DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene

Sandra J. Culp3, Alan R. Warbritton1, Beverly A. Smith, Eddie E. Li2 and Frederick A. Beland

National Center for Toxicological Research, Jefferson, AR 72079 and
1 Pathology Associates International, Jefferson, AR 72079, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Coal tar is a complex mixture containing hundreds of compounds, at least 30 of which are polycyclic aromatic hydrocarbons, including the carcinogen benzo[a]pyrene (BaP). Although humans are exposed to complex mixtures on a daily basis, the synergistic or individual effects of components within a mixture on the carcinogenic process remain unclear. We have compared DNA adduct formation and cell proliferation in mice fed coal tar or BaP for 4 weeks with tumor formation in a 2 year chronic feeding study. Additionally, we have analyzed tumor DNA for mutations in the K-ras, H-ras and p53 genes. In the forestomach of mice fed either coal tar or BaP an adduct indicative of BaP was detected, with adduct levels increasing in a dose-responsive manner. K-ras mutations were detected in the forestomach tumors, with the incidence being similar in mice fed coal tar or BaP. These results suggest that the BaP within coal tar is associated with forestomach tumor induction in coal tar-fed mice. DNA adduct levels in the small intestine were not predictive of tumor incidence in this tissue; instead, the tumors appeared to result from compound-induced cell proliferation at high doses of coal tar. K-ras mutations were detected in lung tumors. Since lung tumors were not increased by BaP, coal tar components other than BaP appear to be responsible for the tumors induced in this tissue. H-ras mutations, primarily occurring at codon 61, were the most common mutation observed in liver tumors induced by coal tar. Since this mutation profile is observed in spontaneous hepatic tumors, components in the coal tar may be promoting the expansion of pre-existing lesions.

Abbreviations: BaP, benzo[a]pyrene; BPDE, (±)-anti-benzo[a]pyrene- trans-7,8-dihydrodiol-9,10-epoxide; BrdU, 5-bromo-2'-deoxyuridine; dG-N2-BPDE, 10{alpha}-(deoxyguanosin-N2-yl)-7{alpha},8ß,9ß-trihydroxy-7,8,9,10-tetrahydro- benzo[a]pyrene; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; PCNA, proliferating cell nuclear antigen.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Complex mixtures that contain carcinogens are widespread in our environment and pose a hazard by potentially increasing the incidence of cancer in humans (1,2). Determining the deleterious biological effects from exposure to complex mixtures presents a greater challenge as compared with single carcinogens because the carcinogenicity of specific components in a mixture may not be additive (3). Coal tar is a complex mixture produced as a by-product of coal gasification and contains 400 identified compounds, including carcinogenic polycyclic aromatic hydrocarbons [e.g. benzo[a]pyrene (BaP)] and aromatic amines (1). Human exposure to coal tar occurs from roofing and road surfacing materials and certain pharmaceutical products.

We recently reported the results of a 2 year chronic bioassay in which female B6C3F1 mice were fed coal tar or BaP (4). The coal tar induced dose-related increases in a wide variety of tumors, including hepatocellular carcinomas, forestomach squamous epithelial papillomas and carcinomas, alveolar/ bronchiolar adenomas and carcinomas and small intestine adenocarcinomas. Mice fed BaP had increased incidences of papillomas and/or carcinomas of the forestomach, esophagus and tongue.

In this study we have evaluated DNA adduct formation and cell proliferation in mice fed coal tar or BaP for 4 weeks in relation to tumor incidence in various tumors from the 2 year feeding study. Additionally, we have analyzed tumors for mutations in the K-ras, H-ras and p53 genes.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Coal tar (CAS registry no. 8007-45-2) Mixture 1 was a composite from seven manufactured gas plant waste sites. Coal tar Mixture 2 was a composite from two of the seven waste sites plus a third site having a very high BaP content. The polycyclic aromatic hydrocarbon composition of the coal tar mixtures was assessed by gas chromatography/mass spectrometry as presented by Culp et al. (4). The BaP content was also analyzed by HPLC with fluorescence detection and found to be 2240 ± 51 (mean ± SD, n = 2) mg BaP/kg coal tar for Mixture 1 and 3669 ± 134 (n = 4) mg BaP/kg coal tar for Mixture 2. Since the HPLC fluorescence method was more amenable to assessing the BaP content of the diets, this technique was used for evaluation of the homogeneity of the coal tar diets. BaP (CAS registry no. 50-32-8, 98.5% pure by gas chromatography with flame ionization detection) was obtained from Chemsyn Science Laboratories (Lenexa, KS).

