Affiliations of authors: L. E. Smith, M. F. Denissenko, G. P. Pfeifer (Department of Biology), W. P. Bennett (Department of Human Genetics), Beckman Research Institute of the City of Hope, Duarte, CA; H. Li, M.-s. Tang, The University of Texas M. D. Anderson Cancer Center, Smithville, TX; S. Amin, American Health Foundation, Valhalla, NY.
Correspondence to: Gerd P. Pfeifer, Ph.D., Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (e-mail: gpfeifer{at}coh.org).
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ABSTRACT |
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INTRODUCTION |
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Lung cancer is the leading cause of cancer death for U.S. women and men (4) and is the most common type of cancer worldwide. Polycyclic aromatic hydrocarbons (PAHs), present in all products of combustion of organic matter (including tobacco smoke), have been suggested as agents responsible for the initiation and development of lung cancer (5). Because they are formed upon combustion of any organic material, they are present in notable concentrations in our environment (6) due to gasoline and diesel engine emissions and industrial sources. However, the largest concentrations of PAHs are inhaled by smokers with the mainstream smoke of cigarettes (7,8).
PAHs are activated by the cytochrome P450 enzymes to form the ultimate carcinogenic diol epoxides, although other pathways of PAH activation exist (9). The diol epoxide PAH metabolites form covalent adducts with DNA, primarily at the exocyclic N2 position of guanine residues and the N6 position of adenines (6). B[a]PDE [(+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide] is the best studied of the activated PAHs. Previous studies with B[a]PDE have demonstrated that the DNA damage spectrum is nonrandom. B[a]PDE induces guanine adducts at mutational hotspots, including codons 157, 248, and 273 of the p53 gene in normal human bronchial epithelial cells (10). B[a]PDE-damage hotspots in exons 5, 7, and 8 correspond to methylated CpG sequences within the p53 gene (11,12). Preferential formation of B[a]PDE adducts is due to enhancement of adduct formation by the 5-methylcytosine base within a CpG sequence (13,14). Furthermore, B[a]PDE adducts in the nontranscribed strand of p53 are repaired two to four times more slowly than those in the transcribed strand (15). The slowly repaired damage hotspots correspond to mutational hotspots observed in human lung cancer. B[a]PDE exposure results in a mutational fingerprint with respect to the types of mutations induced. This bulky carcinogen produces predominantly G-to-T transversions (1620), whereas spontaneous mutations tend to be G-to-A transitions probably arising through spontaneous deamination of methylated cytosines (21,22). The p53 mutation spectrum observed in human lung cancer from smokers is dominated by the presence of G-to-T transversions (approximately 30%40%). This type of mutation is much less frequent in other human cancers, except hepatocellular carcinoma. Of all of the G-to-T transversion mutations in lung tumors, 95% occur on the nontranscribed strand (3,23). In contrast, the p53 mutational spectrum of human lung cancer from nonsmokers demonstrates few hotspots and a much lower frequency of G-to-T transversions (3,23). These data, together with DNA adduct formation at lung cancer mutational hotspots by B[a]PDE (10) and the repair strand bias (15), suggest an etiologic link between benzo[a]pyrene (B[a]P) exposure and human lung cancer.
However, B[a]P is only one of many PAHs found in the complex mixture of chemicals in cigarette smoke (24), with concentrations reported to be as high as 1050 ng per cigarette (6,25). Among other PAHs in tobacco smoke are chrysene, 5-methylchrysene, and 6-methylchrysene at approximately 60, 0.6, and 10 ng per cigarette, respectively (6,25,26). Benzo[c]phenanthrene has been determined to be present in cigarette smoke; however, exact concentrations have not been reported (7). Benzo[g]chrysene has not been reported in cigarette smoke but is present in coal tar and petroleum distillates (27). It is, therefore, found at appreciable levels in the environment and is also likely present in smoke condensate. The activated forms of B[a]P, chrysene, and 6-methylchrysene are planar bay region diol epoxides. The chrysene metabolite (+/-)-anti-chrysene-3,4-diol-1,2-epoxide has been reported to be only weakly mutagenic in Salmonella typhimurium and in Chinese hamster V-79 cells (28) and to be weakly tumorigenic in newborn mouse liver and lung model systems (29); however, the reverse diol epoxide (+/-)-anti-chrysene-1,2-diol-3,4-epoxide (CDE), used in this study, is active (28). The presence of a methyl group adjacent to the bay region in (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) is thought to induce steric hindrance that forces the epoxide ring away from the plane of the molecule and is responsible for its high mutagenicity and carcinogenicity in many model systems (28,3033). In contrast, (+/-)-anti-6-methylchrysene-1,2-diol-3,4-epoxide (6-MCDE) is only weakly mutagenic in many test systems (3032). 6-MCDE is formed in greater amounts than is 5-MCDE in mouse epidermis after exposure to the respective parent PAHs; however, 6-MCDE results in lower levels of DNA adduction (30). These differences have been attributed to decreased initial intercalation of 6-MCDE, increased repair of adducts, or increased detoxification of 6-MCDE by mechanisms such as conjugation with glutathione (32). (+/-)-Anti-benzo[g]chrysene-11,12-diol-13,14-epoxide (B[g]CDE) and (+/-)-anti-benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PDE) are nonplanar fjord region diol epoxides. The nonplanar diol epoxides are more mutagenic and carcinogenic than the planar metabolites. B[c]PDE proved to be mutagenic in bacterial and mammalian assays (34) and is a potent tumor initiator in mouse skin (35). B[c]PDE adducts can occur at both adenine and guanine residues in DNA (36). Both B[c]PDE and B[g]CDE are potent mammary carcinogens in rats (37). B[g]CDE, a tumor initiator in mouse skin (38) and in lung/liver assays in newborn mice (39) and which is also highly mutagenic in the dhfr gene of Chinese hamster ovary cells (40), can likewise form adducts with both adenine and guanine residues (41).
