Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on the tumor initiation, PAHDNA binding and metabolic activation of carcinogenic PAH in mouse epidermis
Charis P. Marston,
Cliff Pereira1,,
Jennifer Ferguson1,,
Kay Fischer2,,
Olaf Hedstrom2,,
Wan-Mohaiza Dashwood and
William M. Baird3,
Department of Environmental and Molecular Toxicology,
1 Department of Statistics and
2 College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA
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Abstract
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Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiationpromotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAHDNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]PDNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiationpromotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAHDNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAHDNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.
Abbreviations: B[a]P, benzo[a]pyrene; B[a]PDE, (+)-anti-benzo[a]pyrene diol-epoxide; B[e]P, benzo[e]pyrene; CYP, cytochrome P450; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE, dibenzo[a,l]pyrene diol-epoxide; DMBA, 7,12-dimethylbenz[a]anthracene; PAH, polycyclic aromatic hydrocarbon(s); SRM, Standard Reference Material 1597; TBA, tumor-bearing animal(s); TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction
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Humans are exposed to complex mixtures of polycyclic aromatic hydrocarbons (PAH), which have been implicated in inducing lung, skin and breast cancer (14). PAH-induced carcinogenesis involves a number of steps including; (i) the enzymatic activation of the PAH into metabolites; (ii) the covalent binding of the PAH metabolites to DNA; and (iii) the induction of mutations that serve to initiate the transformation process as a result of PAHDNA adducts (as reviewed in ref. 5). Understanding the mechanisms of carcinogenesis by complex mixtures of PAH is difficult since humans undergo long-term exposure to low levels of PAH. This is further complicated by the finding that weakly or non-carcinogenic hydrocarbons present in complex mixtures can modify the carcinogenic activity of PAH in rodents (68). Thus, total levels of PAH in complex mixtures may not provide sufficient information to assess the risk that these PAH mixtures pose to humans. However, the assessment of carcinogenic PAH activation within complex mixtures, i.e. metabolism and DNA adduct formation may allow us to predict the ability of that mixture to increase or decrease activation of the carcinogenic PAH present and ultimately to assess the relative carcinogenic risk of that mixture.
To understand the mechanisms of carcinogenesis, it is necessary to determine how hydrocarbons interact in tumor formation and to identify a common property that correlates with potential carcinogenic risk. Metabolic activation of PAH is necessary for their covalent interactions with cellular macromolecules (as reviewed in ref. 9). PAH can covalently bind to DNA, RNA and proteins, but it is the amount of covalent interaction between PAH and DNA that correlates with carcinogenicity (10). Metabolism of the PAH occurs primarily via oxidation by cytochrome P450 (CYP) isozymes to an epoxide, which is hydrolyzed to a diol by epoxide hydrolase and oxidized on the same ring by cytochrome P450 to produce a diol epoxide (11,12). The two predominant CYP isozymes that metabolize PAH are CYP1A1 (11,13) and CYP1B1 (14,15).
Although a number of factors affect the PAH tumor initiation process, the structure of the PAH molecule can play an influential role. The basic structures thought to be involved in determining the carcinogenicity of PAH are a bay-region and a fjord- (hindered-bay) region (Figure 1
). The fjord-region causes repulsive interactions between two opposing hydrogen bonds in this region, distorting the molecule and rendering it out-of-plane (16). Some fjord-region containing PAH have been found to bind more extensively to DNA (17), and thus, render a greater tumorigenic response (18). Determining whether one of these basic structures is capable of inducing a specific CYP isozyme to a greater extent than the other structure and whether the induced isozyme can selectively metabolize a PAH to its carcinogenic metabolites, which can then bind to DNA and induce mutations, will aid in our understanding of PAH-induced carcinogenesis.

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Fig. 1. Structures of the carcinogenic bay-region containing polycyclic aromatic hydrocarbon, benzo[a]pyrene and fjord-region containing polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene.
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Exposure to complex mixtures of PAH is inevitable. One of the best characterized models for studying PAH-induced carcinogenesis and the interactions of PAH with DNA is in mouse epidermis. Co-carcinogenesis is the ability of various PAH to alter the carcinogenic activity of other PAH. It has been demonstrated that PAH present in complex mixtures can serve as co- or anti-carcinogens in mouse skin (6,19) and that a correlation exists between mouse skin tumor induction and the relative carcinogenicity of environmental PAH mixtures in human lung (20). Van Duuren and Goldschmidt (21) found that benzo[e]pyrene (B[e]P), pyrene and fluoranthene acted as co-carcinogens by increasing the carcinogenic activity of benzo[a]pyrene (B[a]P) when applied to mouse skin three times per week for a 1 year period. DiGiovanni et al. (6) demonstrated that B[e]P increased the tumor initiating activity of a single treatment of B[a]P but decreased initiation by 7,12-dimethylbenz[a]anthracene (DMBA) in mouse skin tumor-initiation assays. Therefore, B[e]P can act as either a co-carcinogen or anti-carcinogen depending on the carcinogenic hydrocarbon with which it is applied.
Binding of PAH to DNA in mouse skin has been found to correlate with their relative carcinogenic potency (10). Smolarek et al. (22) studied the effect of B[e]P on the binding of B[a]P or DMBA in mouse skin. They found that at a low dose of B[a]P, low and high doses of B[e]P increased B[a]PDNA binding. In contrast, B[e]P caused a large decrease in DMBADNA binding at all doses tested. Fluoranthene and pyrene have also demonstrated the ability to increase the binding of B[a]P in mouse epidermis (23,24). Thus, the effects of a PAH toward the binding of a carcinogenic PAH to DNA in mouse skin appears to be an indicator of the potential co- or anti-carcinogenic activity of that PAH with that carcinogen.
