Chem. Res. Toxicol. 1997, 10, 1397-1406
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Direct and Hydrogen Peroxide-Induced Chromium(V) Oxidation of Deoxyribose in Single-Stranded and Double-Stranded Calf Thymus DNA Kent D. Sugden* and Karen E. Wetterhahn† Department of Chemistry, Dartmouth College, 6128 Burke Laboratory, Hanover, New Hampshire 03755-3564 Received August 4, 1997X
Oxidative DNA damage by a model Cr(V) complex, [CrO(ehba)2]-, with and without added H2O2, was investigated for the formation of base and sugar products derived from C1′, C4′, and C5′ hydrogen atom abstraction mechanisms. EPR studies with 5,5-dimethylpyrroline N-oxide (DMPO) have shown that Cr(V)-ehba alone can oxidize the spin trap via a direct chromium pathway, whereas reactions of Cr(V)-ehba in the presence of H2O2 generated the hydroxyl radical. Direct (or metal-centered) Cr(V)-ehba oxidation of single-stranded (ss) and double-stranded (ds) calf thymus DNA demonstrated the formation of thiobarbituric acidreactive species (TBARS) and glycolic acid in an O2-dependent manner, consistent with abstraction of the C4′ H atom. A minor C1′ H atom abstraction mechanism was also observed for direct Cr(V) oxidation of DNA, but no C5′ H atom abstraction product was observed. Direct Cr(V) oxidation of ss- and ds-DNA also caused the release of all four nucleic acid bases with a preference for the pyrimidines cytosine and thymine in ds-DNA, but no base release preference was observed in ss-DNA. This base release was O2-independent and could not be accounted for by the H atom abstraction mechanisms in this study. Reaction of Cr(V)-ehba with H2O2 and DNA yielded products consistent with all three DNA oxidation pathways measured, namely, C1′, C4′, and C5′ H atom abstractions. Cr(V)-ehba and H2O2 also mediated a nonpreferential release of DNA bases with the exception of the oxidatively sensitive purine, guanine. Direct and H2O2-induced Cr(V) DNA oxidation had opposing substrate preferences, with direct Cr(V) oxidation favoring ss-DNA while H2O2-induced Cr(V) oxidative damage favored ds-DNA. These results may help explain the carcinogenic mechanism of chromium(VI) and serve to highlight the differences and similarities in DNA oxidation between high-valent chromium and oxygenbased radicals.
Introduction Uptake of chromate, Cr(VI), into cells via the general anion-transport system is followed by formation of the reduced oxidation states of Cr(V), Cr(IV), and Cr(III) and concomitant DNA damage (1). Some of the genotoxic lesions on DNA identified following the reduction of Cr(VI) include Cr-DNA cross-links, Cr-DNA-protein cross-links, single-strand breaks, and alkali-labile sites (2-5). The latter two lesions may arise from an intracellular oxidation process with chromium, while the former two lesions need not involve oxidative processes. The cellular components potentially responsible for the reduction of Cr(VI) are numerous; several relevant species include glutathione (6), hydrogen peroxide (7), ascorbate (8), and NADPH (9). Depending upon the nature of the Cr(VI) reductant, additional species with DNAdamaging potential may be formed, such as carbon-based radicals (8), oxygen-based radicals (7), and thiyl radicals (6). It is the oxidation of DNA by any of these agents during the intracellular reduction Cr(VI) to the stable Cr(III) state that is considered to be responsible for some of the genotoxicity and carcinogenicity associated with this metal. Following uptake and reduction of Cr(VI) by the cell, analysis of accumulation in various organelles has shown
6% to reside in the mitochondria, 25% in the nuclear fraction, and >50% in the cytoplasm (10). While ascorbate and glutathione are considered to be the predominant reductants of Cr(VI) in both the cytoplasmic and nuclear fractions (1), a potential role for hydrogen peroxide may be involved for the mitochondrial fraction of Cr(VI). The relevance of hydrogen peroxide as a potential reductant/oxidant in the cell as a whole is arguable, with overall cellular levels considered to be as low as 10-7-10-9 M (11, 12). However, it is estimated that in mitochondria, the respiratory system converts 1-5% of the total oxygen consumed into hydrogen peroxide (13). This suggests a site-specific relevance for the reduction/oxidation of high-valent chromium by hydrogen peroxide in this organelle since both Cr(VI) and Cr(V) have been shown to react with hydrogen peroxide in vitro to generate oxygen-based radicals (14, 15). The Cr(V)-ehba1 complex, [CrO(ehba)2]-, is a structurally characterized complex with well-defined aqueous properties (16). The EPR properties and ligand environment are similar to that of the unstable Cr(V)-ascorbate complex observed upon reduction of Cr(VI) by ascorbate (8, 17). Unlike the Cr(V)-ascorbate complex, the Cr(V)ehba species does not undergo intramolecular oxidation at neutral pH to generate carbon-based radicals. Another advantageous property of this model complex is the
* To whom correspondence should be addressed. † Deceased June 8, 1997. X Abstract published in Advance ACS Abstracts, November 15, 1997.