Diet preparation
Coal tar diets were prepared by freezing the coal tar mixtures in liquid nitrogen (5) and blending with the appropriate amount of NIH-31 meal (Purina Mills Inc., Richmond, IN). BaP diets were prepared by dissolving the appropriate amount of BaP in acetone (J.T. Baker Inc., Phillipsburg, NJ) and mixing the solution with NIH-31 meal under vacuum using a Patterson-Kelley blender with a liquid-dispersion bar. Diet mixtures were prepared containing: 0.01, 0.03, 0.1, 0.3, 0.6 or 1.0 g coal tar/100 g meal (or percent coal tar) of Mixture 1; 0.03, 0.1 or 0.3% Mixture 2; 0.0005 (5 p.p.m.), 0.0025 (25 p.p.m.) or 0.01% (100 p.p.m.) BaP. Additional groups of mice were fed NIH-31 meal or acetone-treated NIH-31 meal as a control for the coal tar and BaP diets.

Animals
During the studies, recommendations from the NCTR Institutional Animal Care and Use Committee were followed for handling, maintenance, treatment and killing of mice. Female B6C3F1 mice [B6C3F1/Nctr(C57BL/6NxC3H/HeNMTV)], obtained from the breeding colony at the National Center for Toxicological Research, were 5–6 weeks of age at the beginning of treatment. The mice were housed four per cage in polycarbonate cages with micro-isolator bonnets and hardwood chip bedding. Treatment diets and Millipore-filtered tap water were available ad libitum throughout the study. Food consumption and body weight changes were monitored. The animal rooms were maintained on a 12 h light/dark cycle, with a relative humidity of 51.7 ± 1.4% (mean ± SD) and a temperature of 22.8 ± 0.4°C.

Treatment
Four mice per dose group were fed the coal tar or BaP diets described above for 4 weeks. At the end of the feeding period, the animals were killed by carbon dioxide exposure and their liver, lungs, forestomachs and small intestines were immediately excised. Additional groups of four mice per group were fed 0 or 5 p.p.m. BaP for 1, 2, 8, 16 and 32 weeks.

DNA isolation and 32P-post-labeling
After death, DNA was isolated from the forestomach, liver and lungs by the method reported in Culp and Beland (6). Epithelial cells were stripped from the small intestines using the procedure of Westra et al. (7) before DNA was isolated from this tissue. Approximately 10 µg of DNA was 32P-post-labeled using the nuclease P1 enhancement procedure of Reddy and Randerath (8). Adducts were separated by TLC using the methods and solvents described by Culp et al. (9). Areas of radioactivity were measured using a 400E PhosphorImager (Molecular Dynamics, Sunnyvale, CA). DNA was modified with [3H](±)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) (Chemsyn) resulting in one major adduct, 10{alpha}-(deoxyguanosin-N2-yl)-7{alpha},8ß,9ß-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N2-BPDE), at a level of 70 adducts/108 nt (6). This standard was included in each 32P-post-labeling assay. To quantify adduct levels, the amount of 32P incorporated into the experimental samples was compared with the amount incorporated into the standard.

