4-Aminobiphenyl is a major etiological agent of human bladder cancer: evidence from its DNA binding spectrum in human p53 gene

Zhaohui Feng1,*, Wenwei Hu1,*, William N. Rom2, Frederick A. Beland4 and Moon-shong Tang1,2,3,5

1 Department of Environmental Medicine,
2 Department of Medicine and
3 Department of Pathology, New York University School of Medicine, Tuxedo, NY 10987 and
4 Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
4-Aminobiphenyl (4-ABP) is a major etiological agent of human bladder cancer, and its metabolites are able to form DNA adducts that may induce mutation and initiate bladder carcinogenesis. Thirty to sixty percent of human bladder cancer has a mutation in the p53 gene, and the mutational spectrum bears two characteristics: compared with other cancers, the pattern of mutations is more evenly distributed along the p53 gene, and the mutational hotspots occur at both CpG sites, such as codons 175, 248 and 273, and non-CpG sites, such as codons 280 and 285, the latter two being unique mutational hotspots for bladder and other urinary tract cancers. These findings raise the possibility that the special p53 mutational features in human bladder cancer are due to the unique binding spectrum of metabolically activated 4-ABP in bladder cells. To address this question, here we have mapped the 4-ABP–DNA adduct distribution in the p53 gene at the nucleotide sequence level in human bladder cells. We found that, unlike benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide–DNA adduction, which preferentially occurs at CpG sites, 4-ABP–DNA adduction is not biased for CpG sites, and the adducts are more evenly distributed along the p53 gene; nonetheless, the p53 mutational hotspots in bladder cancer at codons 175, 248, 280 and 285 are also the preferential sites for 4-ABP adduct formation. These results strongly suggest that the unique binding spectrum of 4-ABP contributes greatly to the unique mutational spectrum in the p53 gene of human bladder cancer, and provide further molecular evidence to directly link 4-ABP to bladder cancer.

Abbreviations: 4-ABP, 4-aminobiphenyl; BPDE, benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide; dG-C8-4-ABP, N-(2'-deoxyguanosin-8-yl)-4-ABP; N-OH-4-ABP, N-hydroxy-4-aminobiphenyl; N-OH-N-Ac-4-ABP, N-hydroxy-N-acetyl-4-aminobiphenyl; LMPCR, ligation-mediated polymerase chain reaction; PAH, polycyclic aromatic hydrocarbon


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aromatic amine, 4-aminobiphenyl (4-ABP; Figure 1Go), is an environmental and occupational contaminant generated mainly from cigarette smoking, the combustion of fossil fuels and also from rubber, coal, textile and printing processing industries (13). 4-ABP has been shown to be a major etiological agent of human bladder cancer, and also a potent urinary bladder carcinogen in experimental animals (4,5). Cigarette smokers have a 2–10-fold increased incidence of bladder cancer (6), and individuals occupationally exposed to 4-ABP have a high incidence of bladder cancer (7). Significantly higher levels of 4-ABP–DNA adducts have been found in the bladder cancer biopsies from smokers (8), and increased levels of 4-ABP hemoglobin adducts have also been found in the erythrocytes of smokers (9).



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Fig. 1. Chemical structures of 4-ABP, N-OH-4-ABP, N-OH-N-Ac-4-ABP and dG-C8-4-ABP.

 
Metabolically activated electrophilic derivatives of 4-ABP are able to interact with DNA to form adducts that have been found to correlate well with bladder carcinogenesis in both experimental animals (1012) and humans (8,1215). The predominant DNA adduct induced by 4-ABP in vivo is N-(2'-deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP 80%); minor amounts of N-(2'-deoxyadenosine-8-yl)-4-ABP (15%) and 3-(2'-deoxyguanosin-N2-yl)-4-ABP (5%) are also formed (10,16).

The most frequently mutated cancer-associated gene in human bladder cancer is the p53 gene, with mutations occurring in 30–60% of cases (17,18). The p53 mutational spectrum of bladder cancer bears two features that are different from other cancers; first, the mutational frequency is more evenly distributed along the codons in the DNA binding domain of the p53 gene, and second, mutations are not biased for methylated CpG sites. Among the five p53 mutational hotspots of bladder cancer, codons 175, 248 and 273 contain CpG sites and are common mutational hotspots in many human cancers; however, codons 280 and 285 do not contain CpG sites and represent unique mutational hotspots in bladder cancer and other urinary tract cancers (International Agency for Research on Cancer, P53 mutation database.