1 Abbreviations: DMPO, 5,5-dimethylpyrroline N-oxide; TBARS, thiobarbituric acid-reactive species; 5-MF, 5-methylene-2-furanone; ehba, 2-ethyl-2-hydroxybutyric acid; RT, room temperature.
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1398 Chem. Res. Toxicol., Vol. 10, No. 12, 1997
known disproportionation stoichiometry. These properties of the Cr(V)-ehba complex make it an ideal candidate for observing oxidation reactions that occur directly at the metal center and not via a diffusible carbon-based radical species.
Farrell et al. have shown that the Cr(V)-ehba complex can induce frank DNA strand breaks to relax supercoiled plasmid DNA (18). We have previously shown that the Cr(V)-ehba complex can oxidize deoxyribonucleotides in a phosphate-dependent manner by H atom abstraction at the C4′ of the deoxyribose sugar (19). The following study was undertaken to determine whether the Cr(V)ehba complex can oxidize DNA directly by an H atom abstraction mechanism and whether this oxidation is similar to Cr(V)-hydrogen peroxide-induced oxidations of DNA. Specifically, we have looked at qualitative and quantitative differences between direct and hydrogen peroxide-induced Cr(V)-ehba reactions with both singlestranded (ss) and double-stranded (ds) calf thymus DNA by analyzing the oxidative products formed from C1′, C4′, and C5′ H atom abstractions. We have determined that direct Cr(V) oxidations of both ss- and ds-DNA show fundamental differences to that observed for hydrogen peroxide-induced Cr(V)-ehba oxidations. Product analysis has determined that direct Cr(V) oxidation of DNA proceeds via an oxygen-dependent C4′ H atom abstraction mechanism and a minor C1′ H atom abstraction mechanism. No products characteristic of a C5′ H atom abstraction were observed. An oxygen-independent pathway involving base release in direct Cr(V) DNA oxidation was also observed, but that mechanism has yet to be fully elucidated. Oxidative products from all three mechanisms investigated (C1′, C5′, and C4′ H atom abstractions) were observed for hydrogen peroxide-mediatedCr(V) oxidation of DNA. The profile for product generation in the hydrogen peroxide-mediated Cr(V) reaction was consistent with the formation of a diffusible hydroxyl radical species as the primary DNA oxidant.
