Cancer initiation by polycyclic aromatic hydrocarbons results from formation of stable DNA adducts rather than apurinic sites

Victor J. Melendez-Colon1,4, Andreas Luch2, Albrecht Seidel3 and William M. Baird1,5

1 Department of Environmental & Molecular Toxicology and Biochemistry & Biophysics, ALS, Oregon State University, Corvallis, OR 97331, USA,
2 Institute of Toxicology and Environmental Hygiene, Technical University of Munich, 80636 Munich, Germany and
3 Institute of Toxicology, University of Mainz, 55131 Mainz, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with high carcinogenic potencies that have been linked to the etiology of human cancers through their presence in cigarette smoke and environmental mixtures. They are metabolically activated in cells by cytochrome P450 enzymes and/or peroxidases to reactive intermediates that damage DNA. One pathway of activation forms dihydrodiol epoxides that covalently bind to exocyclic amino groups of purines in DNA to form stable adducts. Another pathway involves formation of radical cations that bind to the N7 or C8 of purines to form unstable adducts that depurinate to leave apurinic (AP) sites in DNA. In the present study the proportions of stable DNA adducts and AP sites formed by the carcinogenic PAHs dibenzo[a,l]-pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P) have been investigated in a target tissue for carcinogenesis, mouse epidermis. After topical application of the PAHs on the skin of female SENCAR mice epidermal DNA was isolated and the formation of stable DNA adducts was measured by 33P-postlabeling and HPLC analysis. AP sites in DNA were measured with an aldehyde reactive probe in a slot-blot assay. At both 4 and 24 h after exposure, DB[a,l]P formed significantly higher amounts of stable DNA adducts than DMBA, and B[a]P exhibited the lowest level of binding. In contrast, the number of AP sites present in mice treated with these PAHs was in the order: DMBA > B[a]P >> DB[a,l]P. The level of AP sites was significantly lower than the level of stable adducts for each PAH. The most potent carcinogen, DB[a,l]P, induced the highest level of stable adducts and the lowest level of AP sites in epidermal DNA. These results indicate that stable DNA adducts rather than AP sites are responsible for tumor initiation by carcinogenic PAHs.

Abbreviations: AP, apurinic; ARP, aldehyde reactive probe; B[a]P, benzo[a]pyrene; B[a]PDE, benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide; dA, deoxyadenosine; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE, dibenzo[a,l]pyrene-11,12-dihydrodiol 13,14-epoxide; dG, deoxyguanosine; DMBA, 7,12-dimethylbenz[a]anthracene; DMBADE, 7,12-dimethylbenz[a]anthracene-3,4-dihydrodiol 1,2-epoxide; DMS, dimethyl sulfate; PAH(s), polycyclic aromatic hydrocarbon(s).


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs) induce tumor formation in many tissues in rodents and environmental mixtures of PAHs are implicated in tumor induction also in humans (14). Tumor induction in mouse skin has been extensively used to determine the carcinogenic potency of individual PAHs (1,47). Both benign papillomas and malignant carcinomas can be induced by multiple treatments with carcinogenic PAHs or in a two stage protocol involving initiation by a single treatment with a subcarcinogenic dose of the PAH followed by a promotion twice-weekly with 12-O-tetradecanoylphorbol 13-acetate (5,7,8). Dibenzo[a,l]pyrene (DB[a,l]P) was found to have the highest carcinogenic potency among all PAHs tested to date, including 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) (Figure 1Go), in mouse skin (1,4,6,9).



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Fig. 1. Structures of the potent carcinogens B[a]P, DMBA, and DB[a,l]P.

 
Mouse skin studies have also provided valuable information about the mechanism of tumor induction. Brookes and Lawley (10) compared the covalent binding of various PAHs in mouse skin and demonstrated that the level of binding to DNA correlated with the relative carcinogenic potency of the PAH. The metabolically formed intermediates involved in the binding of PAHs to epidermal DNA were identified as dihydrodiol epoxides (11). (+)-Anti-B[a]P-7,8-dihydrodiol 9,10-epoxide [(+)-anti-B[a]PDE] was identified as the specific stereoisomer responsible for the majority of the binding of B[a]P to DNA (12), predominantly through formation of stable adducts on the exocyclic amino group of deoxyguanosine (dG) [(+)-anti-B[a]PDE–dG adducts] (13).

Studies on metabolic activation of DMBA, first in cells in culture (14) and subsequently in mouse skin (15), demonstrated that the DMBA-3,4-dihydrodiol 1,2-epoxides (DMBADEs) are the ultimate carcinogenic metabolites responsible for covalent binding of DMBA to DNA. The bay-region of DMBA is sterically hindered due to the methyl substituent at position 12 distorting the molecule from planarity (Figure 1Go and ref. 16). The dihydrodiol epoxides of DMBA bind to the exocyclic amino groups of both deoxyadenosine (dA) and dG in DNA, a common property of dihydrodiol epoxides of non-planar PAHs (17). The proportions of adducts formed by the syn- and anti-isomers of DMBADE varied depending on the specific tissue and the time of exposure, but adducts are always formed by both isomers (18).

The hexacyclic PAH DB[a,l]P possesses a fjord region (Figure 1Go) leading to repulsive interaction between the hydrogens at positions 1 and 14 and consequently to an out-of-plane distortion of the molecule (19). Studies with cells in culture and mouse skin demonstrated that DB[a,l]P is stereoselectively activated to the (+)-syn- and (–)-anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDEs) which predominantly bind to dA, and, to a lesser extent, to dG residues in DNA (2022).

