Chromium (VI) induces both bulky DNA adducts and oxidative DNA damage at adenines and guanines in the p53 gene of human lung cells

Abstract

Chromium (VI) [Cr(VI)], a ubiquitous environmental carcinogen, is generally believed to induce mainly mutagenic binary and ternary Cr(III)–deoxyguanosine (dG)-DNA adducts in human cells. However, both adenine (A) and guanine (G) mutations are found in the p53 gene in Cr exposure-related lung cancer. Using UvrABC nuclease and formamidopyrimidine glycosylase (Fpg), and ligation-mediated PCR methods, we mapped the distribution of bulky DNA adducts (BDA) and oxidative DNA damage (ODD) in the p53 gene in Cr(VI)-treated human lung cells. We found that both BDA and ODD formed at 2ʹ-deoxyadenosine (dA) and dG bases. To understand the causes for these Cr-induced DNA damages, we mapped the distribution of BDA adducts and ODD in the p53 gene DNA fragments induced by Cr(III), Cr(VI) and Cr(V), the three major cellular Cr forms. We found that (i) dA at –CA- is a major Cr(VI) binding site followed by -GG- and –G-. Cr(VI) does not bind to –GGG-, (ii) Cr(VI)–DNA binding specificity is distinctly different from the Cr(III)–DNA binding in which –GGG- and –GG- are preferential sites, (iii) Cr(V) binding sites include all of Cr(VI) and Cr(III)–DNA binding sites and (iv) Cr(VI) and Cr(V) induce Fpg-sensitive sites at –G-. Together, these results suggest that Cr(VI) induction of BDA and ODD at dA and dG residues is through Cr(V) intermediate. We propose that these Cr(VI)-induced BDA and ODD contribute to mutagenesis of the p53 gene that leads to lung carcinogenesis.

Introduction

Chromium (VI) [Cr(VI)] is a ubiquitous environmental contaminant. Occupational exposure to Cr(VI) compounds is a well-documented cause of respiratory cancers (1). Although typical Cr(VI)-associated cancer is located in the lung, the risk of nasal cancer is also increased (2–6). Epidemiological studies have revealed that the lung cancer morbidity rate for ex-chromate workers with 9 or more years of exposure is more than 20 times higher than that of non-smokers (7). However, the underlying mechanism of how chromate exposure enhances lung carcinogenesis is unclear.

It has been determined that more than 50% of lung cancers found in cigarette smokers have mutations in the p53 gene and that the p53 mutational hotspots coincide with the preferential DNA-binding sites of major cigarette smoke carcinogens, including polycyclic aromatic hydrocarbons and acrolein (8–10). These results indicate that targeted DNA damage shapes the p53 mutational pattern in lung cancer. By contrast with the p53 mutations in cigarette smoke-related lung cancers, where mutations occur primarily at guanine (-G-) residues, mutations in the p53 gene are equally distributed at both adenine (-A-) and -G- residues in Cr exposure-related lung cancer (11). As Cr(III) and Cr(VI) are produced by many industrial processes such as welding, chrome plating, chrome pigmenting and leather tanning (12), these findings raise the possibility that Cr(III) and Cr(VI) exposure may induce DNA damage at both –A- and –G- positions in the p53 gene.

To address this possibility and to understand the causes that lead to A and G mutagenesis in Cr exposure-related lung cancer, using UvrABC (13) and formamidopyrimidine glycosylase (Fpg) (14), and ligation-mediated PCR (LMPCR) method (8–10), we mapped both bulky DNA adducts (BDA) and oxidative DNA damage (ODD) formed in the p53 gene in human lung cells treated with Cr(VI). We found that Cr-induced BDA and ODD occur at both –A- and –G- residues. To understand how the Cr–DNA interaction generate DNA damage in –A- and –G- residues, we treated DNA fragments with Cr(III), Cr(V) and Cr(VI). We found that (i) Cr(VI) and Cr(V) can form adducts with both A’s and G’s and that Cr(III) forms adducts with G’s only and (ii) Cr(V) and Cr(VI) can induce ODD. Our results suggest that both ODD and bulky 2ʹ-deoxyadenosine (dA)– and 2ʹ-deoxyguanosine (dG)–DNA adducts induced by Cr contribute to the p53 mutations in the Cr exposure-related lung cancer.

