Guanine-Specific Oxidation of Double-Stranded DNA by Cr(VI) and Ascorbic Acid Forms Spiroiminodihydantoin and 8-Oxo-2′-deoxyguanosine

Deoxyribonucleotides

Oligonucleotides used in this study were purchased from Integrated DNA Technologies or Trilink Biotechnologies. The oligonucleotide sequences were d(5′-AGT-TGAGXGGACTTTCCCAGCC-3′), where X denotes G or 8-oxoG, and its complement d(5′-GGCTGGGAAAGTCCCCTCAACT-3′). Purification of the oligonucleotides prior to use was accomplished by HPLC using a Dionex Nucleopac PA-100, 4 mm × 250 mm anion-exchange column employing a linear gradient from 90% mobile phase A (10% aqueous acetonitrile) and 10% mobile phase B (1.5 M ammonium acetate, pH 6.0, in 10% acetonitrile) to 100% mobile phase B over 31 min. Eluting oligonucleotides were monitored at 268 nm. The fraction containing the oligomer was eluted as a single peak. The collected samples were lyophilized and purified into deionized water using a Bio-Rad Micro Bio Spin 6 Column. Pure oligonucleotides were stored at –20 °C until needed.

Synthesis of Spiroiminodihydantoin (Sp) Oligonucleotide Standards

Sp lesions were made at specific sites within the oligonucleotide by reaction of Cr(V) –salen with the 8-oxoG containing oligonucleotide. The Cr(V) –salen synthesis and reaction with DNA were carried out as described previously (26). Briefly, a 20 mM stock solution of the Cr(V) –salen complex in dry acetonitrile was prepared fresh, and a typical reaction mixture contained 10–100 μM DNA and 800 μM Cr(V) –salen. Reactions were allowed to proceed at room temperature for 15 min prior to HPLC purification of the oxidized product. Pure oligonucleotides containing specific lesions were identified by LC–ESI-MS as previously described (26).

Synthesis and Purification of ¹⁶O-Sp and ¹⁸O-Sp Nucleoside Standards

Na₂IrCl₆·6H₂O, Ir(IV), was purchased from Acros Organics. 8-Oxo-2′-deoxyguanosine was purchased from Sigma-Aldrich. Water–¹⁸O (¹⁸O 95%) was purchased from Cambridge Isotope Laboratories, Inc. ¹⁸O-Sp was synthesized by oxidation of 8-oxoG in H₂¹⁸O using Ir(IV) as described previously (28). Briefly, a reaction volume containing 300 μL of 8-oxo-2′-deoxyguanosine (2.5 mM) was incubated for 1 h at 60 °C with Na₂IrCl₆ (5 mM) in 75 mM phosphate buffer (pH 7.4) followed by addition of 30 μL (200 mM; pH 4.8) of Na–Acetate buffer. Excess Ir(IV) was removed by microanion-exchange chromatography, using a column containing 20 mg of DEAE Sephadex A-25 anion-exchange resin. Samples were eluted with four 100 μL washes of deionized (DI) water. Samples were pooled and further purified by HPLC using a Zorbax C18 semipreparation 9.4 mm × 25 cm column with a gradient of 5%/min acetonitrile in water and a flow rate of 1.8 mL/min. Samples were collected and lyophilized to ~50 μL and quantified by UV–vis, ε₂₃₀ = 4900 M⁻¹ cm⁻¹ (37).

DNA Oxidation Using Cr(VI) and Ascorbate

Ascorbate (>99%) and sodium dichromate dihydrate were purchased from Sigma-Aldrich. Purified duplex DNA oligonucleotides (2.5 μM) were oxidized by the addition of a 10:1 molar ratio of ascorbate to Cr(VI) in PBS buffer (pH 7.0). Concentrations of Cr(VI) varied from 3.12 to 50 μM with 31–500 μM ascorbate. Reactions were incubated for a minimum of 1 h at which time UV–vis monitoring at 372 nm has shown complete reduction of Cr(VI) to Cr(III). The oxidized DNA was then purified into DI water using a Bio-Rad Micro Bio-Spin 6 column.