Coal tar is a by-product of the gasification process. It is a complex mixture of hundreds of different compounds, many of which are PAH. Studies have found coal tars to be tumorigenic in mice (2527). Post-labeling and antibody techniques have been utilized to detect PAHDNA adducts in mouse skin as well as in white blood cells and skin biopsy samples of humans exposed to complex mixtures of PAH (2731). However, the methods used in these studies were unable to identify the individual PAHDNA adducts let alone the stereochemical selectivity of activation. Although it is known that complex mixtures of PAH can bind to DNA and initiate tumor formation, it is not clear what specific PAH metabolites are responsible for this activity and how they are modified by the presence of the other weakly or non-carcinogenic PAH. In order to begin to understand the mechanism(s) by which PAH mixtures can alter PAH activation and induction of biological effects, the effects of a complex PAH mixture on the tumor-initiating activity and metabolic activation of carcinogenic PAH to DNA-binding derivatives were investigated.
Standard Reference Material 1597 (SRM), obtained from the National Institute of Standards and Technology, is a natural, combustion-related complex mixture of PAH extracted from a medium crude coke oven coal tar and dissolved in toluene (32). The SRM contains 12 PAH for which certified values of the concentrations have been obtained (33). There are 18 PAH within this mixture of PAH for which only values of non-certified concentrations are available (33). The SRM also contains low amounts of some PAH that have not yet been identified. The advantage of using a standardized complex mixture of PAH is that it is homogeneous and readily available to the scientific community so that inter-laboratory comparison can be obtained. SRM 1597 has been found to be mutagenic using the Ames test, and reference values for this activity have been determined in an international collaborative study between 20 laboratories (34).
The effects of SRM 1597 on the metabolic activation, PAHDNA binding and tumor induction by the bay-region containing B[a]P and the fjord-region containing dibenzo[a,l]pyrene (DB[a,l]P) in mouse epidermis were investigated to understand the mechanisms by which PAH mixtures modify the activity of carcinogenic PAH.
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Materials and methods
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Chemicals
SRM 1597 was obtained from the National Institute of Standards and Technology (Gaithersburg, MD). B[a]P and DB[a,l]P were purchased from Chemsyn Science Laboratories (Lenexa, KS). Re-distilled phenol, RNase (DNase-free) and RNase T1 were obtained from Boehringer-Mannheim (Indianapolis, IN). 12-O-Tetradecanoylphorbol-13-acetate (TPA), nuclease P1, Crotalus atrox venom phosphodiesterase (Type VII), prostatic acid phosphatase, potato apyrase (Grade VII) and proteinase K were purchased from Sigma (St Louis, MO). Cloned T4 polynucleotide kinase was obtained from US Biochemical (Cleveland, OH). [
-33P]ATP was purchased from NEN-Dupont (Boston, MA).
Tumor initiation in mouse epidermis
Female SENCAR mice (NCIFrederick Cancer Research and Development Center, Frederick, MD) 67 weeks of age in the resting phase of their hair-growth cycle were shaved on the dorsal side 2 days prior to initiation. Mice were housed in polycarbonate cages (five mice per cage) and fed Teklad rodent diet (No. 8604, Harlan) and water ad libitum. They were maintained at 72°F on a standard 12 h light/dark cycle with 4060% relative humidity. The SRM (8 g/l coal tar sample in toluene) contains a certified concentration of 95.8 ± 5.8 mg/kg B[a]P (82.9 ± 5.3 µg/ml) (33). SRM treatments were performed using a concentration of 1 mg per 125 µl, which contains 10.4 µg B[a]P. The concentrations of B[a]P and DB[a,l]P were chosen based upon previous studies (18,35,36). Topical treatments with the PAH initiator were carried out 5 min after the SRM treatment and dosed as follows: 10 mice with 200 µl toluene as a vehicle control, 30 mice with 1 mg SRM, 35 mice with 200 nmol (50.4 µg) B[a]P in 100 µl toluene, 35 mice with 1 mg SRM plus 200 nmol B[a]P in 100 µl toluene, 35 mice with 2 nmol (0.6 µg) DB[a,l]P, and 35 mice with 1 mg SRM plus 2 nmol DB[a,l]P. Two weeks after initiation, twice-weekly promotion was begun with TPA at 1 µg/200 µl acetone per mouse for 25 weeks. The mice were examined weekly for skin papillomas. At necropsy, tumors were confirmed by routine histopathology techniques by the Cell and Tissue Analysis Core of the Environmental Health Sciences Center of Oregon State University.
DNA isolation from PAH-treated mouse epidermis
Following acclimatization, shaving and hydrocarbon treatments of the animals (three mice per group) with the PAH as above, the mice were killed by cervical dislocation after 6, 12, 24, 48 or 72 h exposure, and the epidermal cells were harvested by the method of Slaga et al. (37). The skins were treated for 10 min with Nair® depilatory cream to remove any new hair growth, rinsed with cold water and later incubated at 58°C for 30 s. Following an ice bath submersion, the epidermal cells were removed by scraping. For microsomal preparations used in western blot analysis of CYP protein expression, the heat treatment was carried out at 52°C. The epidermal cells from each treated group were pooled and stored at 80°C for DNA isolation or used immediately for microsome preparation.