Experimental Section Materials. Nucleic acid bases, 5,5-dimethylpyrroline Noxide, glycolic acid, triethylamine, calf thymus DNA (type I, highly polymerized), alkaline phosphatase (type VII-L, from bovine intestinal mucosa), P1 nuclease, Chelex 100, and Sephadex DEAE A-25 anion-exchange resin were purchased from Sigma Chemical Co. N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was purchased from the Pierce Chemical Co. Furfural, 1,1,3,3-tetramethoxypropane, ammonium formate, and 2-thiobarbituric acid were obtained from Aldrich Chemical Co. Trichloroacetic acid, 30% hydrogen peroxide, Na2Cr2O7‚2H2O, C18 Sep-Pak Plus cartridges, and HPLC grade methanol and acetonitrile were purchased from Fisher Scientific. Thyminepropenal was obtained from a previous synthetic preparation (19). Synthesis of Cr(V)-ehba. The sodium salt of bis(2-ethyl2-hydroxybutyrato)oxochromate(V) was prepared in a crystalline form using the method of Krumpolc and Rocek (16). Caution: Cr(VI) is a known human carcinogen, and Cr(V) complexes are potentially carcinogenic. Appropriate precautions should be taken in handling these materials.
Sugden and Wetterhahn Synthesis of 5-Methylene-2-furanone (5-MF). 5-Methylene-2-furanone was synthesized and purified as described previously (20). Further purification by HPLC was found to be necessary using system B described below. GC/MS using method B demonstrated an identical mass spectrum to that reported previously for this complex (21) with a molecular ion of m/z 96 and logical mass losses of m/z 68, 54, and 42. HPLC Conditions. HPLC analyses were carried out on a HP-1090 HPLC system with diode array detection at wavelengths of 254 nm for all four nucleic acid bases and 5-methylene-2-furanone. Furfural was quantitated by diode array detection at 275 nm. All HPLC analyses were performed on a Rainen Microsorb-MV 100-Å C18 column, 5-µm particle size (4.6 mm-i.d. × 25-cm length), at a flow rate of 1.0 mL/min. The mobile phases used were system A, a gradient from 100% 100 mM ammonium formate (pH 7.0) to 85% 100 mM ammonium formate:15% MeOH over 10 min with a 3-min hold for detection of all four bases; system B, isocratic, 95% 100 mM triethylammonium acetate:5% acetonitrile (pH 6.5) (22). These afforded the following retention times (in parentheses) for the compounds of interest: system A, Cyt (4.5 min), Gua (7.8 min), Thy (9.3 min), and Ade (12.6 min); system B, furfural (13.9 min) and 5-methylene-2-furanone (15.1 min). The detection limits for 5-MF and furfural in this assay were ∼0.05 µM. Detection limits for the nucleic acid bases Cyt, Gua, and Thy were 0.5 µM, with Ade having a detection limit of 1.0 µM. GC/MS Conditions. GC/MS analyses were carried out using a HP-5890 GC with a HP-5971 mass selective detector as described previously (19). GC conditions were method A, injector temperature of 250 °C, detector temperature of 312 °C, and column temperature gradient of 80-260 °C at 20 °C/min; method B, injector temperature of 150 °C, detector temperature of 312 °C, and column temperature gradient of 80-150 °C at 10 °C/min. Both systems utilized a Supelco SPB-5 column (30 m × 0.2 mm). EPR Conditions. EPR spectra were recorded using a Bruker ESP-300 spectrometer. The spectral parameters were 100-kHz field modulation, 1.0-G modulation amplitude, 5.12ms time constant, 9.769-9.772 microwave frequency, 1 × 105 receiver gain, 2-mW microwave power attenuated at 20 dB, 3380-3580-G sweep width, and 21-s scan time. The signals were averaged over nine scans. Typical reactions were carried out on 0.8-mL volumes at room temperature (RT) in chelexed 100 mM phosphate buffer of the appropriate pH. Measurements were obtained after 80 s with 200 mM spin trap 5,5-dimethylpyrroline N-oxide (DMPO) and 1.0 mM Cr(V)-ehba. For EPR measurements using hydrogen peroxide and ethanol, concentrations of 1.0 mM and 1.0 M, respectively, were used. Measurements were carried out on ca. 100-µL volumes drawn into a capillary tube sealed on one end with Dow-Corning high-vacuum grease and placed in a quartz EPR tube. The g-values were determined in respect to 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), g ) 2.0036. Conditions for Reaction of Cr(V)-ehba with ds- and ssDNA. (A) Determination of TBARS Formation and Base Release. Reactions were carried out