Analysis of Site- and Sequence-Specific Oxidation of DNA by Cr(VI) and Ascorbate

Piperidine-labile cleavage sites on the DNA were analyzed by treating lyophilized samples of the Cr(VI)/Asc-oxidized DNA (2.5 μM) with 100 μL of a 1.0 M solution of freshly distilled piperidine followed by heating at 90 °C for 30 min. Samples were loaded on a 20%, 0.4 mm thickness, 21 cm × 50 cm denaturing (7 M urea) polyacrylamide gel with 4 μL of 80% formamide loading buffer containing 0.05% xylene cyanol and bromophenol blue. Electrophoresis was carried out at 2200 V and 24 mA with 1 × TBE as the running buffer. Visualization of the DNA cleavage products was carried out by autoradiography using Kodak X-Omat Ar-5 film.

Analysis of Trapped Schiff Base Complexes of hOGG1 and mNEIL2

The Cr(VI)/Asc-treated, ³²P-labeled dsDNA (23 μM) was incubated with mNEIL2 or hOGG1 (15 nM) in 60 μL of reaction buffer (20 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1 mM DTT, and 100 μg/mL BSA) in the presence of 50 mM NaCNBH₄ at 37 °C for 90 min. An amount of 25 μL of the trapped enzyme/ DNA complex was removed, and the reaction was stopped by the addition of 5 μL of 6× SDS loading buffer. Samples were heated to 95 °C for 10 min and flash-cooled on ice. Samples were separated on an 8–12% stacking SDS–PAGE gel and visualized by autoradiography.

Enzymatic Digestion of Oxidized DNA

Nuclease P1 and Phosphodiesterase I were purchased from Sigma-Aldrich. Phos-phodiesterase II was purchased from Worthington Biochemical Corporation. Calf intestinal phosphatase was purchased from New England BioLabs. Double-stranded DNA was oxidized using Cr(VI) and ascorbate as described above. Oxidized DNA samples were lyophilized to 100 μL and a final DNA concentration of 34 μM. The DNA was denatured by heating to 95 °C for 5 min and flash-cooled on ice. The 100 μL samples of oxidized, denatured DNA for HPLC-ECD analysis were hydrolyzed using 5 units of nuclease P1 in 20 mM sodium acetate (pH 5.0) buffer, incubated at 37 °C for 1 h. The nucleotides were hydrolyzed to their respective nucleosides by the addition of 5 units of calf intestinal phosphatase in 50 mM Tris-HCl (pH 7.9), incubated at 37 °C for 1 h. The samples of oxidized and denatured DNA for HPLC–ESI-MS analysis were hydrolyzed to the individual nucleotides by the addition of 10 units of phosphodiesterase II (unbuffered), incubated for 3 h at 37 °C. Samples were further hydrolyzed by the addition of 10 units of phosphodiesterase I buffered in 100 mM Tris-HCl, 100 mM NaCl, and 15 mM MgCl₂ (pH 8.9), incubated for 3 h at 37 °C. The resulting base nucleotides were hydrolyzed to their respective nucleosides by the addition of 10 units of calf intestinal phosphatase and incubated for 3 h at 37 °C. Samples for both HPLC–ESI-MS and HPLC-ECD were filtered using a Millipore YM-30 centrifugal filter, lyophilized to dryness, and dissolved in 100 μL of DI H₂O. Deoxyguanosine concentrations were estimated to be 442 μM per hydrolyzed DNA sample at this point.

HPLC–MS Analysis of Oxidized Duplex DNA

Samples were analyzed using HPLC coupled to a Micromass LCT mass spectrometer using electrospray ionization. Separations were performed using a C18 reverse-phase Microsorb MV 2.1 mm × 250 mm column, with a 0.5% acetonitrile/min gradient with aqueous mobile phase and a flow rate of 0.5 mL/min. Mass spectral analysis was performed using selected ion monitoring (SIM) with a cone voltage of 30 V, capillary voltage of 3000 V, desolvation temperature of 250 °C, source temperature of 150 °C, and an aperture of 15.