Materials and methods

Materials

CrCl₃ ^.6H₂O (Sigma) and potassium chromate (K₂CrO₄; Sigma) are the sources for Cr(III) and Cr(VI), respecting CrCl₃ solutions were freshly prepared before each experiment. Sodium bis(2-ethyl-2-hydroxybutyrato)₂oxochromate(V) monohydrate was prepared from anhydrous sodium dichromate and 2-ethyl-2-hydroxybutylic acid in the dehydrated acetone (15). The Cr(V) complex was characterized by absorption spectroscopy: ε_485min = 160M⁻¹cm⁻¹, Σ_510min = 168M⁻¹cm⁻¹, ε_633min = 28.6M⁻¹cm⁻¹, ε_740max = 40.9M⁻¹cm⁻¹. The content of synthesized Cr(V) is 98% based on atomic absorption spectroscopy measurement. Anhydrous sodium dichromate was obtained by heating Na₂Cr₂O₇ (Sigma–Aldrich, St. Louis, MO) at 100°C for 1h. Acetone (Sigma–Aldrich, HPLC grade) was dried over molecular sieves (Fisher, Type 4A). Hexane and 2-ethyl-2-hydroxybutylic acid were purchased from Sigma–Aldrich; pH 4.0- and 5.0-certified buffer solutions were purchased from Fisher (San Diego, CA); [γ-³²P]ATP (3000 Ci/mmol) was purchased from NEN (Boston, MA), and T4 polynucleotide kinase and Taq DNA polymerase were purchased from Promega (Madison, WI). Primers were synthesized by the Midland Certified Reagent Co. (Midland, TX).

Cells culture, Cr(VI) exposure and isolation of Cr-modified genomic DNA

Human adenocarcinoma epithelial lung (A549) cells (CCL-185, ATCC) were grown in minimal essential medium (Gibco BRL, Invitrogen Corporation, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco BRL, Invitrogen Corporation). The cells were grown to 70% confluence in 150cm² cell culture flasks. K₂CrO₄ (10 µM) was added to the culture medium, incubated at 37°C for 1h and then cells were transferred to medium without Cr followed by incubation for 2–24h. Cr-modified genomic DNA was purified from the cells as described (8,16) except that EDTA was omitted from all the solutions.

Determination of Cr-induced Cr–DNA binding and ODD formation and repair

The Cr(VI) treatment-induced BDA, which presumably are binary and ternary Cr(III)–DNA and Cr–ligand–DNA adducts, were detected using UvrABC incision method (17) and atomic absorption spectrophotometry methods (18). Cr(VI) treatment-induced ODD was detected by Fpg incision (14) and by immunochemical detection (19,20). For repair assays, Cr(VI)-treated A549 cells were incubated in Cr(VI)-free growth medium for different times (0, 2, 4, 8 and 24h) after Cr exposure; the genomic DNA was isolated as described above and then the levels of Cr–DNA adducts and ODD in the genomic DNA were measured.

Mapping UvrABC and Fpg-sensitive sites by LMPCR

The UvrA, UvrB, UvrC and Fpg proteins were isolated as described (17,21). The UvrABC reactions were carried out by incubation of Cr-modified genomic DNA (2 µg) at 37°C for 1h, as described previously (13). The Fpg reactions were carried out at 37°C for 15min in a reaction mixture containing 10mM Tris–HCl (pH 7.5), 75mM NaCl, 1mM EDTA, 30ng of Fpg protein and Cr-modified genomic DNA (2 µg) in a total volume of 50 µl. The DNA was further purified by phenol/ether extractions and ethanol precipitated. The method for LMPCR was the same as described (8–10). After LMPCR, the obtained DNA was separated by electrophoresis at 40V/cm in 8% (W/V) denaturing polyacrylamide gels containing 45% (W/V) urea in TBE buffer [50mM Tris–HCl; 50mM sodium borate (pH 8.3) and 10mM EDTA) and electro-transferred to Gene Screen nylon membranes (NEN, Boston, MA). Membranes were hybridized with ³²P-labeled DNA probe and autoradiographed by using Cyclone Phosphor Imager (Packard, Meriden, CT). Band intensity of autoradiograms was quantified by using a ChemiImager. The relative band intensity (RI) was calculated by RI = I_j/I_max, of which I_j is the intensity of each band and I_max is the highest intensity of the bands in the autoradiogram.