In addition to the well-established activation pathway of carcinogenic PAHs catalyzed by cytochrome P450 monooxygenases and microsomal epoxide hydrolase that leads to the formation of dihydrodiol epoxides and subsequently to stable DNA adducts (2326), an alternative activation pathway through radical cations generated by one-electron oxidation has been proposed (27,28). Due to the binding of these intermediates mainly at N7- or C8-positions of purine bases, the resulting adducts are unstable and generate apurinic (AP) sites in the DNA by spontaneous depurination (28). AP sites have also been reported to be produced in DNA by reaction with dihydrodiol epoxides (29) and it has been proposed that these lesions, rather than stable DNA adducts, are responsible for the induction of mutations in critical genes leading to cancer initiation (28,30,31). Based upon analysis of depurinated DNA adducts in vitro, it was reported that DB[a,l]P yields predominantly (85%) unstable adducts formed by both the one-electron oxidation and the dihydrodiol epoxide pathways (29). In contrast, in MCF-7 cells exposed to DB[a,l]P and DB[a,l]PDEs (32) more than 98% of all adducts detected were stable and resulted from the binding of DB[a,l]PDE to DNA. No increase in the number of AP sites in this cellular DNA or DNA reacted with DB[a,l]PDEs in solution was detected using an alkaline cleavage Southern blotting assay to analyze a restriction fragment of the dihydrofolate reductase gene (32).

To determine the role of the formation of stable DNA adducts and AP sites in PAH-dependent carcinogenesis in vivo, both types of lesions present in DNA of a target tissue for PAH carcinogenesis, mouse epidermis, were analyzed after topical treatment with the carcinogens B[a]P, DMBA or DB[a,l]P. Stable DNA adducts were measured by 33P-postlabeling and HPLC analysis; AP sites were quantitated using a highly sensitive slot blot assay with an aldehyde reactive probe (ARP) reagent that binds to aldehyde groups generated within the sugar–phosphodiester backbone at AP sites (33,34). These results permit a quantitative comparison of the proportions of stable adducts and AP sites present in mouse epidermal DNA and allow estimation of the relative contribution of each type of DNA damage induced by PAHs to tumor initiation in this target tissue.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
B[a]P, DMBA and DB[a,l]P were obtained from Chemsyn Science Laboratories (Lenexa, KS). The ARP reagent was synthesized as previously described (35). All chemicals and enzymes used were the same as previously described (32,33,36,37).

Treatment of mouse skin with PAHs
Two days prior to treatment female SENCAR mice (obtained from NCI-Frederick Cancer Center, Frederick, MD), 5–6 weeks of age, were shaved on the dorsal side (five mice per group). In the resting phase of their hair-growth cycle the animals received a topical application of 50, 200 or 400 nmol B[a]P, DMBA or DB[a,l]P, dissolved in 100 µl acetone. Controls were treated with solvent only. After 4 or 24 h the mice were killed by cervical dislocation and the treated areas of skin were promptly excised.

Isolation of epidermal DNA
Epidermal DNA was isolated by a modification of the procedure described by Rho et al. (38). After removal, excised skin was quickly spread dermis side down on a piece of index card and immersed in liquid nitrogen. The epidermis was then separated from the dermis by scraping the frozen skins with a razor blade. The powdered frozen epidermis was added to lysis buffer (10 mM Tris, 1 mM EDTA, 1% SDS; pH 8) and frozen at –80°C until use. After thawing the frozen cell solution, cells were gently homogenized and DNA was then extracted with phenol followed by Sevag (chloroform:isoamyl alcohol, 24:1) extractions, and precipitated with ethanol. The extracted DNA was dissolved in sterilized TE buffer (10 mM Tris, 1 mM EDTA; pH 8) and incubated at 37°C with RNase, and then with Proteinase K. After extraction with Sevag, DNA was reprecipitated and suspended in sterilized TE buffer.

AP site detection by the ARP-slot blot assay
Formation of AP sites was measured using the ARP reagent (Figure 3AGo) in a slot blot assay according to the protocol previously described (33,34) with minor modifications. After isolation, 3 µg DNA was incubated with the ARP reagent and then immobilized on a BAS-85 nitrocellulose membrane using a Minifold II vacuum filter device (Schleicher & Schuell, Keene, NH). The biotin-tagged AP sites were detected using streptavidin-conjugated horseradish peroxidase (Gibco BRL, Gaithersburg, MD). The enzymatic activity on the membrane was visualized using ECL reagents (Amersham, Arlington Heights, IL) and quantified by scanning densitometry and subsequent analysis of the X-ray films using the ImageQuaNTTM Software (Molecular Dynamics, Sunnyvale, CA). The number of AP sites was determined based on comparison to a calibration curve generated with dimethyl sulfate (DMS)-treated DNA of Chinese hamster ovary cells that contained known numbers of AP sites (32). As already described (33), the detection threshold of the ARP-slot blot assay was found to be in the range of 3 AP sites/107 nucleotides.