The mouse epidermal samples from each group were pooled and homogenized in a glass homogenizer containing EDTA, SDS buffer [10 mM Tris, 1 mM Na2EDTA, 1% (w/v) SDS, pH 8]. Homogenates were treated with 50 U/ml RNase, DNase-free and 1000 U/ml RNase T1 at 37°C for 1 h, followed by treatment with 500 µg/ml proteinase K at 37°C for 1 h. DNA was extracted with 25:24:1 phenol:chloroform:isoamyl alcohol, precipitated with 100% ethanol and dissolved in distilled water (38).
33P-post-labeling of DNA adducts
PAH-adducted DNA (10 µg) were digested with 0.6 U nuclease P1, 350 mU prostatic acid phosphatase in nuclease P1 buffer (0.125 M sodium acetate, 30 mM ZnCl2, pH 5.2) at 37°C for 45 min. The volume was reduced to 5 µl and 5'-labeled with 50 µCi [
-33P]ATP, T4 polynucleotide kinase (18 U/0.6 µl) and 2 µl kinase buffer [0.5 M Tris, 8 mM spermidine, 100 mM MgCl2, 100 mM dithiothreitol (DTT), pH 9.6] at 37°C for 1 h. The labeled adducted dinucleotides were digested to mononucleotides with 15 mU snake venom phosphodiesterase and hydrolyzed with 100 mU potato apyrase at 37°C for 1 h. Sep-Pak C18 column chromatography was used as previously described (39) to separate the labeled, adducted mononucleotides from other digestion products. The amount of radioactivity in the samples was determined by scintillation counting and the appropriate aliquot for HPLC analysis was evaporated to ~10 µl under vacuum. The sample was suspended in 100 µl of the appropriate buffers (see next section) in a 1:1 ratio and vortexed to ensure complete recovery of the samples. The samples were then evaporated under vacuum to a final volume of 50 µl and analyzed by reversed-phase HPLC.
HPLC analysis of 33P-post-labeled PAHDNA adducts
PAHDNA adducts were analyzed by reversed-phase HPLC utilizing a 5 µm Symmetry® C18 Cartridge column (4.6 mmx250 mm; Waters, Milford, MA) and an on-line flow detector using a 500 µl flow cell (Radiomatic FLO-ONE BETA; Packard Instruments, Downers Grove, IL). When determining how the SRM affects B[a]PDNA binding, adducts were resolved by elution at 1 ml/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 100% HPLC grade methanol (solvent B). The elution gradient was as follows: 4460% solvent B over 40 min, 6080% solvent B over 10 min, isocratic elution at 80% solvent B over 10 min, and 8044% solvent B over 5 min. When determining how the SRM affects DB[a,l]PDNA binding, adducts were resolved by elution at 1 ml/min with 0.1 M ammonium phosphate, pH 5.5 (solvent A) and 90% HPLC grade methanol/10% acetonitrile (solvent B). The elution gradient was as follows: 4455% solvent B for 50 min, 5580% solvent B over 10 min, and 8044% solvent B over 10 min. The total level of PAHDNA adducts in each sample was calculated based on labeling of a [3H]B[a]PDEDNA standard of known modification which was analyzed together with each set of post-labeling samples (40). For HPLC profile, peaks are defined by the software package used for analysis of the radioactivity elution profile, namely Waters® Millenium software version 2.15. Only peaks which exceeded the background by 200 d.p.m. or more were counted as peaks.
Microsome preparation
Microsomes were prepared as described by Otto et al. (41) with minor modifications. Mouse epidermal samples were homogenized with a steel homogenizer containing microsomal homogenization buffer [0.25 M KPO4, 0.15 M KCl, 10 mM EDTA and 0.25 mM phenylmethylsulfonylfluoride (PMSF)] and centrifuged at 15 000 g for 20 min at 4°C. The supernatant was centrifuged at 100 000 g for 90 min at 4°C, and the pellet was resuspended in microsome dilution buffer (0.1 M KPO4, 20% glycerol, 10 mM EDTA, 0.1 mM DTT and 0.25 mM PMSF). Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL).
Western immunoblotting
Microsomal proteins were separated by 8% SDSPAGE according to Laemmli (42). All lanes were loaded with equal amounts of protein (11.4 µg/µl) as measured by the BCA protein assay and transferred to a nitrocellulose membrane (BioRad). Uniformity of loading was confirmed by staining the nitrocellulose membrane with Ponceau S (Sigma). The membranes were blocked with 5% Carnation® non-fat dried milk in 150 ml PBS-T [PBS with 0.3% (w/v) Tween-20] and 0.2% goat serum.
Mouse CYP1A1 was detected using the anti-mouse monoclonal CYP1A1 antibody 1-36-1 (1:15 000 dilution) provided by Drs S.S.Park (NCIFCRDC, Frederick, MD) and H.Gelboin (NCI, Bethesda, MD). Dr C.R.Jefcoate (University of Wisconsin, Madison, WI) provided the antibody enabling mouse CYP1B1 to be detected. Mouse CYP1B1 was detected using a 1:1500 dilution of a rabbit polyclonal CYP1B1 antibody elicited against recombinant mouse CYP1B1. The positive control used for the detection of CYP1A1 isozyme was Aroclor-induced rat liver S9. Microsomal protein isolated from 10 mM benz[a]anthracene-treated C3H10T1/2 cells were used as positive control for the detection of CYP1B1 protein (14). The immunoreactive proteins were detected by incubating the membrane with peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:15 000 dilution in PBS-T with 0.2% goat serum). The enhanced chemiluminescence detection method was used to detect immunoreactive proteins as described by the manufacturer (Amersham, Arlington Heights, IL).