DNA fragment isolation, 5ʹ end- ³²P-labeling and modifications with Cr(VI), Cr(V) and Cr(III)

Methods for preparing single-5ʹ end ³²P-labeled exons 5 and 7 of the p53 gene DNA fragments were the same as described (22). For Cr(VI) modifications, a single-5ʹ end-³²P-labeled exons 5 and 7 of the p53 gene DNA fragments (2 × 10⁵ cpm, 10ng) in a final volume of 100 µl were incubated with different concentrations of K₂CrO₄ (10 or 20 µM) at 37°C for 30min. In some of the reactions, glutathione at 10-fold higher concentration of K₂CrO₄ was added. For Cr(V) modifications, the same 5ʹ-end-³²P-labeled exons 5 and 7 of the p53 gene fragments were incubated with different concentrations of sodium bis(2-ethy-2-hydroxybutyrato)₂oxochromate (V) monohydrate (0.3–1.9 µM) at 37°C for 30min. Cr(III) modifications were the same as described (13). The Cr(VI)-, Cr(V)- and Cr(III)-modified DNA fragments were purified by phenol/ether extractions and ethanol precipitations, and finally dissolved in water.

Mapping UvrABC and Fpg-sensitive sites in Cr-modified DNA fragments

The BDA formed in the 5ʹ end-³²P-labeled exons 5 or 7 (1 × 10⁵ cpm) DNA fragments modified with Cr(VI), Cr(V) and Cr(III) were mapped by UvrABC incision method, as described (13). The ODD in the same DNA fragments were mapped by Fpg incision method, as described in the method of LMPCR. The DNA fragments were extracted with phenol/ether and precipitated with ethanol, and the resultant DNAs were dissolved in a denaturing dye, heat denatured, separated by electrophoresis and auto-radiographed the same manner as described (13). Band intensity in the autoradiograms was quantified by a ChemiImager.

Results

Cr(VI) treatment induces UvrABC and Fpg-sensitive sites in the genomic DNA of human lung cells

It has been found that Cr(VI) treatment induces Cr(III)–DNA and Cr(III)–ligand–amino acid-conjugated DNA adducts in human lung cells (23). Cr(VI) treatment has also been found to induce oxidative stress (24). Therefore, it is possible that Cr(VI) exposure will induce both BDA and ODD in human lung cells. To test this possibility, we determined the UvrABC and Fpg-sensitive sites formed in human lung cells treated with Cr(VI) with different incubation times. Previously, we have established that the nucleotide excision enzyme complex isolated from Escherichia coli cells is able to incise Cr and Cr–ligand-conjugated BDA specifically and quantitatively (13). It is well established that Fpg is able to incise 7,8-dihydro-8-oxo-2ʹ-deoxyguanosine (8-oxo-dG), the major ODD, and 7,8-dihydro-8-oxo-2ʹ-deoxyadenosine and their imidazole ring open derivatives, N-(2-deoxy-D-pentofuranosyl)-N-(2,6-diamino-4-hydroxy-5-formamidopyrimidine) (Fapy-dG) and N-(2-deoxy-D-pentofuranosyl)-N-(4,6-diamino-5-formamidopyrimidine) (14). As we have found that ODD is insensitive to UvrABC incision and that Fpg is unable to incise BDA, these findings enable us to distinguish Cr treatment-induced BDA and ODD at the DNA sequence level (25). Results in Figure 1A and 1B show that Cr(VI) treatment induces both UvrABC and Fpg-sensitive sites in the genomic DNA indicating that Cr(VI) induces both BDA and ODD and that most of Cr-induced BDA and ODD are not repaired after 24h of incubation. The Cr(VI)-induced Cr–DNA adducts were further measured by atomic absorption spectrophotometry and the results in Figure 1C show that most of Cr(VI)–DNA adducts remain in genomic DNA even after 24h of incubation; these results are consistent with results obtained by using UvrABC incision detection method. The induction of ODD by Cr(VI) treatment was further determined by immunochemical assay using 8-oxo-dG antibodies. As shown in Figure 1D, Cr(VI) treatment induces ODD proportionally to Cr(VI) concentration in A549 cells. Furthermore, the majority of Cr-induced ODD, detected by both the Fpg incision method and the immunochemical method, remain in genomic DNA after 24h of incubation (Figure 1B and 1E). These results together indicate that both BDA and ODD induced by Cr(VI) in human lung cells are refractory to DNA repair mechanisms.