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Fig. 3. Detection of AP sites formed in mouse epidermal DNA after exposure to B[a]P, DMBA or DB[a,l]P. Structure of the ARP reagent (A) used for biotin-tagging of AP sites within epidermal DNA as described in Materials and methods. The slot blot presented (B) depicts signals from epidermal DNA obtained after treatment with 50, 200 or 400 nmol DB[a,l]P (lanes 1, 4 and 7), DMBA (lanes 2, 5 and 8) or B[a]P (lanes 3, 6 and 9). Positive control: DNA containing 142 AP sites/106 nucleotides after reaction with DMS (32).

 
Analysis of stable DNA adducts by 33P-postlabeling and HPLC
Stable PAH-DNA adducts were 33P-postlabeled using the nuclease P1 and prostatic acid phosphatase protocol and subsequently analyzed by reverse-phase HPLC as previously described (32). The B[a]P-DNA adducts were separated by HPLC using a linear gradient consisting of 0.1 M ammonium phosphate buffer (pH 5.5; solvent A) and 100% methanol (solvent B) at a flow rate of 1 ml/min: 44% B for 10 min; from 44 to 49% B over 50 min; from 49 to 58% B over 15 min; and 58% B for 45 min. DMBA–DNA adducts were resolved using a gradient consisting of 0.05 M ammonium phosphate buffer that contained 20 mM tetrabutylammonium phosphate (pH 5.5; solvent A') and solvent B at a flow rate of 1 ml/min: 50% B for 25 min; from 50 to 57% B over 70 min; from 57 to 62% B over 20 min; from 62 to 43% B over 20 min; and 43% B for 20 min. The gradient used for DB[a,l]P-DNA adducts consisted of solvent A and 10% acetonitrile/90% methanol (v/v; solvent B') and a flow rate of 1 ml/min: from 44 to 49% B' over 40 min; from 49 to 55% B' over 60 min; and from 55 to 65% B' over 20 min. The total level of stable PAH–DNA adducts in each sample was calculated based on labeling of a [3H]B[a]PDE–DNA standard of known modification which was analyzed together with each set of postlabeling samples (39).

Statistical analyses
Statistical analyses were conducted using the Student's t-test and the one-way analysis of variance (ANOVA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The stable PAH–DNA adducts present in the epidermal DNA from PAH-treated mice were analyzed by postlabeling the PAH–DNA samples with [33P]ATP and adduct separation on HPLC. Stable DNA adducts were formed in epidermis in a time- and dose-dependent manner (Table IGo). The HPLC profiles of stable adducts formed 4 h after treatment are shown in Figure 2Go. After exposure to B[a]P, mouse epidermis contained only one major DNA adduct that eluted at 45 min (Figure 2Go) and which was identified as the (+)-anti-B[a]PDE–dG adduct by coelution with the standard. Small amounts of dG adducts formed by an isomeric syn-B[a]PDE (40) represented <7% of the total DNA adducts and were found only after treatment with high doses (400 nmol).


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Table I. Levels of stable DNA adducts and AP sites present in epidermal DNA of female SENCAR mice at the indicated times after exposure to 50, 200 or 400 nmol B[a]P, DMBA and DB[a,l]P, respectivelya
 


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Fig. 2. HPLC elution profiles of the 33P-labeled PAH–DNA adducts formed in mouse epidermis 4 h after treatment with 50, 200 or 400 nmol B[a]P, DMBA or DB[a,l]P. The PAH–DNA adducts were isolated, postlabeled and analyzed by reverse-phase HPLC as described in Materials and methods.

 
The stable DMBA–DNA adducts detected in epidermal DNA of mice were formed by the reaction of both syn- and anti-DMBADE with dA or dG (37). The predominant anti-DMBADE–dG and syn-DMBADE–dA adducts eluted around 70 min (Figure 2Go) and represented >70% of all stable adducts. Lower amounts of other DMBADE–dG or –dA adducts were also detected as peaks at 55 (anti-DMBADE–dG), 80 (anti-DMBADE–dA) and 105 min (syn-DMBADE–dA) (37).

The HPLC elution profiles of the DNA adducts found in epidermis after treatment with DB[a,l]P (Figure 2Go) contained one major and three minor peaks previously identified as reaction products between the (–)-anti-DB[a,l]PDE and dA (major adduct at 73 min) or dG (the three adducts eluting between 45 and 65 min; 22). Small amounts of a dA adduct derived from the isomeric (+)-syn-DB[a,l]PDE were also detected (100 min), however, this adduct represented <3% of all stable DB[a,l]P–DNA adducts.

Covalent binding of the ARP reagent (Figure 3AGo) to aldehyde groups generated at AP sites was used to measure the number of these lesions by a biotin-mediated technique. Figure 3BGo shows the band intensities of a typical slot blot assay for the detection of AP sites in epidermal DNA after exposure to B[a]P, DMBA or DB[a,l]P. Visualization of the biotin-tagged AP sites within DNA from mice treated with 50 nmol of the three PAHs resulted in only faint bands with comparable intensities to DNA from solvent-treated animals (Figure 3BGo). Analysis of epidermal DNA from mice exposed to higher doses (200 or 400 nmol) of all three PAHs revealed slightly increased band intensities. However, in all cases the intensity was very low compared with the positive control of DMS-modified DNA containing 142 apurinic sites per 106 nucleotides (Figure 3BGo).