Statistical analysis
All analyses were conducted with SAS version 8.0 (43). Binary responses (mortality and final tumor incidences) were compared between cages and treatments using Fisher's exact test (FET) in the FREQ procedure. After finding no evidence of differences between replicate cages within treatments, comparisons between treatments were made using data pooled over cages.
Time-until-tumor curves were analyzed by proportional hazards (Cox) regression (PHREG procedure) using indicators for treatments or cages, after first graphically assessing that the proportional hazards assumptions was reasonable (LIFETEST procedure). Likelihood ratio tests (LRT) were used for hypothesis testing. The SRM treatment group in the B[a]P study was the only treatment group with indication of significant differences between cages (P = 0.0091, LRT) in their time to tumor response. For the other treatment groups, data were pooled over cages for subsequent analysis (P = 0.34 for all other within-treatment cage comparisons, LRT).
Tumors per tumor bearing animal (TBA) data were analyzed using an extension of the FET for larger two-way contingency tables, also called the FreemanHalton test (43), to detect general departures from the null hypothesis and the Wilcoxon test (WT) for detecting shifts in ordered categories. There were no indications of differences between replicate cages within treatments and data were subsequently pooled over cages for comparing treatments. Exact, permutation P-values were generated in the FREQ procedure (for FET) and the NPAR1WAY procedure (for WT).
Analysis of PAHDNA binding data was conducted on the log transformed scale because of greatly improved residuals on that scale. The ANOVA models included experiment as a blocking factor (three experiments) with a 2 by 5 factorial treatment structure (carcinogen with and without SRM by hours of exposure with five time points). After averaging over any multiple complete post-labelings, there were 30 observations to analyze per carcinogen (B[a]P, DB[a,l]P) using the GLM and MIXED procedures. Residual analyses revealed outliers in the DB[a,l]P data set at 24 and 72 h, but no reason was found to exclude the data. Robust analyses using ranks were also conducted for DB[a,l]P data set to verify that the ANOVA conclusions were not sensitive to the presence of the outliers (Friedman's rank test using the CMH2 and SCORES=RANKS options of the FREQ procedure).
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Results
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Tumor initiation in mouse epidermis
Measures of tumor initiating activity and ultimately of tumorigenesis were obtained from several end-points such as number of tumors per TBA, tumor incidence at end point and time-until-tumor. The effect of the SRM on the tumor initiating activity of B[a]P in mouse skin is illustrated in Figure 2
and Table I
. The tumor latency period between all the PAH treatment groups did not differ greatly (Figure 2A
). Mice initiated with B[a]P first exhibited papillomas during the sixth week of promotion, whereas the groups of mice initiated with the SRM alone and SRM plus B[a]P did not exhibit papillomas until the seventh week of promotion (Figure 2A
). No papillomas were found in the toluene- (vehicle control) initiated group. Compared with B[a]P alone, co-treatment with SRM did not significantly affect tumor incidence, either over time (P = 0.11, Cox regression LRT; Figure 2A
) or specifically at 25 weeks (P = 0.24, FET; Table I
). Among the ninth and 14th weeks of promotion, the number of tumors per TBA identified from mice co-initiated with SRM plus B[a]P were greater than the number of tumors per TBA identified from mice initiated with B[a]P alone (Figure 2B
). The SRM-treated group had a similar number of tumors per TBA as the co-initiated SRM plus B[a]P group in the same time period. By the 15th week of promotion, the number of tumors per TBA were similar for the mice initiated with B[a]P and mice initiated with SRM plus B[a]P, and remained similar until the experiment was terminated at 25 weeks of promotion (Figure 2B
and Table I
).

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Fig. 2. Mouse skin tumor initiationpromotion study to determine the effect of the SRM on the tumor initiating activity of B[a]P. Mice were initiated with either 1 mg SRM, 200 nmol (50.4 µg) B[a]P, 1 mg SRM plus 200 nmol B[a]P, or 200 µl toluene (vehicle control) as described in Materials and methods. Twice weekly promotion with 1 µg TPA was started 2 weeks after the initiation treatment. Mice were checked for skin papillomas throughout a 25 week promotion period. (A) Time course of tumor appearance in mouse skin expressed as the percentage of tumor bearing animals (TBA). (B) Time course of tumor appearance in mouse skin expressed as the number of tumors per TBA. (The decrease in tumors per TBA between consecutive time points are due to the death of a mouse with higher than average tumors per TBA, not due to disappearance of tumors within an animal).
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The tumor initiating activities of SRM, B[a]P and SRM plus B[a]P at the end (25 weeks of promotion) of the mouse skin tumor initiationpromotion study are noted in Table I
. Mortality was understood to be a random event based upon our finding that there was no evidence of mortality rate differences between cages or treatments (P > 0.4 for all comparisons, FET). The toluene-initiated mice exhibited no tumors. With at least 90% of the mice in the three PAH-treatment groups developing at least one tumor, the SRM initiated the formation of 5.3 tumors per TBA. The B[a]P- and the SRM plus B[a]P-initiated groups developed a similar number of 8.9 and 8.8 tumors per TBA, respectively (P = 0.96, WT), and both were more tumorigenic than the SRM (P = 0.02 and P = 0.0042, respectively, WT). All of the tumors were skin papillomas except for one squamous cell carcinoma identified in the group of mice co-initiated with SRM plus B[a]P.