in 0.8-mL volumes in chelexed 100 mM phosphate buffer at a suitable pH and at RT; 2.0 mg/mL ds-DNA was used in the reactions and determined to correspond to ca. 5 mM DNA-PO4 by absorbance at 260 nm using ) 6600 M-1 cm-1. Formation of ss-DNA, by heating ds-DNA for 10 min in a boiling water bath followed by quick cooling on ice, was confirmed by monitoring the hyperchromic shift at 260 nm which was generally 16-20% greater than that of ds-DNA. Both ds- and ss-DNA were reacted with 1.0 mM Cr(V)-ehba from a stock aqueous solution for 1 h at RT. Reactions utilizing hydrogen peroxide were at a final concentration of 1.0 mM. Oxygen-dependent and -independent reactions were assayed in an identical manner but under an oxygen or argon atmosphere (note: the TBARS analyses was carried out in air, and only the 1-h reaction time of Cr(V)-ehba with DNA was carried out under the specified atmosphere). Formation of TBARS was determined using the method of Greenwald et al. (23) using 1,1,3,3-tetramethoxypropane as the standard. TBARS
Chromium(V) Oxidation of Deoxyribose in DNA (thiobarbituric acid-reactive species) assay is a marker of sugar oxidation whereupon aldehydic species from oxidation of the DNA sugar backbone (24) form a chromophore with an absorbance maximum at 532 nm when derivitized with thiobarbituric acid. This particular chromophore is generally considered to be derived from hydrolysis of base propenals formed via C4′ H atom abstraction (25). Release of nucleic acid bases was determined under identical conditions as the TBARS reaction, although a multistep postreaction workup involving isolation and concentration of the free bases was necessary. The 0.8-mL reaction volume was charged on a C18 Sep-Pak Plus cartridge and washed with 1.0 mL of water to remove buffer and excess chromium followed by elution of the free nucleic acid bases and unreacted DNA with 2.0 mL of methanol. The methanol fraction was evaporated in vacuo, redissolved in 500 µL of water, and charged on a 0.5- × 5-cm column of DEAE Sephadex A-25 anion-exchange resin. The column was eluted with 2 × 0.6 mL of water, yielding the uncharged nucleic acid bases which were concentrated in vacuo. The residue was redissolved in 150 µL of water, and 100 µL of the sample was assayed by HPLC using system A. Bases were quantified at 254 nm from standard curves of authentic samples from each of the four nucleic acid bases. Recovery of the bases was between 92% and 100% using this method. Oxygendependent and -independent reactions were assayed in an identical manner, but under either an oxygen or argon atmosphere during the 1-h reaction time only. (B) Determination of Glycolic Acid. Formation of glycolic acid under an oxygen or argon atmosphere was determined in the reaction between 1.0 mM Cr(V)-ehba and ca. 5 mM DNAPO4 in either the presence or absence of 1.0 mM hydrogen peroxide. The reactions were carried out in 1.0-mL volumes at pH 6.5, in 100 mM PO4 buffer at RT for 1 h. Isolation and quantitation of glycolic acid were conducted using the method of McGall et al. (26). Briefly, this method involves the digestion of the DNA by P1 nuclease and alkaline phosphatase followed by concentration of the glycolic acid on an anion-exchange column and formation of the bis-trimethylsilylated derivative. Glycolic acid was then quantitated by GC/MS using method A as described under GC/MS Conditions, above. Recovery of authentic glycolic acid using this method was determined to be ∼80%. (C) Determination of 5-Methylene-2-furanone and Furfural. Formation of 5-MF and furfural was determined from equivalent reaction volumes to those used for the TBARS reaction and base release and was stoichiometrically identical, using ca. 5 mM DNA-PO4, 1.0 mM Cr(V)-ehba, and 1.0 mM hydrogen peroxide where indicated. Following the 1-h reaction at pH 6.5 in 100 mM phosphate buffer at RT, the reaction mixtures were heated at 90 °C for 10 min in a water bath to ensure complete formation of the β-elimination products (5-MF and furfural). The samples were cooled, and 200 µL of the solution was assayed directly by HPLC as described under HPLC Conditions using system B. 5-MF was quantified from a standard curve of a purified synthetic sample measured at 254 nm, while furfural was measured at 275 nm from an authentic sample.