In this study, we have mapped the distribution of adducts formed by the active diol epoxide forms of chrysene, 5-methylchrysene, 6-methylchrysene, benzo[c]phenanthrene, and benzo[g]chrysene (see Fig. 1) in the nontranscribed strand of p53 for exons 5, 7, and 8 in normal human bronchial epithelial cells.
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MATERIALS AND METHODS |
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Racemic diol epoxides of the following PAHs were synthesized as described in the literature: (+/-)-anti-chrysene-1,2-diol-3,4-epoxide (CDE) (6), (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) (42), (+/-)-anti-6-methylchrysene-1,2-diol-3,4-epoxide (6-MCDE) (43), (+/-)-anti-benzo[g]chrysene-11,12-diol-13,14-epoxide (B[g]CDE) (44,45), and (+/-)-anti-benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PDE) (46) (Fig. 1). (+/-)-Anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) was obtained from the National Cancer Institute repository (Midwest Research Institute, Kansas City, MO). The compounds were dissolved in dimethyl sulfoxide (DMSO), and stock concentrations were confirmed by spectrophotometry.
Cell Culture and DNA Modification
Normal human bronchial epithelial cells were cultured in medium recommended by the supplier (Clonetics, San Diego, CA). These cells were treated with various concentrations of the PAH metabolites for 30 minutes at 37°C in the dark. The highest concentrations used (except for those of CDE) produced similar adduct levels in total genomic DNA, as measured after cleavage with the UvrABC excision nuclease complex of Escherichia coli and alkaline agarose gel electrophoresis (approximately one adduct every 5 kilobases [kb]). Controls were treated with the appropriate concentration of DMSO only. After treatment, the cells were harvested and genomic DNA was isolated as previously described (10).
Cleavage of Damaged DNA by UvrABC
Uvr proteins, comprising the nucleotide excision repair complex of E. coli, were purified as described previously (47). Purified DNA was treated with a molar excess of Uvr proteins as described previously (10,47). Cleavage of adducted DNA was confirmed by running 1 µg of treated DNA in 0.6% agarose gels after denaturation in formamide. Under the reaction conditions used, DNA cleavage by UvrABC proteins is quantitative (10,48). Efficient cleavage was achieved with a 10-fold molar excess of UvrABC subunits over DNA (10-kb size). Additional increases in enzyme concentration (up to 40-fold excess) did not result in additional cleavage. After UvrABC treatment, the proteins were removed by phenolchloroform extraction. The cleaved DNA was precipitated and resuspended in TE buffer (i.e., 1 mM TrisHCl [pH 8.0] and 0.1 mM EDTA).
Ligation-Mediated Polymerase Chain Reaction and Quantitation of Data
Ligation-mediated polymerase chain reaction (LMPCR) was performed for exons 5, 7, and 8 of the human p53 gene. The conditions and oligonucleotide primers utilized for LMPCR of these exons were as previously described (49,50),with the following modifications: Longer primers having higher annealing temperatures were used during the initial primer extension step. Primer 5-4D (5'-GGGCCAGACCTAAGAGCAATCAGT) was used for exon 5, primer 7-4B (5'-CAGGGGTCAGCGGCAAGCAGAG) was used for exon 7, and primer 8-4D (5'-AGGCAAGGAAAGGTGATAAAAGTG) was used for exon 8.
