Chromium(VI) exposure enhances polycyclic aromatic hydrocarbonDNA binding at the p53 gene in human lung cells
Zhaohui Feng1,*,
Wenwei Hu1,*,
William N. Rom2,
Max Costa1 and
Moon-Shong Tang1,2,3,4
1 Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY 10987, USA
2 Department of Medicine, New York University School of Medicine, Tuxedo, NY 10987, USA
3 Department of Pathology, New York University School of Medicine, Tuxedo, NY 10987, USA
4 To whom correspondence should be addressed Email: tang{at}env.med.nyu.edu
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Abstract
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Chromium(VI) [Cr(VI)] is a ubiquitous environmental and industrial contaminant. Cr(VI) exposure is strongly associated with a higher incidence of human lung cancer, but the mechanism of Cr(VI) carcinogenicity remains unclear. Cigarette smoking has been known as the prominent cause of lung cancer, and polycyclic aromatic hydrocarbons (PAHs), the major carcinogens in cigarette smoke, have been suggested as being responsible for the initiation and development of lung cancer. It has been reported that lung cancer from workers exposed to Cr(VI) has a high percentage of G to T transversion mutations in the non-transcribed strand of the p53 gene, a hallmark of PAH-induced mutation. Cr(VI) is a weak mutagen although it can induce a high percentage of G to T transversion mutations. These results raise the possibility that Cr(VI) may enhance PAH binding at the p53 gene in lung tissue. To test this possibility, we have determined the effect of Cr(VI) exposure on benzo[a]pyrene diol epoxides (BPDE)DNA binding at total genomic DNA level and at the p53 gene in normal human lung fibroblast cells. We found that in lung cells Cr(VI) pre-exposure does not affect the BPDEDNA binding at the total genomic DNA level or at exons 5, 6 and 9 of the p53 gene; however, it greatly enhances BPDEDNA binding at exons 7 and 8 of the p53 gene, especially at mutational hotspots of lung cancer: codons 248, 273 and 282 of the p53 gene. No enhancement of BPDEDNA binding in the p53 was observed when naked genomic DNA isolated from Cr(VI)-exposed cells was modified with BPDE in vitro. These results suggest that Cr(VI) exposure may enhance chromatin structure-dependent carcinogenDNA binding. This effect may contribute to the synergism of Cr(VI) and BPDE on mutagenesis and cell transformation, and may also contribute to the higher incidence of lung cancer in Cr(VI)-exposed populations.
Abbreviations: BPDE, benzo[a]pyrene diol epoxides; Cr(VI), chromium(VI); Cr(III), chromium(III); LMPCR, ligation-mediated polymerase chain reaction; PAH, polycyclic aromatic hydrocarbon; RI, relative intensity.
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Introduction
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Lung cancer is the leading cause of cancer deaths in the USA and cigarette smoking is acknowledged as the prominent cause of this disease (1,2). Up to 90% of deaths due to lung cancer have been attributed to cigarette smoking (2). Polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke as well as in the environment have been suggested as being responsible for the initiation and development of lung cancer (3). Mutation of the tumor suppressor gene p53 is a major genetic alteration in lung cancer, and over 50% of lung cancers carry a mutation in the p53 gene (46). In smoking-related lung cancer, 40% of the p53 gene mutations are G to T transversions, and 90% of this type of mutation can be attributed to the non-transcribed (coding) strand. G to T transversions have been regarded as a hallmark of PAH-induced mutations in smoking-related lung cancer (46). The p53 mutations in smoking-related lung cancer are concentrated at several CpG sites of codons 157, 158, 245, 248, 273 and 282 along exons 58 (46). Using UvrABC nuclease incision in combination with the ligation-mediated polymerase chain reaction (LMPCR) method, we have found previously that activated metabolites of PAHs in cigarette smoke, including benzo[a]pyrene diol epoxide (BPDE), preferentially form DNA adducts at methylated CpG sites along the p53 gene corresponding to the aforementioned major mutational hotspots in smoking-related lung cancer (68). Furthermore, the pattern of preferential BPDE binding at these CpG sites were determined by the C5 cytosine methylation (911), and BPDE adducts formed at these mutational hotspots were slowly repaired (12).