The mutational spectrum and the kinds of mutations in the p53 gene in human cancers often bear the signature of the cancer etiological agents (1720). Smoking-associated lung cancer is one notable example. In lung cancer of cigarette smokers, the p53 gene mutations are concentrated at several methylated CpG sites (codons 157, 158, 175, 245, 248, 273 and 282), and the major mutation in these mutational hotspots is G to T transversion. This is a signature mutation induced by polycyclic aromatic hydrocarbons (PAHs), the major carcinogens in cigarette smoke that have been suggested to be the primary agents responsible for the initiation and development of lung cancer of smokers (1720). We have found previously that benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide (BPDE), a major metabolite of the cigarette smoke carcinogen benzo[a]pyrene, and dihydrodiol epoxides from other PAHs preferentially form DNA adducts at the methylated CpG sites corresponding to the aforementioned mutational hotspots observed in lung cancer of smokers (21,22). The results strongly suggest that the initial DNA adduct distribution in the p53 gene contributes greatly to the mutational spectrum in human cancers.

The unambiguous role of 4-ABP in bladder carcinogenesis, together with the unique p53 mutational features in bladder cancer, raises the possibility that the unique p53 mutational features in bladder cancer are the result of the unusual DNA binding specificity of metabolically activated 4-ABP in the p53 gene, which may or may not be bladder cell-specific. To address this important question, it is necessary to have a method to assess the 4-ABP–DNA binding specificity at the nucleotide sequence level. We recently have found that Escherichia coli nucleotide excision repair enzyme UvrABC nuclease is able to incise 4-ABP–DNA adducts specifically and quantitatively (23). In this study, we have determined the 4-ABP–DNA adducts distribution at nucleotide sequence level in the p53 gene of both human bladder cells and lung fibroblasts, using UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture, DNA isolation and carcinogen treatment
Human bladder cells (HTB-1 from ATCC) and human lung fibroblasts (CCL-202 from ATCC) were grown in MEM medium supplemented with 10% fetal bovine serum. N-Hydroxy-4-aminobiphenyl (N-OH-4-ABP) was synthesized as described previously (24). N-Hydroxy-N-acetyl-4-aminobiphenyl (N-OH-N-Ac-4-ABP) and BPDE were purchased from Chemsyn Science Laboratories (Lenexa, KS). Stock solutions of N-OH-4-ABP and N-OH-N-Ac-4-ABP were prepared immediately before use by dissolving the compounds in argon-purged ethanol; BPDE was dissolved in dimethyl sulfoxide.

Cells at 50–70% confluency were washed with phosphate buffer and treated with different concentrations of N-OH-N-Ac-4-ABP in serum-free culture medium for 6 h, or different concentrations of BPDE for 30 min, at 37°C in the dark. After treatment, the cells were washed with phosphate buffer and genomic DNA was isolated as described previously (21,22). For in vitro modifications, genomic DNA was isolated from untreated cells and dissolved in 10 mM sodium citrate buffer (pH 5.0). The solution was extensively purged with argon, mixed with different concentrations of N-OH-4-ABP, and incubated at 37°C for 8 h. Decomposition products from N-OH-4-ABP were removed by repeated phenol and diethyl ether extractions. The DNA was then precipitated with ethanol, washed and dried under a vacuum (24).

DNA adduct analysis by 32P-postlabeling technique
32P-Postlabeling analyses were conducted as described previously (24) using 10 µg DNA. Adducts were identified and levels were quantified through comparison to a DNA standard modified with dG-C8-4-ABP at a level of 62 adduct/108 nucleotides (24).

Cleavage of DNA adducts by UvrABC nuclease
UvrABC nuclease, the DNA nucleotide excision repair enzyme complex of E.coli, was purified by the method of Sancar and Rupp (25). Purified genomic DNA was reacted with UvrABC nuclease to cleave the DNA adducts as described previously (26). Briefly, a 10-fold molar excess of UvrABC nuclease was added to DNA (assuming an average size of 14 kb) in a reaction buffer containing 100 mM KCl, 1mM ATP, 10 mM MgCl2, 10 mM Tris–HCl (pH 7.5) and 1 mM EDTA. The reactions were conducted at 37°C for 1 h and were stopped by extracting with phenol and diethyl ether followed by precipitating the DNA with ethanol.