HPLC-ECD Analysis of Oxidized Duplex DNA

HPLC-ECD identification of 8-oxoG was performed using a previously established method (38). 8-OxoG and dG were separated by HPLC with a 4.6 mm × 150 mm reverse-phase YMC basic column and quantified using a CoulArray electrochemical detection system (ESA Inc., Chelmsford, MA). Nucleosides were eluted from the column using an isocratic mobile phase of 100 mM sodium acetate, pH 5.2 in 4% methanol. Potentials of the four coulometric analytical cells of the CoulAray system placed in series were as follows: 50, 125, 175, 200, 250, 380, 500, 700, 785, 850, 890, and 900 mV. Calibration curves were generated from standards of dG ranging from 0.1 to 2 μg and standards of 8-oxoG ranging from 50 to 1000 pg. The amount of 8-oxoG was calculated by comparing the peak area from 50 μL injection of enzymatic hydrolysate of the oxidized DNA sample with the calibration curve. Levels of 8-oxoG were expressed as a percentage of the guanine present in each sample.

Hazardous Materials

Caution: Chromium(VI) is a known human lung carcinogen and Cr(V) is an intermediate metabolite of Cr(VI) reduction. Proper care should be taken in the handling of these materials.

Identification of Guanine Specific Oxidation Sites by Cr(VI)/Ascorbate in Duplex DNA

Duplex DNA with the ³²P-labeled upper strand sequence of 5′-AGT TGA GGG GAC TTT CCC AGC C-3′ was treated with concentrations of Cr(VI) ranging from 3.1 to 50 μM and a 10-fold molar excess of ascorbate (31 – 500 μM). This range of Cr(VI) concentrations are commonly used in the treatment of mammalian cells in culture and, depending on the cell line, display toxicity profiles ranging from 0 to 100% (39, 40). Unidirectional uptake and reduction of Cr(VI) by A549 cells leads to low steady-state cellular exposure to Cr(VI), but cumulative exposures have been observed to be greater than 1 mM intracellular chromium (presumably as Cr(III)) from a 10 μM treatment of Cr(VI) in media (39). Reduction of Cr(VI) by ascorbate in the presence of the DNA was allowed to take place for a minimum of 1 h, at which time absorbance measurements of Cr(VI) at 372 nm showed complete reduction to Cr(III) (data not shown). Piperidine treatment was used to specifically cleave the DNA at the site of lesion formation. The results in Figure 2 showed that treatment of DNA with Cr(VI)/ascorbate resulted in the formation of piperidine labile cleavage sites preferentially at guanines. Lanes 1 and 2 are untreated DNA controls, with and without piperidine, respectively, and show little background cleavage. Lane 3 is the Maxam–Gilbert A/G lane used for sequence-specific cleavage comparisons with the Cr(VI)/Asc reactions. Lanes 4–8 are piperidine-treated duplex DNA samples treated with Cr(VI)/Asc, with lane 4 containing the highest concentration (50 μM/ 500 μM) of Cr(VI)/Asc and decreasing to the lowest concentration in lane 8 (3.1 μM /31 μM). The oxidation of the duplex DNA oligonucleotide by Cr(VI)/Asc indicated that oxidation occurs almost exclusively at guanine and that guanines that are 5′ in a run of guanines (or 5′ in a run of purines such as G⁰) show enhanced oxidation (Figure 2). Similar reactions with ascorbate alone showed little or no cleavage of DNA upon piperidine treatment (data not shown).

Lesion Identification by Reductive Trapping of BER Glycosylases

Oxidized base lesions are recognized by specific BER enzymes that initiate the repair process through base removal and formation of an abasic site. The BER enzyme forms a Schiff base intermediate at the C1′ of the damaged nucleotide sugar that can be covalently trapped as the stable amine using a suitable reductant such as sodium cyanoborohydride (41). BER glycosylases have affinities for a specific lesion or a subset of lesions, and reductive trapping can be used to identify the class of DNA lesions formed in the reaction. The human BER glycosylase hOGG1 has shown specificity toward the recognition of a family of oxidized guanine residues including 8-oxoG in DNA (42). We have shown that the recently characterized BER enzymes, NEIL1 and NEIL2, recognize the further-oxidized lesion of guanine, spiroiminodihydantoin (Sp), but have little affinity for 8-oxoG (36). Duplex DNA containing a single 8-oxoG or Sp lesion was synthesized as described in Materials and Methods. Unmodified DNA, DNA containing a single 8-oxoG lesion, or DNA with a single Sp lesion was incubated with hOGG1 or NEIL2 and trapped with NaCNBH₃. Figure 3 shows a typical gel shift obtained upon trapping of the glycosylases. The results show that NEIL2 was almost exclusively trapped to DNA containing the Sp lesion, and hOGG1 was exclusively trapped to DNA containing the 8-oxoG lesion.