Fig. 1.

Formation and repair of BDA and ODD induced by Cr(VI) treatment in human lung cells. Exponentially growing human lung cells (A549) were exposed to Cr(VI) (10 µM, 1h) and then incubated with fresh medium without Cr(VI) for different times (0–24h). Cells were harvested and genomic DNA was isolated. The BDA was detected by (A) UvrABC incision method and (C) atomic absorption spectrophotometry method. The ODD was detected by (B) Fpg incision method and (D and E) immunochemical method. For (A) and (B), genomic DNA, after treated with UvrABC or Fpg, was denatured, separated by electrophoresis in a 0.5% agarose gel in TBE buffer containing 1 µg/ml ethidium bromide and the gel was visualized by ChemiImager by the same method as described (17). The method for detecting BDA by atomic absorption spectrophotometry was the same as described (18). The method for 8-oxo-dG adduct detection was the same as recommended by the vendor [Japan Institute For the Control of Aging (JalCA), Shizuoka, Japan 437-0122]. For the purpose of comparison, the formation and repair of ODD-induced by H₂O₂ treatment in A549 cells were also determined and shown in (E).

Cr(VI)-induced UvrABC sensitive sites (USS) occur at both dA’s and dG’s in the p53 gene

Cr is a known lung carcinogen (1). It has been found that mutations occur at both –A- and –G- positions in the p53 gene in Cr exposure-related lung cancer (11). Previously, we have established that p53 mutations pattern in tobacco smoke-related lung cancer is shaped by lung cancer etiological agents in tobacco smoke, such as polycyclic aromatic hydrocarbons and acrolein (8–10). These results raise a possibility that Cr exposure induces DNA damage in both –A- and –G- residues in the p53 gene in human lung cells. To test this possibility, we mapped the distribution of both BDA and ODD induced by Cr(VI) exposure in the p53 gene in human lung cells by UvrABC and Fpg incision methods in combination with LMPCR (8–10). Results in Figure 2A and 2C show that the Cr(VI)-induced BDA (UvrABC sensitive sites [USS]) in the p53 exon 7 mainly occurred at –G- positions (55%), in which 90% occur at –NGG- sequences and the rest at sequences with a single –G-. The other 45% of these adducts occurred at –A- positions. It is worth noting that bulky Cr–DNA adducts preferentially occurred at two sequences: -TTGG*G*CC- and –CA*AC- sites. It appears that no significant bulky Cr–DNA adducts formed at sequences other than those formed at these two preferential sites are repaired after 8h incubation (Figure 2A). Using atomic absorption spectroscopy and UvrABC incision methods to measure Cr content, we observed no significant change in Cr content in the genomic DNA isolated from A549 cells with 0–24h incubation after Cr(VI) treatment (Figure 1A and 1C). These results indicate that the Cr(VI) induced bulky Cr–DNA adducts are not repaired during post-treatment incubation.

Fig. 2.