Chromium(VI) [Cr(VI)]-containing compounds are well-known carcinogens (1315). They are carcinogenic to humans, potential inducers of tumors in experimental animals, and can neoplastically transform cells in culture (1318). However, the underlying molecular mechanisms of Cr(VI) carcinogenicity are not well understood. Cr(VI)-containing compounds are widespread in cigarette smoke and environment; and widely used in the chemical industry, artistic paints, anticorrosion paints, electroplating and stainless steel welding (13,18,19). Epidemiological studies have consistently shown that the lower respiratory tract is the target organ of Cr(VI) compound exposure, and occupational exposure to these compounds is strongly associated with a higher incidence of lung cancer (13,16,18). Chromium exists in the environment in two major valence states, Cr(VI) and chromium (III) [Cr(III)], and Cr(VI) is actively transported into cells by the anionic transport system (20). The reduction of Cr(VI) to Cr(III) can lead to the formation of DNAchromium adducts, DNADNA and DNAprotein cross-links, DNACr(III)amino acid ternary complexes and radical-mediated DNA strand breaks (2124).
Humans are frequently exposed to both PAHs and Cr(VI) under environmental and occupational conditions (25). It has been reported that combined treatments of heavy metals, including Cr(VI), with other DNA damaging agents, including cigarette smoke or BPDE, have either a synergistic or additive effects on cell toxicity, DNA damage, mutation frequency and morphological transformation in mammalian cells (2629). It has also been reported that lung cancer from workers with occupational exposure to Cr(VI) have a high incidence of G to T transversion mutations in the non-transcribed strand of the p53 gene (30). It is known that G to T transversion is a hallmark of PAH-induced mutation in lung cancer and PAHs are the major carcinogens in cigarette smoke as well as environmental pollutant (46). Cr(VI) also induce high percentage of G to T transversions (22), however, in comparison with PAHs, Cr(VI) is a weak mutagen (1315). Together these results raise the possibility that BPDE may have a higher binding affinity toward Cr(VI)-exposed DNA and this may enhance the susceptibility of the p53 gene in lung tissue to PAH-induced mutations. In this report, using the UvrABC nuclease incision method in combination with the LMPCR technique, we tested this possibility by determining the effect of Cr(VI) exposure on BPDEDNA binding at the sequence level along the p53 gene and at the total genomic DNA level in normal human lung and skin fibroblast cells.
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Materials and methods
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Cells, cell culture and Cr(VI) exposure
The normal human lung fibroblast cells (CCL-202, ATCC, Manassas, VA) were grown in minimal essential medium supplemented with 10% fetal bovine serum; the normal human skin fibroblast cells (CRL-2076, ATCC) were grown in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum according to the vendor's instruction. Cells were grown to 50% confluence in 150-mm dishes. Different concentrations of potassium chromate, K2CrO4 (Sigma, Saint Louis, MO), were added to the culture medium and incubated at 37°C for 48 h. After treatment, K2CrO4 was removed and cells were incubated in fresh culture medium.
BPDE treatment of cells
Cells with or without K2CrO4 pre-exposure were treated with different concentrations of BPDE or [3H]BPDE (2210 mCi/mmol) (Chemsyn Science Laboratories, Lenexa, KS) in culture medium without serum, and incubated at 37°C for 30 min in the dark as described previously (7,12). After treatment, BPDE was removed and cells were lysed for DNA isolation.