Figure 3
and Table II
illustrate the effects of the SRM on the initiating activity of DB[a,l]P in mouse skin. Papillomas were initially detected in the two groups of mice initiated with DB[a,l]P alone and SRM plus DB[a,l]P during the sixth week of promotion (Figure 3A
). Papillomas were not initially detected until the eighth week of promotion in the group of mice initiated with SRM alone (Figure 3A
). A papilloma was detected in the toluene-initiated group during the 11th week of promotion. Compared with DB[a,l]P alone, there was some evidence that co-treatment with SRM affected tumor incidence over time (P = 0.0527, LRT; Figure 2A
), although not specifically at 25 weeks when nearly all mice had tumors (P = 0.9, FET; Table II
). Mice initiated with DB[a,l]P alone had a consistently greater number of tumors per TBA compared with mice initiated with either SRM plus DB[a,l]P or SRM alone from the seventh week of promotion until the final 25th week of promotion. This same effect was also detected between the SRM plus DB[a,l]P and the SRM only groups.

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Fig. 3. Mouse skin tumor initiationpromotion study to determine the effect of the SRM on the tumor initiating activity of DB[a,l]P. Mice were initiated with either 1 mg SRM, 2 nmol (0.6 µg) DB[a,l]P, 1 mg SRM plus 2 nmol DB[a,l]P, or 200 µl toluene (vehicle control) as described in Materials and methods. Twice weekly promotion with 1 µg TPA was started 2 weeks after the initiation treatment. Mice were checked for skin papillomas throughout a 25 week promotion period. (A) Time course of tumor appearance in mouse skin expressed as the percentage of TBA. (B) Time course of tumor appearance in mouse skin expressed as the number of tumors per TBA.
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The tumor initiating activities of SRM, DB[a,l]P and SRM plus DB[a,l]P at the end (25 weeks of promotion) of the mouse skin tumor initiationpromotion study are noted in Table II
. Mortality was understood to be a random event after finding no evidence of mortality rate differences between cages or treatments (P > 0.8 for all comparisons, FET). All of the tumors were determined to be skin papillomas. The toluene-initiated group exhibited 1 tumor per TBA at 25 weeks of promotion. With at least 96% of the mice in the three PAH-treatment groups developing at least one tumor, the SRM, DB[a,l]P and SRM plus DB[a,l]P groups initiated the formation of 4.1, 8.1 and 5.2 tumors per TBA, respectively. DB[a,l]P was considerably more tumorigenic than the SRM plus DB[a,l]P or the SRM alone (P = 0.006 and P < 0.0001, respectively, WT). SRM plus DB[a,l]P was also statistically more tumorigenic than the SRM alone (P = 0.03, WT).
PAHDNA binding in mouse epidermis
In order to determine if the effect of the SRM on the tumorigenic activity of the two PAH correlated to PAHDNA binding, mice were treated in the same manner as the mouse skin tumor initiationpromotion studies, and the PAHDNA adducts were 33P-post-labeled and analyzed by reversed-phase HPLC. Adduct levels were quantitated after 6, 12, 24, 48 and 72 h exposure. HPLC profiles detected no DNA adduct formation from untreated nor toluene- (vehicle control) treated mice (data not shown).
The HPLC elution profiles of 33P-post-labeled PAHDNA adducts formed in mouse skin are depicted after 12 and 24 h exposure to PAH in Figures 4 and 5
. Although there are over 100 different PAH in the SRM, the HPLC profile of the DNA from mice treated with the SRM had only four to seven major peaks, which indicated the formation of relatively few different PAHDNA adducts. One peak of interest eluted around 25 (Figure 4
) or 22 min (Figure 5
). The B[a]P HPLC profile gave evidence that this peak represents the (+)-anti-B[a]P diol-epoxide (B[a]PDE) deoxyguanosine adduct (39). The importance of this peak is that the (+)-anti-B[a]PDE metabolite is the stereoisomeric metabolite responsible for the majority of the tumor initiating activity of B[a]P (44). Mouse skin treated with B[a]P exhibited typical HPLC profiles with the (+)-anti-B[a]PDEdG adduct eluting around 25 min (Figure 4
). The (+)-anti-B[a]PDEdG peak was the only significant adduct resolved by HPLC analysis in the SRM plus B[a]P co-treated mice.

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Fig. 4. Reversed-phase HPLC elution profiles of 33P-post-labeled SRM, B[a]P and SRM plus B[a]PDNA adducts formed in mouse epidermis after (A) 12 h and (B) 24 h exposure to 1 mg SRM, 200 nmol (50.4 µg) B[a]P, or 1 mg SRM plus 200 nmol B[a]P. Total PAHDNA adducts formed after 12 h exposure to SRM, B[a]P or SRM plus B[a]P were 6.31, 39.80 and 28.48 pmol adducts per mg DNA (pmol/mg DNA), respectively. Total PAHDNA adducts formed after 24 h exposure to SRM, B[a]P or SRM plus B[a]P were 3.11, 14.37 and 18.76 pmol/mg DNA, respectively.