Mapping the distribution of UvrABC (A) and Fpg (B)-sensitive sites in exon 7 of the p53 gene of human lung cells exposed to Cr(VI). The same UvrABC or Fpg-treated genomic DNA as described in Figure 1 were subjected to LMPCR, and the resultant DNA was separated by electrophoresis, as described (17). Symbols: C, C + T and A + G are Maxam–Gilbert sequencing products (39). G and A following the codon number represent the adducted purines in the codon. (C) Quantifications. The RI of UvrABC and Fpg incision band resulted from cells without repair incubation (time ‘0’) were scanned and calculated.

Cr(VI)-induced Fpg-sensitive sites occur at both dA’s and dG’s in the p53 gene

Results in Figure 2B and 2C show that Fpg-sensitive sites occur at both dA (40%) and dG (60%) positions in p53 exon 7 sequence. The majority of oxidized dG occurred at –NGG- sequences (70%) and the others occurred at –G- positions. Similar to bulky Cr–DNA adducts, no significant ODD were repaired after 24h incubation. This result is consistent with the results in Figure 1B and 1E, which show that no significant amount of Cr(VI)-induced ODD in genomic DNA is repaired. These results are somewhat unexpected, as it has been found that H₂O₂-induced ODD are efficiently repaired within a relatively short period time (26). Results in Figure 1E show that indeed the majority of ODD induced by H₂O₂ was repaired within 8h of incubation. These results indicate that the co-existence of Cr-induced BDA and ODD lead to inefficient repair of Cr(VI)-induced ODD or Cr(VI)-induced ODD was further oxidized to forms such as spiroiminodihydantoin and guanidinohydantoin, which are no longer a substrate of hOGG1 (27,28). It is worth noting that a significant amount of Cr induced BDA and ODD occur at codons 245 (-CGG-) and 249 (-AGG-), the two mutational hotspots in Cr-related lung cancer (Figure 2A and 2B) (11,29) .

Cr(VI) induces USS at dG and dA positions, but Cr(III) induces USS only at dG positions in vitro

Previously, we have found that Cr(III) with and without ligands can form BDA that are formed exclusively at –G- positions and preferentially at –NGG- sequences (13). These results are in stark contrast with the current observation that Cr(VI)-induced BDA formed at both –A- and –G- positions (Figure 2A). We have previously found, by using Fourier transformed infrared difference spectroscopy, that Cr(VI) but not Cr(III) can bind with DNA at –A- as well as at –G- residues (30). These results raise the possibility that inside of cells, a high valency state (or some) Cr(VI) is maintained, rather than being reduced to Cr(III), to interact with genomic DNA to form Cr–dA and Cr–dG adducts. To test this possibility, we incubated Cr(VI) and Cr(III) in water with the exons 5 or 7 DNA fragments of the p53 gene and then determined the Cr–DNA binding sites using the UvrABC incision method (13). The results in Figure 3 show that Cr(VI) induces Cr–DNA adducts, not only at G positions but also at –A- positions, and that the distributions of Cr(VI)-induced Cr–DNA adducts in exons 5 and 7 of the p53 gene are distinctly different from Cr(III)–DNA binding, which only occurs at –G- positions [cf. lane 7 versus lanes 9 and 12 in Figure 3A (exon 5), and lane 6 versus lanes 8 and 10 in Figure 3A (exon 7)]. Cr(VI)-induced DNA binding is significantly enhanced in the presence of glutathione [cf. lanes 9 and 12 versus lanes 10 and 13 in Figure 3A (exon 5)], but was not affected by the presence of free radical scavenging Tris–HCl buffer [cf. lane 12 versus lane 14 in Figure 3A (exon 5)].

Fig. 3.