Analysis for specific glycosylase trapping to Cr(VI)/Asc-treated duplex DNA was carried out using 15, 25, and 50 μM Cr(VI) and a 10-fold excess of ascorbate. Each sample was incubated with NaCNBH₃ and hOGG1 or NEIL2 in a manner identical to that in Figure 3. Trapping with NEIL2 under these conditions exhibited a distinct gel shift, whereas hOGG1 showed essentially no trapping (Figure 4). Since hOGG1 is relatively specific for the 8-oxoG lesion and NEIL2 has little or no affinity for 8-oxoG (see Figure 3 above), this result suggested that the majority of the lesions formed from the oxidation of duplex DNA by Cr(VI)/Asc was not 8-oxoG but a further-oxidized guanine lesion such as Sp.

Further experiments were carried out on the NEIL2 trapping to determine whether there was a dose dependence for lesion formation from the Cr(VI)/Asc oxidation reaction. Reaction of Cr(VI)/Asc and normal duplex DNA were carried out with Cr(VI) concentrations ranging from 1.5 τ0 50 μM and a 10-fold molar excess of ascorbate, followed by NaCNBH₃ trapping of the Schiff base intermediates as described above. Figure 5 panels A and B show the gel shifts for trapped NEIL2 and the densitometric analysis of each shift, respectively. A clear dose-dependent response of increased NEIL2 trapping with Cr(VI)/Asc was observed.

Identification and Quantification of 8-Oxogua-nine (8-oxoG) in Duplex DNA

8-OxoG is considered an intermediate in the formation of further-oxidized guanine lesions such as Sp. As such, it was considered that residual 8-oxoG should be observable in Cr(VI)/ Asc-treated DNA. DNA oligonucleotides were oxidized with Cr(VI) concentrations ranging from 0 to 50 μM with a 10-fold molar excess of ascorbate. The DNA (2.5 μM) was digested and analyzed for 8-oxoG using HPLC coupled with electrochemical detection (ECD). The elution time for 8-oxoG was typical for this assay at approximately 12 min. The 8-oxoG peak was monitored on the electrochemical detector at 175 mV, 200 mV, and 250 mV, as described previously (38). A standard curve was prepared and used to quantify 8-oxoG formation in this assay (data not shown). Figure 6 shows the formation of 8-oxoG as a percentage of total guanine concentration with increasing Cr(VI) concentrations. The data points represent the mean ± standard deviation from the measurement of three independently prepared samples. The results of these experiments showed increasing 8-oxoG formation with increasing Cr(VI)/Asc concentrations. The highest concentration on Cr(VI)/Asc yielded an approximately 32-fold increase in 8-oxoG over background.

Identification of Spiroiminodihydantoin (Sp) in Chromium-Oxidized Duplex DNA

Enzymatic digests of Cr(VI)/Asc-oxidized, duplex DNA oligonucleotides were carried out as described and analyzed by LC–ESI-MS for the formation of Sp. The retention time of Sp at a 0.4 mL/min flow rate using C18 reverse-phase chromatography was ×6.0 min with the other nucleosides eluting in the typical order of dC, dG, dT, and dA. A stable isotope-labeled standard, ¹⁸O-Sp, was synthesized as described previously (37) and identified by its characteristic mass spectral pattern as shown in Figure 7. Known concentrations of the ¹⁸O-Sp were spiked into the DNA digest of Cr(VI)/Asc-treated DNA as an internal standard to allow quantification. SIM was used to identify Sp formation in the DNA digests using the characteristic masses for the base, B + H₂, or that of the nucleoside, M + H, and M + Na ions of Sp at m/z 184, 300, and 322, for ¹⁶O-Sp, and those at m/z 186, 302, and 324, for ¹⁸O-Sp. Figure 8 shows the LC/MS chromatogram ion current profiles at m/z 322 and 324 of a 2.5 μM DNA sample treated with 50 μM Cr(VI) and 500 μM ascorbate. The exact mass calculation of the M + Na peak for ¹⁶O-Sp using the ¹⁸O-Sp internal standard was 322.0764 compared to a calculated mass of 322.0770 to yield a 1.9 ppm mass accuracy. The identical retention times for ¹⁶O-Sp and ¹⁸O-Sp coupled with the exact mass calculations show the formation of spiroiminodihydantoin in the reaction of duplex DNA with Cr(VI) and ascorbate under these conditions.