DNA isolation
Genomic DNA was isolated as described previously (7,31) except EDTA was omitted from all the solutions as it has been reported to chelate trivalent chromium with high affinity (32,33). Briefly, cells were lysed with lysing buffer (0.5% SDS, 10 mM Tris, pH 7.5, 10 mM NaCl, 100 µg/ml proteinase K) at room temperature for 2 h. RNA was removed by treatment with RNase A (50 µg/ml) at 37°C for 1 h followed by repeated phenol and diethyl ether extractions. The DNA was then ethanol precipitated and dissolved in 10 mM Tris (pH 7.5) (32,33).
Measurement of total BPDEDNA adduct formation in cells
To determine the amount of total BPDEDNA adduct formation in cells, normal human lung and skin fibroblast cells were pre-exposed with or without different concentrations of K2CrO4 for 48 h, then K2CrO4 was removed and cells were treated with 1 or 2 µM [3H]BPDE (2210 mCi/mmol) at 37°C for 30 min in the dark. After treatment, cells were lysed and genomic DNA was isolated as described above. The known amount (20 µg) of each purified genomic DNA was mixed thoroughly with LSC cocktail (Fisher Scientific Co., Pittsburgh, PA) and the [3H] d.p.m. was determined in a 1219 RACKBETA scintillation counter (LKB Wallac, Turku, Finland). The number of BPDE-induced DNA adducts in 10 kb genomic DNA was calculated based on the specific activity of the [3H]BPDE.
BPDE modification of genomic DNA in vitro
Genomic DNA isolated from cells with or without K2CrO4 pre-exposure was modified with BPDE in vitro as described previously (34). Briefly, purified genomic DNA was reacted with 1 or 2 µM BPDE at 25°C for 2 h, and the reaction was stopped by repeated phenol and diethyl ether extractions. DNA was then ethanol precipitated, and dissolved in 10 mM Tris (pH 7.5).
Cleavage of BPDEDNA adducts by UvrABC nuclease
UvrABC nuclease, the nucleotide excision repair complex of Escherichia coli, was purified from the E.coli K12 strain CH296 carrying plasmids pUNC 45 (uvrA), pUNC21 (uvrB) or pDR3274 (uvrC) (35). These plasmids and strain CH296 were kindly provided by Dr A.Sancar at University of North Carolina, Chapel Hill, NC. The purification procedures were the same as described previously (35). Purified genomic DNA was treated with UvrABC nuclease (a 10-fold molar excess of nuclease over 10 kb DNA) at 37°C for 1 h as described previously (7,8,36). Under these reaction conditions, the cleavage at BPDEDNA adducts by UvrABC nuclease is specific and quantitative (37,38), and increasing the enzyme concentration did not result in additional cleavage. After UvrABC nuclease treatment, proteins were removed by repeated phenol and diethyl ether extractions. The resultant DNA was then ethanol precipitated, and dissolved in 10 mM Tris (pH 7.5) for LMPCR.
LMPCR to map BPDEDNA adduct distribution in the p53 gene
The accurate same amount of each DNA sample cleaved with UvrABC nuclease was used for LMPCR to map BPDEDNA adduct distribution along exons 59 in the p53 gene. Untreated control genomic DNA was subjected to MaxamGilbert sequencing reaction (39) and followed by LMPCR to serve as a sequencing ladder. Oligonucleotide primers for LMPCR of exons 59 of human p53 gene were the same as described previously (40). LMPCR was done as described (7,8,40) with slight modifications (31,41). 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 as an internal standard. After LMPCR, equivalent counts of 32P, representing equal amounts of template DNA were loaded into each lane of the sequencing gel. The resultant DNA was separated by electrophoresis in 8% denaturing polyacrylamide gels, and electro-transferred to GeneScreen nylon membranes (NEN, Boston, MA). Membranes were hybridized with 32P-labeled DNA probes specific for each exon. The membranes were exposed to a Cyclone PhosphorImager (Packard, Meriden, CT) first and then to films. All experiments were done independently three times and gave very similar results. The calculation of relative intensity (RI) of DNA adduct formation at different codons in the p53 gene was the same as described previously (41). In brief, the intensities of the well-separated UvrABC nuclease incision bands and the MaxamGilbert A + G sequencing reaction bands were quantified with the Cyclone PhosphorImager. After subtracting the background from the control lanes (DNA isolated from untreated control cells and cut with UvrABC nuclease), the intensities of carcinogenDNA 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 MaxamGilbert A + G bands. The RI was calculated based on RI = Ij/Imax, where Ij is the intensity of each UvrABC incision band and Imax is the UvrABC incision band with the highest intensity in an autoradiograph.