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Fig. 5. Reversed-phase HPLC elution profiles of 33P-post-labeled PAHDNA adducts formed in mouse epidermis after (A) 12 h and (B) 24 h exposure to 1 mg SRM, 2 nmol (0.6 µg) DB[a,l]P, or 1 mg SRM plus 2 nmol DB[a,l]P. Total PAHDNA adducts formed after 12 h exposure to SRM, DB[a,l]P or SRM plus DB[a,l]P were 1.78, 3.44 and 0.98 pmol adducts per mg DNA (pmol/mg DNA), respectively. Total PAHDNA adducts formed after 24 h exposure to SRM, DB[a,l]P or SRM plus DB[a,l]P were 4.93, 9.98 and 0.64 pmol/mg DNA, respectively. Based upon comparison of elution times to those reported in Ralston et al. (49) and the use of the same diol epoxide adduct markers, the DB[a,l]P peak labeled 1 was formed by reaction of the syn diol epoxide with dA. Peak 3 resulted from reactions of (+)-syn diol epoxide with dA. Peak 4 resulted from reactions of (-)-anti diol epoxide with dA.
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DB[a,l]P was actively metabolized to fjord-region diol epoxides. Four major DB[a,l]PDEDNA adducts eluted at 32, 40, 42 and 50 min, as well as a minor adduct eluting around 22 min (Figure 5
). HPLC elution profiles illustrated that metabolism of DB[a,l]P to form DNA adducts was inhibited when the mice were co-treated with the SRM plus DB[a,l]P (Figure 5
).
The levels of PAHDNA adducts formed in mouse skin treated with either the SRM, B[a]P or co-treatment with the SRM and B[a]P are plotted in Figure 6A
. The greatest level of DNA adduct formation in the SRM-treated mice, 2.1 pmol PAH adducts per mg DNA (pmol/mg DNA), was quantitated at 12 h exposure. There was strong evidence of a significant difference in the levels of PAHDNA adducts between the B[a]P-treated mice and the SRM plus B[a]P co-treated mice when averaged over the time points (main effect, P < 0.0001, ANOVA). However, there was some evidence that the difference between B[a]P and the co-treatment changed with hours of exposure (interaction, P = 0.079, ANOVA) leading to comparisons within each exposure time. The B[a]P-treated mice had the greatest level of B[a]PDNA adducts at 12 h (15.7 pmol/mg DNA). At this early exposure, the SRM significantly decreased the total level of B[a]PDNA adducts from 15.7 pmol/mg DNA found in mice treated with only B[a]P to 7.8 pmol/mg DNA total PAHDNA adducts in the mice treated with both SRM plus B[a]P (contrast, P = 0.0225, ANOVA). The major (+)-anti-B[a]PDEDNA adduct was also decreased by the SRM in a similar manner (12.3 to 4.0 pmol/mg DNA) (data not shown). The two extreme exposure times of 6 and 72 h also resulted in a significant reduction of PAHDNA binding upon co-treatment of SRM plus B[a]P compared with B[a]P alone (contrast, P = 0.0002 and 0.0064, respectively, ANOVA). However, a reducing effect of the SRM towards B[a]PDNA binding was not observed at either the 24 or 48 h exposure times (contrast, P = 0.2761 and 0.4671, respectively, ANOVA).

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Fig. 6. The effect of exposure time on the level of PAHDNA adducts in mouse epidermis. Mice were treated, and DNA was isolated, 33P-post-labeled and analyzed by reversed-phase HPLC as described in Materials and methods. Points are means with standard error bars for the log transformed data from n = 3 separate experiments. (A) Mouse skin was exposed to SRM, B[a]P or SRM plus B[a]P. The SRM significantly decreased the level of B[a]PDEDNA binding over time of exposure (main effect, P < 0.0001, ANOVA) and specifically at 6, 12 and 72 h (P = 0.0002, 0.0224 and 0.0064, respectively). Data are from one complete post-labeling per experiment (where post-labeling efficiency was at least 25%). From the ANOVA model (see Statistical methods) the standard error of a difference between any two means = 0.12. (B) Mouse skin was exposed to SRM, DB[a,l]P or SRM plus DB[a,l]P. Significant decreases in PAHDNA adduct levels by the SRM towards DB[a,l]PDEDNA binding were noted over time of exposure (main effect, P < 0.0006, ANOVA and rank test). Log transformed data are averaged over two complete post-labelings per experiment. From the ANOVA model, the standard error of a difference = 0.156, however, two outliers question the validity of the model (see Statistical methods).
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The data in Figure 6B
represents the level of PAHDNA adducts in mouse skin treated with either SRM, DB[a,l]P or a co-treatment of SRM plus DB[a,l]P. In this study, the maximum level of PAHDNA binding calculated in the SRM treated mice was 3.9 pmol/mg DNA after 12 h exposure. The greatest levels of DB[a,l]PDNA adducts were detected at 12 and 24 h exposure (levels >2.5 pmol/mg DNA persisted through 48 h). The SRM substantially influenced the level of PAHDNA binding of DB[a,l]P. A significant decrease in PAHDNA adduct formation was observed in the SRM plus DB[a,l]P treatment group compared with DB[a,l]P treatment alone when averaged over the time points (main effect, P < 0.0006, ANOVA and rank test). Unlike the B[a]P study, there was no evidence that the effect of the addition of the SRM to the DB[a,l]P treatment changed with the hour of exposure (interaction, P > 0.61, ANOVA and rank test on differences) rendering comparisons within exposure times between DB[a,l]P alone and SRM plus DB[a,l]P unnecessary. Not only were the level of adducts lower in the group of mice treated with SRM plus DB[a,l]P compared with those mice treated with only DB[a,l]P, but the SRM decreased the level of PAHDNA adducts in the group of mice treated with SRM plus DB[a,l]P to levels even lower than those found in the DNA from mice treated with the SRM alone suggesting that the DB[a,l]P inhibited SRM metabolism to adduct formation as well (Figure 6B
).
