although the TPA treatments are identical for both carcinogens (43 , 44) .4 There are three more possible explanations for this phenomenon: (a) activated DMBA diol epoxides may not form DNA adducts at codons 12 and 13 efficiently; (b) the adducts formed in these sequences are repaired swiftly and efficiently; and (c) only certain species of metabolically activated DMBADE are able to react with codon 61 of the H-ras gene. Mapping the distributions of anti- and syn-DMBADE-DNA adducts along the H-ras gene may allow us to determine which of these possibilities is correct.

In conclusion, the current data show that the major pathway for the metabolic activation of DMBA leading to its tumor-initiating activity on mouse skin involves formation of both the bay-region syn- and anti-DMBADEs. Both bay-region DMBADEs react with dAdo residues and initiate skin papillomas with A182-T mutations in codon 61 of H-ras gene. Furthermore, although alternate routes for the metabolic activation of DMBA may exist, they seem, based on the current data, to contribute only a minor component of the biological activity of DMBA on mouse skin.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants ES03124, ES08389 (to M-s. T.), CA36979, CA79442 (to J. D.), CA45293 (to R. M.), CA76262 (to T. J. S.), and Grant CH3995 from the Tobacco Research Council (to M-s. T.). Back

2 To whom requests for reprints should be addressed, at Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: (914) 731-3585; Fax: (914) 351-3492: E-mail: tang{at}env.med.nyu.edu Back

3 The abbreviations used are: DMBA, 7,12-dimethylbenz[a]anthracene; PAH, polycyclic aromatic hydrocarbon; dAdo, deoxyadenosine; TPA, 12-O-tetradecanoylphorbol-13-acetate; (±)anti-DMBADE, (±) 1ß,2ß-epoxy-3ß,4{alpha}-dihydroxyl-1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene; (±)syn-DMBADE, (±) 1{alpha},2{alpha}-epoxy-3ß,4{alpha}dihydroxyl-1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene; dGuo, deoxyguanosine; THDMBA, 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene; B(a)p, benzo(a)pyrene. Back

4 J. DiGiovanni, unpublished data. Back

Received 3/10/00. Accepted 8/ 9/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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