Quantification of Spiroiminodihydantoin (Sp) in Duplex DNA

The quantification of Sp in the Cr(VI)-oxidized, duplex DNA was performed by LC–ESI-MS using ¹⁸O-Sp as an internal standard. A calibration plot was obtained to judge the response of the mass spectrometer to known concentrations of ¹⁶O-Sp and ¹⁸O-Sp. Samples containing 0.1 nmoles of ¹⁸O-Sp were spiked with increasing amounts of ¹⁶O-Sp from 0.01 to 5.12 nmols. The ratio of the currents of these ions was plotted as a function of the concentration of ¹⁶O-Sp yielding a linear calibration plot as shown in Figure 9.

Sp, formed from the oxidation reaction in DNA by Cr(VI)/ascorbate, was measured with known amounts of spiked ¹⁸O-Sp. The ions at m/z 322 and 324 (M + Na for ¹⁶O-Sp and ¹⁸O-Sp, respectively) were monitored. Duplex DNA (2.5 μM), oxidized by the greatest amount of Cr(VI)/Asc (50 μM/500 μM), converted 0.341 ± 0.013 nmoles or 1.55 ± 0.06% of all guanines to the further-oxidized lesion Sp. The lowest concentration of Cr(VI)/ Asc used in this study, 3.1 μM/31 μM, converted 0.069 ± 0.01 nmoles or 0.315 ± 0.05% of all guanines to the further-oxidized lesion Sp. A small amount of Sp and 8-oxoG was observed without Cr(VI) treatment. We have attributed this formation to the rigorous DNA digestion procedure necessary for Sp analysis or the presence of small amounts of contaminating, redox-active metals. Figure 10 shows a graphical representation of Sp formation with respect to 8-oxoG formation at the concentrations of Cr(VI) and ascorbate used in this study. The formation of Sp was observed to be approximately 20-fold greater than that of 8-oxoG.

We have observed that Sp within DNA was refractory to normal enzymatic digestion reactions which necessitated the inclusion of a more rigorous digestion cocktail. Further, the enhanced polarity of Sp versus other nucleosides creates technical difficulties in the separation by HPLC from the hydrolyzed DNA matrix. These factors are likely to cause inherent errors in the quantification of Sp, and it is likely that the amount of Sp may be greater than that observed. However, these results clearly show that Sp can be formed directly from guanine by Cr(VI)/Asc oxidation in duplex DNA and, under these conditions, was significantly more prevalent than 8-oxoG.

Reduction of Cr(VI) by Ascorbate

Previous EPR studies have characterized the Cr(VI)/Asc system used in this study in terms of the reactive intermediates produced during reduction (12). The reduction of Cr(VI) with a 10-fold molar excess of ascorbate has been observed to initially form a transient Cr(IV) species and the oxidized form of ascorbate, dehydroascorbate (DHA), followed by a second reduction reaction of Cr(IV) with Asc to give rise to the ascorbyl radical, Asc^•−, and a kinetically inert Cr(III) species as shown in eqs 1 and 2 below:

On the basis of these results, we postulate that the species responsible for oxidation of guanine in DNA to form Sp and 8-oxoG is the Cr(IV) species formed in eq 1. Cr(IV) is a prime candidate for the DNA oxidant in this system since it is known to be highly oxidizing with standard reduction potentials for the chelate-stabilized Cr(IV)/Cr(III) couple estimated at 1.33–1.37 V (43). Cr(III) and Asc^•− are generally less reactive. Under these conditions, no oxygen radicals have been observed (12), suggesting that the mechanism involves a direct, metal-mediated oxidation of DNA. The formation of Sp directly from guanine in DNA has previously only been observed for carbonate radical anions (44). To our knowledge, this study is the first example of Sp formation arising discretely from guanine in duplex DNA from reaction with a known carcinogenic metal.