Adduct confirmation
[3H]BPDE-modified DNA and forestomach DNA from mice fed coal tar was 32P-post-labeled as described above. The 32P-post-labeled adducts co-eluting on the TLC plates with dG-N2-BPDE were extracted with 2 M ammonium acetate (pH 7.5):isopropanol (1:1). The solvent was evaporated and the residue was reconstituted in 50 mM potassium phosphate (pH 7.2) buffer. mAb 8E11 [kindly provided by Dr Regina Santella and described in Santella et al. (10)], which has specificity for dG-N2-BPDE, was bound to Gamma Bind Sepharose (Pharmacia, Piscataway, NJ) and added to each sample. The samples were vortexed for 20 min, centrifuged for 30 s at 10 000 g and the supernatant discarded. The adduct/mAb 8E11/Gamma Bind complex was rinsed twice with phosphate buffer and the adducts were extracted from the antibody with dimethylsulfoxide:methanol (1:1). The extract was evaporated, reconstituted in phosphate buffer and analyzed by HPLC using a 4.6 mmx25 cm Zorbax 5 µm phenyl column that was eluted with the following solvent system at 1 ml/min: 0–30 min, a convex gradient (Waters curve 2) of 0–100% solvent B; 30–45 min, 100% solvent B (solvent B = 50% methanol/50% solvent A; solvent A = 500 mM ammonium phosphate, pH 4.0). One minute fractions were collected and radioactivity was measured by liquid scintillation counting.

Cell proliferation
Four mice per group were fed 0.01, 0.3, 0.6 or 1.0% Mixture 1, 0.3% Mixture 2, 5 or 100 p.p.m. BaP or control diet for 4 weeks. Thirty minutes before being killed, the mice were given a single i.p. injection of 100 mg/kg body wt 5-bromo-2'-deoxyuridine (BrdU) (Sigma Chemical Co., St Louis, MO). The BrdU was dissolved with heating in Dulbecco's phosphate-buffered saline (Gibco BRL, Gaithersburg, MD), pH 7.1.

Small intestine and forestomach tissues were fixed in 10% neutral buffered formalin for 48 h and routinely processed. The labeling indices (S phase) in the target tissues was determined by immunohistochemical localization of BrdU (1113). Tissue sections were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol into phosphate-buffered saline. Endogenous peroxidase was quenched with 3% H2O2 containing 0.1% sodium azide. The sections were immersed in a 0.1% trypsin proteolytic enzyme (Sigma Chemical Co.) solution for 20 min at 37°C. A routine streptavidin procedure was performed, beginning with application of 0.5% casein to block non-specific binding of subsequent antibody and sequential incubation of sections with a mouse anti-BrdU mAb (clone Bu20a; Dako Corp., Carpinteria, CA), biotinylated goat anti-mouse F(ab1)2 IgG (Boehringer-Mannheim, Indianapolis, IN) and streptavidin-conjugated horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA). The BrdU-positive cells were visualized by incubating the sections in 3,3'-diaminobenzidine hydrochloride chromogen followed by counterstaining with Mayer's hematoxylin. The procedure was the same for locating proliferating cell nuclear antigen (PCNA), except that the slides were immersed in Antigen Retrieval solution (BioGenex Laboratories, San Ramon, CA) and incubated with 0.5% casein block, monoclonal mouse anti-human PCNA (clone PC10, 1:9000; Dako Corp.), biotinylated goat anti-mouse F(ab1)2 IgG and peroxidase-conjugated streptavidin.

The stained slides were analyzed with the point counting feature of an image analysis system (Optimas Corporation, Bothell, WA). The cell proliferation index for the small intestine and forestomach was determined by counting the number of labeled cells per mm of epithelium; ~1 mm of epithelium tissue was counted.