Results Stability of Cr(V)-ehba in Phosphate Buffer and Radical Formation. Stability measurements were carried out using EPR to monitor the loss of the Cr(V)ehba signal under the conditions used in the DNA oxidation studies. Complete decay of the 1.0 mM Cr(V)ehba signal was observed in 100 mM phosphate buffer for the pH range of 6.5-7.5 after 1 h at RT (data not shown). A significant Cr(V)-ehba signal remained after 1 h for pH’s less than 6.0. The initial decay of the Cr(V)-ehba signal was enhanced at all pH’s with the addition of 1.0 mM hydrogen peroxide, although a small Cr(V)-ehba signal, presumably due to in situ reduction
Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1399
Figure 1. EPR spectra of the DMPO radical species formed upon reaction of Cr(V)-ehba in 100 mM phosphate buffer, pH 7.5: (trace A) reaction of 1.0 mM Cr(V)-ehba with 1.0 mM H2O2 and 200 mM DMPO; (trace B) reaction of 1.0 mM Cr(V)-ehba with 1.0 mM H2O2, 200 mM DMPO, and 1.0 M ethanol; (trace C) reaction of 1.0 mM Cr(V)-ehba with 200 mM DMPO and 1.0 M ethanol; (trace D) reaction of 1.0 mM Cr(V)-ehba with 200 mM DMPO. The pound sign (#) denotes the 4-line signal arising from the DMPO-OH• adduct with AN ) AH ) 14.9 G; the asterisk (*) indicates the 6-line signal of the DMPO-CHOH-CH3• adduct with AN ) 15.8 G and AH ) 22.8 G.
of the Cr(VI) disproportionation product by hydrogen peroxide, was observed to be continually present. To determine whether oxygen radical species (primarily hydroxyl radical, OH•) may be responsible for Cr(V)induced DNA oxidation, EPR studies with the spin trap DMPO were carried out. The reaction of 1.0 mM Cr(V)ehba with 200 mM DMPO produced an observable radical signal in the pH range of 6.5-7.5. At these pH’s the formation of the 1:2:2:1 DMPO-OH• adduct with AN ) AH ) 14.9 G was observed, with the greatest signal intensity at pH 7.5 (Figure 1, trace D). This same adduct was observed at a much higher intensity when the reaction was carried out in the presence of 1.0 mM hydrogen peroxide (Figure 1, trace A). The addition of 1.0 M ethanol as a hydroxyl radical trap to the reaction of Cr(V)-ehba and DMPO alone did not show the expected splitting associated with the R-hydroxyethyl adduct when OH• is present (Figure 1, trace C). However, when the reaction mixture contained hydrogen peroxide as well as Cr(V)-ehba and DMPO, the 6-line spectra of the R-hydroxyethyl adduct of DMPO, DMPO-CHOH-CH3• with AN ) 15.8 G and AH ) 22.8 G (Figure 1, trace B), was observed. At pH’s below 6.5, shorter-lived DMPO-OH• radicals were only observed in those reactions containing hydrogen peroxide. The EPR spectra of Cr(V)-ehba alone or with hydrogen peroxide and DMPO were unaffected when carried out under argon (data not shown). TBARS Formation in the Reaction of DNA and Cr(V)-ehba. Oxidation reactions of calf thymus DNA by the Cr(V)-ehba complex were carried out in demetalated 100 mM phosphate buffer. Formation of TBARS in the reaction of ca. 5 mM DNA-PO4 with 1.0 mM Cr(V)-ehba was found to be maximal in the pH range of 6.0-6.5 for both ss- and ds-DNA (Figure 2A). Formation of TBARS in ss-DNA was determined to be 24% greater at pH 6.5 than in ds-DNA. Above pH 6.5, the formation of TBARS decreased rapidly for both ss- and ds-DNA. Attempts to maximize this reaction by increasing chromium concentrations above 1.0 mM failed to significantly enhance TBARS formation.