The levels of AP sites and stable adducts detected in epidermal DNA from mice after exposure to B[a]P, DMBA or DB[a,l]P are reported in Table IGo and illustrated in Figure 4Go. Exposure to 50 nmol of any PAH for 4 h caused no detectable increase in the level of AP sites. However, a slight increase in the level of AP sites was observed after treatment with 200 or 400 nmol B[a]P and DMBA—about 1 and 2 AP sites per 106 nucleotides, respectively (Table IGo, Figure 4AGo). These values were significantly elevated compared with DNA from acetone-treated animals (P < 0.05) and were above the detection threshold of the assay used (~0.3 AP sites/106 nucleotides). In contrast, after 4 h of exposure to DB[a,l]P no statistically significant increase in the number of AP sites in epidermal DNA was found at any dose tested (P > 0.1) (Table IGo, Figure 4AGo). Stable PAH–DNA adduct levels 4 h after exposure to B[a]P, DMBA or DB[a,l]P were dose-dependent and statistically significant increases in the number of lesions were observed for all PAHs [4–11 (B[a]P), 5–15 (DMBA) and 8–21 (DB[a,l]P) adducts per 106 nucleotides] (Table IGo, Figure 4BGo).



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Fig. 4. Comparison of the levels of AP sites and stable adducts present in epidermal DNA after treatment of mice with increasing doses of B[a]P (plain bars), DMBA (shaded bars) or DB[a,l]P (hatched bars). Levels of AP sites (A) and stable DNA adducts (B) 4 h after exposure to 50, 200 or 400 nmol PAH. Levels of AP sites (C) and stable DNA adducts (D) 24 h after exposure to 50 or 400 nmol PAH. Data represent mean ± SD (n = 3).

 
After 24 h of exposure the levels of AP sites in epidermal DNA of B[a]P- and DMBA-treated mice were significantly higher than those present after 4 h of exposure (P < 0.001). The levels ranged from 4 to 5 (B[a]P) and 5 to 6 (DMBA) AP sites per 106 nucleotides (Table IGo, Figure 4CGo). In contrast, epidermal DNA from DB[a,l]P-treated animals contained only low levels of AP sites (1–3 AP sites/106 nucleotides) (Table IGo, Figure 4CGo). These values were significantly higher than the acetone-control group (P < 0.05), but significantly lower than the levels induced by B[a]P or DMBA (P < 0.0001). Large increases in the level of stable DNA adducts were observed 24 h after exposure (Table IGo, Figure 4DGo). The amounts of stable PAH–DNA adducts/106 nucleotides were: B[a]P (1422), DMBA (2132), DB[a,l]P (2943). Thus, after exposure to a dose of 50 nmol DB[a,l]P the ratio of stable DNA adducts to AP sites was in the range of ~32:1, but significantly lower after exposure to DMBA or B[a]P (4:1).


    Discussion
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 Abstract
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 Materials and methods
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 References
 
The pathways of metabolic activation and the types of DNA damage induced by three carcinogenic PAHs in mouse epidermis were evaluated by analysis of the formation of stable adducts and AP sites in the target tissue DNA. Previous investigations provided evidence for both types of DNA damage induced by PAHs but their etiological role during initiation of tumorigenesis is still a matter of debate (26,28,30,41).

The present results demonstrate that B[a]P, DMBA and DB[a,l]P formed stable DNA adducts in epidermal DNA of mice in a dose- and time-dependent manner (Table IGo, Figure 4Go). This was observed at doses of PAHs which have been shown to initiate the formation of high levels of skin tumors in these animals (1,4,6). The hexacyclic DB[a,l]P, the most potent carcinogen among these three PAHs (6), formed the highest levels of stable DNA adducts in mouse epidermis. This compound initiates tumor formation at doses as low as 1.33 and 1 nmol in female SENCAR (42) and Swiss mice (6), respectively. Analysis of the stable DNA adducts revealed that epidermal DNA of DB[a,l]P-treated mice contained mainly (–)-anti-DB[a,l]PDE–DNA adducts with a small proportion (3%) of (+)-syn-DB[a,l]PDE–DNA adducts, a result that confirms previous analyses of DB[a,l]P–DNA adducts in mouse skin using a 35S-postlabeling technique (21). Epidermal DNA from skin of treated mice contained high levels of stable B[a]P– and DMBA–DNA adducts that resulted from activation through bay region dihydrodiol epoxides (Table IGo, Figure 4Go). The levels of stable dihydrodiol epoxide–DNA adducts were in the order DB[a,l]P > DMBA > B[a]P at all doses and times tested.

Analysis of the presence of AP sites in epidermal DNA from mice exposed to each of these three carcinogenic PAHs (Table IGo, Figure 4Go) indicates that AP lesions surpassed the background level only after treatment with high doses (200 and 400 nmol) or after prolonged exposure times (24 h). For the most potent carcinogen, DB[a,l]P, almost no increase in AP sites was detected in epidermal DNA from mice exposed to a dose of 50 nmol at either 4 or 24 h. Only after exposure to the highest dose tested (400 nmol) were low levels of AP sites (~2–3/106 nucleotides) detected (Table IGo, Figure 4CGo). Application of DB[a,l]P at doses >100 nmol has been found to be highly toxic to mouse skin and caused a severe dose-dependent inflammatory response (43) and an inverse relationship between dose and tumor incidence (1). Comparison of the proportions of stable adducts and AP sites formed by DB[a,l]P in epidermal DNA after 24 h of exposure indicates that <6% of all lesions were AP sites (Table IGo, Figure 4Go). In the case of B[a]P and DMBA, stable DNA adducts accounted for >80% of the total DNA lesions at all doses and times of exposure.