Cr(VI)-, Cr(V)- and Cr(III)-induced DNA adducts formation in exons 5 and 7 were identified by the UvrABC incision method. Exons 5 and 7 DNA fragments with single-5ʹ end-³²P-labeled were modified (A) with Cr(VI) (10 and 20 µM), in the absence or presence of glutathione (0.1 and 0.2mM) and 10mM Tris–HCl (pH 7.5), (B) Cr(V) (0.3 and 1.9 µM for exon 5 and 0.3 µM for exon 7), and (A and B) with Cr(III) (3 µM), and then treated with UvrABC. The resultant DNAs were denatured and separated by electrophoresis, as described (13). Symbols are the same as in Figure 1. Note: ~30 times higher concentration of Cr(VI) than Cr(V) is needed for DNA modification to produce the same level of UvrABC incision.

Cr(V) induces USS at dG and dA positions

We have shown that Cr(III) forms USS at –G- residues only (13); however, the results in Figure 3 show Cr(VI) induces USS at 
both –A- and –G- residues; these results together indicate that Cr(III) is not an intermediate for Cr(VI)–DNA interactions. One possible pathway for Cr(VI)–DNA interactions is Cr(VI) reduction to Cr(V) followed by Cr(V) binding to dA’s and dG’s to form Cr(V)–dA and Cr(V)–dG adducts, respectively. To investigate this possibility, we incubated chemically synthesized Cr(V) with the ³²P-labeled exons 5 and 7 of DNA fragments. The results in Figure 4C and 4D show that (i) Cr(V)–DNA interactions produce all binding bands that are produced by Cr(VI)–DNA interactions, including those at both –A- and –G- positions, and (ii) Cr(V)–DNA interactions produce more DNA binding bands than those that are produced by Cr(VI) or Cr(III) interactions. We interpret these results to mean that Cr(V)–DNA binding bands are the sum of the Cr(VI)–DNA binding bands, plus Cr(III)–DNA binding bands (Figure 4), and that in the presence of DNA, Cr(V) indeed binds to –A-and –G- residues but that some of the Cr(V) was disproportionate into Cr(VI) and Cr(III) (31), subsequently forming Cr(VI)– and Cr(III)–DNA adducts. This interpretation is further strengthened by the results in Figure 5, which show that under acidic buffer, in which Cr(V) is relatively stable, Cr(V)-induced DNA binding bands that maintain dA adduct bands but reduced dG adduct bands (cf lane 3 versus lanes 6 and 8).

Fig. 4.

Distribution of Cr(III)–DNA, Cr(VI)–DNA and Cr(V)–DNA adducts in exon 5 (A, B, C) and exon 7 (E, F, G). (D) Distribution of Cr(V)–DNA adduct formed at pH 4.0 in exon 5. The band intensity shown in Figures 3 and 5 were scanned, and the RI was calculated. Sequences and codon are depicted on the x-axis.

Fig. 5.

Sequence preference of Cr(V)-induced DNA adducts formation under acidic buffer conditions. Exon 5 DNA fragments with single-5ʹ end-³²P-labeled were modified with Cr(V) (0.6 µM) under acidic buffer conditions (pH 4 and 5), treated with UvrABC and the resultant DNAs were denatured and separated by electrophoresis the same as in Figure 3. Symbols are the same as in Figure 3.

Cr(VI) and Cr (V) induce Fpg-sensitive sites at dG positions

The above results suggest that Cr(VI) interacts with DNA directly and/or indirectly through non-Cr(III) intermediates. Our hypothesis is that weakly bound Cr(VI) is transformed into Cr(V), which then interacts with an –A- and –G- to form Cr(V)–dA DNA and Cr(V)–dG DNA adducts. If this hypothesis is correct, then ODD most probably occurs at –G- and –GG- positions because the oxidative potentials at these sites are relatively low (32). A possible mechanism is that Cr(VI) and Cr(V) produce reactive oxygen species, presumably hydroxyl radicals through Fenton-like reaction to oxidize –G- residues (33,34). To test this possibility, we determined the location of Fpg-sensitive sites in Cr(VI)- and Cr(V)-treated DNA fragments. The results in Figure 6 show that (i) both Cr(V) and Cr(VI) induce Fpg-sensitive sites almost exclusively at –G- positions; and that these sites preferentially occur at –GG- sequences, and (ii) both Cr(V) and Cr(VI) induce a similar, if not identical, Fpg-sensitive site pattern. These results are consistent with the aforementioned proposed pathway that is Cr(VI) interacts with DNA and produces Cr–dA-DNA and Cr–dG-DNA adducts via conversion to Cr(V) intermediate.