1400 Chem. Res. Toxicol., Vol. 10, No. 12, 1997
Figure 2. pH dependence for the formation of TBARS in the reaction of Cr(V)-ehba and DNA, with and without added H2O2: (A) reaction of 1.0 mM Cr(V)-ehba with 5 mM ds- (b) or ss- (O) DNA in 100 mM phosphate buffer at RT and ambient O2 pressure; (B) reaction of 1.0 mM Cr(V)-ehba and 1.0 mM H2O2 with 5 mM ds- (b) or ss- (O) DNA in 100 mM phosphate buffer at RT and ambient O2 pressure.
Identical Cr(V)-ehba/DNA reactions as above were carried out with addition of 1.0 mM hydrogen peroxide to the reaction mixture (Figure 2B). The formation of TBARS was nearly 5-fold greater for the reaction of Cr(V)-ehba with hydrogen peroxide and DNA than in the reaction between Cr(V)-ehba and DNA alone. ss-DNA showed the greatest formation of TBARS with Cr(V)-ehba alone versus ds-DNA. With the addition of hydrogen peroxide, an opposite substrate preference was observed, with ds-DNA being more reactive than ss-DNA. Base Release in the Reaction of DNA with Cr(V)ehba. Loss of nucleic acid DNA bases occur through labilization of the glycosidic bond which can arise through both oxidative and nonoxidative processes. H atom abstraction is an oxidative process which can occur at any carbon on deoxyribose to yield free DNA bases. Base release in the reaction of ss- and ds-DNA with Cr(V)ehba was measured by HPLC (Figure 3A), under conditions identical with that for the TBARS reaction described above. The pH dependence for total base release in the reaction of Cr(V)-ehba with ds- and ss-DNA showed a similar reaction profile to that of the TBARS reaction (data not shown). Base release was maximal at more acidic pH and decreased at higher pH. However, substantial base release was observed even at the highest pH tested, pH 7.5. As well, ss-DNA showed a significant increase in total base release over that observed for dsDNA in the reaction with Cr(V)-ehba alone. The reaction profile between Cr(V)-ehba and hydrogen peroxide with ds- and ss-DNA for base release was identical with that observed for the TBARS reaction. The reactivity was lower at higher pH, and a substrate
Sugden and Wetterhahn
Figure 3. Typical HPLC chromatograms for products formed from sugar oxidation: (A) HPLC chromatogram, using HPLC system A from the Experimental Section, showing release of all four nucleic acid bases after treatment of 5 mM ss-DNA with 1.0 mM Cr(V)-ehba and 1.0 mM H2O2; (B) partial HPLC chromatogram showing the formation of 5-MF and furfural after treatment of 5 mM ss-DNA with 1.0 mM Cr(V)-ehba and 1.0 mM H2O2, using HPLC system B from the Experimental Section.