Tumor mutation analysis
Small intestine, forestomach, lung and liver tumors were obtained from mice removed from the 2 year bioassay. The treatment and pathology of the mice have been described previously in detail by Culp et al. (4). The tumors were dissected free of surrounding tissue; one half was subjected to histopathological examination and DNA was isolated from the remaining portion using an AutoGen 540 (AutoGen Inc., Framingham, MA) automated nucleic acid extractor. For each DNA sample, K-ras exons 1 and 2, H-ras exons 1 and 2 and p53 exons 5, 6, 7 and 8 were amplified by PCR. A 50 µl reaction mixture contained 200 µM each of dATP, dCTP, dGTP and dTTP, 250 nM appropriate primers (Table IGo), 1.5 U AmpliTaq Gold DNA polymerase (Perkin Elmer, Foster City, CA) and AmpliTaq buffer (50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin and 10 mM Tris–HCl, pH 8.3). A 40 bp GC-clamp was attached to the 5'-end of all forward primers (14). Samples were preheated to 94°C for 10 min and cycled for 35 rounds of 1 min at 94°C, 1 min at 62°C (58°C for K-ras) and 1 min at 72°C. A final extension period of 7 min at 72°C was added at the end of the reaction. All amplified fragments were prescreened by denaturing gradient gel electrophoresis using a modified procedure of Mittelstaedt et al. (15). Briefly, the acrylamide gels (9.6%) were run overnight at 90 V and 64°C, stained with ethidium bromide and photographed under UV illumination. H-ras exon 2, K-ras exon 1 and p53 exons 6 and 7 were run on 30–55% gradient gels; H-ras exon 1 on 40–55% gels; K-ras exon 2 on 20–45% gels; p53 exons 5 and 8 on 40–65% gels. Resolved heteroduplex bands (containing putative DNA mutations) were excised from the gels, and DNA was extracted (15) and amplified under the conditions described above except that the appropriate forward primers (Table IGo) contained an M13 plasmid sequence (–21M13, 5'-TGT AAA ACG GCC AGT-3') at the 5'-end. PCR products, carrying a –21M13 tag on the 5'-end were cycled using an ABI PrismTM Dye Primer Cycle Sequencing Kit (Perkin Elmer), using fluorescent –21M13 primers. The DNA was purified using a Centri-Sep column (Princeton Separations Inc., Adelphia, NJ) and sequenced on an Applied Biosystems Model 373A automated DNA sequencer.


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Table I. PCR primers for mouse H-ras and K-ras exons 1 and 2 and p53 exons 5–8
 
Statistical analyses
Average slopes for dose versus adduct levels were calculated by linear regression analysis. When necessary, data were transformed to maintain a normal distribution or equal variance. The average slopes among the various treatments were compared by ANOVA followed by Tukey's test. ANOVA and Dunnett's test were used to compare adduct levels in a particular dose with the levels in the control group. ANOVA and Tukey's test were used to compare the PCNA and BrdU results for cell replication.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Forestomach DNA adducts
In a 2 year bioassay, an increased incidence of forestomach tumors was observed in female mice fed either BaP or coal tar (4). In this study we have examined DNA from the forestomach of female mice fed BaP or coal tar for 4 weeks. Figure 1Go shows images from 32P-post-labeling analyses of DNA reacted with [3H]BPDE (Figure 1A and DGo) and forestomach DNA from a mouse fed 100 p.p.m. BaP (Figure 1B and EGo). The major adduct observed in the forestomach DNA co-eluted on PEI–cellulose TLC plates with dG-N2-BPDE. In mice fed coal tar, 32P-post-labeling of DNA from the forestomach gave a diffuse adduct pattern with a pronounced diagonal zone of radioactivity, as exemplified by Figure 1C and FGo, which shows the analysis from a mouse fed 1.0% Mixture 1. Within the diagonal zone, a number of discrete adducts were visible, including one with the same elution characteristics as dG-N2-BPDE. The radioactivity from this latter region was extracted from the PEI plate and bound to mAb 8E11. After washing the antibody, bound radioactivity was eluted with a mixture of dimethyl sulfoxide and methanol and analyzed by HPLC. A major adduct was observed (Figure 2BGo) with the same retention time as the 32P-post-labeled dG-N2-BPDE standard that had been subjected to the same extraction and binding procedure (Figure 2AGo). Approximately 43% of the radioactivity from the adduct area on the TLC plate co-eluted with the dG-N2-BPDE standard.



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Fig. 1. Phosphorimages of (A) DNA reacted with [3H]BPDE, (B) forestomach DNA from a mouse fed 100 p.p.m. BaP for 4 weeks and (C) forestomach DNA from a mouse fed 1.0% coal tar Mixture 1 for 4 weeks. (C) is representative of the images obtained from liver, lung and small intestine DNA of mice fed coal tar. (DF) The respective 3-dimensional images of phosphorimages generated using the UNIX program Cantata, courtesy of William B.Melchior Jr. The arrow indicates dG-N2-BPDE. The origins are circled.