In a study on metabolic activation of DB[a,l]P in vivo, it has been reported that 99% of all DB[a,l]P adducts detected in mouse skin were unstable and released from the sugar–phosphodiester DNA backbone by depurination (44). Because DB[a,l]P has been shown to induce H-ras mutations in this tissue (45) it was proposed that the resulting AP sites are responsible for tumor initiation in mouse skin. Devanesan et al. (46) reported that 99% of all PAH-adducted purines in mouse skin exposed to DMBA are lost from the DNA by depurination. The activation of B[a]P in mouse skin has been reported to result in the formation of >70% depurinating B[a]P–DNA adducts (47). In rat mammary glands, another target tissue of carcinogenic PAHs, 52 and 97% of all adducts formed after exposure to DMBA and DB[a,l]P, respectively, were found to be unstable and released from the DNA by depurination (48,49).

At present the biological consequences of the formation of depurinating adducts by PAHs are unknown. AP sites are rapidly repaired in mammalian cells (50). It has been estimated that the highly efficient repair capacity of living mammalian cells is enzymatically competent for repairing more than 10 000 AP sites spontaneously formed per day in each individual cell (51). The failure to detect AP sites at levels above the spontaneous background upon exposure to low tumor-initiating doses of B[a]P, DMBA and DB[a,l]P (Table IGo, Figure 4AGo) indicates that depurinating adducts do not increase the level of AP sites present in epidermal DNA above the background level. This may result from efficient repair of AP sites formed by depurinating adducts. Recent studies on the repair of PAH-induced DNA lesions provided evidence that a very fast and efficient base excision repair pathway may be responsible for the removal of AP sites induced by PAHs (52,53). It was demonstrated that dihydrodiol epoxides from strong carcinogens such as B[a]P induced a small fraction of labile but a large fraction of stable and persistent DNA adducts in vitro. In contrast, treatment with dihydrodiol epoxides from weak carcinogens such as chrysene resulted in a time-dependent accumulation of AP sites in DNA which should be effectively repaired by the base excision repair pathway (5254). Although AP sites may be strongly mutagenic in the absence of an efficient repair system and stable adducts tend to be mutagenic only in a particular percentage depending upon the specific adduct structure and its sequence context, the absence of an increase in AP sites in DNA of a target tissue treated with cancer-initiating doses of strong carcinogenic PAHs suggests that the biological consequences of depurinating adducts are unlikely to be an important factor in tumor induction by PAHs.

Formation of stable PAH–DNA adducts can lead to the induction of mutations that activate protooncogenes or inactivate tumor suppressor genes as an important event during tumor initiation (5557). Activation of the H-ras protooncogene may be involved in tumor initiation in mouse skin by various carcinogenic PAHs (5,58). Nucleotide transversions within codons 12 (G->T) or 61 (A->T) of cellular H-ras have frequently been found as activating mutations upon exposure to carcinogenic PAHs such as B[a]P (58), DMBA (5,59,60) or DB[a,l]P (45). Exposure to B[a]P and DB[a,l]P mainly results in the induction of G->T and A->T transversions, respectively, whereas DMBA treatment caused both kinds of mutations in these two codons (5,45,58,59). These mutational events correlate with the preferential binding of metabolically formed dihydrodiol epoxides of B[a]P and DB[a,l]P to dG [(+)-anti-B[a]PDE–dG adduct] and dA [(–)-anti-DB[a,l]PDE–dA adduct], respectively (Figure 2Go). However, transversions within the H-ras gene may also correlate with the types of mutations expected at AP sites formed by depurinating adducts (28,31). The type of mutations formed is not sufficient to distinguish the nature of PAH-induced DNA damage responsible for their induction.

The present study demonstrates that topical exposure of mice to the potent carcinogens B[a]P, DMBA and DB[a,l]P results in a dose-dependent formation of high levels of stable dihydrodiol epoxide–DNA adducts but only low levels of AP sites within epidermal DNA. The generation of increased levels of stable DNA adducts in the order DB[a,l]P > DMBA > B[a]P correlates with the order of carcinogenic potency of these PAHs in mouse skin (6). DB[a,l]P, the compound previously found to be the most potent skin carcinogen among all PAHs tested, formed the highest level of stable adducts and the lowest level of AP sites. These results indicate that the genomic damage induced by stable dihydrodiol epoxide–DNA adducts rather than the formation of AP sites is responsible for cancer initiation in mouse skin by PAHs.


    Acknowledgments
 
This work was supported by grants CA40228 and CA28825 from the National Cancer Institute, Department of Health and Human Services (W.M.B.), and by the Deutsche Forschungsgemeinschaft (SFB 302) (A.S.).