Cytochrome P450 1A1 and 1B1 protein expression
Western immunoblotting with antibodies against CYP1A1 and CYP1B1 was utilized to determine whether a complex mixture of PAH induced CYP protein expression (Figures 7 and 8
). After 12 and 24 h exposure, microsomes were isolated from mice topically treated as previously described. Although adduct level trends among the treatment groups differed between the 12 and 24 h exposure times, a similar trend was seen in CYP protein expression at both these times. The SRM induced both CYP1A1 and CYP1B1 protein expression (Figures 7 and 8
). Both the B[a]P-treated mice and the co-treated SRM plus B[a]P treated mice also induced CYP1A1 and CYP1B1 expression (Figure 7
). Although the mice treated with DB[a,l]P alone did not induce either CYP protein at the above treatment concentration, mice treated with the SRM plus DB[a,l]P induced both CYP1A1 and CYP1B1 expression (Figure 8
). These results suggested that the effect of the SRM on adduct formation and tumor initiating activity of the two carcinogenic PAH was not due to the absence of the two PAH metabolizing enzymes, CYP1A1 and CYP1B1.

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Fig. 7. Protein expression of CYP1A1 and CYP1B1 in mouse epidermis treated with 1 mg SRM, 200 nmol (50.4 µg) B[a]P, or 1 mg SRM plus 200 nmol B[a]P. Protein (50 µg) from isolated microsomes were loaded on each lane. (A & B) CYP1A1 expression at (A) 12 and (B) 24 h exposure. The `+ control' lane represents 25 µg Aroclor-induced rat liver S9. (C & D) CYP1B1 expression at (C) 12 and (D) 24 h exposure. The `+ control' lane represents 50 µg microsomal protein isolated from benz[a]anthracene-treated C3H10T1/2 cells.
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Fig. 8. Protein expression of CYP1A1 and CYP1B1 in mouse epidermis treated with 1 mg SRM, 2 nmol (0.6 µg) DB[a,l]P, or 1 mg SRM plus 2 nmol DB[a,l]P. Protein (50 µg) from isolated microsomes were loaded on each lane. (A & B) CYP1A1 expression at (A) 12 and (B) 24 h exposure. The `+ control' lane represents 25 µg Aroclor-induced rat liver S9. (C & D) CYP1B1 expression at (C) 12 and (D) 24 h exposure. The `+ control' lane represents 50 µg microsomal protein isolated from benz[a]anthracene-treated C3H10T1/2 cells.
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Discussion
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The effect of a complex mixture of PAH on the metabolic activation and DNA binding of two structurally different carcinogenic PAH was investigated to elucidate the mechanism(s) by which the mixture is able to modify the activation of the carcinogenic PAH present in that mixture. B[a]P has long been identified as a major carcinogenic component of coal tar (45). Although DB[a,l]P is not an identified PAH within the SRM, it has been identified in smoky coal tar (46) and air particulates (47).
As seen in other mouse tumorigenesis studies (2527), the SRM itself initiated a substantial number of tumors. Mice treated with 200 nmol (50.4 µg) B[a]P developed approximately nine tumors per TBA. Since the 1 mg dose of SRM only contained 10.4 µg B[a]P, the number of tumors in the SRM treated group of mice do not appear to be solely due to the B[a]P within the mixture. Interestingly, the SRM plus DB[a,l]P group exhibited a significantly reduced number of mouse skin papillomas compared with those induced by DB[a,l]P alone but had no significant effect on the number of tumors induced by B[a]P (Tables I and II
). In order to determine the mechanisms behind the results of the tumor studies, the ability of the SRM to modify PAHDNA binding and CYP expression were examined.
HPLC profiles of B[a]P-treated mice with or without the SRM looked quite similar (Figure 4
). The differences, if any, in PAHDNA adduct levels between the SRM plus B[a]P-treated mice and the B[a]P-treated mice occurred in the amount of metabolism of B[a]P to the (+)-anti-B[a]PDE, thereby affecting the level of the (+)-anti-B[a]PDEdG adduct, as denoted by the peak eluting at ~25 min (Figure 4
). HPLC profiles of DB[a,l]P- versus SRM plus DB[a,l]P-treated mice varied considerably (Figure 5
). The DB[a,l]PDNA adduct peaks were identified by co-chromatography with DB[a,l]PDE adduct standards as previously described (50). The metabolic activation of DB[a,l]P was significantly altered by the presence of the SRM. Previous studies have demonstrated that DB[a,l]P is stereoselectively metabolized to the (+)-syn- and ()-anti-DB[a,l]P diol epoxides (DB[a,l]PDE) which predominantly bind to deoxyadenosine and to a lesser extent, deoxyguanosine (48). The presence of the SRM with DB[a,l]P treatment significantly decreased all of the DB[a,l]PDEDNA adducts. The HPLC profile indicated that the adducts that were present upon co-treatment with SRM plus DB[a,l]P were barely discernible from the background. This severe inhibitory effect on PAH metabolism resulted in a lower level of PAHDNA adducts in the SRM plus DB[a,l]P treated group compared with the SRM alone (Figure 6B
).
The formation of PAHDNA adducts are essential for PAH-induced tumorigenesis (10). Surprisingly, the SRM decreased B[a]PDNA binding in the SRM plus B[a]P co-treated group when averaged across all time points (Figure 6A
), yet the SRM did not decrease the tumorigenic response of B[a]P (Table I
). These results indicate that adduct levels may not be the primary factor in determining carcinogenic potency of a complex mixture. Persistence of certain low level PAHDNA adducts may also play an important role.