Mapping DNA adducts with LMPCR
A known quantity of purified DNA was treated with UvrABC nuclease or subjected to Maxam–Gilbert sequencing reactions (27), and followed by LMPCR. The conditions and oligonucleotide primers utilized for LMPCR are the same as described previously with one modification (21,22): a specific amount of 32P-labeled linearized pBR322 plasmid DNA (~20 000 d.p.m.) was added to each sample at the beginning of the reaction to serve as an internal standard. The resultant PCR products were separated by electrophoresis in 8% denaturing polyacrylamide gels, then electro-transferred to nylon membranes and hybridized with 32P-labeled probes. The membranes were exposed initially to a PhosphorImager (Cyclone, Packard) and then to film. Each experiment was repeated three times, with very similar results being obtained. The calculation of relative intensity of DNA adduct formation at different codons in the p53 gene was same as described previously with slight modification (21,22). In brief, the intensities of the well-separated UvrABC incision bands and the Maxam–Gilbert A + G sequencing reaction bands (>15 bands) were quantified with the PhosphorImager. After subtracting the background from the control lanes (DNA isolated from untreated cells and cut with UvrABC, or DNA isolated from cells treated with carcinogens but not reacted with UvrABC), the relative intensities of carcinogen–DNA adduct formation were calculated by normalization of the intensity per nucleotide (band intensity/nucleotide length of the band) of UvrABC incision bands to the intensity per nucleotide of the Maxam–Gilbert A + G bands. We used various concentrations of carcinogens for treating cells in this study, and found that concentrations did not affect qualitatively the carcinogen–DNA binding patterns. Thus, for the sake of clarity, results from one concentration of each carcinogen were used for the quantitative determinations.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The major adducts formed in human bladder cells treated with N-OH-N-Ac-4-ABP are the same as in DNA treated with N-OH-4-ABP
Previously, we established a method using UvrABC nuclease incision to detect DNA adducts formed in the p53 DNA fragments modified with N-OH-4-ABP (Figure 1Go), a major proximate metabolite of the human bladder carcinogen 4-ABP (23). However, when human bladder cells were cultured with N-OH-4-ABP at concentrations up to 2 mM, we were unable to detect DNA adduct formation in the p53 gene using the UvrABC incision method in combination with LMPCR. It appeared that under the cell culture conditions, the short half-life of N-OH-4-ABP, combined with its high reactivity, prevented the compound from reaching nucleus before it was consumed either through degradation or interaction with other cellular macromolecules. To overcome this problem we sought a more stable activated derivative of 4-ABP that would induce same kinds of DNA adducts produced by N-OH-4-ABP. Toward this end we treated the cells with N-OH-N-Ac-4-ABP (Figure 1Go) and analyzed the adducts formed in the genomic DNA using a 32P-postlabeling technique. These results were compared with those obtained from reacting N-OH-ABP with DNA.

When N-OH-4-ABP (100 µM) was reacted with DNA, one major adduct was observed at a level of 95 adducts/105 nucleotides (Figure 3BGo). This adduct has previously been identified as dG-C8-4-ABP (Figure 1Go; 10,16,24) and is the same adduct that is detected in urinary bladder tissue DNA from individuals exposed to 4-ABP (8,13,28,29). Incubation of human bladder cells with N-OH-N-Ac-4-ABP (50 µM) resulted in the formation of the same DNA adduct at a level of 4 adducts/105 nucleotides (Figure 3CGo). This may have occurred through deacetylation to N-OH-4-ABP and subsequent reaction of the N-OH-4-ABP with the genomic DNA. Alternatively, there could have been a sequential O-acetylation to give N-acetoxy-N-acetyl-4-aminobiphenyl followed by a N-deacetylation to produce N-acetoxy-4-aminobiphenyl (Figure 1Go). A similar reaction sequence has been demonstrated for the activation of the analogous arylhydroxamic acid N-hydroxy-2-acetylaminofluorene in Chinese hamster ovary cells (30).



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Fig. 3. 32P-Postlabeling autoradiograms of human genomic DNA incubated in the (A) absence or (B) presence of N-OH-4-ABP (100 µM) and (C) of DNA from human bladder cells incubated with N-OH-N-Ac-4-ABP (50 µM). The location of dG-C8-4-ABP is indicated.