 


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Fig. 2. HPLC separation of 32P-post-labeled DNA adducts extracted from the area co-eluting with dG-N2-BPDE on PEI–cellulose TLC plates and then bound to mAb 8E11. (A) The major adduct, dG-N2-BPDE, obtained from reacting DNA with BPDE, eluted at 28 min. (B) Adducts from DNA from a mouse fed 1.0% coal tar Mixture 1 for 4 weeks.

 
The levels of dG-N2-BPDE in the forestomach increased linearly throughout the dose range of coal tar Mixtures 1 and 2 (Figure 3Go). The slopes of the dG-N2-BPDE concentration curves did not differ between Mixtures 1 and 2. In mice fed BaP, the level of dG-N2-BPDE also increased in a linear manner (r2 = 0.78, P < 0.001) with the amount of BaP consumed (Figure 3Go) and was ~6-fold lower than observed in mice fed coal tar. Additional mice were fed 5 p.p.m. BaP and the levels of dG-N2-BPDE were assessed at 1, 2, 4, 8, 16 and 32 weeks. As shown in Figure 4Go, the adduct level reached a plateau after ~4 weeks of feeding BaP.



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Fig. 3. Amount of BaP consumed per day versus the concentration of dG-N2-BPDE in the forestomachs of mice fed BaP ({blacktriangleup}) or coal tar containing BaP for 4 weeks. The data are presented as the mean ± SD of four mice. CT1, coal tar Mixture 1 ({blacksquare}); CT2, coal tar Mixture 2 (•).

 


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Fig. 4. Concentration of dG-N2-BPDE in the forestomach of mice fed 5 p.p.m. BaP for up to 32 weeks. The data are presented as the mean ± SD of four mice.

 
In the mice fed coal tar, total adduct levels in the radioactive diagonal zone were also measured (Table IIGo) and were quantified through comparison with the dG-N2-BPDE standard (see Materials and methods). The total adduct levels in the forestomach increased in a linear manner (r2 = 0.84, P < 0.001 for Mixture 1; r2 = 0.59, P = 0.001 for Mixture 2) and were ~3-fold greater than the levels of dG-N2-BPDE (compare Figure 3Go and Table IIGo).


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Table II. Total DNA adduct levels in tissues of female B6C3F1 mice fed coal tar Mixture 1 or 2 for 4 weeksa
 
Lung, liver and small intestine DNA adducts
Total adduct levels were measured in the lung, liver and small intestine of mice fed coal tar for 4 weeks. In the 2 year bioassay, an increased incidence of tumor formation was observed in these tissues in mice fed coal tar but not BaP (4). 32P-post-labeling of the lung, liver and small intestine DNA from coal tar-fed mice gave a diffuse adduct pattern with a diagonal zone of radioactivity, similar to that observed in the forestomach (Figure 1CGo). The DNA adduct levels in these tissues did not differ significantly between coal tar Mixtures 1 and 2 (Table IIGo). In the lung and liver, the DNA adduct levels increased with dose (r2 = 0.84, P < 0.001) up to the 0.6% dose (Table IIGo), with the levels being ~7- and 2-fold greater, respectively, than those observed in the forestomach. The total adduct levels in the small intestine increased with dose (r2 = 0.70, P < 0.001) up to the 0.6% dose, whereupon there was a decrease such that the binding at the 1% dose was similar to that found with the 0.3% treatment (Table IIGo).

Cell proliferation study
Cell proliferation was measured, using both the BrdU and PCNA methods, in the small intestine of mice fed 0.01, 0.3, 0.6 and 1.0% Mixture 1, 0.3% Mixture 2 and control diet for 4 weeks. Sections were taken at 0.7, 7, 9, 12 and 17 cm from the stomach and at 1.7 and 17 cm from the large intestine. There were no significant differences in the number of S phase cells among the sections, thus the data from all the sections were combined. When assessed by measuring BrdU incorporation, there were significant increases in the number of S phase cells in mice fed 0.6 and 1.0% Mixture 1 and 0.3% Mixture 2 (Figure 5AGo). Nearly identical results were obtained using the PCNA technique (Figure 5BGo).