Although the SRM decreased PAHDNA adducts when co-treated with B[a]P, the SRM may be activating an oncogene or deactivating a tumor suppressor gene. Either of these two responses would make it possible for increased tumor formation, thus allowing for a decrease in adduct formation in the co-treated SRM plus B[a]P group compared with B[a]P alone in spite of the similar tumorigenic response between the two treatment groups. One, therefore, might have expected a similar effect of the SRM towards DB[a,l]P. However, metabolic activation of DB[a,l]P by CYP enzymes, as noted below, may play a more important role in determining the effect of the SRM on DB[a,l]PDNA adduct formation and tumor initiation.
Another possible mechanism by which the SRM may be affecting the activation of B[a]P and DB[a,l]P is through the expression or activity of CYP1A1 and/or CYP1B1. These CYP enzymes are often present in many tissues at low levels. However, they are inducible by planar xenobiotics such as PAH through the aryl hydrocarbon (Ah) receptor (50). Chaloupka et al. (51) demonstrated that a mixture of PAH from coal tar exhibited non-additive (synergistic) activities as an Ah receptor agonist. Western immunoblotting of microsomal proteins from mouse epidermis demonstrated that the SRM induced both CYP1A1 and CYP1B1 (Figures 7 and 8
). Therefore, the SRM did not block activation by inhibiting CYP1A1 and CYP1B1 protein expression.
Relating CYP protein levels to metabolism of the PAH forming PAHDNA adducts reveals that the expression of the CYP protein does not always account for the observed PAHDNA adduct level. Although the SRM did induce CYP1A1 and CYP1B1 protein expression, the adduct levels found in the SRM treated mice were not higher than those quantitated in mice treated with either carcinogenic PAH, which did not induce either CYP isoform to the same extent as the SRM alone. These results indicate that the CYP enzymes metabolized many of the various PAH in the SRM to non-DNA binding (non-tumorigenic) metabolites.
Kim et al. (52) demonstrated that B[a]P can be metabolized to the ultimate carcinogen, the (+)-anti-B[a]PDE, by both CYP1A1 and CYP1B1. They found that metabolism of B[a]P by CYP1B1 occurred at a slower rate than by CYP1A1. Work done by Luch et al. (15) determined that CYP1A1 metabolizes DB[a,l]P to form DB[a,l]PDEDNA adducts as well as several highly polar adducts. CYP1B1, instead, catalyzed the exclusive formation of DB[a,l]PDEDNA adducts.
Our data have revealed that both CYP1A1 and CYP1B1 protein levels were increased in the presence of SRM. This increased level of CYP enzymes had an effect on the metabolism to PAHDNA binding metabolites but not the tumor initiating activity of the bay-region B[a]P. In addition, increased CYP1A1 and CYP1B1 protein expression with SRM decreased the level of the fjord-region containing DB[a,l]PDEDNA adducts, which correlates with its lower tumor initiating potency when DB[a,l]P and SRM are co-treated. These findings suggest that the metabolic activity of CYP1B1 for DB[a,l]P was inhibited by components of the SRM. The SRM induced CYP isozyme expression, yet CYP1B1 activity was inhibited. DB[a,l]P metabolism to DB[a,l]PDEDNA adducts can occur exclusively through activation by CYP1B1 (15). On the other hand, CYP1A1 efficiently metabolizes B[a]P to its ultimate carcinogenic metabolite (52). Inhibition of CYP1B1 activity by the SRM would account for the decreased level of PAHDNA adducts resulting in decreased tumor initiation of DB[a,l]P, yet this inhibition would not affect B[a]P to the same extent.
In conclusion, it was found that SRM significantly reduced PAHDNA adduct formation of B[a]P yet had no effect on tumor initiation by B[a]P. The SRM also decreased PAHDNA adduct levels of DB[a,l]P in mouse skin and diminished its tumorigenic activity. Interestingly, the SRM did induce expression of the two PAH metabolizing enzymes, CYP1A1 and CYP1B1.
Our results are consistent with the hypothesis that (i) persistence of certain PAHDNA adducts, not just adduct levels, may play an important role in determining carcinogenic potency of a complex mixture (B[a]P versus SRM plus B[a]P) and (ii) a major determinant of the carcinogenic activity of complex mixtures may be the ability of PAH within the mixture to inhibit the activation of carcinogenic PAH by the induced CYP isozymes (DB[a,l]P versus SRM plus DB[a,l]P). CYP1B1 may be playing an important role in the mechanism of PAH-induced tumorigenesis of complex mixtures. Future studies will be aimed at assessing the ability of the SRM to inhibit the activity of the induced metabolizing enzymes as well as determining the effects of a complex mixture of PAH on the metabolic activation and DNA binding utilizing human enzymes.
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Notes
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3 To whom correspondence should be addressed Email: william.baird{at}orst.edu 
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Acknowledgments
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This work was supported by grant CA 28825, DHHS, from the National Cancer Institute. We would like to thank Dr George Bailey for his helpful discussions at the Carcinogenesis Research Core meetings. The Environmental Health Sciences Center of Oregon State University funded by Center Grant ES00210 for the histopathology of all tumor samples through the Center's Cell and Tissue Analysis Core, as well as the statistical analysis through the Center's Statistics Core.
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Received July 5, 2000;
revised February 7, 2001;
accepted February 22, 2001.