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Results
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Chromium(VI) exposure enhances BPDEDNA adduct formation at exons 7 and 8 of the p53 gene
To test whether Cr(VI) exposure could enhance the susceptibility of DNA to PAH binding in the human p53 gene, cultured normal human lung and skin fibroblast cells were pre-exposed to different concentrations of K2CrO4 for 48 h, and then treated with BPDE for 30 min. The BPDEDNA adduct distribution was mapped along the coding (non-transcribed) strand of exons 59 in the p53 gene by UvrABC nuclease incision in combination with the LMPCR method.
Results in Figure 1 show that, in exon 7 of the p53 gene, BPDE binds preferentially at codon 248, a CpG site and a mutational hotspot in smoking-related lung cancer, in both types of human cells with and without Cr(VI) pre-exposure. However, BPDEDNA binding at this codon is enhanced significantly, at least 23 fold, in cells pre-exposed to Cr(VI) as compared with cells without Cr(VI) pre-exposure. The enhancement of BPDE binding is also seen at other non-CpG sites like codons 242 and 244. Furthermore, the enhancement of BPDE binding at these codons seems directly related to the Cr(VI) dose. There are noticeable UvrABC incision bands in genomic DNA isolated from cells treated with Cr(VI) only (Figure 1A and B, lanes 5 and 6). These bands could be due to UvrABC nuclease cleavage of chromiumDNA adducts as we have found that UvrABC nuclease is able to incise Cr(III)DNA adducts (unpublished observation). The intensities of these putative Cr(III)DNA adduct-induced UvrABC incision bands are, however, much weaker than BPDEDNA adduct-induced UvrABC incision bands (Figure 1A and B, compare lanes 5 and 6 with lanes 9).


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Fig. 1. The effect of Cr(VI) pre-exposure on BPDEDNA adduct formation in exon 7 of the p53 gene in normal human lung (A) and skin (B) fibroblast cells. Cells were pre-exposed to different concentrations of K2CrO4 for 48 h and then treated with 1 µM BPDE for 30 min. Genomic DNA was isolated, and treated with UvrABC nuclease. BPDEDNA adduct distribution was mapped by the LMPCR method as described in Materials and methods. (A and B) Typical autoradiographs; (C and D) quantifications. UvrABC nuclease incision bands corresponding to the codons of interest are labeled at the right-hand side of the panel. G, A + G and T + C are MaxamGilbert sequencing reaction. The RI of UvrABC nuclease incision bands were calculated as described in Materials and methods. Results represent three independent experiments.
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Results similar to those found in exon 7 were also observed in exon 8 of the p53 gene (Figure 2). In both types of cells, codon 273, another CpG site and a major mutational hotspot in smoking-related lung cancer, stands out as a preferential BPDE binding site in cells without Cr(VI) pre-exposure. Cr(VI) exposure greatly enhances BPDEDNA binding not only at codon 273 but also at other codons, such as codons 281, 282 and 293, and the enhancement also appears to be related to the Cr(VI) dose.


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Fig. 2. The effect of Cr(VI) pre-exposure on BPDEDNA adduct formation in exon 8 of the p53 gene in normal human lung (A) and skin (B) fibroblast cells. The same UvrABC nuclease-treated genomic DNA as described in Figure 1 was used to map BPDEDNA adduct distribution in exon 8 of the p53 gene by the LMPCR method. (A and B) Typical autoradiographs; (C and D) quantifications. UvrABC nuclease incision bands corresponding to codons of interest are labeled at the right-hand side of the panel. A + G and T + C are MaxamGilbert sequencing reaction. The RI of UvrABC nuclease incision bands were calculated as described in Materials and methods. Results represent three independent experiments.