The mechanism of Sp formation directly from guanine is a four-electron process with Sp being the terminal product that predominates at pH 7 or greater (45). 8-OxoG is the two electron intermediate in this oxidation process which is further oxidized to the 5-hydroxy-8-oxoguanine species, 5-OH-8-oxoG, and ultimately Sp. Figure 11 shows the putative reaction scheme for the formation of Sp from guanine with sequential one-electron oxidation by Cr(IV). A second terminal product of guanine oxidation, guanidinohydantoin (Gh), may also be formed during this oxidation reaction. However, these products are generally formed under more acidic conditions, while this study was carried out at neutral pH (45). We have not observed Gh under the current oxidation conditions, but we have yet to optimize the HPLC–ESI-MS parameters for identification of this product. It is also important to note that the oxidation process used here shows, for the first time, guanine-specific products caused by the direct oxidation of unmodified dsDNA by Cr(VI) without the introduction of exogenous H₂O₂.

Guanine-Specific Cleavage by Cr(VI)/Asc in Duplex DNA

A number of studies on the reaction of Cr(VI) with DNA have shown a bias toward reaction with guanine (46). These results have often been interpreted as the formation of chromium adducts with guanine through a phosphodiester backbone-liganded intermediate (47). However, no adduct of chromium with the guanine base has ever been discretely identified. We suggest that guanine-specific reactions in DNA give rise primarily to further-oxidized lesions such as Sp, although formation of these ternary adducts cannot be ruled out in our system.

Sp is known to be considerably more piperidine labile than 8-oxoG and has shown the ability to induce profound arrests in polymerase processing (24). The enhancement in cleavage that we have observed in guanines that are 5′ in a run of purines also supports this oxidative mechanism. We postulate that many of the effects of Cr(VI) treatment observed in cellular systems may be adequately explained by the formation of these further-oxidized lesions.

Formation of 8-OxoG in the Cr(VI)/Asc Oxidation Reaction

8-Oxo-2′ -deoxyguanine, 8-oxoG, is a well-characterized biomarker of oxidative damage in cellular and in vitro systems (48, 49). The formation of 8-oxoG in DNA has been observed with a vast array of oxidizing species, and its formation has been extensively followed using HPLC–MS, GC–MS, HPLC-ECD, and immuno-assays. 8-OxoG itself is mildly mutagenic and forms G:C → T:A transversion mutations approximately half the time during DNA replication in vitro (22, 30–33) but only about 5% of the time in vivo (31). The hOGG1 base excision repair pathway is the primary route for the excision and repair of 8-oxoG in human DNA, and genetic polymorphisms of this repair enzyme have been associated with increased risk of lung cancer (50). However, Ogg1 knockout mice have shown no change in the observable phenotype relative to wild-type mice, although a slight increase in mutation rate was observed in some tissues (51, 52). This lack of phenotype has been attributed to redundancy in base excision repair. The formation of 8-oxoG in DNA following an oxidant insult is undoubtedly an important consequence of DNA oxidation. However, the focus on 8-oxoG as the primary oxidized lesion of guanine may arise more from its ease of detection than from its biological significance.

8-OxoG has been shown to be sensitive to further oxidation, especially when compared to the parent guanine base (23, 24). The lowered reduction potential of this oxidized base with respect to guanine allows the lesion to act as a thermodynamic sink for electron donation in redox reactions and serve as a hot spot for further oxidation within a DNA sequence (25). The thermodynamics of 8-oxoG oxidation would thus suggest that a secondary oxidation of this product would predominate and result in the formation of a different subset of oxidized lesions, many of which may be more deleterious than the 8-oxoG lesion. A number of further-oxidized lesions of guanine have been identified arising from 8-oxoG oxidation in vitro which include that lesion studied here, spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh), imidazalone, oxazolone, and oxaluric acid, to name a few (28, 29, 37, 42). We have previously shown that 8-oxoG lesions in DNA are prone toward specific further oxidation by high-valent chromium, Cr(V), complexes at the 8-oxoG site leading to the formation of Sp and Gh. Sp and Gh have proven to be much more mutagenic than 8-oxoG, causing G:C → T:A and G:C → C:G transversion mutations at a significantly greater frequency (30–33). As noted previously, the reaction of Cr(VI) with DNA in the presence of ascorbate can also form Cr/Asc/DNA ternary adducts (47). These ternary adducts have been shown to give rise to similar G:C → T:A transversion mutations as those observed for Sp and 8-oxoG (53).