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Fig. 5. Extent of cell proliferation, as indicated by the percentage of S phase cells, in the small intestines of mice fed 0.01, 0.3, 0.6 or 1.0% coal tar Mixture 1 (CT1) or 0.3% Mixture 2 (CT2) for 4 weeks. The percentage S phase cells was determined by BrdU incorporation (A) or PCNA (B). The data are expressed as the mean ± SD from four mice. *Significantly different from the control group.

 
Cell proliferation was measured in the forestomach of mice fed coal tar and also from mice fed 5 and 100 p.p.m. BaP. When assessed using BrdU incorporation, significant increases in S phase cells were observed in mice fed 1.0% Mixture 1 and 5 and 100 p.p.m. BaP (not shown). These changes were not detected when assayed using PCNA.

Tumor mutation study
DNA was isolated from tumor tissue from mice fed coal tar or BaP for 2 years and examined for mutations in the K-ras, H-ras and p53 genes (Table IIIGo). Of the 31 forestomach tumors analyzed from BaP-fed mice, 21 (68%) had K-ras mutations, eight in codon 12 and 13 in codon 13. Each of these was a G->C or T transversion. There were also three (10%) H-ras mutations at codon 13 and three (10%) p53 mutations, and again these were G->C or T transversions. In the forestomach tumors from mice fed coal tar, 8/15 (53%) had K-ras mutations, five in codon 12 and three in codon 13. Each of these was a G->C or T transversion, with the exception of a single G->A transition. Two (13%) H-ras mutations were observed in codon 61, both being A->T transversions. Two (13%) p53 transversion mutations involving G were also detected.


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Table III. K-ras, H-ras and p53 mutations in tumors from mice fed coal tar Mixture 1 or 2 or BaP
 
A significant increase in lung, small intestine and liver tumors was observed only in the mice fed coal tar. Of the 15 lung tumors analyzed, nine (60%) contained K-ras mutations, five in codon 12 and four in codon 13 (Table IIIGo). Each mutation involved a G and both transitions and transversions occurred. H-ras and p53 mutations were not detected in the lung. Of the 22 small intestine tumors analyzed for mutations, two (9%) contained K-ras mutations, while five (23%) had p53 mutations (Table IIIGo). Each of these involved a G:C base pair. Thirteen (35%) of the 37 liver tumors analyzed had mutations in H-ras, one in codon 13 and 12 in codon 61. Eight of the 12 codon 61 mutations involved an A:T base pair. There were also two (5%) K-ras mutations and four (11%) p53 mutations.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have compared the relationships between DNA adduct levels and cell proliferation from short-term studies of mice fed various doses of coal tar or BaP and the tumor incidence in mice treated for 2 years.

DNA adduct concentrations were measured in the forestomachs of mice fed BaP or coal tar for 4 weeks to determine if the levels were predictive of tumor incidence for either treatment. With both treatments, an adduct indicative of BaP (i.e. dG-N2-BPDE) was detected and the adduct levels increased in a dose-related manner (Figure 3Go). In coal tar-fed mice, the dG-N2-BPDE levels were 6-fold higher than indicated by the BaP content of the coal tar. This may be due to components in the coal tar other than BaP enhancing the metabolic activation of BaP. Alternatively, the formation of adducts with chromatographic characteristics similar to BaP-induced adducts (i.e. dG-N2-BPDE) may occur. Additional support for the role of dG-N2-BPDE in forestomach tumorigenesis for both treatments was obtained by analysis of the tumors for activated oncogenes. K-ras mutations were observed in 68 and 53%, respectively, of the forestomach tumors in mice fed BaP or coal tar (Table IIIGo). These mutations were divided nearly equally between codons 12 and 13 and all involved G:C base pairs, which is consistent with adduct formation at deoxyguanosine (i.e. dG-N2-BPDE).