preference for ds-DNA was observed. The overall yield of free bases was significantly greater at every pH tested for this reaction in comparison to the reaction with Cr(V)-ehba alone. Reactions at pH 6.5 were chosen for probing base release specificity, formation of sugar products, and oxygen dependence since this represented a compromise between maximal oxidative activity and physiologically relevant conditions. Specificity of Base Release. Determination of the specificity of base release in the reaction of Cr(V)-ehba with ss- and ds-DNA at pH 6.5 showed the release of all four DNA bases (Figure 4A). With ds-DNA, no specific base was targeted for release, but a general trend was observed that favored the release of the pyrimidines, thymine and cytosine, over the two purines, guanine and adenine. In ds-DNA, this trend was consistent for the full range of pH’s tested, 5.5-7.5. Overall, the pyrimidine DNA base cytosine was released in the greatest amount at all pH’s in ds-DNA. Reaction of Cr(V)-ehba alone with ss-DNA, under identical conditions as that for ds-DNA, displayed an increase in the release of all four DNA bases but unlike ds-DNA showed no preference for release of a specific base (Figure 4A). The reaction of Cr(V)-ehba with ds- and ss-DNA at pH 6.5 in the presence of hydrogen peroxide showed a roughly 8-fold increase in base release over Cr(V)-ehba alone (Figure 4B). With the exception of guanine, all
Chromium(V) Oxidation of Deoxyribose in DNA
Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1401
Table 1. Products from C4′ H Atom Abstraction in the Reaction between Cr(V)-ehba and DNAa glycolic acid (µM)b
TBARS (µM)b
reaction
atmosphere
ds-DNA
ss-DNA
ds-DNA
ss-DNA
Cr(V) Cr(V) Cr(V) + H2O2 Cr(V) + H2O2
O2 argon O2 argon
2.5 ( 1.7 1.3 ( 0.4 11.3 ( 3.4 5.6 ( 1.7
2.8 ( 0.7 1.4 ( 1.1 9.9 ( 2.3 5.5 ( 2.1
3.1 ( 0.3 1.0 ( 0.1 16.7 ( 0.2 17.7 ( 0.3
3.8 ( 0.3 0.6 ( 0.3 16.0 ( 0.4 15.7 ( 0.3
a All reactions were carried out in 100 mM phosphate buffer, pH 6.5, at RT for 1 h in either an oxygen or argon atmosphere with 5.0 mM DNA. b Mean ( SD (n ) 3).
Table 2. Oxygen Dependence for Release of Free Nucleic Acid Bases in the Reaction of Cr(V)-ehba and DNAa total base release (µM)b reaction
atmosphere
ds-DNA
ss-DNA
Cr(V) Cr(V) Cr(V) + H2O2 Cr(V) + H2O2
O2 argon O2 argon
7.5 ( 0.6 6.4 ( 0.2 58.5 ( 10.2 89.6 ( 5.5
11.2 ( 1.4 11.2 ( 1.3 46.2 ( 4.0 78.7 ( 11.8
a All reactions were carried out in 100 mM phosphate buffer, pH 6.5, at RT for 1 h in either an oxygen or argon atmosphere with 5.0 mM DNA. b Mean ( SD (n ) 3).
Figure 4. Specificity of release of nucleic acid bases in the reaction between Cr(V)-ehba and DNA, with and without H2O2: (A) reaction of 1.0 mM Cr(V)-ehba with 5 mM ds- or ssDNA in 100 mM phosphate buffer, pH 6.5, at RT; (B) reaction of 1.0 mM Cr(V)-ehba and 1.0 mM H2O2 with 5 mM ds- or ssDNA in 100 mM phosphate buffer, pH 6.5, at RT.