Identification of 8-oxoG in Cr(VI)-treated systems in vitro had previously only been observed with addition of hydrogen peroxide to reaction mixtures of Cr(VI). In vivo, the formation of 8-oxoG has been reported as present in some cellular systems in low levels and absent in other studies. The results of this study may shed light on this contradiction, and we propose that as a labile intermediate of guanine oxidation, 8-oxoG, is either repaired or preferentially oxidized in DNA to further-oxidized lesions such as Sp. Thus, the 8-oxoG species would always be at low or even undetectable levels intracellulary.

Taken together, these data suggest that 8-oxoG may be an excellent and readily measurable biomarker of general oxidation, but the actual lesions responsible for the initiation of carcinogenesis may be the more mutagenic, further-oxidized lesions such as the Sp observed in these studies.

Sp Formation in the Cr(VI)/Asc Reaction With Unmodified Duplex DNA

The primary focus of this study was to identify and quantify the formation of the further-oxidized guanine lesions, spiroiminodihydantoin (Sp), arising from oxidation of guanine in unmodified duplex DNA. Clearly, the formation of these further-oxidized lesions from oxidative attack on guanine in DNA instead of in an 8-oxoG-modified DNA strand was necessary to establish their potential relevance in biological systems. To identify these lesions as arising in unmodified DNA from Cr(VI) oxidation, we exploited the specific affinity of trapping by the base excision repair enzyme NEIL2 and have further unambiguously identified their formation using HPLC–ESI-MS. The NEIL family of genes (Nei-like) have only recently been identified, and their affinities for oxidatively damaged bases are still being defined. We have recently shown that NEIL1 and NEIL2 both have high affinities for the further-oxidized guanine lesions Sp and Gh but little or no affinity for 8-oxoG in duplex DNA (36). When Sp formation was compared with 8-oxoG in this study, the prevalence of the glycosylase recognition was specific for further-oxidized lesions and not 8-oxoG, suggesting that Sp was formed at significantly greater levels than 8-oxoG.

The quantification of Sp by HPLC–ESI-MS is neither trivial nor ideal. Sp oligonucleotides have proven difficult to hydrolyze to their individual nucleosides. The generation of a tetrahedral sp³-hybridized carbon in Sp should significantly perturb DNA structure which slows enzymatic hydrolysis. Therefore, a cocktail of enzymes must be used to achieve digestion, while at the same time, the use of buffers must be minimized to give a clean mass spectrometric signal. An alternative method of chemical hydrolysis using HF in pyridine to hydrolyze the Sp base from oxidized DNA has been developed and successfully utilized by Ravanat and Cadet (54, 55). Finally, the retention time of Sp on a typical reverse-phase column is poor due to the polarity of Sp, leading to problematic identification/quantification of this species. It is likely, based on the rapidly increasing literature examples of Sp formation in a variety of different systems, that this may be a ubiquitous lesion at least as common as 8-oxoG. However, the poor enzymatic digestion and chromatographic behavior of this lesion may be a factor in it being overlooked in many of the systems. Therefore, while the quantification of Sp achieved here is not ideal, our approach has clearly shown the formation and existence of Sp by Cr(VI) oxidation in unmodified dsDNA and has shown that Sp is formed in quantities much greater than 8-oxoG in vitro.

Conclusion

The reaction of the human carcinogen Cr(VI) with the endogenous reductant ascorbate exhibits guanine-specific oxidation in normal duplex DNA. The primary lesion that was observed in this system was the further-oxidized lesion spiroiminodihydantoin, Sp. The formation of Sp, even at the lowest concentrations of Cr(VI) and ascorbate, was much greater than that of 8-oxoG (10–20-fold). This work suggest that the primary lesion responsible for initiation of carcinogenesis by Cr(VI) may be Sp, and the use of this species as a biomarker for Cr(VI) exposure may be more relevant than 8-oxoG. Further work to identify the formation of Sp in Cr(VI)-treated cellular systems is necessary to confirm the physiological significance of this lesion.