    Notes
 
4 Present address: Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA Back

5 To whom correspondence should be addressed Email: william.baird{at}orst.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Cavalieri,E.L., Higginbotham,S., RamaKrishna,N.V.S., Devanesan,P.D., Todorovic,R., Rogan,E.G. and Salmasi,S. (1991) Comparative dose-response tumorigenicity studies of dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene and two dibenzo[a,l]pyrene dihydrodiols in mouse skin and rat mammary gland. Carcinogenesis, 12, 1939–1944.[Abstract]
  2. Grimmer,G. (1983) Environmental Carcinogens: Polycyclic Aromatic Hydrocarbons. CRC Press, Boca Raton, FL.
  3. Harvey,R.G. (1991) Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity. Cambridge University Press, Cambridge, UK.
  4. LaVoie,E.J., He,Z.-M., Meegalla,R.L. and Weyand,E.H. (1993) Exceptional tumor-initiating activity of 4-fluorobenzo[j]fluoranthene on mouse skin: comparison with benzo[j]fluoranthene, 10-fluoro-benzo[j]fluoranthene, benzo[a]pyrene, dibenzo[a,l]pyrene and 7,12-dimethylbenz[a]anthracene. Cancer Lett., 70, 7–14.[ISI][Medline]
  5. DiGiovanni,J. (1992) Multistage carcinogenesis in mouse skin. Pharmac. Ther., 54, 63–128.[ISI][Medline]
  6. Higginbotham,S., RamaKrishna,N.V.S., Johansson,S.L., Rogan,E.G. and Cavalieri,E.L. (1993) Tumor-initiating activity and carcinogenicity of dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene at low doses in mouse skin. Carcinogenesis, 14, 875–878.[Abstract]
  7. DuBowski,A., Johnston,D.A., Rupp,T., Beltran,L., Conti,C.J. and DiGiovanni,J. (1998) Papillomas at high risk for malignant progression arising both early and late during two-stage carcinogenesis in SENCAR mice. Carcinogenesis, 19, 1141–1147.[Abstract]
  8. Schmidt,R. and Hecker,E. (1989) Biological assays for irritant, tumor-initiating and tumor-promoting activities II. Standardized initiation/promotion protocol and semiquantitative estimation of promoting (or initiating) potencies in skin of NMRI mice. J. Cancer Res. Clin. Oncol., 115, 516–524.[ISI][Medline]
  9. Cavalieri,E.L., Rogan,E.G., Higginbotham,S., Cremonesi,P. and Salmasi,S. (1989) Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a]pyrenes: the very potent environmental carcinogen dibenzo[a,l]pyrene. J. Cancer Res. Clin. Oncol., 115, 67–72.[ISI][Medline]
  10. Brookes,P. and Lawley,P.D. (1964) Evidence for the binding of polynuclear aromatic hydrocarbons to the nucleic acids of mouse skin: relation between carcinogenic power of hydrocarbons and their binding to deoxyribonucleic acid. Nature, 202, 781–784.[ISI]
  11. Sims,P., Grover,P.L., Swaisland,A., Pal,K. and Hewer,A. (1974) Metabolic activation of benzo[a]pyrene proceeds by a diol-epoxide. Nature, 252, 326–328.[ISI][Medline]
  12. Kooreda,M., Moore,P.D., Wislocki,P.G., Levin,W., Conney,A.H., Yagi,H. and Jerina,D.M. (1978) Binding of benzo[a]pyrene 7,8-diol 9,10-epoxides to DNA, RNA, and protein of mouse skin occurs with high stereoselectivity. Science, 199, 778–781.[ISI][Medline]
  13. Meehan,T. and Calvin,M. (1979) Double-stranded DNA stereoselectively binds benzo[a]pyrene diol epoxides. Nature, 277, 410–412.[ISI][Medline]
  14. Moschel,R.C., Baird,W.M. and Dipple,A. (1977) Metabolic activation of the carcinogen 7,12-dimethylbenz[a]anthracene for DNA binding. Biochem. Biophys. Res. Commun., 76, 1092–1098.[ISI][Medline]
  15. Bigger,C.A.H., Sawicki,J.T., Blake,D.M., Raymond,L.G. and Dipple,A. (1983) Products of binding of DMBA to DNA in mouse skin. Cancer Res., 43, 5647–5651.[Abstract]
  16. Rabinowitz,J.R., Little,S.B. and Lewis-Bevan,L. (1996) The effect of crowding in the bay/fjord region on the structure and reactivities of polycyclic aromatic hydrocarbons and their metabolites: quantum mechanical studies. Polycyclic Aromat. Comp., 11, 237–244.[ISI]
  17. Dipple,A., Pigott,M., Moschel,R.C. and Costantino,N. (1983) Evidence that binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse embryo cell cultures results in extensive substitution of both adenine and guanine residues. Cancer Res., 43, 4132–4135.[Abstract]
  18. Cheng,S.C., Prakash,A.S., Pigott,M.A., Hilton,B.D., Roman,J.M., Lee,H., Harvey,R.G. and Dipple,A. (1988) Characterization of 7,12-dimethylbenz[a]anthracene-adenine nucleoside adducts. Chem. Res. Toxicol., 1, 216–221.[ISI][Medline]
  19. Katz,A.K., Carrell,H.L. and Glusker,J.P. (1998) Dibenzo[a,l]pyrene (dibenzo[def,p]chrysene): fjord-region distortions. Carcinogenesis, 19, 1641–1648.[Abstract]