Fig. 6.

Mapping the distribution of Cr(VI)- and Cr(V)-induced ODD in (A) exon 5 and (B and C) exon 7 of the p53 gene. Exons 5 and 7 DNA fragments with single-5ʹ end-³²P-labeled were modified with Cr(VI) (10, 20 and 30 µM for exon 5 and 10 and 20 µM for exon 7), Cr(V) (5, 10 and 20 µM) or H₂O₂ (100 μM), treated with Fpg, and the resultant DNAs were denatured and separated by electrophoresis, in the same manner as in Figure 3. Symbols are the same as in Figure 3. (D) Quantifications of the Cr(VI)- and Cr(V)-induced oxidative damage in exons 5 and 7. The RIs were calculated in the same manner as in Figure 2.

Discussion

In in vitro condition, we have found that although Cr(III)–DNA modifications produce only BDA at dG’s, Cr(VI)– and Cr(V)–DNA modifications produce not only BDA at both dA’s and dG’s but also ODD at dG positions. We also found that both BDA and ODD formed at dG’s and dA’s in Cr(VI)-treated lung cells. Based on these results, we propose that within cells, Cr(VI) maintain high valence (VI or V), and that these high-valent Cr molecular react with DNA to produce bulky Cr(III)–DNA adducts and ODD via the following pathway: Cr(VI) interacts with genomic DNA first to produce oxidized form of dG’s, dA’s and Cr(V). In addition, it is probable that Cr(V) is formed by reduction of Cr(VI) with other intracellular reductants such as a glutathione (33). Cr(V) is subsequently transferred to N-7 of intact or oxidized dA’s and dG’s to form Cr(V)–dA–DNA and Cr(V)–dG–DNA complexes. The Cr in the Cr(V)–DNA adducts further undergo intracellular 2-electron reduction and are converted to a stable and final Cr(III)–DNA adduct.

Using the Fpg incision method to identify ODD, we found that in water solution, Cr(VI) caused DNA oxidation almost exclusively at dG’s (Figure 6). This oxidation was greatly reduced when DNA was in the Tris–HCl buffer (data not shown), suggesting that hydroxyl radicals produced via Fenton-like reaction can mediate the oxidation (33,34). It has been reported that reactions of Cr(VI) or Cr(V) with DNA, in the presence or absence of hydrogen peroxide (H₂O₂), produce 8-hydroxy-dG, which is a tautomer of 8-oxo-dG (33,34). Moreover, reaction of Cr(VI) with DNA was found to form hydroxyl radicals, which can interact with dG to produce 8-oxo-dG and Fapy-dG (35). Recent studies showed that the interactions of hydroxyl radicals with DNA produced 8-hydroxy-guanine neutral radical adducts, which were subsequently converted to 8-oxo-dG and Fapy-dG through one-electron oxidation and reduction, respectively (36). Together, these results suggest that Cr(VI) causes oxidative and reductive damage mainly at dG’s in in vitro modifications through hydroxyl radicals. However, direct oxidation of –G- residues by Cr(VI) to produce Cr(V) cannot be ruled out. Inside cells, the direct oxidation of –G- may occur more preferably due to intracellular radical scavengers. Intriguingly, substantial levels of dA’s in exon 7 of p53 gene were oxidized in vivo, but these effects were not observed under the in vitro Cr(VI)-DNA modification protocols. It is possible that dA’s in chromosomal DNA have lower oxidative potential than dA’s in naked DNA.