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Chromium(VI) exposure does not affect BPDEDNA adduct formation in exons 5, 6 and 9 of the p53 gene or the total BPDEDNA adduct formation in cells
The UvrABC nuclease-cleaved DNA samples used for mapping BPDEDNA adduct distribution in exons 7 and 8 were also used to map BPDEDNA adduct distribution along exons 5, 6 and 9 in the p53 gene. However, the enhancement of BPDEDNA binding observed in exons 7 and 8 of the p53 gene in cells pre-exposed to Cr(VI) was not observed in exons 5, 6 or 9 of the same gene. Results in Figure 3 show that codons 152, 154, 156 and 157 of exon 5 in the p53 gene are preferential BPDE binding sites for both types of human cells with and without Cr(VI) pre-exposure; however, Cr(VI) pre-exposure does not change the extent of binding at these sites. In exon 6 (Figure 4) or 9 (data not shown) of the p53 gene, there are no preferential BPDE binding sites; only codon 192 in exon 6 shows moderate BPDE binding, and Cr(VI) pre-exposure does not change the extent of binding at these two exons. These results suggest that the enhancing effect of Cr(VI) on BPDEDNA binding is DNA sequence-dependent and limited to certain regions of genomic DNA. To further test this possibility, we determined the BPDEDNA adduct formation at the total genomic DNA level in cells pre-exposed to Cr(VI). Normal human lung and skin cells were pre-exposed to different concentrations of Cr(VI) for 48 h and then treated with [3H]BPDE for 30 min. Genomic DNA was isolated and total [3H]BPDEDNA adduct formation at the genomic DNA level was determined. The results in Figure 5 show no obvious difference in BPDEDNA adduct formation at the total genomic DNA level between cells with and without Cr(VI) pre-exposure. Together these results indicate that the enhancing effect of Cr(VI) exposure on BPDEDNA binding is limited to certain regions of genomic DNA or certain DNA sequences.


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Fig. 3. The effect of Cr(VI) pre-exposure on BPDEDNA adduct formation in exon 5 of the p53 gene in normal human lung and skin fibroblast cells. The same UvrABC nuclease-treated genomic DNA as described in Figures 1 and 2 was used to map BPDEDNA adduct distribution in exon 5 of the p53 gene by the LMPCR method. (A and B) Typical autoradiographs; (C and D) quantifications. UvrABC nuclease incision bands corresponding to the codons of interest are labeled at the right-hand side of the panel. G, A + G and T + C are MaxamGilbert sequencing reaction. The RI of UvrABC nuclease incision bands was calculated as described in Materials and methods. Results represent three independent experiments.
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Fig. 4. The effect of Cr(VI) exposure on BPDEDNA adduct formation in exon 6 of the p53 gene in normal human lung (A) and skin (B) fibroblast cells. The same UvrABC nuclease-treated genomic DNA as described in Figures 1 and 2 was used for mapping BPDEDNA adduct distribution in exon 6 of the p53 gene by the LMPCR method. UvrABC nuclease incision bands corresponding to the codons of interest are labeled at the right-hand side of the panel. A + G and T + C are MaxamGilbert sequencing reaction.
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Fig. 5. The effect of Cr(VI) pre-exposure on total BPDEDNA adduct formation at the genomic DNA level in normal human lung and skin fibroblast cells. Cells were pre-exposed to K2CrO4 for 48 h, and then treated with [3H]BPDE for 30 min. The genomic DNA was isolated, and the [3H] d.p.m. of a known amount of purified genomic DNA was determined by a scintillation counter. The number of BPDEDNA adducts in 10 kb genomic DNA was calculated based on the specific activity of the [3H]BPDE as described in Materials and methods. Results represent three independent experiments.