Although BaP is known to induce forestomach tumors in mice (1720), the mutation spectra in BaP-induced tumors has not been examined. Forestomach tumors induced in CDF1 mice by the heterocyclic aromatic amine 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) have been shown to contain H-ras mutations involving a G->T transversion in the second base of codon 13 (21,22). MeIQ differs from BaP in that the primary adduct detected in the forestomach is substituted at C8 of deoxyguanosine (23) as compared to N2 of deoxyguanosine, the site of substitution for BaP. MeIQ also induces p53 mutations in the forestomach (24) and, as with BaP and coal tar, these involve base substitutions at deoxyguanosine.

In contrast to the forestomach tumors, small intestine adenocarcinomas were observed only in mice fed 0.6 or 1.0% coal tar in the 2 year bioassay (4). DNA adduct levels were determined in the small intestine of mice fed coal tar for 4 weeks and although the maximum tumorigenic response (88%) was observed at the 1% dose, the adduct concentration was highest with the 0.6% treatment (Table IIGo). As a consequence, the adduct level associated with the 61% small intestine tumor incidence at 0.6% coal tar was approximately three times greater than that associated with an 88% tumor incidence at 1.0% coal tar. Furthermore, the adduct levels at the 1.0% coal tar dose were very similar to the adduct levels from the 0.3% dose (Table IIGo), where no small intestine tumors were observed. One mechanism for the induction of tumors at high doses is an increase in cell proliferation as a result of toxic insult (25). With 2-acetylaminofluorene, for example, an increase in bladder tumors in mice is only seen at high doses of the carcinogen and this has been attributed to a synergy between DNA adduct formation and 2-acetylaminofluorene-induced cell proliferation (26,27). Since DNA adduct levels in the small intestine were markedly non-linear (Table IIGo), we assessed the extent of cell proliferation in the small intestine of mice fed coal tar. A nearly 50% increase in the number of S phase cells was observed in mice fed 0.6 or 1.0% coal tar for 4 weeks compared with controls (Figure 5Go).

Although proliferation in the small intestine is relatively rapid (28), an increase in labeling index of this tissue has been noted in mice expressing a p53 null genotype (p53–/–) following exposure to high doses of {gamma}-irradiation (29). This increase in cell proliferation was accompanied by a decrease in the apoptotic response that would normally accompany the toxic insult (29,30). A similar response may be occurring in the mice exposed to high doses of coal tar because 23% of the small intestine tumors showed mutations in the p53 tumor suppressor gene (Table IIIGo). Thus, our data suggest that both compound-induced cell proliferation and compound-induced genotoxicity may act in synergy to increase the small intestine tumorigenic response in mice fed coal tar.

In the 2 year bioassay (4), lung tumors were elevated in mice fed coal tar but not BaP. The induction of lung tumors in certain strains of mice, notably A/J, has been attributed to the presence of three susceptibility loci (Pas genes), one of which is K-ras (31). K-ras mutations were detected in 60% of the lung tumors analyzed from mice fed coal tar and these were distributed approximately equally between codons 12 and 13 (Table IIIGo). Mutations at codons 12 and 13 of K-ras have been reported in spontaneous lung tumors from B6C3F1 mice (32), however, all of these involved the first base of either codon, while with the coal tar-treated mice all of the mutations in codon 12 occurred at the second base (Table IIIGo). In addition, codon 61 mutations were also observed in the spontaneous tumors (32), but these were not found in lung tumors from the mice fed coal tar. These results suggest that the lung tumors result from genotoxic compounds contained in the coal tar and are not due entirely to coal tar promoting the expression of spontaneous lung tumors. This is an important observation because it is the lung tumors in coal tar-fed mice that have been used to estimate the risk associated with exposures to coal tar mixtures (33).