The extension was performed with the use of a 16:1 combination of the Vent exo- and Vent polymerases (New England Biolabs, Inc., Beverly, MA) with a thermocycler protocol of 3 minutes at 95°C, 3 minutes at the primer annealing temperature, and 15 minutes at 72°C. After elongation of the primer, DNA was precipitated in the presence of glycogen. The primer-extended fragments were ligated as previously described (4951). Ligated fragments were polymerase chain reaction (PCR) amplified after ethanol precipitation with Amplitaq Gold DNA polymerase (The Perkin-Elmer Corp., Foster City, CA) according to the manufacturer`s directions. The amplified PCR products were separated by denaturing gel electrophoresis, electroblotted to a Genescreen Plus (Du Pont NEN, Boston, MA) nylon membrane, and hybridized with 32P-labeled p53-specific probes as previously described (51). All LMPCR experiments were repeated at least once and gave very similar results. The nylon membranes were exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Radioactivity was measured in all PAH metabolite-specific bands of the sequencing gel that could be fully resolved. The radioactivity of each band was quantified after background subtraction with the use of lanes that were treated with UvrABC, but no carcinogen. To correct for DNA sequence-dependent LMPCR amplification efficiencies, we normalized the increases in the intensity of a radioactive signal from a treated sample by dividing this signal by the corresponding MaxamGilbert control band. We then calculated the relative intensity of each band in a particular exon by dividing the corrected damage signal at each nucleotide by the band of greatest intensity produced by each particular compound in a specific exon. The relative intensity was then plotted as a function of the nucleotide sequence for codons within exons 5, 7, and 8. Only one concentration of PAH diol epoxides was used for quantitation, but the LMPCR-damage patterns are quite reliable and dose independent as long as a sufficient adduct frequency of more than one adduct every 10 kb is provided.
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RESULTS |
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Fig. 3, A, demonstrates the profile of PAH adducts produced within the nontranscribed strand of exon 5 after a single 30-minute exposure of cells to each activated PAH compound. The relative intensity of damage for each compound and previously obtained data for B[a]PDE damage within exon 5 are summarized in Fig. 3
, B. Codons 154, 156, 157, 158, and 159 demonstrated the overall highest accumulation of DNA adducts after exposure to the PAH metabolites. These codons all contain 5-methylcytosines within a CpG sequence and were also sites of greatest damage induced by B[a]PDE (10) (Fig. 3
, B). In addition, codons 164, 171, and 175 demonstrate substantial levels of DNA adducts, depending on the specific agent. CDE was weakly effective at producing DNA adducts at codon 156, with lesser amounts of adducts found at codons 154 and 158 (Fig. 3
, A). 5-MCDE produced adducts at codons 154, 156, 157, 158 (highest), 159, 171, and 175. 6-MCDE adducts were formed primarily at codons 154 (highest), 157, 158, 159, 164, 170, 171, and 175. B[g]CDE adducts occurred primarily at codons 158 (highest), 175, 159, and 156. B[c]PDE adducts were observed primarily at codons 158 (highest), 175, 154, 156, and 159. Codon 158 was more selectively targeted by 5-MCDE, B[c]PDE, and B[g]CDE relative to B[a]PDE and 6-MCDE. The adduct profiles of the fjord region PAHs B[c]PDE and B[g]CDE were more similar to each other than to the profiles of the bay region PAHs. DNA damage at codons 154, 157, and 158 is of particular interest, since mutations at these positions are common in lung cancer (Fig. 2
) but are rare in other tumors. The effective order of total DNA adduct formation at comparable concentrations of the diol epoxides within the nontranscribed strand of exon 5 was CDE < 6-MCDE < B[c]PDE < 5-MCDE = B[g]CDE (Fig. 3
, A; data not shown).
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DISCUSSION |
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All PAH compounds demonstrated strong adduction at guanines in codons 157 and 158 of exon 5, codons 245 and 248 of exon 7, and codon 273 of exon 8, which are prominent hotspots for mutation in human lung cancer. Mutations in lung cancer are not particularly common at codon 237, another strong PAH-binding site, but G-to-T transversions at the guanine of this codon are seen frequently in breast tumors (54). The reason could be that the PAHs that damage codon 237 are not abundant in cigarette smoke or that this mutation is not selected in lung cancer. The damaged guanines in codons 157, 158, 245, 248, and 273 are all within methylated CpG dinucleotides. The effect of cytosine methylation on the targeting of DNA damage by the PAHs investigated in this study is not yet well defined. Previous data from our studies (13), using methylated and unmethylated plasmids containing exons 5, 7, or 8 of p53, demonstrated that the presence of 5-methylcytosine in a CpG sequence enhanced B[a]PDE binding. A subsequent study (14) demonstrated that cytosine methylation resulted in increased binding of B[g]CDE to guanines in CpG sequences. The data presented here suggest that the presence of methylated cytosines in DNA-damage hotspot sequences may contribute to the binding of other activated PAHs. No specific sequence rules, other than a preference for methylated CpG sequences, could be identified.