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The effect of chromatin structure on Cr(VI) treatment enhanced BPDEDNA binding
There are two possibilities to account for the results that Cr(VI) exposure enhances BPDEDNA binding in exons 7 and 8 but not in exon 5, 6 or 9 of the human p53 gene. (i) The effect of Cr(VI) exposure on BPDEDNA binding could be due to the direct chromiumDNA interactions or chromium-induced DNADNA crosslinks which enhance the susceptibility of DNA toward BPDE binding. If this is the case, then the enhancement of BPDEDNA binding by Cr(VI) exposure would be dependent on the DNA sequence at which chromium binds, i.e. the effect would be DNA-sequence dependent. (ii) The enhancing effect of Cr(VI) exposure on BPDEDNA binding could be due to chromium-induced changes in nucleoproteinDNA complex, which may change the local chromatin structure (42). It has been shown that BPDE has a much higher binding affinity toward DNA at the replication fork and linker DNA regions than toward DNA at the nucleosomal core particles and overall bulk genomic DNA (43,44). In order to distinguish between these two possibilities, we isolated genomic DNA from lung and skin fibroblast cells with or without Cr(VI) pre-exposure, modified the naked genomic DNA with BPDE in vitro, and then mapped the BPDEDNA adduct distribution in the exon 7 and 8 of the p53 gene by UvrABC nuclease incision in combination with the LMPCR method. Results in Figure 6 show there is no enhancement of BPDEDNA binding in exons 7 (Figure 6A) or 8 (Figure 6B) of the p53 gene when naked genomic DNA isolated from Cr(VI) pre-exposed cells was modified with BPDE in vitro as compared with DNA from cells without Cr(VI) pre-exposure. These results suggest that the enhancement of BPDEDNA binding in exons 7 and 8 of the p53 gene in cells with Cr(VI) pre-exposure is probably caused by the effect of chromium on local chromatin structure rather than by direct chromiumDNA interactions.

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Fig. 6. BPDEDNA adduct distribution in exons 7 (A) and 8 (B) of the p53 gene in BPDE-modified genomic DNA isolated from normal human lung fibroblast cells with and without Cr(VI) pre-exposure. Cells were pre-exposed to K2CrO4 for 48 h, and genomic DNA was isolated. Purified genomic DNA was modified with 1 µM BPDE in vitro and BPDEDNA adduct distribution in exons 7 and 8 of the p53 gene was mapped by the the UvrABC nuclease incision in combination with the LMPCR method. UvrABC nuclease incision bands corresponding to the codons of interest are labeled at the right-hand side of the panel. A + G and T + C are MaxamGilbert sequencing reaction.
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Discussion
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Cr(VI) is a ubiquitous environmental and industrial contaminant. Chromium accumulation in lung tissue is found in workers with occupational exposure to Cr(VI) and cigarette smokers, and workers with occupational exposure to Cr(VI) have a higher incidence of lung cancer (13,15,16), which strongly suggest that Cr(VI) contamination is involved in human lung carcinogenesis. However, the underlying molecular mechanisms of Cr(VI) carcinogenicity are not well understood.
Using the APRT+
APRT- forward mutation system, we have found that although Cr(VI) is a weak mutagen, however, mutation frequency synergistically increases in mammalian cells pre-exposed to a low concentration (0.3 µM) of Cr(VI) followed by BPDE treatment (unpublished data from this lab). Three possibilities could account for this effect of Cr(VI) enhanced BPDE-induced mutagenesis. Intracellular chromium may affect the efficiency of BPDE-adduct formation, the repair efficiency of BPDEDNA adducts, or the fidelity of translesion synthesis. Our current results demonstrate that Cr(VI) treatment indeed enhances BPDEDNA adduct formation at exons 7 and 8 of the p53 gene in human cells (Figures 1 and 2). However, these results by no means exclude the other two possibilities.