The DNA adduct levels in the lungs of mice fed BaP (data not shown) were ~100-fold less than those observed in mice fed coal tar (Table IIGo). A similar relationship has been observed previously in male B6C3F1 mice fed coal tar or BaP (6). The relatively low level of DNA adduct formation may contribute to the lack of lung tumors in B6C3F1 mice fed BaP. In contrast to feeding studies, when neonatal B6C3F1 mice are injected i.p. with coal tar or BaP, similar DNA adduct levels are obtained in the lung, however, neither coal tar nor BaP induced lung tumors in this model (34). BaP induces lung tumors in A/J mice (35,36) and this is associated with K-ras mutations (36). Nonetheless, the BaP-induced K-ras mutations are predominantly located in codon 12 (31,36) and contrast with the codon 12 and 13 mutations found in mice fed coal tar (Table IIIGo). This suggests that although BaP has the potential to induce lung tumors in mice, the lung tumors resulting from coal tar are due to additional genotoxic components in the mixture. This conclusion is supported by the observation that lung tumor incidence in A/J mice administered coal tar is greater than predicted by the BaP content of the coal tar (35,37).

Although liver tumors were induced in mice fed coal tar, the incidence was less than that observed with lung tumors (4). This may be a reflection of the lower levels of DNA adducts found in liver compared with lung (Table IIIGo). Higher DNA adduct levels in lung compared with liver have also been observed in male B6C3F1 mice fed coal tar (6). Approximately 35% of the liver tumors from the coal tar-fed mice had H-ras mutations, with the majority of these occurring in codon 61 (Table IIIGo). Spontaneous liver tumors in B6C3F1 mice are associated with H-ras mutations at codon 61, with the majority being CAA->AAA, CAA->CGA and CAA->CTA (38). Since these types of mutations were observed in liver tumors from mice fed coal tar, it is likely that the coal tar is promoting the expansion of spontaneous tumors.

In the 2 year feeding study BaP did not increase the incidence of liver tumors (4). BaP is hepatocarcinogenic when administered to neonatal B6C3F1 mice (34,39) and this has been associated with the induction of K-ras mutations (39). Hepatic tumors are also induced when neonatal mice are treated with coal tar (34); however, the oncogene changes in these tumors have not yet been determined.

In summary, in the forestomach of mice fed either coal tar or BaP an adduct consistent with BaP was detected, with the adduct levels increasing with dose. Because the incidence and type of K-ras mutations detected in the forestomach tumors were similar in mice fed either coal tar or BaP, the BaP component of coal tar (or similar acting polycyclic aromatic hydrocarbons) appears to be responsible for forestomach tumor induction. DNA adduct levels in the small intestine were not predictive of tumor incidence in this tissue; instead the tumors appear to result from compound-induced cell proliferation that only occurs at high doses of coal tar. Although K-ras mutations were detected in lung tumors from coal tar-fed mice, BaP alone did not increase lung tumor frequency. Thus, coal tar components other than BaP appear to be responsible for the tumors induced in this tissue. H-ras mutations, primarily at codon 61, were the most common mutation observed in liver tumors induced by coal tar. Since this mutation profile is also present in spontaneous hepatic tumors, it is possible that components in the coal tar may be promoting the expansion of pre-existing lesions.


    Notes
 
2 Present address: Zeneca, PO Box 15458, Wilmington, DE 19850, USA Back

3 To whom correspondence should be addressed at: HFT-110, NCTR, 3900 NCTR Road, Jefferson, AR 72079, USA Email: sculp{at}nctr.fda.gov Back


    Acknowledgments
 
We thank Dan Nestorick and Larry Rushing for conducting the chemical analyses of the coal tar mixtures, Donna Norton for assistance with immunohistochemical staining, Howard Durrett for assistance with animal care and Betty Spadoni for computer support. Preliminary results of this manuscript were presented at the 15th International Symposium on Polycyclic Aromatic Compounds, Belgirate, Italy (Polycyclic Aromatic Compounds, 11, 161–168, 1996). This research was conducted through a Cooperative Research and Development Agreement between the National Center for Toxicological Research, Jefferson, AR, and the Electric Power Research Institute, Palo Alto, CA (CRADA no. U34570).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 19, 2000; revised March 13, 2000; accepted March 29, 2000.