 
The DNA adduct distribution in the p53 gene in human bladder cells treated with N-OH-N-Ac-4-ABP
Having established that the kind of DNA adducts formed in human bladder cells treated with N-OH-N-Ac-4-ABP are the same as that in genomic DNA reacted with N-OH-4-ABP, we then used the UvrABC incision method in combination with LMPCR to map the adduct distribution in coding strand along exons 5, 7 and 8 of the p53 gene in the bladder cells treated with N-OH-N-Ac-4-ABP (Figure 4Go). For comparative purposes, we also mapped the distribution of BPDE–DNA adducts in the p53 gene of the same bladder cells treated with BPDE (Figure 5Go). The results show that the 4-ABP–DNA adduct distribution in the exons 5, 7 and 8 of the p53 gene in the bladder cells is dramatically different from the distribution of BPDE–DNA adducts. The DNA adducts of 4-ABP appears more evenly distributed along the three exons of the p53 gene as compared with the BPDE–DNA adducts. In exon 5 of the p53 gene, preferential 4-ABP–DNA adduct formation was found at codons 152, 154 and 157, and a significant amount of adduct formation was also found at 158, 159 and 175. Except for codon 155, all these codons contain CpG sites. In exon 7, 4-ABP–DNA adducts formed at CpG site in codon 248 were slightly higher than in other regions; however, there were also substantial 4-ABP adducts at non-CpG sites in codons 238, 239, 240, 242, 244 and 249. In exon 8, 4-ABP–DNA adducts were clustered around codons 280–285, with codon 280 giving the highest level of adduction. This region contains no CpG sites, except at codon 282. 4-ABP–DNA adduct formation was barely detected at codon 273, a CpG site and a major mutational hotspot in many human cancers including bladder cancer. The results in Figure 4Go show that among the five p53 gene mutational hotspots in human bladder cancer, codons 175, 248, 280 and 285 were the preferential sites for 4-ABP adduct formation. The results also show that 4-ABP–DNA adduction was not biased toward CpG sites. Although binding did occur at codons containing CpG sequences (such as codons 154, 156, 157, 175 and 248), substantial binding also occurred at non-CpG sequences (such as codons 155, 238, 242, 244, 280, 281 and 285). In contrast, BPDE–DNA adducts preferentially formed at only CpG sites in codons 154,156, 157 of exon 5, codon 248 of exon 7 and codon 273 of exon 8 in the p53 gene in the bladder cells (Figure 5Go). This distribution of BPDE–DNA in the p53 gene of bladder cells is very similar to what we found previously human lung cells (21,22).




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Fig. 4. DNA adduct distribution along the coding strand of exons 5, 7 and 8 of the p53 gene in human bladder cells treated with N-OH-N-Ac-4-ABP. Cultured human bladder cells were treated with N-OH-N-Ac-4-ABP (50 and 100 µM), and the genomic DNA was isolated. The resultant DNA was reacted with UvrABC nuclease and followed by LMPCR as described in Material and methods. (A) Typical autoradiographs and (B) quantification. Shown are adduct distribution in the coding strand of (a) exon 5, (b) exon 7 and (c) exon 8. The codon numbers corresponding to UvrABC incision bands are labeled on the right of the panels. AG and TC are Maxim–Gilbert sequencing reaction. In (B) the concentration of N-OH-N-Ac-4-ABP used for quantification was 100 µM. The intensity of N-OH-N-Ac-4-ABP-induced bands was quantified by PhosphorImager, and the relative intensity (RI) was calculated as described in Material and methods.

 



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Fig. 5. DNA adduct distribution along the coding strand of exons 5, 7 and 8 of the p53 gene in human bladder cells treated with BPDE. Cultured human bladder cells were treated with BPDE (1 µM), and the genomic DNA was isolated. The resultant DNA was reacted with UvrABC nuclease and followed by LMPCR as described in Materials and methods. (A) Typical autoradiographs, and (B) quantification. Shown are the adduct distribution in the coding strand of (a) exon 5, (b) exon 7 and (c) exon 8. The codon numbers corresponding to UvrABC incision bands are labeled on the right of the panels. AG and TC are Maxim–Gilbert sequencing reaction. The intensity of BPDE-induced bands was quantified by PhosphorImager, and the relative intensity (RI) was calculated as described in Materials and methods.

 
The 4-ABP–DNA adduct distribution in the p53 gene of human lung fibroblasts treated with N-OH-N-Ac-4-ABP
The unique features of 4-ABP–DNA adduct distribution in the p53 gene in bladder cells could be the result of the unique DNA binding property of activated 4-ABP or the unique property of bladder cells. To address this question, we mapped the distribution of 4-ABP–DNA adducts along the p53 gene of human lung fibroblasts incubated with N-OH-N-Ac-4-ABP. The results in Figure 6Go demonstrate that the 4-ABP–DNA adduct distribution pattern in the p53 gene of human lung fibroblasts was almost identical to that found in the bladder cells. These results clearly demonstrate that the specific binding spectrum of 4-ABP in bladder cells is due to the intrinsic binding specificity of 4-ABP and is not restricted to bladder cells only.