Intriguingly, we observed no differences in the total BPDEDNA adduct formation in genomic DNA from cells with and without Cr(VI) pre-exposure (Figure 5). These results suggest the enhancing effect of Cr(VI) pre-exposure on BPDEDNA binding may be limited to certain regions of genomic DNA; this interpretation was further supported by the observation that BPDEDNA adduct formation in exons 5, 6 and 9 of the p53 was not affected by Cr(VI) pre-exposure (Figures 3 and 4).
It has been reported that certain carcinogenic metals such as nickel and arsenate can cause hypermethylation at CpG sites (4547). Previously, we have shown that C5 cytosine methylation at CpG sites greatly enhances BPDE binding at CpG sites and affects BPDE binding at surrounding sequences (911). These findings raise one possibility that the enhancing effect of Cr(VI) on BPDEDNA binding in cells is due to hypermethylation at CpG sites caused by Cr(VI) exposure. In this study, the extent of cytosine methylation was determined by the MaxamGilbert sequencing reaction method (39,48). Because hydrazine is unable to modify 5-C-methylated cytosines; both the 5' and 3' phosphodiester bonds of each methylated cytosine are refractory to piperidine hydrolysis and no cytosine ladders are observed at methylated cytosines (39,48). We found that most of the CpG sites in exons 7 (such as codon 248) and 8 (such as codons 273 and 282) of the p53 gene of the lung and skin fibroblasts are heavily methylated, which is the same as reported by Pfeifer and his colleagues (49). Furthermore, we did not observe obvious change in methylation pattern between genomic DNA isolated from Cr(VI)-treated or untreated cells (data not shown). Together these results lead us to propose that Cr(VI) may induce changes in nucleoproteinDNA complex, which may change the local chromatin structure and affect DNA susceptibility toward BPDE binding. Consistent with this interpretation are the results in Figure 6 which show that the level of BPDE binding at exons 7 or 8 of the p53 gene is the same in the naked genomic DNA isolated from cells with or without Cr(VI) pre-exposure. This interpretation also provides a reasonable explanation for the results in Figures 1 and 2, which show that the enhancement of BPDEDNA binding in the p53 gene is found along almost the whole region of exons 7 and 8, including CpG and non-CpG sites. Exons 7 (110 bp) and 8 (137 bp) are separated by intron 7 of 350 bp; therefore, these two exons cannot be in the same nucleosome. It is possible that Cr(VI) may affect the nucleoproteinDNA structures that encompass these two exons, and consequently, affect the BPDEDNA binding affinity in the whole regions. It has been reported that a significant amount of ternary chromiumDNA adducts were lost during standard DNA purification from cells (50). Our results, therefore, do not necessarily exclude the possibility that the direct chromiumDNA interactions or chromium-induced DNADNA crosslinks could also enhance the susceptibility of DNA toward BPDE binding.
The reason why Cr(VI) pre-exposure only enhances BPDE-DNA binding at exons 7 and 8 but not exons 5, 6 or 9 of the p53 gene is unclear. Our results predict a high proportion of the p53 mutations in lung cancer from workers with occupational Cr(VI) exposure occur at the coding strand of exons 7 and 8. Interestingly, two reports found that mutations in the p53 gene in lung cancer from workers with Cr(VI) exposure were either in the coding strand of exons 7 or 8 (30,51). Determining the nucleoprotein content and structure at these different exons of the p53 gene in lung cells may shed light on this intriguing phenomenon.
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Notes
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*Both authors contributed equally. 
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Acknowledgments
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We wish to thank Dr Tang Yen-Yee for critical review. This work was supported by Public Health Service grants ES03124, ES08389 (M.-s.T.), ES10344 (M.C), MO100096 and UOICA86137 (W.R.) from the National Institute of Health, and Service Core Support from NIEHS grant ES10344 and ES00260.
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Received October 14, 2002;
revised January 10, 2003;
accepted January 21, 2003.