Using UvrABC incision to identify bulky Cr–DNA damage, the results in Figures 3 and 4 show that Cr(VI) preferentially binds at –CA- sites. We hypothesize that this occurs via transformation of Cr(VI) to Cr(V), which then reacts with N7 of dA. Three lines of evidence support this hypothesis: (i) it has been found that the DNA binding ability of Cr(V) is one order of magnitude higher than that for Cr(VI); (ii) Cr(IV) is unstable and re-oxidized to Cr(V) by excessive Cr(VI) in the reaction mixtures (37) and (iii) the sequence specificity of Cr(V)–dA binding is identical to Cr(VI)–dA binding. It is, therefore, probable that Cr(VI) is reduced by DNA and that the resulting Cr(V) is subsequently transferred to N7 of the dA to form a Cr(V)–dA adduct, which eventually converts to stable Cr(III)–dA.

The Cr(V) binding sites encompass all Cr(VI) and Cr(III) binding sites (Figures 3 and 4). Cr(III)-induced DNA binding is limited to dG’s and preferentially occurs at –NGG- sites. By contrast, Cr(VI)- and Cr(V)-induced DNA binding occurs at both dG’s and dA’s. Interactions of Cr(V) with DNA in neutral aqueous solution retain a similar Cr–DNA binding pattern as that by interactions of Cr(III) with DNA (Figures 3 and 4). However, in acidic conditions (pH 4–5), which contains the Cr(III)-chelating agent, 10mM potassium hydrogen phthalate (38), the Cr(III)-induced Cr–dG adducts (codons 146 and 171) disappeared, but Cr–dA and Cr–dG adducts at codons 174 (Figures 3 and 4) remained. Together, these results suggest that a Cr(VI)-induced Cr–DNA binding pattern is unlikely to be derived from Cr(III). Instead, the Cr–dG (codons 146 and 171) binding comes from Cr(III) that formed from disproportionation of Cr(V) (31). It should be noted that affinity of Cr(V) to dG is higher than Cr(VI) to dG (Figure 3B). Together, these results suggest that Cr(VI) is reduced by DNA into Cr(V), which interact with N7 of purines to form Cr–dG- and Cr–dA-DNA adducts.

We found that Cr(VI) treatment induces USS at –CAA- and –CA- sequences in p53 codons 234 and 253 positions in human lung cells. These sequences are also USS in purified p53 exon 7 DNA fragments modified with high-valent Cr, Cr(VI) and Cr(V), in the cell-free system (Figures 3–5). It is probable that those bulky damages are Cr–DNA adducts. If this is the case, these results indicate that inside cells, the interactions of high-valent Cr species with genomic DNA not only form Cr–dG adducts but also form Cr–dA adducts as well. Most, if not all, of Cr(VI)-induced bulky Cr–DNA adduct formation sites overlapped with Cr(VI)-induced ODD sites. For example, both bulky Cr–DNA adducts and ODD occur at codons 245 and 249, the two lung cancer p53 mutational hotspots (11,29). Furthermore, both bulky Cr–DNA adducts and oxidized dG and dA can cause G:C→T:A and A:T→T:A transversions, which have been found in chromate-induced lung cancer (11,29).

Our results also show that BDA and ODD induced by Cr(VI) treatment in both genomic DNA and at the p53 sequence level are repaired poorly (Figures 1 and 2). Taken together, these results suggest that inside of the cells, Cr(VI) and Cr(V) interact with adenines and guanines of genomic DNA to cause oxidative and bulky damage, which are poorly repaired and consequently can induce mutations and subsequently triggers lung carcinogenesis.

Funding

National Institutes of Health (CA114541, ES014641, CA99007 and ES00260).

Glossary

Abbreviations:

BDA

bulky DNA adducts

Cr(VI)

chromium (VI)

dA

2ʹ-deoxyadenosine; dG, 2ʹ-deoxyguanosine

Fapy-dG

N-(2-deoxy-D-pentofuranosyl)-N-(2,6-diamino-4-hydroxy-5-formamidopyrimidine)

Fpg

formamidopyrimidine glycosylase

LMPCR

ligation-mediated PCR

ODD

oxidative DNA damage

8-oxo-dG

7,8-dihydro-8-oxo-2ʹ-deoxyguanosine

RI

relative intensity

USS

UvrABC-sensitive sites