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Fig. 6. DNA adduct distribution along the coding strand of exons 7 and 8 of the p53 gene in human lung fibroblast cells treated with N-OH-N-Ac-4-ABP. Cultured human lung fibroblast cells were treated with N-OH-N-Ac-4-ABP (100 µM), and the genomic DNA was isolated. The resultant DNA was reacted with UvrABC nuclease and followed by LMPCR as described in Materials and methods. Shown are adduct distribution in the coding strand of (a) exon 7 and (b) exon 8. The codon numbers corresponding to UvrABC incision bands are labeled on the right of the panels. AG and TC are Maxim–Gilbert sequencing reaction.

 
The role of chromatin structure in determining 4-ABP–DNA adduct distribution in the p53 gene
Chromatin structure can have a significant influence on the distribution of DNA adducts, such as cyclobutane dimers and (64)photoproducts induced by ultraviolet light (31). To determine the possible effects of chromatin structure in affecting the 4-ABP–DNA adduct distribution, we modified genomic DNA isolated from bladder cells with N-OH-4-ABP and then mapped the 4-ABP–DNA adduct distribution in the p53 gene. The results in Figure 7Go show that 4-ABP–DNA adduct distribution in the p53 gene in the naked genomic DNA modified with N-OH-4-ABP was almost identical to that obtained from bladder cells treated with N-OH-N-Ac-4-ABP. These results indicate that chromatin structure does not play an important role for the unique feature of 4-ABP–DNA adduct distribution in the p53 gene of the human cells, rather, it is determined by DNA binding specificity of the N-OH-4-ABP and the human genomic context.



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Fig. 7. DNA adduct distribution along the coding strand of exons 5, 7 and 8 of the p53 gene in genomic DNA modified with N-OH-4-ABP. Genomic DNA, isolated from human bladder cells, was modified with N-OH-4-ABP (50 and 100 µM), and reacted with UvrABC nuclease followed by LMPCR as described in Materials and methods. The codon numbers corresponding to UvrABC incision bands are labeled on the right of the panels. AG and TC are Maxim–Gilbert sequencing reaction.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have found previously that UvrABC nuclease, a nucleotide excision repair enzyme complex isolated from E.coli cells, is able to incise specifically and quantitatively the p53 DNA fragments modified with N-OH-4-ABP (23). The results from our 32P-postlabeling study show that bladder cells treated with N-OH-N-Ac-4-ABP form the same major DNA adduct (dG-C8-4-ABP) as found in purified genomic DNA treated with N-OH-4-ABP. These findings validate our approach using the UvrABC incision method combined with LMPCR to map the 4-ABP–DNA adduct distribution at the nucleotide sequence level in human cells treated with N-OH-N-Ac-4-ABP. The DNA adducts detected by the UvrABC-LMPCR method show that 80% are guanine adducts and 20% are adenine adducts. Although an adenine adduct was not detected by 32P-postlabeling analyses in the current study, the UvrABC-LMPCR data are consistent with distribution of adduct obtained upon reacting N-OH-4-ABP with DNA (10,16,24).

The most frequently mutated gene in human cancer is p53 gene, with >50% of human cancers containing such a mutation. Most of the mutations in the p53 gene are located at the DNA binding domain of the p53 protein and distributed along more than 200 codons in this domain. Interestingly, >30% of the p53 gene mutations occur at CpG sites in codons 157, 175, 245, 248, 273 and 282. All of the cytosines of CpG sites in the coding region of the p53 gene are methylated in a variety of tissues (32). In previous studies, we have found that many carcinogens including PAHs, N-acetoxy-2-acetylaminofluorene and aflatoxin B1 preferentially form DNA adducts at methylated CpG sites (21,22,3335). Furthermore, the adducts formed in these CpG sites are poorly repaired (36). These findings suggest that mutational hotspots at methylated CpG sites in the p53 gene may be a consequence of preferential carcinogen binding at these sites. The results also provide a logic explanation of mutational hotspots in the p53 gene in human cancers, particularly for lung cancer resulting from cigarette smoking.