bases were released in equivalent amounts, with ss-DNA showing less base release than that observed with dsDNA. Free guanine was observed in significantly lower concentrations for both ds- and ss-DNA in these reactions. Oxygen Dependence for the Formation of TBARS and Glycolic Acid. An intermediate in the formation of base propenals is the C4′-peroxy radical which is formed from the addition of oxygen to the C4′ radical of deoxyribose (27). Thus, TBARS formation should be an oxygen-dependent product. Table 1 shows that in the reaction between Cr(V)-ehba and both ds- and ss-DNA, at pH 6.5, the formation of TBARS is indeed oxygendependent, with a 3-4-fold increase observed under an oxygen versus argon atmosphere. Identical reactions with hydrogen peroxide showed a 5-6-fold increase in the formation of TBARS over Cr(V)-ehba alone (Table 1). However, TBARS formation was not oxygen-dependent under these conditions, yielding equivalent levels of TBARS under either oxygen or argon atmospheres. The use of chromate, Cr(VI), did not generate significant TBARS in this assay, and neither Cr(V)-ehba alone nor Cr(V)-ehba in the presence of hydrogen peroxide de-
graded a synthetic base propenal, trans-thyminepropenal, under these reaction conditions. A C4′ oxygen-dependent pathway involving the formation of TBARS/base propenals should also yield a threecarbon sugar fragment. This three-carbon fragment, which remains associated with the DNA backbone, is 3′phosphoglycolate and can be enzymatically cleaved to yield glycolic acid (28). Table 1 shows the formation of glycolic acid in the reaction between both Cr(V)-ehba alone and Cr(V)-ehba with added hydrogen peroxide toward ca. 5 mM ss- or ds-DNA. Quantitation of glycolic acid was hampered by a large background level of glycolic acid in DNA controls. However, the results were consistent with the TBARS results, namely, that glycolic acid formation was substantially greater in Cr(V)-ehba reactions with added hydrogen peroxide than with Cr(V)-ehba alone and that an increase in glycolic acid yield was observed under oxygen. The trend of higher oxidation levels in ss-DNA for Cr(V)-ehba alone, versus higher oxidation levels of ds-DNA for Cr(V)-ehba with hydrogen peroxide, was consistent with that observed for the TBARS reaction and base release. However, the oxygen dependence observed for glycolic acid formation in the reactions containing hydrogen peroxide did not correlate with the oxygen independence of TBARS formation in the reaction of Cr(V)-ehba and hydrogen peroxide with DNA. Oxygen Dependence on Total Base Release. The oxygen dependence for total base release was probed by reacting Cr(V)-ehba and DNA under either an oxygen or argon atmosphere. Cr(V)-ehba alone, when reacted at pH 6.5 with either ds- or ss-DNA, demonstrated no oxygen dependence for release of free nucleic acid bases (Table 2). When Cr(V)-ehba was reacted with ds- or ssDNA in the presence of hydrogen peroxide, an actual increase in total base release was observed under an argon atmosphere versus an oxygen atmosphere for both DNA substrates (Table 2). Both ds- and ss-DNA showed significantly greater base release in the presence of hydrogen peroxide over that observed for Cr(V)-ehba alone. Once again, the opposing pattern of substrate preferences whereby reactions with Cr(V)-ehba alone favor ss-DNA and reactions of Cr(V)-ehba with hydrogen peroxide favor ds-DNA was observed. Formation of 5-MF and Furfural. The reaction of Cr(V)-ehba with ds- and ss-DNA at pH 6.5 with and
1402 Chem. Res. Toxicol., Vol. 10, No. 12, 1997 Table 3. Oxygen Dependence for Formation of 5-MF and Furfural in the Reaction of Cr(V)-ehba and Hydrogen Peroxide with DNAa product 5-MF 5-MF furfural furfural
atmosphere O2 argon O2 argon
ds-DNAb 0.16c
1.73 ( 0.72 ( 0.04c 1.74 ( 0.13c 0.83 ( 0.03c
ss-DNAb 0.74 ( 0.01c 0.41 ( 0.01c 0.19 ( 0.01c 0.18 ( 0.05c
a All reactions were carried out in 100 mM phosphate buffer, pH 6.5, at RT for 1 h in either an oxygen or argon atmosphere with 5.0 mM DNA. b Mean ( SD (n ) 3). c Concentration (µM) of oxidized DNA products.
without added hydrogen peroxide was investigated for formation of the sugar fragments associated with C1′ and C5′ H atom abstraction mechanisms. The product of a C1′ H atom abstraction is the sugar fragment, 5-MF, and a free nucleic acid base (21). The product of a C5′ H atom abstraction is furfural and a free nucleic acid base (22). Both of the sugar fragments were readily resolved and quantified by HPLC (Figure 3B). The reaction of Cr(V)ehba alone with ds- and ss-DNA at pH 6.5 demonstrated no measurable furfural and only minor amounts of 5-MF (