  20. Ralston,S.L., Lau,H.H.S., Seidel,A., Luch,A., Platt,K.L. and Baird,W.M. (1994) The potent carcinogen dibenzo[a,l]pyrene is metabolically activated to fjord-region 11,12-diol 13,14-epoxides in human mammary carcinoma MCF-7 cell cultures. Cancer Res., 54, 887–890.[Abstract]
  21. Ralston,S.L., Lau,H.H.S., Seidel,A., Luch,A., Platt,K.L. and Baird,W.M. (1994) Identification of dibenzo[a,l]pyrene-DNA adducts formed in cells in culture and in mouse skin. Polycyclic Aromat. Comp., 6, 199–206.
  22. Ralston,S.L., Seidel,A., Luch,A., Platt,K.L. and Baird,W.M. (1995) Stereoselective activation of dibenzo[a,l]pyrene to (–)-anti(11R,12S,13S,14R)- and (+)-syn(11S,12R,13S,14R)-11,12-diol-13,14-epoxides which bind extensively to deoxyadenosine residues of DNA in the human mammary carcinoma cell line MCF-7. Carcinogenesis, 16, 2899–2907.[Abstract]
  23. Guengerich,F.P. and Shimada,T. (1991) Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem. Res. Toxicol., 4, 391–407.[ISI][Medline]
  24. Guengerich,F.P. (1992) Metabolic activation of carcinogens. Pharmacol. Ther., 54, 17–61.[ISI][Medline]
  25. Hall,M. and Grover,P.L. (1990) Polycyclic aromatic hydrocarbons: metabolism, activation and tumour initiation. In Cooper,C.S. and Grover,P.L. (eds.) Chemical Carcinogenesis and Mutagenesis, Vol. I. Springer-Verlag, Heidelberg, pp. 327–372.
  26. Szeliga,J. and Dipple,A. (1998) DNA adduct formation by polycyclic aromatic hydrocarbon dihydrodiol epoxides. Chem. Res. Toxicol., 11, 1–11.[ISI][Medline]
  27. Cavalieri,E.L. and Rogan,E.G. (1984) Role of radical cations in aromatic hydrocarbon carcinogenesis. Environ. Health Perspect., 64, 69–84.
  28. Cavalieri,E.L. and Rogan,E.G. (1995) Central role of radical cations in metabolic acitivation of polycyclic aromatic hydrocarbons. Xenobiotica, 25, 677–688.[ISI][Medline]
  29. Li,K.-M., Todorovic,R., Rogan,E.G., Cavalieri,E.L., Ariese,F., Suh,M., Jankowiak,R. and Small,G.J. (1995) Identification and quantitation of dibenzo[a,l]pyrene-DNA adducts formed by rat liver microsomes in vitro: preponderance of depurination adducts. Biochemistry, 34, 8043–8049.[ISI][Medline]
  30. Cavalieri,E.L. and Rogan,E.G. (1996) The primary role of apurinic sites in tumor initiation. Polycyclic Aromat. Comp., 10, 251–258.[ISI]
  31. Chakravarti,D., Pelling,J.C., Cavalieri,E.L. and Rogan,E.G. (1995) Relating aromatic hydrocarbon-induced DNA adducts and c-Ha-ras mutations in mouse skin papillomas: the role of apurinic sites. Proc. Natl Acad. Sci. USA, 92, 10422–10426.[Abstract]
  32. Melendez-Colon,V.J., Smith,C.A., Seidel,A., Luch,A., Platt,K.L. and Baird,W.M. (1997) Formation of stable adducts and absence of depurinating DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[a,l]pyrene or its diol epoxides. Proc. Natl Acad. Sci. USA, 94, 13542–13547.[Abstract/Free Full Text]
  33. Melendez-Colon,V.J., Luch,A., Seidel,A. and Baird,W.M. (1999) Comparison of cytochrome P450 and peroxidase dependent metabolic activation of the potent carcinogen dibenzo[a,l]pyrene in human cell lines: formation of stable DNA adducts and absence of a detectable increase in apurinic sites. Cancer Res., 59, 1412–1416.[Abstract/Free Full Text]
  34. Nakamura,J., Walker,V.E., Upton,P.B., Chiang,S., Kow,Y.W. and Swenberg,J. (1998) Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions. Cancer Res., 58, 222–225.[Abstract]
  35. Kubo,K., Ide,H., Wallace,S.S. and Kow,Y.W. (1992) A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion. Biochemistry, 31, 3703–3708.[ISI][Medline]
  36. Einolf,H.J., Amin,S., Yagi,H., Jerina,D.M. and Baird,W.M. (1996) Benzo[c]phenanthrene is activated to DNA-binding diol epoxides in the human mammary carcinoma cell line MCF-7 but only limited activation occurs in mouse skin. Carcinogenesis, 17, 2237–2244.[Abstract]
  37. Lau,H.H.S., Coffing,S.L., Lee,H., Harvey,R.G. and Baird,W.M. (1995) Stereoselectivity of activation of 7,12-dimethylbenz[a]anthracene-3,4-dihydrodiol to the anti-diol epoxide metabolite in a human mammary carcinoma MCF-7 cell-mediated V79 cell mutation assay. Chem. Res. Toxicol., 8, 970–978.[ISI][Medline]
  38. Rho,O., Bol,D.K., You,J., Beltran,L., Rupp,T. and DiGiovanni,J. (1996) Altered expression of insulin-like growth factor I and its receptor during multistage carcinogenesis in mouse skin. Mol. Carcinog., 17, 62–69.[ISI][Medline]
  39. Lau,H.H.S. and Baird,W.M. (1991) Detection and identification of benzo[a]pyrene–DNA adducts by [35S]phosphorothioate labeling and HPLC. Carcinogenesis, 12, 885–893.[Abstract]