Our results strongly suggest that DNA damage contributes greatly to shaping the p53 mutational spectrum in human cancers. This explanation raises the intriguing question of why codon 249 (-GAGG-) in liver cancer and codons 280 (-GAGA-) and 285 (-AGAG-) in bladder cancer are the mutational hotspots as these codons do not contain CpG sequences and the Gs in these codons are not within CpG sequences? It is possible that the etiological agent for liver cancer may preferentially bind at codon 249 and etiological agents for bladder cancer may preferentially bind at codons 280 and 285. However, previous studies have found that the aflatoxin B1, the major etiological agent for liver cancer, binds only modestly at codon 249 (37). In addition, it has been found that only the codon 249-mutated p53 gene product has a strong dominant-negative effect in liver cells (38). This indicates that growth selection plays an important role in shaping the p53 mutational spectrum in liver cancer. By the same token, the unique p53 mutational spectrum in bladder cells may be shaped either by the etiological agents that bind preferentially at codons 280 and 285 or could be due to mutations at codons 280 and 285 having a stronger dominant-negative effect in bladder cells than mutations at other codons. This is a very important question for understanding bladder carcinogenesis and may affect approaches for the prevention, risk assessment and therapy used in bladder cancer. Our current results do not exclude the possibility that the p53 gene product containing mutations at codons 280 and/or 285 may have a stronger dominant-negative effect than a p53 gene product containing mutations at other codons; nonetheless, our data do show that 4-ABP, a major etiological agent of bladder cancer, binds strongly at codons 280 and 285 and suggest that 4-ABP-induced DNA damage plays a crucial role in shaping the p53 mutational spectrum in bladder cancer.

The chemistry of preferential 4-ABP–DNA adduct formation at codons 280 and 285, two non-CpG sites, remains to be understood. The uniqueness of 4-ABP adduct formation is further manifested by the failure of 4-ABP to form DNA adducts at the methylated CpG site in codon 273, which is a preferential binding site for many carcinogens including PAHs and AFB1 (21,22,34,37) and also a major mutational hotspot in many human cancers including bladder cancer. These results suggest that carcinogens other than 4-ABP are responsible for the mutation at this codon in bladder cancer. There are a number of carcinogens present in tobacco smoke and other environmental pollutants that could bind to codon 273 and trigger bladder carcinogenesis.

Cigarette smoke generates a substantial amount of 4-ABP and metabolically activated 4-ABP preferentially binds at codons 280 and 285 of the p53 gene but, intriguingly, mutations in these two codons rarely occur in the lung cancer. 4-ABP–DNA adducts have been detected in human lung tissue (29,39), but the levels did not differ between smokers and non-smokers (39). This observation, coupled with the finding that 4-ABP is poorly activated by lung tissue, led to the suggestion that the 4-ABP–DNA adducts detected in lung tissue do not arise from 4-ABP but rather from 4-nitrobiphenyl, a widespread air pollutant (39). Our results are consistent with this hypothesis because sites of preferential binding of 4-ABP in the p53 gene of lung cells are not the major sites from mutation observed in lung cancer. PAHs are 20–50-fold more abundant than 4-ABP in cigarette smoke (40), and benzo[a]pyrene, a major PAH carcinogen in cigarette smoke, is activated by lung microsomes 100–1000 fold better than 4-ABP (39). This may explain why the p53 mutational spectrum in lung cancer closely parallels the binding spectrum of metabolically activated PAHs.

In summary, our results strongly suggest that the unique binding specificity of 4-ABP in the p53 gene greatly contributes to the unusual p53 mutational spectrum in human bladder cancer, which further substantiates the hypothesis that 4-ABP is a major etiological agent of human bladder.



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Fig. 2. Mutational spectrum in exons 5–8 of the p53 gene in human bladder cancer. The numbers were obtained from the June 2001 update of the International Agency for Research on Cancer TP53 mutation database with a total number of 627 mutations (http//www.p53.iarc.fr/p53DataBase.htm). The labeled codons have a mutation frequency >3% of total 627 mutations.

 

    Notes
 
* These two authors contributed equally Back

5 To whom correspondence should be addressed Email: tang{at}env.med.nyu.edu Back


    Acknowledgments
 
We wish to thank Dr Aziz Sancar for generous gifts of uvr plasmids and the bacterial strains. This work was supported by Public Health Service grants ES03124, ES08389, ES10344 (M.-s.T.), MO100096 and UO1CA86137 (W.R.) from the National Institute of Health, and service core support from NIEHS Center grants ES10344 and ES00260.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 13, 2002; revised June 20, 2002; accepted June 24, 2002.