Of the six most dominant lung cancer mutational hotspots that contain guanine bases (codons 157, 158, 245, 248, 249, and 273; Fig. 2), five are prominent PAH adduct-binding sites. The only exception is codon 249. This codon may represent a special case, where mutations may be frequent as a consequence of strong selection rather than preferential adduct formation [see also (55)]. Of the five strongest adduct-binding sites in exon 5 (codons 154, 156, 157, 158, and 159), two (codons 157 and 158) are prominent mutational hotspots in lung tumors and one (codon 154) is a moderate hotspot. Although they form high levels of adducts, codons 156 and 159 are not commonly mutated in lung tumors. At codons 156 and 159, a G-to-T transversion was described only once, each in a glioblastoma and a breast cancer, respectively (54), indicating that this type of mutation rarely may be selected at these codons. In exon 7, the three codons showing the highest levels of adduction (codons 237, 245, and 248) are mutational hotspots in breast cancer (codon 237) or lung cancer (codons 245 and 248). In exon 8, extremely high levels of adducts are seen at codon 273 (Fig. 5
, A). This adduct distribution is largely similar to the lung cancer mutational spectrum, where codon 273 is the most commonly mutated site along the entire p53 sequence (Fig. 2
). Moderate levels of adducts also form at codon 290, which is at the 3' end of the DNA-binding domain of the p53 protein, and mutations here probably are not selected. In summary, it appears that the p53 mutational spectrum in lung cancer is largely adduct driven, but selection determines which of the preferentially targeted codons are sampled into the tumor mutation database.
The sterically hindered fjord region diol epoxides, B[g]CDE and B[c]PDE, also bound substantially to adenines in codons 236, 239, 240, and 246 in exon 7, although the levels of adduction were less than the quantities achieved at heavily damaged guanines. The majority of the mutations in the lung tumor database occur at guanines and are most frequently G-to-T transversions. Codons that have been reported to have frequent mutations at adenines in the human lung cancer database are 163 and 179. Codon 179, a prominent mutational hotspot in lung cancers (Fig. 2), does not contain a guanine on the nontranscribed strand and, therefore, is not an important target for DNA damage induced by B[a]PDE and chrysene metabolites. We observed little binding by B[g]CDE and B[c]PDE at the codon 179 sequence. The mutations observed at codon 179 are A-to-G transitions, a type of mutation that can be induced by fjord region PAHs (56,57). However, A-to-T transversion is a more common type of mutation induced by these compounds (56,57), which is rare (5% of all mutations) in the lung cancer database (3,54). An alternative hypothesis regarding the production of A-to-G transitions is exposure to nitric oxide (58). The adenine nucleotide most substantially damaged by fjord region PAHs occurs in the second position of codon 236. This lesion most likely would produce an A-to-T transversion, TAC (Tyr) to TTC (Phe), but this mutation has never been reported in the p53 database of more than 10000 entries; therefore, it is likely that this event is not selected during tumorigenesis.
Although this study has focused on PAHs as a major class of carcinogenic components of cigarette smoke, it does not exclude the possibility that other carcinogens present in smoke, such as NNK [i.e., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] (via pyridyloxobutyl adducts) or aromatic and heterocyclic amines, may have a similar sequence specificity.
We (10) previously reported that B[a]PDE-induced DNA damage occurred at several lung cancer mutational hotspots in the p53 gene. In this article, DNA damage was mapped for the diol epoxide forms of other PAHs, which may be present in the complex mixture of cigarette smoke. The DNA damage induced by these compounds maps, at least in part, to the same codons previously reported for B[a]PDE. The data show that B[a]PDE is a reasonable model compound for the strongest sites of adduction; however, other PAH metabolites probably contribute to the total load of adducts in the p53 gene of smokers and, therefore, may contribute to the mutational spectrum observed in human lung tumors.
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NOTES |
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Present address: M.-s. Tang, Department of Environmental Medicine, New York University, Tuxedo.
Supported by grant 6RT-0361 (to G. P. Pfeifer) from the University of California Tobacco Related Disease Research Program and by Public Health Service grant ES03124 (to M.-s.Tang) from the National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services.
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Manuscript received October 19, 1999; revised February 22, 2000; accepted March 7, 2000.
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