  40. Lau,H.H.S. and Baird,W.M. (1994) Separation and characterization of post-labeled DNA adducts of stereoisomers of benzo[a]pyrene-7,8-diol-9,10-epoxide by immobilized boronate chromatography and HPLC analysis. Carcinogenesis, 15, 907–915.[Abstract]
  41. Baird,W.M. and Ralston,S.L. (1997) Carcinogenic polycyclic aromatic hydrocarbons. In Sipes,I.G., McQueen,C.A. and Gandolfi,A.J. (eds) Comprehensive Toxicology, Vol. 12. Cambridge University Press, Cambridge, UK, pp. 171–200.
  42. Gill,H.S., Kole,P.L., Wiley,J.C., Li,K.-M., Higginbotham,S., Rogan,E.G. and Cavalieri, E.L. (1994) Synthesis and tumor-initiating activity in mouse skin of dibenzo[a,l]pyrene syn- and anti-fjord-region diolepoxides. Carcinogenesis, 15, 2455–2460.[Abstract]
  43. Casale,G.P., Higginbotham,S., Johansson,S.L., Rogan,E.G. and Cavalieri,E.L. (1997) Inflammatory response of mouse skin exposed to the very potent carcinogen dibenzo[a,l]pyrene: a model for tumor promotion. Fund. Appl. Toxicol., 36, 71–78.[ISI][Medline]
  44. Li,K.-M., Todorovic,R., Cavalieri,E.L., Rogan,E.G., Jankowiak,R. and Small,G. (1997) Depurinating DNA adducts detected in mouse skin treated with dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, (±)-anti-DB[a,l]P diol epoxide (DE) or (±)-syn-DB[a,l]PDE. Proc. Am. Assoc. Cancer Res., 38, 127.
  45. Chakravarti,D., Mailander,P., Franzen,J., Higginbotham,S., Cavalieri,E.L. and Rogan,E.G. (1998) Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR-RFLP method. Oncogene, 16, 3203–3210.[ISI][Medline]
  46. Devanesan,P.D., RamaKrishna,N.V.S., Padmavathi,N.S., Higginbotham,S., Rogan,E.G., Cavalieri,E.L., Marsch,G.A., Jankowiak,R. and Small,G.J. (1993) Identification and quantitation of 7,12-dimethylbenz[a]anthracene-DNA adducts formed in mouse skin. Chem. Res. Toxicol., 6, 364–371.[ISI][Medline]
  47. Rogan,E.G., Devanesan,P.D., RamaKrishna,N.V.S., Higginbotham,S., Padmavathi,N.S., Chapman,K., Cavalieri,E.L., Jeong,H., Jankowiak,R. and Small,G.J. (1993) Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin. Chem. Res. Toxicol., 6, 356–363.[ISI][Medline]
  48. Todorovic,R., Ariese,F., Devanesan,P.D., Jankowiak,R., Small,G.J., Rogan,E.G. and Cavalieri,E.L. (1997) Determination of benzo[a]pyrene- and 7,12-dimethylbenz[a]anthracene-DNA adducts formed in rat mammary glands. Chem. Res. Toxicol., 10, 941–947.[ISI][Medline]
  49. Todorovic,R., Li,K.-M., Rogan,E.G. and Cavalieri,E.L. (1996) Identification and quantitation of DNA adducts of benzo[a]pyrene (BP), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P) formed in rat mammary glands. Proc. Am. Assoc. Cancer Res., 37, 119.
  50. Wood,R.D. (1996) DNA repair in eukaryotes. Annu. Rev. Biochem., 65, 135–167.[ISI][Medline]
  51. Loeb,L.A. (1989) Endogenous carcinogenesis: molecular oncology into the twenty-first century – Presidential address. Cancer Res., 49, 5489–5496.[ISI][Medline]
  52. Braithwaite,E., Wu,X. and Wang,Z. (1998) Repair of DNA lesions induced by polycyclic aromatic hydrocarbons in human cell-free extracts: involvement of two excision repair mechanisms in vitro. Carcinogenesis, 19, 1239–1246.[Abstract]
  53. Braithwaite,E., Wu,X. and Wang,Z. (1999) Repair of DNA lesions: mechanisms and relative repair efficiencies. Mutat. Res., 424, 207–219.[ISI][Medline]
  54. Friedberg,E.C., Walker,G.C. and Siede,W. (1995) DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, DC.
  55. Cooper,C.S. (1990) The role of oncogene activation in chemical carcinogenesis. In Cooper,C.S. and Grover,P.L. (eds) Chemical Carcinogenesis and Mutagenesis, Vol. II. Springer-Verlag, New York, NY, pp. 319–352.
  56. Denissenko,M.F., Pao,A., Tang,M.-S. and Pfeifer,G.P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in p53. Science, 274, 430–432.[Abstract/Free Full Text]
  57. Prahalad,A.K., Ross,J.A., Nelson,G.B., Roop,B.C., King,L.C., Nesnow,S. and Mass,M.J. (1997) Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung. Carcinogenesis, 18, 1955–1963.[Abstract]
  58. Colapietro,A.M., Goodell,A.L. and Smart,R.C. (1993) Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels. Carcinogenesis, 14, 2289–2295.[Abstract]
  59. Brown,K., Buchmann,A. and Balmain,A. (1990) Carcinogen-induced mutations in the mouse skin c-Ha-ras gene provide evidence of multiple pathways for tumor progression. Proc. Natl Acad. Sci. USA, 87, 538–542.[Abstract]
  60. Quintanilla,M., Brown,K., Ramsden,M. and Balmain,A. (1986) Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature, 322, 78–80.[ISI][Medline]
Received April 8, 1999; revised July 8, 1999; accepted July 9, 1999.